基于RNA-Seq的稻瘟病菌Δznf1突变体的表达谱分析

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岳晓凤, 阙亚伟, 王政逸. 基于RNA-Seq的稻瘟病菌Δznf1突变体的表达谱分析[J]. , 2016, 49(17): 3347-3358 https://doi.org/10.3864/j.issn.0578-1752.2016.17.009
YUE Xiao-feng, QUE Ya-wei, WANG Zheng-yi. Analysis of RNA-Seq-Based Expression Profiles of Δznf1 Mutants in Magnaporthe oryzae[J]. Scientia Acricultura Sinica, 2016, 49(17): 3347-3358 https://doi.org/10.3864/j.issn.0578-1752.2016.17.009

0 引言

【研究意义】稻瘟病菌（Magnaporthe oryzae）侵染水稻引起的稻瘟病是水稻上的重要病害,每年可造成10%—30%的稻谷产量损失,严重威胁世界各水稻产区粮食生产安全^[1]。由于经济的重要性和分子遗传的可操作性,稻瘟病菌-水稻互作机制已成为研究病原菌-寄主植物互作的模式系统^[1-3]。对稻瘟病菌致病分子机理的深入了解不仅有助于新的稻瘟病防治策略的研发,有效控制病害流行,而且对其他植物病原真菌的研究及其病害防治具有重要的指导意义。【前人研究进展】稻瘟病菌通过形成一种特异性的侵染结构——附着胞侵入寄主细胞^[4]。当分生孢子着落在稻株表面后,在适宜的水分条件下萌发产生芽管,芽管顶端迅速膨大并逐渐分化形成附着胞。成熟的附着胞黑色素化并通过甘油等溶质的积累而产生强大膨压（高达8 MPa）。在膨压的作用下,附着胞产生足够的机械压力驱动稻瘟病菌成功穿透寄主表皮进行侵染,随后鳞茎状的侵染菌丝在细胞内和细胞间扩展。侵染3—5 d后水稻叶片表面形成可见的坏死病斑^[1,5-6]。研究表明,cAMP-PKA信号途径调控早期表面信号识别和附着胞分化,外源添加cAMP能诱导分生孢子在亲水表面形成附着胞^[7-12]。Pmk1-MAPK信号途径调控附着胞成熟、穿透及扩展^[11-14]。PMK1缺失突变体不能形成附着胞,不能穿透寄主表皮,对寄主致病性完全丧失^[13]。Mst12是位于Pmk1下游的转录因子,参与调控侵染钉形成和穿透。Δmst12能形成成熟的附着胞,但不能穿透寄主表皮而完全丧失对寄主的致病性^[15-17]。由于MST12不是附着胞发育与成熟所必需,因此研究者们认为还存在其他受Pmk1调控的转录因子参与附着胞的形成。近期,笔者实验室研究发现了一个C₂H₂型锌指结构转录因子Znf1,其可能位于cAMP-PKA和Pmk1-MAPK信号途径下游,在附着胞介导的稻瘟病菌侵染过程中有重要作用^[18]。Δznf1在营养生长、色素沉积以及有性生殖等方面与野生型菌株没有明显的差异,但Δznf1芽管顶端不能分化形成成熟的附着胞,不能穿透寄主表皮,即使是在划伤或针刺的大麦和水稻叶片表面都不能产生病斑。在稻瘟病菌附着胞发育阶段,Znf1受Pmk1调控,并且同Δpmk1能响应外源cAMP刺激类似,外源cAMP或IBMX能诱导Δznf1芽管顶端分化形成囊泡状的膨大结构。但酵母双杂交实验表明Znf1与Pmk1没有直接的互作^[18]。【本研究切入点】Znf1下游调控的基因尚不明确,Znf1对附着胞相关基因的调控以及与Pmk1的关系有待挖掘,而且对Znf1作用机制的深入研究能够加深对稻瘟病菌附着胞调控机理的认识。本研究通过高通量测序技术,从转录水平上解析Znf1的调控机制。【拟解决的关键问题】利用RNA-Seq（RNA-sequencing）技术对稻瘟病菌野生菌株Guy11和Δznf1突变体进行表达谱分析和差异表达基因筛选,以期为进一步鉴定和研究受Znf1调控的基因提供基础数据。此外,通过比较Δznf1和Δpmk1中的差异表达基因,筛选受Znf1和Pmk1共同调控的基因,深入了解Znf1与Pmk1的关系以及鉴定新的与稻瘟病菌附着胞形成相关基因,从而为完善稻瘟病菌致病分子机理提供数据。

1 材料与方法

1.1 供试材料

RNA-Seq所用的稻瘟病菌野生型Guy11、Δznf1和Δpmk1突变体均为笔者实验室保存或研究获得的菌株。供试菌株在CM液体培养基中振荡培养48 h（28℃,180 r/min）后,2层纱布过滤收集菌丝,经无菌水冲洗数遍后,将水分压干,液氮速冻后储存于-80℃保存备用。每个菌株3个重复。CM培养基配方：10 g·L^-1 glucose, 2 g·L^-1 peptone,1 g·L^-1yeast extract,1 g·L^-1 casamino acids,0.1%（v/v）trace elements,0.1%（v/v）vitamin supplement,6 g·L^-1NaNO₃,0.5 g·L^-1 KCl,0.5 g·L^-1 MgSO₄,1.5 g·L^-1 KH₂PO₄,pH 6.5。

1.2 RNA提取及质量检测

2015年9月样品组织送至深圳华大基因科技服务中心,RNA-Seq测序在该中心进行。采用Trizol法分别提取各菌株的菌丝样品总RNA,并使用DnaseI去除样品中的DNA杂质。通过1%琼脂糖凝胶电泳,以及NanoDrop ND-2000和Agilent 2100对RNA样品进行浓度、纯度和完整性等检测。OD值标准：OD_260/280≥ 1.8,OD_260/230≥1.8;RIN（RNA完整系数）≥8,28S/18S≥1。为了提高试验的准确性,将3个生物学重复样品分别提取RNA,然后等量混合作为一个样本用于RNA-Seq。

1.3 文库构建及RNA-Seq测序

用带有Oligo（dT）的磁珠富集mRNA,向得到的mRNA中加入适量打断试剂高温条件下使其片断化,再以片断化后的mRNA为模板,合成cDNA,经过磁珠纯化、末端修复、3′末端加碱基A、加测序接头后,进行PCR扩增,从而完成整个文库制备工作。构建好的文库用Agilent 2100 Bioanalyzer和ABI StepOne Plus Real-Time PCR System进行质量和产量检测,文库质控合格后进行Illumina HiSeq^TM 2000测序分析,测序反应进行一次。

1.4 基因定量分析及差异基因筛选

基因表达量用FPKM（Fragments Per Kilobase of exon per Million fragments mapped）法计算^[19]。根据野生菌株Guy11和突变体Δznf1或Δpmk1中基因的FPKM值来比较不同样品间的基因差异表达。在本试验分析中,差异基因筛选标准为错误发现率（false discovery rate,FDR）≤0.001且差异倍数在2倍以上即∣log₂（样品间基因表达量的比值）∣≥1。

1.5 差异基因GO功能注释及KEGG Pathway富集分析

将所有差异表达基因向Gene Ontology（GO）数据库（http://www.geneontology.org/）的各个term映射,计算每个term的基因数目,然后应用超几何检验,找出与整个稻瘟病菌基因组相比,在差异表达基因中显著富集的GO条目。得到差异基因的GO注释后,利用WEGO软件对差异基因进行GO功能分类统计^[20]。Pathway显著性富集分析以KEGG Pathway为单位^[21],应用GO富集原理可以得出差异表达基因中显著性富集的Pathway。

1.6 实时荧光定量PCR（qRT-PCR）

为了验证RNA-Seq结果的可靠性,随机选择10个差异表达基因进行qRT-PCR检测,引物见表1。通过Trizol法提取CM液体培养基振荡培养48 h的菌丝总RNA,用反转录试剂盒PrimeScript®RT Reagent Kit With gDNA Eraser（TaKaRa）合成cDNA。采用GoTag^®qPCR Master Mix（Promega）试剂,一个反应体系为20 μL,3个重复。qRT-PCR反应在BIO-RAD CFX96^TM Real-Time System仪上运行。反应程序为：95℃ 30 s,95℃5 s,56℃ 15 s,72℃ 20 s,39个循环。以稻瘟病菌β-tubulin（MGG_00604）为内参基因,通过2^-△△CT法计算基因相对表达水平^[22]。通过3次独立重复试验计算基因相对表达的平均值和标准差。
Table 1
表1
表1本研究qRT-PCR引物
Table 1qRT-PCR primers used in the study

名称Name	序列Sequence (5′-3′)	名称Name	序列Sequence (5′-3′)
Tublin-F	CATACGGTGACCTGAACTAC	Tublin-R	CCATGAAGAAGTGCAGACG
10422-F	TGTCGCCGATCAACACAAGC	10422-R	CATCCGAGTCTCCAAAGTCC
01391-F	AGGACCGACACTACCGTTTC	01391-R	TGCGGTGCAGTTGGCTCAATC
03825-F	ACCGAAGTCGAGATTGTGCG	03825-R	CGCGCTTAAAGTAGTTACGG
09000-F	GGCACGGAGACTACAACTAC	09000-R	ACTTTCGCCTGGTTGATCAG
09994-F	CGATCACTCTCGGTATCTTCC	09994-R	AGGATCGGGAAGAACATGGC
05719-F	AGGACGCTGAGAAGAACACC	05719-R	GCTTCTTGGAGACACCCTTC
03329-F	GCTCTTCCGACTCATTGACG	03329-R	TCTCCTGCAGGTCGAACTTG
02246-F	TGAGGAGTCCAAGCAGCAC	02246-R	TCCTTGGCAGACTCCTTGG
08944-F	CCTGACCAATGGCAACAAGC	08944-R	TTGTCGACGGAGCCCTCAA
11084-F	CCTCCATGGTTCAGGTCAG	11084-R	GTCTCCTCGTGATTCTTGTC

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2 结果

2.1 表达谱测序及测序质量评估

为了深入了解稻瘟病菌中受转录因子ZNF1调控的基因,对稻瘟病菌野生型菌株Guy11以及ZNF1基因缺失突变体菌株Δznf1进行表达谱分析。收集菌株Guy11和Δznf1在液体CM培养基中培养2 d的菌丝,并分别进行样品Total RNA抽提。NanoDrop ND-2000及Agilent 2100对Total RNA的质检结果表明,样品RNA的浓度、纯度及完整性等各项指标达到建库测序要求。进而构建测序文库,文库质控合格后利用Illumina HiSeq^TM 2000进行测序。
测序得到的原始图像数据经base calling转化的序列数据称为raw reads。将低质量序列以及接头序列等杂质raw reads过滤后得到可用于数据分析的clean reads。菌株Guy11和Δznf1分别获得22 100 589和22 074 094个clean reads,分别占原始数据的98.85%和98.72%。对数据质量评估合格后（Q20>95%）,将clean reads比对到水稻稻瘟病菌基因组序列（http://www.broadinstitute.org/annotation/genome/magnaporthe_grisea/MultiHome.html）。比对结果显示,Guy11和Δznf1分别有92.63%和92.37%的clean reads能有效的比对到参考基因组上,并且具有唯一比对位点的reads数分别占比对到基因组上总reads数的89.20%和87.88%（表2）,表明样品测序质量较高。
Table 2
表2
表2样品RNA-Seq序列与参考基因组的比对统计
Table 2Statistical analysis of RNA-Seq reads mapping to the reference genome

Reads比对统计 Statistics of reads mapping	Guy11		Δznf1
Reads比对统计 Statistics of reads mapping	Reads数目 Reads number	百分比 Percentage (%)	Reads数目 Reads number	百分比 Percentage (%)
总reads Total reads	22100589	100.00	22074094	100.00
比对上的reads Total mapped reads	20471630	92.63	20389943	92.37
完美匹配的reads Perfect match reads	17561189	79.46	17578874	79.64
唯一位点匹配的reads Unique match reads	19712923	89.20	19399539	87.88
多位点匹配的reads Multi-position match reads	758707	3.43	990404	4.49
未比对上的reads Total unmapped reads	1628959	7.37	1684151	7.63

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测序饱和度在一定程度上可以评估测序数据量是否达到要求。由图1所示,在测序量（reads数量）较小时,样品检测到的基因数均随着测序量的增加而随之上升,当测序量达到5 M左右后,其增长速度趋于平缓,说明检测到的基因数趋于饱和。菌株Guy11和Δznf1均产生不低于20 M的clean reads,因此可以认为测序检测到的基因数能够基本覆盖细胞表达的全部基因,测序量已达到要求。

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图1RNA-Seq测序饱和度分析
-->Fig. 1Analysis of sequencing saturation
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2.2 Δznf1差异表达基因的筛选

为鉴定受Znf1调控相关基因,对野生型Guy11和Δznf1表达谱进行比较,获得差异表达基因（differentially expressed genes,DEGs）。根据基因的表达量（FPKM值）计算该基因在两个样品间的差异表达倍数,通过控制错误发现率FDR来决定P-value的域值。以∣log₂ratio (Δznf1/Guy11)∣≥1（即倍数差异在2倍以上）,并且FDR≤0.001为筛选标准,共获得709个差异表达基因,其中上调表达基因299个,∣log₂ratio∣值>5（即上调倍数大于32）的有22个,下调表达基因410个,∣log₂ratio∣值>5的有12个（图2-A、2-B;附表1）。

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图2Δznf1差异表达基因分析A：Δznf1差异基因数目统计Statistical analysis of DGEs in Δznf1;B：基因表达量的散点图。横、纵轴分别为Guy11和Δznf1基因表达量的对数值,橙色点表示上调基因,蓝色点表示下调基因,褐色是非显著性差异基因Scatter plot of gene expression. The horizontal and vertical axis were the lg (FPKM) value for all genes in Guy11 and Δznf1, respectively. Orange points represented up-regulated genes, blue points represented down-regulated genes, and brown points represented not significant genes;C：差异基因GO功能分类 GO categories of the DEGs
-->Fig. 2Analysis of differentially expressed genes in Δznf1
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Table S1
附表1
附表1Δznf1菌丝中差异表达基因(|log₂ratio|≥1且 FDR＜0.001)
Table S1Differentially expressed genes in the mycelia of Δznf1(|log₂ratio|≥1 and FDR＜0.001)

基因编号 Gene ID	Log₂ratio (znf1/Guy11)	上调-下调 Up-Down-Regulation	基因描述 Gene description
MGG_11300	16.40789841	Up	hypothetical protein (213 nt)
MGG_15270	12.58871464	Up	predicted protein (441 nt)
MGG_00585	11.93663794	Up	predicted protein (354 nt)
MGG_14301	11.60269887	Up	calpastatin (393 nt)
MGG_07669	11.58871464	Up	predicted protein (351 nt)
MGG_02236	11.29920802	Up	conserved hypothetical protein (366 nt)
MGG_09844	11.03617361	Up	predicted protein (564 nt)
MGG_08940	10.69696753	Up	predicted protein (873 nt)
MGG_05104	10.58871464	Up	predicted protein (576 nt)
MGG_14376	10.39874369	Up	conserved hypothetical protein (2001 nt)
MGG_08946	10.2644426	Up	predicted protein (711 nt)
MGG_08352	9.980139578	Up	isoflavone reductase family protein (999 nt)
MGG_05553	9.797661526	Up	DUF895 domain membrane protein (1446 nt)
MGG_13037	9.550746785	Up	conserved hypothetical protein (1692 nt)
MGG_12423	9.344295908	Up	choline monooxygenase (1308 nt)
MGG_09972	9.276124405	Up	conserved hypothetical protein (1605 nt)
MGG_15318	9.252665432	Up	conserved hypothetical protein (1392 nt)
MGG_00018	9.022367813	Up	integral membrane protein (1650 nt)
MGG_02058	8.965784285	Up	mitochondrial chaperone BCS1 (1701 nt)
MGG_08944	6.118774553	Up	predicted protein (429 nt)
MGG_09659	5.502500341	Up	S-(hydroxymethyl)glutathione dehydrogenase (1233 nt)
MGG_11084	5.112842837	Up	TPR domain-containing protein (2241 nt)
MGG_05551	4.584962501	Up	conserved hypothetical protein (1521 nt)
MGG_15149	4.584962501	Up	conserved hypothetical protein (1521 nt)
MGG_02067	4.024585638	Up	conserved hypothetical protein (738 nt)
MGG_08723	3.855717944	Up	conserved hypothetical protein (972 nt)
MGG_09645	3.749534268	Up	fatty acid synthase (6381 nt)
MGG_08431	3.488286481	Up	conserved hypothetical protein (1044 nt)
MGG_08950	3.485426827	Up	high-affinity glucose transporter (1395 nt)
MGG_05747	3.48174178	Up	predicted protein (426 nt)
MGG_14871	3.466699619	Up	bifunctional molybdenum cofactor biosynthesis protein (801 nt)
MGG_04437	3.37914914	Up	heat shock protein 78 (2421 nt)
MGG_12501	3.378511623	Up	conserved hypothetical protein (1374 nt)
MGG_13924	3.356380304	Up	serine palmitoyltransferase 2 (2250 nt)
MGG_08943	3.331514144	Up	conserved hypothetical protein (957 nt)
MGG_05084	3.328187085	Up	conserved hypothetical protein (636 nt)
MGG_01989	3.266786541	Up	predicted protein (1665 nt)
MGG_07246	3.249359469	Up	conserved hypothetical protein (600 nt)
MGG_02060	3.245873855	Up	aromatic amino acid aminotransferase (1143 nt)
MGG_10934	3.177120503	Up	predicted protein (381 nt)
MGG_08962	3.126031175	Up	conserved hypothetical protein (546 nt)
MGG_14195	3.117512049	Up	predicted protein (417 nt)
MGG_13912	3.10433666	Up	conserved hypothetical protein (1482 nt)
MGG_09100	3.064822001	Up	cutinase (645 nt)
MGG_13116	3.062735755	Up	conserved hypothetical protein (1260 nt)
MGG_13923	3.062685268	Up	predicted protein (582 nt)
MGG_09221	3.03562391	Up	predicted protein (3054 nt)
MGG_08430	3.011713421	Up	retinol dehydrogenase 12 (933 nt)
MGG_03395	2.967185849	Up	predicted protein (471 nt)
MGG_06753	2.921840937	Up	predicted protein (465 nt)
MGG_00822	2.888354644	Up	predicted protein (912 nt)
MGG_10110	2.884080713	Up	conserved hypothetical protein (441 nt)
MGG_07952	2.865316383	Up	predicted protein (516 nt)
MGG_10158	2.857980995	Up	QDE-2-interacting protein (1713 nt)
MGG_05896	2.815494267	Up	predicted protein (309 nt)
MGG_07979	2.776103988	Up	DUF563 domain-containing protein (1554 nt)
MGG_07953	2.759123954	Up	bifunctional P-450:NADPH-P450 reductase (3258 nt)
MGG_08948	2.756784894	Up	predicted protein (666 nt)
MGG_02234	2.748451166	Up	conserved hypothetical protein (537 nt)
MGG_11445	2.727691395	Up	hypothetical protein (750 nt)
MGG_03411	2.677599742	Up	riboflavin transporter MCH5 (1443 nt)
MGG_03158	2.604862058	Up	pumilio-family RNA binding repeat protein (2328 nt)
MGG_10954	2.597562538	Up	isotrichodermin C-15 hydroxylase (1479 nt)
MGG_05132	2.590701406	Up	conserved hypothetical protein (1086 nt)
MGG_08817	2.585869528	Up	conserved hypothetical protein (357 nt)
MGG_02059	2.523561956	Up	conserved hypothetical protein (9060 nt)
MGG_03983	2.514368617	Up	Hsp70 nucleotide exchange factor FES1 (654 nt)
MGG_02350	2.463656204	Up	conserved hypothetical protein (927 nt)
MGG_08371	2.452126818	Up	predicted protein (810 nt)
MGG_00080	2.451025299	Up	hypothetical protein (429 nt)
MGG_10109	2.438920966	Up	conserved hypothetical protein (465 nt)
MGG_09138	2.42053125	Up	glutathione S-transferase II (672 nt)
MGG_09632	2.396352936	Up	conserved hypothetical protein (1044 nt)
MGG_07404	2.387023123	Up	tripeptidyl-peptidase 1 (1806 nt)
MGG_09139	2.38669835	Up	laccase-1 (1794 nt)
MGG_06759	2.364189581	Up	heat shock protein 80 (2109 nt)
MGG_10271	2.342392197	Up	conserved hypothetical protein (3960 nt)
MGG_05554	2.329422631	Up	conserved hypothetical protein (1401 nt)
MGG_02436	2.296338285	Up	conserved hypothetical protein (459 nt)
MGG_10415	2.270089163	Up	predicted protein (2403 nt)
MGG_05564	2.254813899	Up	predicted protein (753 nt)
MGG_06459	2.200152894	Up	heat shock protein HSP98 (2781 nt)
MGG_09474	2.179085001	Up	hypothetical protein (504 nt)
MGG_09241	2.178803153	Up	predicted protein (801 nt)
MGG_02235	2.174370906	Up	oxidoreductase (996 nt)
MGG_07949	2.170357399	Up	conserved hypothetical protein (879 nt)
MGG_10108	2.167556766	Up	conserved hypothetical protein (789 nt)
MGG_08947	2.165337732	Up	cytochrome P450 (1626 nt)
MGG_03115	2.145452682	Up	predicted protein (1401 nt)
MGG_05819	2.131244533	Up	conserved hypothetical protein (690 nt)
MGG_10205	2.111202562	Up	isoleucyl-tRNA synthetase (1269 nt)
MGG_09381	2.104530583	Up	conserved hypothetical protein (474 nt)
MGG_03495	2.096526924	Up	predicted protein (528 nt)
MGG_05824	2.068901305	Up	UDP-glucuronosyl/UDP-glucosyltransferase (1299 nt)
MGG_06958	2.06641867	Up	hsp70-like protein (1956 nt)
MGG_07954	2.03876957	Up	epoxide hydrolase 2 (1017 nt)
MGG_11021	2.02307246	Up	conserved hypothetical protein (2061 nt)
MGG_02372	2.018018704	Up	hypothetical protein (729 nt)
MGG_08424	2.0138058	Up	endo-1,4-beta-xylanase I (696 nt)
MGG_07615	2.006853671	Up	protoporphyrinogen oxidase (693 nt)
MGG_06298	1.989920444	Up	hypothetical protein (1176 nt)
MGG_07646	1.983511877	Up	alpha-glucuronidase (2592 nt)
MGG_10912	1.959738155	Up	mycocerosic acid synthase (7641 nt)
MGG_07219	1.949373927	Up	conidial yellow pigment biosynthesis polyketide synthase (6528 nt)
MGG_14351	1.94240208	Up	glycosyltransferase (1269 nt)
MGG_03471	1.939458458	Up	predicted protein (1176 nt)
MGG_14703	1.937959253	Up	conserved hypothetical protein (387 nt)
MGG_14340	1.934604368	Up	predicted protein (324 nt)
MGG_14375	1.902364997	Up	conserved hypothetical protein (609 nt)
MGG_15049	1.871675903	Up	predicted protein (1944 nt)
MGG_02267	1.866733469	Up	predicted protein (1044 nt)
MGG_10683	1.849665727	Up	predicted protein (1479 nt)
MGG_03238	1.848445436	Up	zinc finger protein ZPR1 (1533 nt)
MGG_02095	1.839416707	Up	conserved hypothetical protein (996 nt)
MGG_08378	1.824913293	Up	cytochrome P450 oxidoreductase GliF (1605 nt)
MGG_04511	1.821029859	Up	MFS transporter (1461 nt)
MGG_07946	1.811554911	Up	conserved hypothetical protein (1494 nt)
MGG_12697	1.798601538	Up	pheromone-regulated membrane protein 10 (2973 nt)
MGG_05505	1.795986108	Up	autophagy protein (Atg22) (1704 nt)
MGG_01410	1.791820579	Up	glutathione S-transferase Gst3 (1035 nt)
MGG_02043	1.777192952	Up	BTB/POZ domain-containing protein (510 nt)
MGG_14675	1.772956761	Up	hypothetical protein (180 nt)
MGG_06616	1.762500686	Up	high-affinity methionine permease (1833 nt)
MGG_11754	1.757836032	Up	heavy metal tolerance protein (2163 nt)
MGG_02096	1.757618704	Up	glycoside hydrolase (2478 nt)
MGG_14523	1.75482247	Up	hypothetical protein (366 nt)
MGG_02002	1.744835012	Up	conserved hypothetical protein (1281 nt)
MGG_02069	1.739398362	Up	glyoxalase/bleomycin resistance protein/dioxygenase (396 nt)
MGG_10573	1.729422654	Up	predicted protein (1473 nt)
MGG_06156	1.727406766	Up	kelch repeat protein (2379 nt)
MGG_14621	1.724556807	Up	conserved hypothetical protein (279 nt)
MGG_15465	1.720557962	Up	hypothetical protein (729 nt)
MGG_11336	1.703898264	Up	conserved hypothetical protein (774 nt)
MGG_07608	1.696188456	Up	predicted protein (375 nt)
MGG_07216	1.691742463	Up	versicolorin reductase (825 nt)
MGG_10921	1.684958291	Up	conserved hypothetical protein (201 nt)
MGG_05515	1.669026766	Up	predicted protein (588 nt)
MGG_08180	1.665397624	Up	conserved hypothetical protein (1125 nt)
MGG_13764	1.664251411	Up	bilirubin oxidase (1890 nt)
MGG_13110	1.662130361	Up	4-hydroxyacetophenone monooxygenase (1914 nt)
MGG_08989	1.660472735	Up	conserved hypothetical protein (816 nt)
MGG_00681	1.647698256	Up	conserved hypothetical protein (1215 nt)
MGG_15364	1.641546029	Up	predicted protein (537 nt)
MGG_09998	1.637095305	Up	hypothetical protein (282 nt)
MGG_09647	1.633461018	Up	2-succinylbenzoate-CoA ligase (1506 nt)
MGG_13019	1.631716269	Up	predicted protein (690 nt)
MGG_11680	1.620690205	Up	AAA family ATPase (2112 nt)
MGG_10327	1.616246789	Up	conserved hypothetical protein (2121 nt)
MGG_15048	1.612022843	Up	NAD-dependent deacetylase sirtuin-5 (1074 nt)
MGG_08949	1.604478457	Up	hypothetical protein (1233 nt)
MGG_00791	1.603109847	Up	Lactamase_B domain containing protein (1077 nt)
MGG_05046	1.579829654	Up	adenine phosphoribosyltransferase (3519 nt)
MGG_03034	1.574139531	Up	conserved hypothetical protein (1581 nt)
MGG_06234	1.567215347	Up	predicted protein (426 nt)
MGG_04627	1.566815154	Up	conserved hypothetical protein (2787 nt)
MGG_00287	1.56664049	Up	predicted protein (528 nt)
MGG_08519	1.565654069	Up	aflatoxin B1 aldehyde reductase member 3 (1002 nt)
MGG_12714	1.563004767	Up	conserved hypothetical protein (930 nt)
MGG_14872	1.551770974	Up	calpain-9 (2604 nt)
MGG_08941	1.543130572	Up	predicted protein (615 nt)
MGG_13562	1.541985428	Up	conserved hypothetical protein (1593 nt)
MGG_01454	1.541813265	Up	conserved hypothetical protein (720 nt)
MGG_01364	1.531668886	Up	conserved hypothetical protein (2925 nt)
MGG_08980	1.528536579	Up	stress-induced-phosphoprotein 1 (1755 nt)
MGG_05418	1.526747887	Up	predicted protein (852 nt)
MGG_11991	1.516744272	Up	predicted protein (252 nt)
MGG_15420	1.515463451	Up	predicted protein (498 nt)
MGG_07766	1.514203204	Up	predicted protein (372 nt)
MGG_03396	1.501631699	Up	conserved hypothetical protein (2049 nt)
MGG_04736	1.500318333	Up	conserved hypothetical protein (912 nt)
MGG_15188	1.496902147	Up	conserved hypothetical protein (567 nt)
MGG_13118	1.49523234	Up	predicted protein (555 nt)
MGG_02065	1.492089159	Up	kinesin light chain (2622 nt)
MGG_05968	1.483038711	Up	endoribonuclease L-PSP (411 nt)
MGG_02977	1.479542529	Up	predicted protein (1809 nt)
MGG_02854	1.47566256	Up	conserved hypothetical protein (942 nt)
MGG_05555	1.474172623	Up	xenobiotic compound monooxygenase (1476 nt)
MGG_08726	1.47279708	Up	hypothetical protein (588 nt)
MGG_00809	1.472177608	Up	predicted protein (1770 nt)
MGG_06957	1.449286928	Up	dienelactone hydrolase family protein (852 nt)
MGG_11779	1.447112965	Up	conserved hypothetical protein (5598 nt)
MGG_07402	1.436863862	Up	conserved hypothetical protein (465 nt)
MGG_13253	1.434425855	Up	choline dehydrogenase (1977 nt)
MGG_08373	1.427917291	Up	conserved hypothetical protein (552 nt)
MGG_01439	1.425133377	Up	inorganic phosphate transporter PHO84 (2352 nt)
MGG_13839	1.42203958	Up	predicted protein (408 nt)
MGG_14692	1.420240156	Up	conserved hypothetical protein (1233 nt)
MGG_00832	1.402635441	Up	cytochrome P450 (1605 nt)
MGG_15168	1.402503146	Up	predicted protein (1179 nt)
MGG_15151	1.40118504	Up	hypothetical protein (900 nt)
MGG_06779	1.394108478	Up	NADP-dependent mannitol dehydrogenase (816 nt)
MGG_08486	1.385724603	Up	beta-lactamase family protein (1698 nt)
MGG_09019	1.3852473	Up	secretory phospholipase A2 (561 nt)
MGG_03585	1.381283373	Up	predicted protein (420 nt)
MGG_08429	1.381090167	Up	serin endopeptidas (2595 nt)
MGG_04258	1.375077953	Up	predicted protein (423 nt)
MGG_08677	1.358748368	Up	predicted protein (1065 nt)
MGG_02252	1.352329115	Up	tetrahydroxynaphthalene reductase (852 nt)
MGG_07078	1.347104217	Up	DUF775 domain-containing protein (609 nt)
MGG_08434	1.34533795	Up	conserved hypothetical protein (3564 nt)
MGG_15466	1.339875928	Up	conserved hypothetical protein (327 nt)
MGG_15150	1.339875928	Up	conserved hypothetical protein (327 nt)
MGG_02056	1.337755578	Up	predicted protein (1062 nt)
MGG_05590	1.337107236	Up	zeaxanthin epoxidase (1197 nt)
MGG_09131	1.334601925	Up	alpha/beta hydrolase (861 nt)
MGG_09168	1.330645312	Up	predicted protein (732 nt)
MGG_00156	1.330364932	Up	conserved hypothetical protein (966 nt)
MGG_10272	1.329656431	Up	conserved hypothetical protein (864 nt)
MGG_00963	1.327804661	Up	predicted protein (2850 nt)
MGG_02648	1.327556301	Up	interferon-induced GTP-binding protein Mx (2259 nt)
MGG_07607	1.303355989	Up	conserved hypothetical protein (477 nt)
MGG_10207	1.295674855	Up	predicted protein (1449 nt)
MGG_05574	1.292180751	Up	conserved hypothetical protein (1539 nt)
MGG_05419	1.290864083	Up	predicted protein (2484 nt)
MGG_08451	1.288093497	Up	predicted protein (333 nt)
MGG_08029	1.281729267	Up	conserved hypothetical protein (942 nt)
MGG_11682	1.281189416	Up	phenylacetone monooxygenase (1674 nt)
MGG_10450	1.276331228	Up	conserved hypothetical protein (3099 nt)
MGG_00399	1.268384642	Up	DUF833 domain-containing protein (1191 nt)
MGG_07146	1.262639835	Up	glycosyl hydrolase (2286 nt)
MGG_00282	1.258104518	Up	conserved hypothetical protein (2610 nt)
MGG_08104	1.254630144	Up	41 kDa peptidyl-prolyl cis-trans isomerase (1131 nt)
MGG_01951	1.25334101	Up	4-coumarate-CoA ligase 2 (2010 nt)
MGG_05012	1.248761682	Up	predicted protein (453 nt)
MGG_00814	1.246729595	Up	Hsp90 co-chaperone AHA1 (984 nt)
MGG_01420	1.239727032	Up	conserved hypothetical protein (1524 nt)
MGG_11409	1.239290946	Up	predicted protein (363 nt)
MGG_15105	1.235134723	Up	predicted protein (795 nt)
MGG_07367	1.22804954	Up	conserved hypothetical protein (831 nt)
MGG_10911	1.22795486	Up	cupin domain-containing protein (597 nt)
MGG_08158	1.217117479	Up	predicted protein (993 nt)
MGG_05978	1.216910356	Up	conserved hypothetical protein (564 nt)
MGG_05913	1.213091603	Up	hypothetical protein (657 nt)
MGG_10716	1.196161698	Up	conserved hypothetical protein (540 nt)
MGG_04757	1.193739826	Up	conserved hypothetical protein (1485 nt)
MGG_05059	1.193074321	Up	scytalone dehydratase (519 nt)
MGG_04346	1.189968987	Up	sterol 24-C-methyltransferase (1173 nt)
MGG_15230	1.186570067	Up	hypothetical protein (207 nt)
MGG_05611	1.181656367	Up	hypothetical protein (243 nt)
MGG_09384	1.160059648	Up	conserved hypothetical protein (1542 nt)
MGG_05486	1.158382958	Up	conserved hypothetical protein (1053 nt)
MGG_08990	1.15218757	Up	predicted protein (882 nt)
MGG_07571	1.142006602	Up	LysM domain-containing protein (429 nt)
MGG_07227	1.141693504	Up	hypothetical protein (468 nt)
MGG_06155	1.137106032	Up	hypothetical protein (1782 nt)
MGG_00834	1.136488996	Up	hypothetical protein (987 nt)
MGG_03822	1.128882769	Up	peptidase family T4 (1185 nt)
MGG_14857	1.126377286	Up	conserved hypothetical protein (1050 nt)
MGG_15342	1.12210343	Up	predicted protein (1407 nt)
MGG_08459	1.118055762	Up	ent-kaurene oxidase (1422 nt)
MGG_02114	1.117237675	Up	conserved hypothetical protein (2439 nt)
MGG_02064	1.115477217	Up	conserved hypothetical protein (1167 nt)
MGG_02105	1.112563575	Up	conserved hypothetical protein (915 nt)
MGG_05985	1.110182918	Up	conserved hypothetical protein (2136 nt)
MGG_03005	1.109080361	Up	conserved hypothetical protein (1332 nt)
MGG_08007	1.107108893	Up	conserved hypothetical protein (762 nt)
MGG_00046	1.097054684	Up	conserved hypothetical protein (2739 nt)
MGG_11608	1.096844325	Up	laccase-2 (1794 nt)
MGG_15462	1.095964604	Up	hypothetical protein (222 nt)
MGG_03442	1.092775669	Up	conserved hypothetical protein (702 nt)
MGG_01596	1.088810859	Up	DNA damage response protein kinase DUN1 (2025 nt)
MGG_14651	1.08767481	Up	conserved hypothetical protein (696 nt)
MGG_04242	1.081893423	Up	bax Inhibitor family protein (786 nt)
MGG_00121	1.08161991	Up	conserved hypothetical protein (537 nt)
MGG_00292	1.079165336	Up	conserved hypothetical protein (612 nt)
MGG_09664	1.075597619	Up	beta-mannosidase (2739 nt)
MGG_06131	1.073159746	Up	conserved hypothetical protein (720 nt)
MGG_08612	1.072360223	Up	predicted protein (1851 nt)
MGG_05940	1.070943772	Up	short chain dehydrogenase (780 nt)
MGG_07577	1.070389328	Up	conserved hypothetical protein (1821 nt)
MGG_02061	1.067002855	Up	conserved hypothetical protein (2025 nt)
MGG_07315	1.066011253	Up	predicted protein (411 nt)
MGG_00078	1.063578796	Up	conserved hypothetical protein (1188 nt)
MGG_10613	1.054447784	Up	retinol dehydrogenase 11 (975 nt)
MGG_08142	1.052911531	Up	conserved hypothetical protein (1305 nt)
MGG_05567	1.05288558	Up	conserved hypothetical protein (573 nt)
MGG_06610	1.051720297	Up	lipase (1203 nt)
MGG_03098	1.049687124	Up	conserved hypothetical protein (984 nt)
MGG_09328	1.048429433	Up	conserved hypothetical protein (1113 nt)
MGG_07818	1.043795118	Up	conserved hypothetical protein (2484 nt)
MGG_11355	1.041169189	Up	predicted protein (1995 nt)
MGG_01489	1.04029647	Up	conserved hypothetical protein (2940 nt)
MGG_14061	1.039647809	Up	oxalate decarboxylase oxdC (1503 nt)
MGG_10896	1.038395379	Up	allantoate permease (1578 nt)
MGG_05656	1.035868248	Up	ankyrin repeat protein (5400 nt)
MGG_00591	1.034995421	Up	predicted protein (1677 nt)
MGG_04569	1.029318937	Up	NADPH dehydrogenase (1329 nt)
MGG_00738	1.025321666	Up	zinc-binding alcohol dehydrogenase domain-containing protein 2 (1020 nt)
MGG_13360	1.021398037	Up	conserved hypothetical protein (1653 nt)
MGG_02355	1.018265026	Up	predicted protein (687 nt)
MGG_10097	1.017080484	Up	intracellular hyphae protein 1 (489 nt)
MGG_08020	1.014080909	Up	endoglucanase-4 (729 nt)
MGG_13119	1.009407453	Up	predicted protein (1401 nt)
MGG_12222	1.009179427	Up	F-box domain-containing protein (1383 nt)
MGG_02445	1.008878151	Up	predicted protein (801 nt)
MGG_05045	1.008787304	Up	GTP cyclohydrolase I (777 nt)
MGG_10893	1.00877021	Up	conserved hypothetical protein (1548 nt)
MGG_01692	1.008721271	Up	conserved hypothetical protein (1206 nt)
MGG_05880	1.00264149	Up	glycerol uptake facilitator protein (1173 nt)
MGG_14668	-12.80976813	Down	hypothetical protein (228 nt)
MGG_11005	-12.36632221	Down	conserved hypothetical protein (1368 nt)
MGG_14997	-12.14210706	Down	hypothetical protein (219 nt)
MGG_14426	-11.69696753	Down	conserved hypothetical protein (609 nt)
MGG_14841	-11.62113611	Down	predicted protein (342 nt)
MGG_07726	-11.50183718	Down	conserved hypothetical protein (369 nt)
MGG_15120	-11.15481811	Down	hypothetical protein (399 nt)
MGG_09144	-10.96578428	Down	predicted protein (450 nt)
MGG_14926	-10.92925841	Down	predicted protein (531 nt)
MGG_10040	-10.12928302	Down	ferulic acid esterase A (1023 nt)
MGG_01975	-9.252665432	Down	predicted protein (2526 nt)
MGG_09490	-8.965784285	Down	predicted protein (1701 nt)
MGG_15409	-4.635588574	Down	predicted protein (432 nt)
MGG_03044	-4.310142181	Down	conserved hypothetical protein (1101 nt)
MGG_02942	-4.161723188	Down	conserved hypothetical protein (1749 nt)
MGG_07807	-4.138216142	Down	conserved hypothetical protein (648 nt)
MGG_09436	-4.034269902	Down	conserved hypothetical protein (303 nt)
MGG_09879	-3.992938336	Down	conserved hypothetical protein (438 nt)
MGG_03349	-3.849175098	Down	auxin Efflux Carrier superfamily (1314 nt)
MGG_00766	-3.619372938	Down	conserved hypothetical protein (558 nt)
MGG_00824	-3.572889668	Down	phenylacetone monooxygenase (1890 nt)
MGG_10751	-3.440572591	Down	peroxisomal copper amine oxidase (2025 nt)
MGG_01298	-3.423211431	Down	malic acid transport protein (1323 nt)
MGG_07857	-3.413850584	Down	conserved hypothetical protein (1125 nt)
MGG_10650	-3.408267701	Down	hypothetical protein (291 nt)
MGG_03825	-3.273018494	Down	isotrichodermin C-15 hydroxylase (1617 nt)
MGG_05250	-3.245205238	Down	conserved hypothetical protein (2520 nt)
MGG_10593	-3.2410081	Down	predicted protein (456 nt)
MGG_14237	-3.214407437	Down	predicted protein (288 nt)
MGG_09994	-3.174512271	Down	lipid phosphate phosphatase 1 (1140 nt)
MGG_03329	-3.169891506	Down	small heat shock protein (633 nt)
MGG_05165	-3.15264701	Down	conserved hypothetical protein (1533 nt)
MGG_02290	-3.141142101	Down	conserved hypothetical protein (858 nt)
MGG_08416	-3.070389328	Down	lipase 1 (1674 nt)
MGG_01391	-3.070166018	Down	ent-kaurene oxidase (1569 nt)
MGG_07713	-3.06833713	Down	predicted protein (453 nt)
MGG_09418	-3.02188266	Down	conserved hypothetical protein (429 nt)
MGG_11915	-2.901295133	Down	predicted protein (393 nt)
MGG_08971	-2.886958693	Down	conserved hypothetical protein (573 nt)
MGG_08326	-2.881995905	Down	conserved hypothetical protein (1416 nt)
MGG_10322	-2.87570589	Down	cortical patch protein (765 nt)
MGG_02336	-2.814444347	Down	isoflavone reductase family protein (981 nt)
MGG_09134	-2.765762647	Down	hypothetical protein (357 nt)
MGG_04171	-2.721732314	Down	predicted protein (1050 nt)
MGG_14581	-2.718799594	Down	hypothetical protein (744 nt)
MGG_07348	-2.697353738	Down	predicted protein (420 nt)
MGG_04075	-2.688913465	Down	lambda-crystallin (993 nt) Note(s): contains exonic Ns
MGG_10713	-2.660707862	Down	conserved hypothetical protein (813 nt)
MGG_02329	-2.649306721	Down	isotrichodermin C-15 hydroxylase (1632 nt)
MGG_00733	-2.605714662	Down	conserved hypothetical protein (1026 nt)
MGG_07354	-2.579487371	Down	glutathionylspermidine synthase (1296 nt)
MGG_12800	-2.531615468	Down	conserved hypothetical protein (1257 nt)
MGG_01152	-2.496200352	Down	conserved hypothetical protein (549 nt)
MGG_07871	-2.493092449	Down	conserved hypothetical protein (726 nt)
MGG_08498	-2.492768803	Down	isotrichodermin C-15 hydroxylase (1506 nt)
MGG_07697	-2.470862199	Down	superoxide dismutase (642 nt)
MGG_07556	-2.435386145	Down	conserved hypothetical protein (672 nt)
MGG_04699	-2.425911543	Down	C2H2 finger domain-containing protein FlbC (1179 nt)
MGG_12865	-2.412767326	Down	conserved hypothetical protein (1344 nt)
MGG_06337	-2.409390936	Down	conserved hypothetical protein (3441 nt)
MGG_00040	-2.400827014	Down	high-affinity glucose transporter ght2 (1713 nt)
MGG_11876	-2.393623513	Down	sugar transporter STL1 (1722 nt)
MGG_01811	-2.370837695	Down	conserved hypothetical protein (3165 nt)
MGG_01368	-2.345597001	Down	conserved hypothetical protein (714 nt)
MGG_10714	-2.315031003	Down	cyanide hydratase (1071 nt)
MGG_03739	-2.30256277	Down	predicted protein (2823 nt)
MGG_03977	-2.298702913	Down	hypothetical protein (1389 nt)
MGG_01369	-2.295885623	Down	alpha/beta hydrolase fold-3 domain-containing protein (1044 nt)
MGG_10429	-2.277318632	Down	conserved hypothetical protein (963 nt)
MGG_06513	-2.271567134	Down	hypothetical protein (369 nt)
MGG_05804	-2.269857074	Down	phospholipase D p1 (2574 nt)
MGG_13655	-2.268229568	Down	conserved hypothetical protein (1416 nt)
MGG_09445	-2.266500219	Down	conserved hypothetical protein (1437 nt)
MGG_07088	-2.257691984	Down	hypothetical protein (315 nt)
MGG_12086	-2.254241287	Down	conserved hypothetical protein (759 nt)
MGG_05798	-2.248888349	Down	conserved hypothetical protein (651 nt)
MGG_09473	-2.24858331	Down	hypothetical protein (354 nt)
MGG_09000	-2.230954435	Down	protein kinase domain-containing protein (2160 nt)
MGG_10268	-2.209640685	Down	MAP kinase kinase PBS2 (2058 nt)
MGG_10498	-2.201633861	Down	conserved hypothetical protein (1044 nt)
MGG_08405	-2.190222416	Down	retinol dehydrogenase 13 (969 nt)
MGG_06545	-2.168411949	Down	conserved hypothetical protein (2358 nt)
MGG_08439	-2.161473337	Down	predicted protein (846 nt)
MGG_13198	-2.159984689	Down	predicted protein (270 nt)
MGG_09261	-2.13827611	Down	hypothetical protein (969 nt)
MGG_02340	-2.118644496	Down	cell wall glycosyl hydrolase YteR (1218 nt)
MGG_04254	-2.107640723	Down	conserved hypothetical protein (621 nt)
MGG_01431	-2.102966579	Down	predicted protein (462 nt)
MGG_03469	-2.083854051	Down	predicted protein (714 nt)
MGG_02943	-2.079420312	Down	predicted protein (1011 nt)
MGG_15283	-2.073777926	Down	NAD dependent epimerase/dehydratase family protein (804 nt)
MGG_01542	-2.072756342	Down	endo-1,4-beta-xylanase (996 nt)
MGG_00127	-2.070866331	Down	saccharopine dehydrogenase (1290 nt)
MGG_05719	-2.05779793	Down	30 kDa heat shock protein (723 nt)
MGG_09851	-2.05288558	Down	predicted protein (1029 nt)
MGG_08277	-2.051977291	Down	predicted protein (867 nt)
MGG_12366	-2.051002903	Down	hypothetical protein (162 nt)
MGG_07356	-2.050626073	Down	predicted protein (1701 nt)
MGG_02850	-2.044818504	Down	conserved hypothetical protein (846 nt)
MGG_10503	-2.038908043	Down	mannitol-1-phosphate 5-dehydrogenase (1203 nt)
MGG_21009	-2.035128223	Down	Magnaporthe grisea 70-15 mitochondria cytochrome c oxidase subunit I cox1 (1599 nt)
MGG_02709	-2.016440916	Down	conserved hypothetical protein (465 nt)
MGG_10456	-2.014882446	Down	hypothetical protein (450 nt)
MGG_15428	-2.012748493	Down	para-nitrobenzyl esterase (1674 nt)
MGG_03764	-2	Down	salicylate hydroxylase (1395 nt)
MGG_08059	-1.99175344	Down	conserved hypothetical protein (615 nt)
MGG_15427	-1.991282783	Down	predicted protein (705 nt)
MGG_10497	-1.989731665	Down	blue light-inducible protein Bli-3 (627 nt)
MGG_02803	-1.972238488	Down	hypothetical protein (207 nt)
MGG_04251	-1.96800463	Down	phosphate transporter family protein (1782 nt)
MGG_10107	-1.950287788	Down	caleosin domain-containing protein (750 nt)
MGG_04561	-1.942052651	Down	DNA repair family protein (912 nt)
MGG_12471	-1.921390165	Down	predicted protein (735 nt)
MGG_02302	-1.920565533	Down	HhH-GPD family base excision DNA repair protein (1128 nt)
MGG_09001	-1.915662644	Down	hypothetical protein (474 nt)
MGG_00114	-1.914793825	Down	conserved hypothetical protein (594 nt)
MGG_00677	-1.902180799	Down	endoglucanase-1 (774 nt)
MGG_06865	-1.901041095	Down	hypothetical protein (450 nt)
MGG_00993	-1.89630787	Down	vacuolar iron transporter Ccc1 (1014 nt)
MGG_03643	-1.885795281	Down	conserved hypothetical protein (3816 nt)
MGG_15068	-1.872299651	Down	hypothetical protein (399 nt)
MGG_11813	-1.864915077	Down	predicted protein (738 nt)
MGG_00126	-1.862496476	Down	conserved hypothetical protein (2886 nt)
MGG_01373	-1.846069502	Down	arabinose-proton symporter (1728 nt)
MGG_14292	-1.812914447	Down	aminopeptidase Y (1440 nt)
MGG_03341	-1.801095932	Down	galactose-proton symporter (1716 nt)
MGG_07806	-1.800230488	Down	conserved hypothetical protein (1230 nt)
MGG_04203	-1.799556551	Down	4-coumarate-CoA ligase 1 (1671 nt)
MGG_10730	-1.793549123	Down	calcium-transporting ATPase 3 (3285 nt)
MGG_01544	-1.788997336	Down	pisatin demethylase (1464 nt)
MGG_09852	-1.788893386	Down	sugar transporter STL1 (1707 nt)
MGG_08883	-1.771558534	Down	conserved hypothetical protein (2241 nt)
MGG_03987	-1.767305023	Down	predicted protein (1773 nt)
MGG_14146	-1.764469109	Down	hypothetical protein (384 nt)
MGG_09828	-1.763810581	Down	vacuolar conductance protein (1959 nt)
MGG_10515	-1.745603911	Down	conserved hypothetical protein (966 nt)
MGG_04165	-1.74159068	Down	conserved hypothetical protein (3669 nt)
MGG_02752	-1.734749061	Down	hypothetical protein (1071 nt)
MGG_03575	-1.73033967	Down	conserved hypothetical protein (372 nt)
MGG_02893	-1.727525065	Down	conserved hypothetical protein (468 nt)
MGG_10147	-1.727109398	Down	multidrug resistance protein fnx1 (1719 nt)
MGG_02641	-1.724508709	Down	conserved hypothetical protein (873 nt)
MGG_14591	-1.715023041	Down	cytochrome P450 3A24 (1617 nt)
MGG_05358	-1.714428705	Down	LYR family protein (267 nt)
MGG_06567	-1.708110731	Down	conserved hypothetical protein (996 nt)
MGG_09323	-1.703160095	Down	phenylacetone monooxygenase (1944 nt)
MGG_08581	-1.701809149	Down	conserved hypothetical protein (1278 nt)
MGG_08770	-1.700439718	Down	conserved hypothetical protein (1539 nt)
MGG_09260	-1.697890657	Down	integral membrane protein (495 nt)
MGG_05750	-1.695030732	Down	conserved hypothetical protein (933 nt)
MGG_13798	-1.686291456	Down	predicted protein (915 nt)
MGG_07966	-1.684741955	Down	phosphate transporter family protein (1767 nt)
MGG_09818	-1.683468858	Down	conserved hypothetical protein (1359 nt)
MGG_02929	-1.675273272	Down	phosphotransferase enzyme family protein (789 nt)
MGG_10359	-1.673771768	Down	conserved hypothetical protein (1248 nt)
MGG_09116	-1.672705395	Down	xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (2433 nt)
MGG_04257	-1.672160943	Down	secretory lipase family protein (1269 nt)
MGG_02117	-1.671216173	Down	conserved hypothetical protein (462 nt)
MGG_07890	-1.667424661	Down	aldehyde dehydrogenase 3I1 (1482 nt)
MGG_05164	-1.665356281	Down	conserved hypothetical protein (1704 nt)
MGG_02246	-1.659294698	Down	conserved hypothetical protein (258 nt)
MGG_02332	-1.661898167	Down	conserved hypothetical protein (810 nt)
MGG_01376	-1.657020649	Down	tyrosine-protein phosphatase 1 (3420 nt)
MGG_01945	-1.653849412	Down	predicted protein (729 nt)
MGG_03336	-1.651154546	Down	LEA domain-containing protein (405 nt)
MGG_08994	-1.649813645	Down	conserved hypothetical protein (726 nt)
MGG_10973	-1.647520814	Down	conserved hypothetical protein (570 nt)
MGG_10422	-1.646850887	Down	C6 transcription factor OefC (2112 nt)
MGG_09985	-1.628174568	Down	hypothetical protein (1347 nt)
MGG_14578	-1.620712526	Down	predicted protein (855 nt)
MGG_04628	-1.615245068	Down	cytochrome P450 51 (1548 nt)
MGG_07816	-1.607682577	Down	predicted protein (486 nt)
MGG_13428	-1.605463615	Down	mitochondrial phosphate carrier protein (945 nt)
MGG_12530	-1.59724083	Down	histidine kinase G7 (3768 nt)
MGG_01798	-1.587742596	Down	conserved hypothetical protein (906 nt)
MGG_09351	-1.580096746	Down	aspergillopepsin-F (1293 nt)
MGG_12664	-1.579226131	Down	conserved hypothetical protein (777 nt)
MGG_06181	-1.578735926	Down	predicted protein (1539 nt)
MGG_03246	-1.5776577	Down	conserved hypothetical protein (456 nt)
MGG_01463	-1.576467707	Down	phosphate metabolism protein 7 (2682 nt)
MGG_10092	-1.574527048	Down	hypothetical protein (1086 nt)
MGG_09857	-1.574236094	Down	sorbitol dehydrogenase (1116 nt)
MGG_00097	-1.569986896	Down	conserved hypothetical protein (972 nt)
MGG_01260	-1.566081696	Down	serine/threonine-protein kinase psk1 (1770 nt)
MGG_08547	-1.55844129	Down	serine/threonine-protein kinase srk1 (1872 nt)
MGG_09954	-1.557777671	Down	conserved hypothetical protein (3147 nt)
MGG_08552	-1.555690119	Down	hypothetical protein (351 nt)
MGG_10394	-1.553362167	Down	conserved hypothetical protein (618 nt)
MGG_10053	-1.552972386	Down	conserved hypothetical protein (867 nt)
MGG_15228	-1.538419915	Down	predicted protein (354 nt)
MGG_05897	-1.535135392	Down	predicted protein (462 nt)
MGG_06956	-1.531940639	Down	predicted protein (636 nt)
MGG_05988	-1.5261405	Down	lipid phosphate phosphatase 2 (1239 nt)
MGG_05993	-1.524431255	Down	conserved hypothetical protein (2223 nt)
MGG_00830	-1.519972879	Down	conserved hypothetical protein (1908 nt)
MGG_00033	-1.51815913	Down	conserved hypothetical protein (1605 nt)
MGG_06347	-1.514836414	Down	conserved hypothetical protein (441 nt)
MGG_04922	-1.512820264	Down	alpha-N-arabinofuranosidase 2 (1083 nt)
MGG_01069	-1.510674464	Down	hypothetical protein (312 nt)
MGG_05692	-1.509837762	Down	conserved hypothetical protein (681 nt)
MGG_14793	-1.507927117	Down	hypothetical protein (306 nt)
MGG_12612	-1.502500341	Down	drug resistance transporter (1887 nt)
MGG_11560	-1.501217831	Down	taurine catabolism dioxygenase TauD (1125 nt)
MGG_09511	-1.49128203	Down	2-nitropropane dioxygenase (990 nt)
MGG_07270	-1.48244442	Down	fatty aldehyde dehydrogenase (1647 nt)
MGG_14681	-1.477507164	Down	conserved hypothetical protein (1110 nt)
MGG_15174	-1.476305437	Down	predicted protein (342 nt)
MGG_14951	-1.471998252	Down	conserved hypothetical protein (3174 nt)
MGG_04248	-1.471121655	Down	conserved hypothetical protein (2223 nt)
MGG_05140	-1.465508537	Down	conserved hypothetical protein (1086 nt)
MGG_03245	-1.461904104	Down	aldose 1-epimerase (1215 nt)
MGG_00815	-1.451773288	Down	predicted protein (738 nt)
MGG_01993	-1.450147629	Down	predicted protein (306 nt)
MGG_10315	-1.443282704	Down	hydrophobin (339 nt)
MGG_13651	-1.435823052	Down	high-affinity glucose transporter (1605 nt)
MGG_09091	-1.434294618	Down	adiponectin receptor protein 1 (903 nt)
MGG_01456	-1.433441793	Down	conserved hypothetical protein (1674 nt)
MGG_10269	-1.424797693	Down	auxin Efflux Carrier superfamily (1818 nt)
MGG_04594	-1.422470257	Down	conserved hypothetical protein (1410 nt)
MGG_12899	-1.422161934	Down	DNA ligase 4 (3006 nt)
MGG_06260	-1.418952549	Down	conserved hypothetical protein (2727 nt)
MGG_03491	-1.418713157	Down	conserved hypothetical protein (1458 nt)
MGG_07211	-1.418645279	Down	conserved hypothetical protein (1482 nt)
MGG_08697	-1.414463064	Down	conserved hypothetical protein (3480 nt)
MGG_09533	-1.412206292	Down	predicted protein (501 nt)
MGG_09551	-1.403741594	Down	chitin synthase 3 (2673 nt)
MGG_12804	-1.398733231	Down	conserved hypothetical protein (2241 nt)
MGG_05871	-1.397592365	Down	integral membrane protein (1896 nt)
MGG_00268	-1.396421821	Down	conserved hypothetical protein (2049 nt)
MGG_09447	-1.392923114	Down	predicted protein (684 nt)
MGG_06364	-1.390057478	Down	hypothetical protein (1398 nt)
MGG_10452	-1.388423507	Down	predicted protein (756 nt)
MGG_09817	-1.388052876	Down	minor extracellular protease vpr (2664 nt)
MGG_05051	-1.385121921	Down	predicted protein (864 nt)
MGG_05114	-1.380370367	Down	hypothetical protein (1662 nt)
MGG_06863	-1.379399163	Down	conserved hypothetical protein (1134 nt)
MGG_09435	-1.37926127	Down	conserved hypothetical protein (1719 nt)
MGG_05722	-1.378511623	Down	conserved hypothetical protein (1755 nt)
MGG_02401	-1.374703835	Down	conserved hypothetical protein (1950 nt)
MGG_01673	-1.373458396	Down	conserved hypothetical protein (1788 nt)
MGG_00899	-1.372926068	Down	tubulin-specific chaperone C (1056 nt)
MGG_13008	-1.370486472	Down	predicted protein (384 nt)
MGG_04835	-1.367605669	Down	conserved hypothetical protein (1008 nt)
MGG_15050	-1.367277082	Down	conserved hypothetical protein (1203 nt)
MGG_11945	-1.360317981	Down	candidapepsin precursor (1560 nt)
MGG_06866	-1.3594841	Down	conserved hypothetical protein (759 nt)
MGG_06781	-1.357079427	Down	conserved hypothetical protein (1365 nt)
MGG_03392	-1.357010554	Down	cytochrome P450 11B1, mitochondrial precursor (1527 nt)
MGG_03941	-1.350682695	Down	conserved hypothetical protein (897 nt)
MGG_09444	-1.34612779	Down	methylitaconate delta2-delta3-isomerase (1278 nt)
MGG_10727	-1.342332724	Down	conserved hypothetical protein (1569 nt)
MGG_10585	-1.340360376	Down	monooxygenase (1008 nt)
MGG_10401	-1.335858602	Down	predicted protein (882 nt)
MGG_14590	-1.327050328	Down	conserved hypothetical protein (1344 nt)
MGG_09875	-1.326221123	Down	CAS1 domain-containing protein 1 (828 nt)
MGG_15134	-1.324180547	Down	conserved hypothetical protein (1029 nt)
MGG_01612	-1.320419788	Down	hypothetical protein (1818 nt)
MGG_05938	-1.319541502	Down	2,2-dialkylglycine decarboxylase (1383 nt)
MGG_06888	-1.315767594	Down	glutamine synthetase (978 nt)
MGG_02081	-1.3152412	Down	conserved hypothetical protein (1638 nt)
MGG_01025	-1.314815369	Down	predicted protein (717 nt)
MGG_01311	-1.313320922	Down	nuclear elongation and deformation protein 1 (2298 nt)
MGG_12302	-1.313253761	Down	predicted protein (414 nt)
MGG_14205	-1.306570073	Down	conserved hypothetical protein (1107 nt)
MGG_08776	-1.306195169	Down	major myo-inositol transporter iolT (1899 nt)
MGG_04952	-1.303477942	Down	predicted protein (522 nt)
MGG_11239	-1.303475332	Down	RING finger protein (2259 nt)
MGG_03550	-1.29736681	Down	CDP-diacylglycerol-serine O-phosphatidyltransferase (729 nt)
MGG_15162	-1.295210757	Down	conserved hypothetical protein (1119 nt)
MGG_15012	-1.283048195	Down	conserved hypothetical protein (645 nt)
MGG_09271	-1.282656701	Down	conserved hypothetical protein (1092 nt)
MGG_07521	-1.281401333	Down	conserved hypothetical protein (1578 nt)
MGG_02273	-1.280405227	Down	hypothetical protein (576 nt)
MGG_13552	-1.271732585	Down	conserved hypothetical protein (1617 nt)
MGG_07887	-1.269888077	Down	4-hydroxyacetophenone monooxygenase (1701 nt)
MGG_14925	-1.267759394	Down	hypothetical protein (180 nt)
MGG_00520	-1.267642607	Down	conserved hypothetical protein (357 nt)
MGG_13624	-1.266786541	Down	ABC transporter CDR4 (4644 nt)
MGG_08731	-1.258589746	Down	conserved hypothetical protein (2601 nt)
MGG_02583	-1.256908303	Down	mitochondrial ATP-binding cassette sub-family B member 6 (2385 nt)
MGG_07455	-1.252739522	Down	sorbitol utilization protein SOU2 (879 nt)
MGG_00819	-1.252476214	Down	niemann-Pick C1 protein precursor (3588 nt)
MGG_00426	-1.246736876	Down	short-chain dehydrogenase/reductase SDR (819 nt)
MGG_00747	-1.246377062	Down	palmitoyltransferase PFA4 (1488 nt)
MGG_05800	-1.242628195	Down	predicted protein (1188 nt)
MGG_00745	-1.241375525	Down	zn-dependent hydrolases (1233 nt)
MGG_00016	-1.24082967	Down	2-epi-5-epi-valiolone synthase (1461 nt)
MGG_01094	-1.238748592	Down	conserved hypothetical protein (3573 nt)
MGG_10059	-1.236333192	Down	Gelsolin repeat-containing protein (1281 nt)
MGG_00623	-1.233852328	Down	hexokinase-1 (1686 nt)
MGG_02439	-1.231528369	Down	2-nitropropane dioxygenase (1074 nt)
MGG_03029	-1.229698609	Down	24 kDa metalloproteinase precursor (1074 nt)
MGG_01822	-1.228057747	Down	mitogen-activated protein kinase HOG1 (1074 nt)
MGG_00528	-1.225885735	Down	sterol esterase 2 (2268 nt)
MGG_04621	-1.224401746	Down	conserved hypothetical protein (1791 nt)
MGG_09562	-1.222058619	Down	conserved hypothetical protein (1608 nt)
MGG_01195	-1.221596452	Down	acetylesterase (732 nt)
MGG_10106	-1.217591435	Down	conserved hypothetical protein (1002 nt)
MGG_11308	-1.209658931	Down	zinc-binding alcohol dehydrogenase domain-containing protein 1 (1047 nt)
MGG_10400	-1.209426856	Down	glucan 1,3-beta-glucosidase (972 nt)
MGG_09639	-1.206598264	Down	cell wall alpha-1,3-glucan synthase ags1 (6756 nt)
MGG_01139	-1.203413624	Down	dihydroxy-acid dehydratase (1797 nt)
MGG_07482	-1.200572679	Down	conserved hypothetical protein (924 nt)
MGG_07856	-1.20029865	Down	pisatin demethylase (1542 nt)
MGG_04126	-1.197056128	Down	sulfate permease 2 (2370 nt)
MGG_10058	-1.195351422	Down	predicted protein (813 nt)
MGG_05976	-1.190497206	Down	transesterase (1218 nt)
MGG_03475	-1.189435005	Down	predicted protein (339 nt)
MGG_09802	-1.185601801	Down	conserved hypothetical protein (1734 nt)
MGG_13261	-1.181160664	Down	conserved hypothetical protein (738 nt)
MGG_14157	-1.17995933	Down	conserved hypothetical protein (1362 nt)
MGG_03070	-1.174154936	Down	epoxide hydrolase domain-containing protein (1233 nt)
MGG_08521	-1.172603107	Down	conserved hypothetical protein (1242 nt)
MGG_10525	-1.170024378	Down	conserved hypothetical protein (2520 nt)
MGG_01467	-1.169925001	Down	transmembrane protein 34 (1689 nt)
MGG_09238	-1.168480139	Down	conserved hypothetical protein (879 nt)
MGG_04177	-1.166309419	Down	conserved hypothetical protein (5589 nt)
MGG_04345	-1.16083999	Down	cytochrome P450 17A1 (1368 nt)
MGG_10083	-1.14543044	Down	endoglucanase 3 (1131 nt)
MGG_09371	-1.145365428	Down	phosphatidate cytidylyltransferase (1143 nt)
MGG_06593	-1.143703628	Down	endo-1,4-beta-xylanase 2 (771 nt)
MGG_05521	-1.142717043	Down	conserved hypothetical protein (1707 nt)
MGG_15245	-1.137629446	Down	conserved hypothetical protein (1161 nt)
MGG_14746	-1.136383802	Down	PH domain-containing protein (3543 nt)
MGG_11210	-1.134496375	Down	beta-glucosidase 1 (2631 nt)
MGG_06383	-1.133974216	Down	conserved hypothetical protein (717 nt)
MGG_02928	-1.131083275	Down	conserved hypothetical protein (1902 nt)
MGG_01883	-1.129721257	Down	conserved hypothetical protein (1632 nt)
MGG_09216	-1.129532813	Down	conserved hypothetical protein (456 nt)
MGG_01393	-1.126896424	Down	hypothetical protein (2214 nt)
MGG_08257	-1.126617775	Down	fatty acid transporter protein (1908 nt)
MGG_00190	-1.125277221	Down	conserved hypothetical protein (1263 nt)
MGG_06755	-1.124478665	Down	conserved hypothetical protein (1419 nt)
MGG_00829	-1.122877586	Down	alpha/beta hydrolase (972 nt)
MGG_08983	-1.121015401	Down	catechol 1,2-dioxygenase 1 (960 nt)
MGG_00562	-1.120456325	Down	NADPH-dependent 1-acyldihydroxyacetone phosphate reductase (855 nt)
MGG_13946	-1.12042304	Down	conserved hypothetical protein (1026 nt)
MGG_13334	-1.118919584	Down	general amino acid permease AGP2 (1731 nt)
MGG_08555	-1.118181426	Down	predicted protein (1470 nt)
MGG_10856	-1.11064934	Down	hypothetical protein (222 nt)
MGG_00939	-1.110191576	Down	conserved hypothetical protein (1425 nt)
MGG_15327	-1.102653177	Down	predicted protein (1059 nt)
MGG_07458	-1.10240544	Down	conserved hypothetical protein (2628 nt)
MGG_05499	-1.101830675	Down	predicted protein (822 nt)
MGG_11422	-1.096263655	Down	predicted protein (501 nt)
MGG_07369	-1.095194126	Down	conserved hypothetical protein (1269 nt)
MGG_07476	-1.095180364	Down	conserved hypothetical protein (2565 nt)
MGG_14632	-1.094090285	Down	hypothetical protein (216 nt)
MGG_02804	-1.092397138	Down	DNA repair protein rad16 (2883 nt)
MGG_09955	-1.089511202	Down	hypothetical protein (345 nt)
MGG_12317	-1.089306541	Down	hypothetical protein (864 nt)
MGG_01427	-1.088881281	Down	SET1 complex component ash2 (1704 nt)
MGG_07993	-1.08604406	Down	predicted protein (417 nt)
MGG_14639	-1.081068424	Down	predicted protein (1437 nt)
MGG_11597	-1.079777841	Down	glucosamine-fructose-6-phosphate aminotransferase (2121 nt)
MGG_02259	-1.077846845	Down	predicted protein (1590 nt)
MGG_12466	-1.074085046	Down	predicted protein (174 nt)
MGG_02444	-1.07290822	Down	1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase 1 (3051 nt)
MGG_09478	-1.072623134	Down	hypothetical protein (561 nt)
MGG_13805	-1.072440564	Down	predicted protein (1032 nt)
MGG_07257	-1.072244257	Down	hypothetical protein (1407 nt)
MGG_08453	-1.071790683	Down	conserved hypothetical protein (1473 nt)
MGG_04002	-1.069186812	Down	hypothetical protein (204 nt)
MGG_08856	-1.069185403	Down	membrane-bound O-acyltransferase domain-containing protein 1 (1662 nt)
MGG_03351	-1.065406996	Down	glycosyl hydrolase (2391 nt)
MGG_10537	-1.064899775	Down	conserved hypothetical protein (1269 nt)
MGG_08774	-1.064388508	Down	chitin deacetylase (1107 nt)
MGG_01208	-1.062644511	Down	conserved hypothetical protein (639 nt)
MGG_12329	-1.061240946	Down	DNA excision repair protein ERCC-8 (1533 nt)
MGG_01653	-1.056904511	Down	nuclear transcription factor Y subunit B-3 (615 nt)
MGG_13944	-1.054479897	Down	hypothetical protein (288 nt)
MGG_06026	-1.054080814	Down	conserved hypothetical protein (1905 nt)
MGG_07734	-1.053901938	Down	M-phase inducer phosphatase (1710 nt)
MGG_01528	-1.052273413	Down	serine/threonine-protein phosphatase PP2A catalytic subunit (1320 nt)
MGG_02574	-1.050021593	Down	conserved hypothetical protein (2709 nt)
MGG_09949	-1.046379935	Down	oxysterol-binding protein (1572 nt)
MGG_13157	-1.044105332	Down	acetate kinase (1299 nt)
MGG_03649	-1.043931126	Down	protein phosphatase 1 regulatory subunit SDS22 (1146 nt)
MGG_09199	-1.042885813	Down	repressible acid phosphatase (1374 nt)
MGG_02423	-1.03922131	Down	ER lumen protein retaining receptor 2 (645 nt)
MGG_03219	-1.038280996	Down	conidiophore development protein hymA (963 nt)
MGG_04682	-1.037803847	Down	predicted protein (1278 nt)
MGG_00203	-1.037786759	Down	conserved hypothetical protein (1881 nt)
MGG_05714	-1.037259619	Down	conserved hypothetical protein (3984 nt)
MGG_01390	-1.031395196	Down	MFS hexose transporter (1533 nt)
MGG_00593	-1.029202535	Down	liver carboxylesterase (2088 nt)
MGG_06926	-1.029146346	Down	cyclopropane-fatty-acyl-phospholipid synthase (1056 nt)
MGG_04159	-1.027834208	Down	vacuolar calcium ion transporter (1749 nt)
MGG_01872	-1.024833422	Down	hypothetical protein (513 nt)
MGG_06331	-1.022966687	Down	conserved hypothetical protein (963 nt)
MGG_07569	-1.022779869	Down	cellobiose dehydrogenase (1728 nt)
MGG_10423	-1.022063736	Down	cellulase family protein (1269 nt)
MGG_08662	-1.018657186	Down	predicted protein (372 nt)
MGG_08201	-1.018223292	Down	DUF636 domain-containing protein (411 nt)
MGG_07872	-1.017921908	Down	conserved hypothetical protein (477 nt)
MGG_10492	-1.017731319	Down	mitochondrial carnitine carrier (1038 nt)
MGG_08367	-1.017292236	Down	conserved hypothetical protein (1071 nt)
MGG_00588	-1.017142968	Down	CIA30 family protein (702 nt)
MGG_12671	-1.016753905	Down	DUF718 domain-containing protein (447 nt)
MGG_13350	-1.016571188	Down	conserved hypothetical protein (1236 nt)
MGG_10482	-1.015788886	Down	conserved hypothetical protein (1218 nt)
MGG_03990	-1.013649062	Down	alpha-1,2 glucosyltransferase ALG10 (1983 nt)
MGG_02982	-1.011866587	Down	averantin oxidoreductase (1569 nt)
MGG_02542	-1.008331275	Down	predicted protein (2499 nt)
MGG_05527	-1.006418938	Down	peptidyl-tRNA hydrolase (648 nt)
MGG_08550	-1.005932956	Down	lipolytic enzyme (1083 nt)
MGG_00631	-1.005719087	Down	conserved hypothetical protein (1851 nt)
MGG_01550	-1.005566676	Down	neuronal calcium sensor 1 (624 nt)
MGG_09816	-1.004576686	Down	conserved hypothetical protein (933 nt)
MGG_07789	-1.000708419	Down	3-phytase (2256 nt)

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为分析DEGs与哪些生物学功能显著相关,利用GO注释及GO功能富集分析,对差异表达基因进行功能分类（图2-C）。结果显示,在细胞组分（cellular component）、分子功能（molecular function）和生物过程（biological process）3类功能注释的DEGs分别有118、299和308个。此外,细胞组分、分子功能和生物过程分别包含8、11和16个功能分类（图2-C）。其中,在细胞组分分组中,DEGs在细胞（cell; GO：0005623）和细胞区域（cell part;GO：0044464）类所占比例最高,大分子复合物（macromolecular complex;GO：0032991）类所占比例最低;在分子功能分组中,催化活性（catalytic activity;GO：0003824）和结合（binding;GO：0005488）类,DEGs所占比例最高,在结构分子活性（structural molecule activity;GO：0005198）和营养库活性类（nutrient reservoir activity;GO：0045735）所占比例最低;在生物过程分组中,DEGs在代谢过程（metabolic process;GO：0008152）和细胞过程（cellular process;GO：0009987）类所占比例最高,而在生物过程负调控（negative regulation of biological process;GO：0048519）类所占比例最低。
通过Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway富集分析确定了DEGs参与的最主要生化代谢途径和信号转导途径^[23],发现共有143个DEGs具有KEGG注释。结合注释结果,进行pathway富集分析,发现这些DEGs位于65个pathway中（附表2）。其中,DEGs位于代谢途径（metabolic pathways;pathway ID：ko04146）的数目最多（41个）,其次位于次生代谢物质生物合成（biosynthesis of secondary metabolites;pathway ID：ko01110）（24个）,甘油磷脂代谢（glycerophospholipid metabolism;pathway ID： ko00564）（6个）,酵母MAPK信号通路（MAPK signaling pathway-yeast;pathway ID：ko04011）（5个）,内质网对蛋白质的加工（protein processing in endoplasmic reticulum;pathway ID：ko04141）（5个）,酵母细胞周期（cell cycle-yeast;pathway ID：ko04111）（5个）等途径中（附表2）。
Table S2
附表2
附表2143个差异表达基因的代谢途径
Table S2Pathways of the 143 differentially expressed genes

通路编号 Pathway ID	描述 Description	Pathway注释的差异基因数 Number of DEGs with pathway annotation (143)	P-value
ko00130	Ubiquinone and other terpenoid-quinone biosynthesis	3 (2.1%)	0.01565294
ko01110	Biosynthesis of secondary metabolites	24 (16.78%)	0.03312813
ko00564	Glycerophospholipid metabolism	6 (4.2%)	0.04582877
ko00051	Fructose and mannose metabolism	5 (3.5%)	0.05442695
ko00360	Phenylalanine metabolism	4 (2.8%)	0.0633638
ko00100	Steroid biosynthesis	4 (2.8%)	0.07559576
ko00650	Butanoate metabolism	5 (3.5%)	0.07874347
ko04650	Natural killer cell mediated cytotoxicity	1 (0.7%)	0.09034359
ko00311	Penicillin and cephalosporin biosynthesis	1 (0.7%)	0.09034359
ko00591	Linoleic acid metabolism	3 (2.1%)	0.106024
ko00565	Ether lipid metabolism	2 (1.4%)	0.1186817
ko03420	Nucleotide excision repair	4 (2.8%)	0.1267428
ko00640	Propanoate metabolism	3 (2.1%)	0.1478173
ko00500	Starch and sucrose metabolism	5 (3.5%)	0.1640955
ko04011	MAPK signaling pathway - yeast	5 (3.5%)	0.1640955
ko00400	Phenylalanine, tyrosine and tryptophan biosynthesis	2 (1.4%)	0.1671517
ko00910	Nitrogen metabolism	3 (2.1%)	0.1704372
ko00071	Fatty acid metabolism	3 (2.1%)	0.2306433
ko00300	Lysine biosynthesis	2 (1.4%)	0.2705874
ko00780	Biotin metabolism	1 (0.7%)	0.2822858
ko00010	Glycolysis / Gluconeogenesis	3 (2.1%)	0.2940734
ko04141	Protein processing in endoplasmic reticulum	5 (3.5%)	0.3101778
ko01100	Metabolic pathways	41 (28.67%)	0.3105893
ko04146	Peroxisome	4 (2.8%)	0.3110427
ko04144	Endocytosis	3 (2.1%)	0.3198399
ko03410	Base excision repair	2 (1.4%)	0.3400542
ko00230	Purine metabolism	6 (4.2%)	0.3404561
ko04113	Meiosis - yeast	5 (3.5%)	0.3848884
ko00250	Alanine, aspartate and glutamate metabolism	2 (1.4%)	0.3908593
ko00380	Tryptophan metabolism	3 (2.1%)	0.3968992
ko00062	Fatty acid elongation	1 (0.7%)	0.4064083
ko00290	Valine, leucine and isoleucine biosynthesis	1 (0.7%)	0.4064083
ko00310	Lysine degradation	2 (1.4%)	0.4557394
ko04111	Cell cycle - yeast	5 (3.5%)	0.4957104
ko04070	Phosphatidylinositol signaling system	1 (0.7%)	0.5091888
ko00061	Fatty acid biosynthesis	1 (0.7%)	0.5319913
ko00562	Inositol phosphate metabolism	1 (0.7%)	0.5319913
ko00240	Pyrimidine metabolism	3 (2.1%)	0.5418366
ko00561	Glycerolipid metabolism	2 (1.4%)	0.5452004
ko02010	ABC transporters	1 (0.7%)	0.6131495
ko03020	RNA polymerase	1 (0.7%)	0.6131495
ko00860	Porphyrin and chlorophyll metabolism	1 (0.7%)	0.6311514
ko00770	Pantothenate and CoA biosynthesis	1 (0.7%)	0.6311514
ko03430	Mismatch repair	1 (0.7%)	0.6483211
ko00410	beta-Alanine metabolism	1 (0.7%)	0.6483211
ko00520	Amino sugar and nucleotide sugar metabolism	2 (1.4%)	0.6592972
ko00790	Folate biosynthesis	1 (0.7%)	0.6803152
ko01040	Biosynthesis of unsaturated fatty acids	1 (0.7%)	0.6803152
ko00480	Glutathione metabolism	1 (0.7%)	0.6803152
ko00510	N-Glycan biosynthesis	1 (0.7%)	0.7094172
ko00270	Cysteine and methionine metabolism	1 (0.7%)	0.7094172
ko00620	Pyruvate metabolism	1 (0.7%)	0.7358868
ko03022	Basal transcription factors	1 (0.7%)	0.7482092
ko00052	Galactose metabolism	1 (0.7%)	0.7482092
ko00460	Cyanoamino acid metabolism	1 (0.7%)	0.7482092
ko00280	Valine, leucine and isoleucine degradation	1 (0.7%)	0.7599605
ko00260	Glycine, serine and threonine metabolism	1 (0.7%)	0.7711671
ko03030	DNA replication	1 (0.7%)	0.792045
ko00340	Histidine metabolism	1 (0.7%)	0.8017632
ko00330	Arginine and proline metabolism	1 (0.7%)	0.8830261
ko03015	mRNA surveillance pathway	1 (0.7%)	0.8937462
ko03018	RNA degradation	1 (0.7%)	0.9034902
ko03010	Ribosome	2 (1.4%)	0.9042958
ko00350	Tyrosine metabolism	1 (0.7%)	0.9508671
ko03013	RNA transport	1 (0.7%)	0.988561

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2.3 稻瘟病菌部分已知致病相关基因受Znf1调控

对Δznf1 RNA-Seq数据分析发现大约有20个已知的参与稻瘟病菌形态分化与致病性的基因受Znf1调控（表3）。其中,转录因子编码基因HOX7、FLBC以及脂质磷酸磷酸酶基因LPP2和LPP3在Δznf1中显著下调表达。此外,参与细胞渗透调节的MAPK（mitogen-activated protein kinase）基因OSM1和MAPKK基因PBS2,以及细胞壁形成相关基因CHS3和AGS1的表达也明显下调。然而,与一氧化氮合成和致病相关基因NOL3,参与黑色素合成相关基因ALB1、BUF1和RSY1以及编码热激蛋白40的MHF21的表达水平明显上调,说明在营养生长阶段Znf1对这些基因的转录可能起负调控作用。数据结果表明,这些与稻瘟病菌侵染相关形态分化、致病过程以及代谢相关基因可能直接或间接受Znf1调控。
Table 3
表3
表3稻瘟病菌部分致病相关基因在Δznf1表达谱数据中的分析
Table 3Analysis of Δznf1 RNA-Seq data for several pathogenicity-related genes in M. oryzae

基因编号 Gene ID	描述 Description	Log₂ ratio (Δznf1/Guy11)	参考文献 Reference
MGG_09994	脂质磷酸磷酸酶1 Lipid phosphate phosphatase 1 (Lpp3)	-3.2	[24]
MGG_04699	C₂H₂锌指结构蛋白FlbC C₂H₂ finger domain-containing protein FlbC (FlbC)	-2.4	[25]
MGG_12865	保守假定蛋白 Conserved hypothetical protein (Hox7)	-2.4	[16]
MGG_10268	丝裂原活化蛋白激酶激酶PBS2 MAP kinase kinase PBS2 (Pbs2)	-2.2	[26]
MGG_02246	保守假定蛋白 Conserved hypothetical protein (Con6)	-1.7	[27]
MGG_05988	脂质磷酸磷酸酶2 Lipid phosphate phosphatase 2 (Lpp2)	-1.5	[24]
MGG_10315	疏水蛋白 Hydrophobin (Mpg1)	-1.4	[28]
MGG_05871	膜整合蛋白 Integral membrane protein (Pth11)	-1.4	[29]
MGG_09551	几丁质合成酶3 Chitin synthase 3 (Chs3)	-1.4	[30]
MGG_01822	丝裂原活化蛋白激酶HOG1 MAP kinase HOG1 (Osm1)	-1.2	[31]
MGG_09639	细胞壁α-1,3-葡聚糖合成酶Ags1 Cell wall alpha-1,3-glucan synthase Ags1 (Ags1)	-1.2	[32]
MGG_10492	线粒体肉碱载体蛋白 Mitochondrial carnitine carrier (Crc1)	-1.0	[33]
MGG_07953	双功能 P450:NADPH-P450 还原酶 Bifunctional P-450:NADPH-P450 reductase (Nol3)	2.8	[34]
MGG_07219	分生孢子黄色素合成聚酮合酶 Conidial yellow pigment biosynthesis polyketide synthase (Alb1)	2.0	[35-36]
MGG_02252	四羟基萘还原酶 Tetrahydroxynaphthalene reductase (Buf1)	1.4	[35-36]
MGG_08180	保守假定蛋白 Conserved hypothetical protein (Mhf21)	1.7	[37]
MGG_05059	小柱孢酮脱水酶 Scytalone dehydratase (Rsy1)	1.2	[35-36]
MGG_06131	保守假定蛋白 Conserved hypothetical protein (bZIP14)	1.1	[38]

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2.4 受Znf1和Pmk1共同调控基因的分析

筛选Znf1和Pmk1共调控基因可为进一步有效鉴定Znf1下游调控基因以及明确Znf1与Pmk1的关系提供数据支持。因此,在同等条件下对Δpmk1进行了表达谱分析。将Δznf1与Δpmk1中的DEGs比较分析发现,Δznf1的410个下调表达基因中,有201个（约50%）基因在Δpmk1中的表达同样为下调;而Δznf1的299个上调基因中,有116个（约40%）基因在Δpmk1中也上调（图3,附表3）。比如,与在野生菌株Guy11中相比,LPP3以及HOX7都在Δznf1和Δpmk1中有相似的下调表达倍数,疏水蛋白基因MPG1在Δpmk1中显著下调表达（log₂ratio为-7.4）,但在Δznf1 中MPG1仅下调表达2.5倍（log₂ratio为-1.4）。功能未知基因MGG_14997和MGG_03044在Δznf1和Δpmk1中均显著下调,log₂ratio（Δznf1/Guy11）和log₂ratio（Δpmk1/Guy11）分别为-12.1、-12.1、-4.3、-3.6,说明这两个基因受Znf1和Pmk1共同调控,且调控水平相似（附表3）。试验结果表明,Znf1可能作为Pmk1 MAPK信号途径下游的转录因子与Pmk1共同调控许多基因的转录过程。

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图3Δznf1和Δpmk1中共同差异表达基因统计
-->Fig. 3Statistical analysis of differentially expressed genes in Δznf1 and Δpmk1
-->

Table S3
附表3
附表3Δznf1和Δpmk1菌丝中共同差异表达基因(|log₂ratio|≥1且 FDR<0.001)
Table S3Differentially expressed genes both in the mycelia of Δznf1和Δpmk1 (|log₂ratio|≥1且 FDR<0.001)

基因编号 Gene ID	Log₂ratio (Δpmk1/Guy11)	Log₂ratio (Δznf1/Guy11)	上调-下调 Up-Down-Regulation	描述 Description
MGG_15270	12.4252159	12.58871464	Up	predicted protein (441 nt)
MGG_00585	11.09671515	11.93663794	Up	predicted protein (354 nt)
MGG_05104	11.17990909	10.58871464	Up	predicted protein (576 nt)
MGG_13037	8.906890596	9.550746785	Up	conserved hypothetical protein (1692 nt)
MGG_12423	11.02236781	9.344295908	Up	choline monooxygenase (1308 nt)
MGG_09972	10.79766153	9.276124405	Up	conserved hypothetical protein (1605 nt)
MGG_00018	13.81478272	9.022367813	Up	integral membrane protein (1650 nt)
MGG_08723	4.664689693	3.855717944	Up	conserved hypothetical protein (972 nt)
MGG_14871	4.337869639	3.466699619	Up	bifunctional molybdenum cofactor biosynthesis protein (801 nt)
MGG_04437	4.430042663	3.37914914	Up	heat shock protein 78 (2421 nt)
MGG_12501	5.266786541	3.378511623	Up	conserved hypothetical protein (1374 nt)
MGG_05084	3.820733952	3.328187085	Up	conserved hypothetical protein (636 nt)
MGG_01989	4.266786541	3.266786541	Up	predicted protein (1665 nt)
MGG_13116	1.015941544	3.062735755	Up	conserved hypothetical protein (1260 nt)
MGG_09221	3.070389328	3.03562391	Up	predicted protein (3054 nt)
MGG_06753	3.721099189	2.921840937	Up	predicted protein (465 nt)
MGG_10110	2.338801913	2.884080713	Up	conserved hypothetical protein (441 nt)
MGG_07952	1.819845866	2.865316383	Up	predicted protein (516 nt)
MGG_10158	3.303780748	2.857980995	Up	QDE-2-interacting protein (1713 nt)
MGG_05896	2.714653577	2.815494267	Up	predicted protein (309 nt)
MGG_03158	3.718818247	2.604862058	Up	pumilio-family RNA binding repeat protein (2328 nt)
MGG_05132	5.17194685	2.590701406	Up	conserved hypothetical protein (1086 nt)
MGG_02059	1.432959407	2.523561956	Up	conserved hypothetical protein (9060 nt)
MGG_03983	2.586844348	2.514368617	Up	Hsp70 nucleotide exchange factor FES1 (654 nt)
MGG_02350	2.331205908	2.463656204	Up	conserved hypothetical protein (927 nt)
MGG_10109	2.445516154	2.438920966	Up	conserved hypothetical protein (465 nt)
MGG_09138	5.678738771	2.42053125	Up	glutathione S-transferase II (672 nt)
MGG_07404	1.943416472	2.387023123	Up	tripeptidyl-peptidase 1 (1806 nt)
MGG_09139	5.358628777	2.38669835	Up	laccase-1 (1794 nt)
MGG_06759	2.044335357	2.364189581	Up	heat shock protein 80 (2109 nt)
MGG_10271	2.478047297	2.342392197	Up	conserved hypothetical protein (3960 nt)
MGG_02436	2.489221598	2.296338285	Up	conserved hypothetical protein (459 nt)
MGG_10415	2.795180208	2.270089163	Up	predicted protein (2403 nt)
MGG_06459	2.679268795	2.200152894	Up	heat shock protein HSP98 (2781 nt)
MGG_07949	2.485303261	2.170357399	Up	conserved hypothetical protein (879 nt)
MGG_10108	1.898958651	2.167556766	Up	conserved hypothetical protein (789 nt)
MGG_03115	3.651338443	2.145452682	Up	predicted protein (1401 nt)
MGG_06958	2.5667168	2.06641867	Up	hsp70-like protein (1956 nt)
MGG_02372	1.805673786	2.018018704	Up	hypothetical protein (729 nt)
MGG_08424	2.698303974	2.0138058	Up	endo-1,4-beta-xylanase I (696 nt)
MGG_14351	2.667083195	1.94240208	Up	glycosyltransferase (1269 nt)
MGG_14340	1.43300209	1.934604368	Up	predicted protein (324 nt)
MGG_15049	2.29820072	1.871675903	Up	predicted protein (1944 nt)
MGG_02267	4.455568979	1.866733469	Up	predicted protein (1044 nt)
MGG_10683	3.971543554	1.849665727	Up	predicted protein (1479 nt)
MGG_03238	2.754089772	1.848445436	Up	zinc finger protein ZPR1 (1533 nt)
MGG_02095	3.391566506	1.839416707	Up	conserved hypothetical protein (996 nt)
MGG_08378	2.876341532	1.824913293	Up	cytochrome P450 oxidoreductase GliF (1605 nt)
MGG_04511	2.095611912	1.821029859	Up	MFS transporter (1461 nt)
MGG_12697	2.509796533	1.798601538	Up	pheromone-regulated membrane protein 10 (2973 nt)
MGG_05505	1.13606155	1.795986108	Up	autophagy protein (Atg22) (1704 nt)
MGG_01410	4.081388327	1.791820579	Up	glutathione S-transferase Gst3 (1035 nt)
MGG_02043	3.251588514	1.777192952	Up	BTB/POZ domain-containing protein (510 nt)
MGG_14675	2.084092679	1.772956761	Up	hypothetical protein (180 nt)
MGG_06616	1.146841388	1.762500686	Up	high-affinity methionine permease (1833 nt)
MGG_02096	2.378511623	1.757618704	Up	glycoside hydrolase (2478 nt)
MGG_02002	2.710465401	1.744835012	Up	conserved hypothetical protein (1281 nt)
MGG_14621	5.47952576	1.724556807	Up	conserved hypothetical protein (279 nt)
MGG_11336	1.68772515	1.703898264	Up	conserved hypothetical protein (774 nt)
MGG_05515	4.516015147	1.669026766	Up	predicted protein (588 nt)
MGG_08989	2.02379212	1.660472735	Up	conserved hypothetical protein (816 nt)
MGG_13019	1.288469453	1.631716269	Up	predicted protein (690 nt)
MGG_11680	3.174174474	1.620690205	Up	AAA family ATPase (2112 nt)
MGG_00791	2.343677678	1.603109847	Up	Lactamase_B domain containing protein (1077 nt)
MGG_04627	2.017921908	1.566815154	Up	conserved hypothetical protein (2787 nt)
MGG_08519	4.717022911	1.565654069	Up	aflatoxin B1 aldehyde reductase member 3 (1002 nt)
MGG_12714	3.215904085	1.563004767	Up	conserved hypothetical protein (930 nt)
MGG_13562	2.936230497	1.541985428	Up	conserved hypothetical protein (1593 nt)
MGG_01454	2.096343869	1.541813265	Up	conserved hypothetical protein (720 nt)
MGG_01364	3.189777559	1.531668886	Up	conserved hypothetical protein (2925 nt)
MGG_08980	1.781051303	1.528536579	Up	stress-induced-phosphoprotein 1 (1755 nt)
MGG_11991	2.58090102	1.516744272	Up	predicted protein (252 nt)
MGG_07766	1.874973469	1.514203204	Up	predicted protein (372 nt)
MGG_04736	2.432603231	1.500318333	Up	conserved hypothetical protein (912 nt)
MGG_05968	1.502985604	1.483038711	Up	endoribonuclease L-PSP (411 nt)
MGG_02977	1.278859373	1.479542529	Up	predicted protein (1809 nt)
MGG_02854	2.907591239	1.47566256	Up	conserved hypothetical protein (942 nt)
MGG_08726	1.570262889	1.47279708	Up	hypothetical protein (588 nt)
MGG_00809	1.45172962	1.472177608	Up	predicted protein (1770 nt)
MGG_11779	1.443301351	1.447112965	Up	conserved hypothetical protein (5598 nt)
MGG_07402	1.446512239	1.436863862	Up	conserved hypothetical protein (465 nt)
MGG_09019	1.860437934	1.3852473	Up	secretory phospholipase A2 (561 nt)
MGG_07078	2.571377145	1.347104217	Up	DUF775 domain-containing protein (609 nt)
MGG_00156	4.616608127	1.330364932	Up	conserved hypothetical protein (966 nt)
MGG_00963	2.119999776	1.327804661	Up	predicted protein (2850 nt)
MGG_05574	2.463885759	1.292180751	Up	conserved hypothetical protein (1539 nt)
MGG_08029	1.175849835	1.281729267	Up	conserved hypothetical protein (942 nt)
MGG_11682	2.598873395	1.281189416	Up	phenylacetone monooxygenase (1674 nt)
MGG_00282	2.187627003	1.258104518	Up	conserved hypothetical protein (2610 nt)
MGG_08104	1.480588488	1.254630144	Up	41 kDa peptidyl-prolyl cis-trans isomerase (1131 nt)
MGG_01951	2.831080992	1.25334101	Up	4-coumarate-CoA ligase 2 (2010 nt)
MGG_08158	1.466117407	1.217117479	Up	predicted protein (993 nt)
MGG_05978	1.070591968	1.216910356	Up	conserved hypothetical protein (564 nt)
MGG_05913	2.441623161	1.213091603	Up	hypothetical protein (657 nt)
MGG_04757	1.824771975	1.193739826	Up	conserved hypothetical protein (1485 nt)
MGG_04346	1.993886716	1.189968987	Up	sterol 24-C-methyltransferase (1173 nt)
MGG_05611	2.44908666	1.181656367	Up	hypothetical protein (243 nt)
MGG_00834	1.225326394	1.136488996	Up	hypothetical protein (987 nt)
MGG_14857	1.379032546	1.126377286	Up	conserved hypothetical protein (1050 nt)
MGG_02105	2.84595717	1.112563575	Up	conserved hypothetical protein (915 nt)
MGG_03005	1.209718591	1.109080361	Up	conserved hypothetical protein (1332 nt)
MGG_08007	1.599353593	1.107108893	Up	conserved hypothetical protein (762 nt)
MGG_14651	1.68921535	1.08767481	Up	conserved hypothetical protein (696 nt)
MGG_04242	1.610501769	1.081893423	Up	bax Inhibitor family protein (786 nt)
MGG_09664	1.329214449	1.075597619	Up	beta-mannosidase (2739 nt)
MGG_06131	1.292159087	1.073159746	Up	conserved hypothetical protein (720 nt)
MGG_05940	3.869107257	1.070943772	Up	short chain dehydrogenase (780 nt)
MGG_10613	1.170277265	1.054447784	Up	retinol dehydrogenase 11 (975 nt)
MGG_08142	3.902461751	1.052911531	Up	conserved hypothetical protein (1305 nt)
MGG_03098	2.155595633	1.049687124	Up	conserved hypothetical protein (984 nt)
MGG_09328	3.202101303	1.048429433	Up	conserved hypothetical protein (1113 nt)
MGG_04569	3.895608462	1.029318937	Up	NADPH dehydrogenase (1329 nt)
MGG_00738	1.481889031	1.025321666	Up	zinc-binding alcohol dehydrogenase domain-containing protein 2 (1020 nt)
MGG_02355	1.947266884	1.018265026	Up	predicted protein (687 nt)
MGG_02445	2.016601967	1.008878151	Up	predicted protein (801 nt)
MGG_01692	1.496153234	1.008721271	Up	conserved hypothetical protein (1206 nt)
MGG_14668	-3.298015475	-12.80976813	Down	hypothetical protein (228 nt)
MGG_11005	-2.198904068	-12.36632221	Down	conserved hypothetical protein (1368 nt)
MGG_14997	-12.14210706	-12.14210706	Down	hypothetical protein (219 nt)
MGG_15409	-1.630445271	-4.635588574	Down	predicted protein (432 nt)
MGG_05719	-3.374964882	-2.05779793	Down	30 kDa heat shock protein (723 nt)
MGG_03044	-3.610359278	-4.310142181	Down	conserved hypothetical protein (1101 nt)
MGG_02942	-1.468094002	-4.161723188	Down	conserved hypothetical protein (1749 nt)
MGG_07807	-1.828888084	-4.138216142	Down	conserved hypothetical protein (648 nt)
MGG_09879	-3.161857344	-3.992938336	Down	conserved hypothetical protein (438 nt)
MGG_00766	-2.934617277	-3.619372938	Down	conserved hypothetical protein (558 nt)
MGG_07857	-4.96133838	-3.413850584	Down	conserved hypothetical protein (1125 nt)
MGG_10650	-5.107637884	-3.408267701	Down	hypothetical protein (291 nt)
MGG_03825	-9.764871591	-3.273018494	Down	isotrichodermin C-15 hydroxylase (1617 nt)
MGG_05250	-1.389007222	-3.245205238	Down	conserved hypothetical protein (2520 nt)
MGG_09994	-2.903078982	-3.174512271	Down	lipid phosphate phosphatase 1 (1140 nt)
MGG_02290	-3.545532356	-3.141142101	Down	conserved hypothetical protein (858 nt)
MGG_01391	-5.276496666	-3.070166018	Down	ent-kaurene oxidase (1569 nt)
MGG_09418	-1.14038108	-3.02188266	Down	conserved hypothetical protein (429 nt)
MGG_08971	-3.422138556	-2.886958693	Down	conserved hypothetical protein (573 nt)
MGG_03329	-2.624176135	-3.169891506	Down	small heat shock protein (633 nt)
MGG_08326	-2.827301286	-2.881995905	Down	conserved hypothetical protein (1416 nt)
MGG_10322	-1.926158373	-2.87570589	Down	cortical patch protein (765 nt)
MGG_09134	-6.916311635	-2.765762647	Down	hypothetical protein (357 nt)
MGG_04171	-2.600160554	-2.721732314	Down	predicted protein (1050 nt)
MGG_14581	-2.126487637	-2.718799594	Down	hypothetical protein (744 nt)
MGG_07348	-2.099094415	-2.697353738	Down	predicted protein (420 nt)
MGG_10713	-4.412028749	-2.660707862	Down	conserved hypothetical protein (813 nt)
MGG_02329	-2.776515741	-2.649306721	Down	isotrichodermin C-15 hydroxylase (1632 nt)
MGG_00733	-2.378276173	-2.605714662	Down	conserved hypothetical protein (1026 nt)
MGG_12800	-2.149777312	-2.531615468	Down	conserved hypothetical protein (1257 nt)
MGG_01152	-2.06786603	-2.496200352	Down	conserved hypothetical protein (549 nt)
MGG_07871	-3.074075099	-2.493092449	Down	conserved hypothetical protein (726 nt)
MGG_08498	-4.181479228	-2.492768803	Down	isotrichodermin C-15 hydroxylase (1506 nt)
MGG_07697	-2.947300243	-2.470862199	Down	superoxide dismutase (642 nt)
MGG_12865	-1.990940661	-2.412767326	Down	conserved hypothetical protein (1344 nt)
MGG_11876	-1.968229221	-2.393623513	Down	sugar transporter STL1 (1722 nt)
MGG_01368	-2.284535475	-2.345597001	Down	conserved hypothetical protein (714 nt)
MGG_10714	-5.989415578	-2.315031003	Down	cyanide hydratase (1071 nt)
MGG_03977	-1.306035559	-2.298702913	Down	hypothetical protein (1389 nt)
MGG_01369	-3.381956573	-2.295885623	Down	alpha/beta hydrolase fold-3 domain-containing protein (1044 nt)
MGG_10429	-1.969063833	-2.277318632	Down	conserved hypothetical protein (963 nt)
MGG_05804	-3.656239914	-2.269857074	Down	phospholipase D p1 (2574 nt)
MGG_13655	-2.325562743	-2.268229568	Down	conserved hypothetical protein (1416 nt)
MGG_09445	-1.745816512	-2.266500219	Down	conserved hypothetical protein (1437 nt)
MGG_07088	-1.983069604	-2.257691984	Down	hypothetical protein (315 nt)
MGG_09473	-2.923306997	-2.24858331	Down	hypothetical protein (354 nt)
MGG_09000	-11.72280753	-2.230954435	Down	protein kinase domain-containing protein (2160 nt)
MGG_08405	-4.502500341	-2.190222416	Down	retinol dehydrogenase 13 (969 nt)
MGG_06545	-1.491190941	-2.168411949	Down	conserved hypothetical protein (2358 nt)
MGG_08439	-2.822122796	-2.161473337	Down	predicted protein (846 nt)
MGG_13198	-2.155937856	-2.159984689	Down	predicted protein (270 nt)
MGG_01542	-1.740912779	-2.072756342	Down	endo-1,4-beta-xylanase (996 nt)
MGG_09851	-1.605426603	-2.05288558	Down	predicted protein (1029 nt)
MGG_12366	-1.629422558	-2.051002903	Down	hypothetical protein (162 nt)
MGG_07356	-3.355480655	-2.050626073	Down	predicted protein (1701 nt)
MGG_10503	-1.83865173	-2.038908043	Down	mannitol-1-phosphate 5-dehydrogenase (1203 nt)
MGG_21009	-1.862947248	-2.035128223	Down	mitochondria cytochrome c oxidase subunit I cox1 (1599 nt)
MGG_02709	-4.08516956	-2.016440916	Down	conserved hypothetical protein (465 nt)
MGG_15428	-1.279201609	-2.012748493	Down	para-nitrobenzyl esterase (1674 nt)
MGG_03764	-1.207232614	-2	Down	salicylate hydroxylase (1395 nt)
MGG_08059	-3.573950442	-1.99175344	Down	conserved hypothetical protein (615 nt)
MGG_02803	-1.994865686	-1.972238488	Down	hypothetical protein (207 nt)
MGG_04251	-1.062442879	-1.96800463	Down	phosphate transporter family protein (1782 nt)
MGG_09001	-5.224001674	-1.915662644	Down	hypothetical protein (474 nt)
MGG_00114	-2.379300001	-1.914793825	Down	conserved hypothetical protein (594 nt)
MGG_00993	-3.513254318	-1.89630787	Down	vacuolar iron transporter Ccc1 (1014 nt)
MGG_03643	-1.63169993	-1.885795281	Down	conserved hypothetical protein (3816 nt)
MGG_01373	-1.56802005	-1.846069502	Down	arabinose-proton symporter (1728 nt)
MGG_07806	-5.134649527	-1.800230488	Down	conserved hypothetical protein (1230 nt)
MGG_04203	-1.078328361	-1.799556551	Down	4-coumarate-CoA ligase 1 (1671 nt)
MGG_01544	-2.897819605	-1.788997336	Down	pisatin demethylase (1464 nt)
MGG_14146	-13.98726401	-1.764469109	Down	hypothetical protein (384 nt)
MGG_09828	-1.149050919	-1.763810581	Down	vacuolar conductance protein (1959 nt)
MGG_02752	-2.678576833	-1.734749061	Down	hypothetical protein (1071 nt)
MGG_05358	-1.222148207	-1.714428705	Down	LYR family protein (267 nt)
MGG_09323	-2.623386485	-1.703160095	Down	phenylacetone monooxygenase (1944 nt)
MGG_09260	-1.5966815	-1.697890657	Down	integral membrane protein (495 nt)
MGG_13798	-14.07430874	-1.686291456	Down	predicted protein (915 nt)
MGG_07966	-1.256055394	-1.684741955	Down	phosphate transporter family protein (1767 nt)
MGG_02929	-1.30712216	-1.675273272	Down	phosphotransferase enzyme family protein (789 nt)
MGG_10359	-2.127489735	-1.673771768	Down	conserved hypothetical protein (1248 nt)
MGG_04257	-1.213955584	-1.672160943	Down	secretory lipase family protein (1269 nt)
MGG_07890	-1.490184276	-1.667424661	Down	aldehyde dehydrogenase 3I1 (1482 nt)
MGG_02332	-5.967706597	-1.661898167	Down	conserved hypothetical protein (810 nt)
MGG_01376	-1.203750015	-1.657020649	Down	tyrosine-protein phosphatase 1 (3420 nt)
MGG_01945	-1.348094132	-1.653849412	Down	predicted protein (729 nt)
MGG_10973	-2.155358888	-1.647520814	Down	conserved hypothetical protein (570 nt)
MGG_10422	-6.573787425	-1.646850887	Down	C6 transcription factor OefC (2112 nt)
MGG_09985	-2.729103476	-1.628174568	Down	hypothetical protein (1347 nt)
MGG_14578	-1.750480401	-1.620712526	Down	predicted protein (855 nt)
MGG_13428	-1.222885988	-1.605463615	Down	mitochondrial phosphate carrier protein (945 nt)
MGG_01798	-1.143330494	-1.587742596	Down	conserved hypothetical protein (906 nt)
MGG_12664	-1.927149435	-1.579226131	Down	conserved hypothetical protein (777 nt)
MGG_06181	-2.643544222	-1.578735926	Down	predicted protein (1539 nt)
MGG_01463	-1.577173699	-1.576467707	Down	phosphate metabolism protein 7 (2682 nt)
MGG_10092	-3.783980414	-1.574527048	Down	hypothetical protein (1086 nt)
MGG_00097	-2.915493244	-1.569986896	Down	conserved hypothetical protein (972 nt)
MGG_10394	-4.911891277	-1.553362167	Down	conserved hypothetical protein (618 nt)
MGG_10053	-2.443347895	-1.552972386	Down	conserved hypothetical protein (867 nt)
MGG_15228	-2.91083778	-1.538419915	Down	predicted protein (354 nt)
MGG_05897	-1.372405891	-1.535135392	Down	predicted protein (462 nt)
MGG_00830	-2.72106473	-1.519972879	Down	conserved hypothetical protein (1908 nt)
MGG_00033	-2.15631457	-1.51815913	Down	conserved hypothetical protein (1605 nt)
MGG_04922	-2.498173488	-1.512820264	Down	alpha-N-arabinofuranosidase 2 (1083 nt)
MGG_05692	-2.055684133	-1.509837762	Down	conserved hypothetical protein (681 nt)
MGG_14681	-2.293223008	-1.477507164	Down	conserved hypothetical protein (1110 nt)
MGG_15174	-1.676243008	-1.476305437	Down	predicted protein (342 nt)
MGG_05140	-1.039275149	-1.465508537	Down	conserved hypothetical protein (1086 nt)
MGG_00815	-4.545589327	-1.451773288	Down	predicted protein (738 nt)
MGG_01993	-4.432702494	-1.450147629	Down	predicted protein (306 nt)
MGG_10315	-7.432355637	-1.443282704	Down	hydrophobin (339 nt)
MGG_04594	-1.06210985	-1.422470257	Down	conserved hypothetical protein (1410 nt)
MGG_06260	-3.62058641	-1.418952549	Down	conserved hypothetical protein (2727 nt)
MGG_07211	-1.990331352	-1.418645279	Down	conserved hypothetical protein (1482 nt)
MGG_05871	-2.888578717	-1.397592365	Down	integral membrane protein (1896 nt)
MGG_00268	-1.301002256	-1.396421821	Down	conserved hypothetical protein (2049 nt)
MGG_09447	-3.113863284	-1.392923114	Down	predicted protein (684 nt)
MGG_06364 c2h2	-2.305329222	-1.390057478	Down	hypothetical protein (1398 nt)
MGG_10452	-1.258362966	-1.388423507	Down	predicted protein (756 nt)
MGG_05051	-1.568568062	-1.385121921	Down	predicted protein (864 nt)
MGG_05114	-1.957295549	-1.380370367	Down	hypothetical protein (1662 nt)
MGG_06863	-1.446733214	-1.379399163	Down	conserved hypothetical protein (1134 nt)
MGG_00899	-1.038913905	-1.372926068	Down	tubulin-specific chaperone C (1056 nt)
MGG_13008	-4.824004051	-1.370486472	Down	predicted protein (384 nt)
MGG_04835	-1.101212251	-1.367605669	Down	conserved hypothetical protein (1008 nt)
MGG_11945	-1.370706095	-1.360317981	Down	candidapepsin precursor (1560 nt)
MGG_06781	-1.471499338	-1.357079427	Down	conserved hypothetical protein (1365 nt)
MGG_03392	-3.572023445	-1.357010554	Down	cytochrome P450 11B1, mitochondrial precursor (1527 nt)
MGG_03941	-2.031093294	-1.350682695	Down	conserved hypothetical protein (897 nt)
MGG_15134	-1.513608912	-1.324180547	Down	conserved hypothetical protein (1029 nt)
MGG_01612	-2.97012947	-1.320419788	Down	hypothetical protein (1818 nt)
MGG_06888	-4.18999471	-1.315767594	Down	glutamine synthetase (978 nt)
MGG_02081	-1.13884463	-1.3152412	Down	conserved hypothetical protein (1638 nt)
MGG_01025	-1.566072607	-1.314815369	Down	predicted protein (717 nt)
MGG_12302	-3.623436649	-1.313253761	Down	predicted protein (414 nt)
MGG_14205	-1.897111208	-1.306570073	Down	conserved hypothetical protein (1107 nt)
MGG_07521	-2.054563812	-1.281401333	Down	conserved hypothetical protein (1578 nt)
MGG_02273	-8.206429711	-1.280405227	Down	hypothetical protein (576 nt)
MGG_13552	-1.036846026	-1.271732585	Down	conserved hypothetical protein (1617 nt)
MGG_07887	-2.492280498	-1.269888077	Down	4-hydroxyacetophenone monooxygenase (1701 nt)
MGG_07455	-1.525919085	-1.252739522	Down	sorbitol utilization protein SOU2 (879 nt)
MGG_00819	-1.540981307	-1.252476214	Down	niemann-Pick C1 protein precursor (3588 nt)
MGG_00016	-1.47720632	-1.24082967	Down	2-epi-5-epi-valiolone synthase (1461 nt)
MGG_10059	-2.754979047	-1.236333192	Down	Gelsolin repeat-containing protein (1281 nt)
MGG_00528	-1.060745796	-1.225885735	Down	sterol esterase 2 (2268 nt)
MGG_01195	-1.057252482	-1.221596452	Down	acetylesterase (732 nt)
MGG_01139	-1.261454236	-1.203413624	Down	dihydroxy-acid dehydratase (1797 nt)
MGG_07482	-1.788337696	-1.200572679	Down	conserved hypothetical protein (924 nt)
MGG_07856	-1.736965594	-1.20029865	Down	pisatin demethylase (1542 nt)
MGG_04126	-3.325886108	-1.197056128	Down	sulfate permease 2 (2370 nt)
MGG_10058	-1.574549094	-1.195351422	Down	predicted protein (813 nt)
MGG_05976	-1.212222528	-1.190497206	Down	transesterase (1218 nt)
MGG_03475	-4.5325338	-1.189435005	Down	predicted protein (339 nt)
MGG_09802	-2.41739292	-1.185601801	Down	conserved hypothetical protein (1734 nt)
MGG_03070	-3.115573215	-1.174154936	Down	epoxide hydrolase domain-containing protein (1233 nt)
MGG_08521	-1.865223321	-1.172603107	Down	conserved hypothetical protein (1242 nt)
MGG_10525	-1.593134174	-1.170024378	Down	conserved hypothetical protein (2520 nt)
MGG_04345	-3.438620981	-1.16083999	Down	cytochrome P450 17A1 (1368 nt)
MGG_10083	-1.254364811	-1.14543044	Down	endoglucanase 3 (1131 nt)
MGG_09371	-1.111678983	-1.145365428	Down	phosphatidate cytidylyltransferase (1143 nt)
MGG_06593	-4.186067212	-1.143703628	Down	endo-1,4-beta-xylanase 2 (771 nt)
MGG_05521	-1.41214922	-1.142717043	Down	conserved hypothetical protein (1707 nt)
MGG_11210	-1.409390936	-1.134496375	Down	beta-glucosidase 1 (2631 nt)
MGG_01883	-1.599360469	-1.129721257	Down	conserved hypothetical protein (1632 nt)
MGG_06755	-1.610663011	-1.124478665	Down	conserved hypothetical protein (1419 nt)
MGG_00829	-2.409350363	-1.122877586	Down	alpha/beta hydrolase (972 nt)
MGG_08983	-2.207727034	-1.121015401	Down	catechol 1,2-dioxygenase 1 (960 nt)
MGG_13946	-1.113996771	-1.12042304	Down	conserved hypothetical protein (1026 nt)
MGG_13334	-2.139981199	-1.118919584	Down	general amino acid permease AGP2 (1731 nt)
MGG_00939	-2.060144203	-1.110191576	Down	conserved hypothetical protein (1425 nt)
MGG_14632	-5.437663374	-1.094090285	Down	hypothetical protein (216 nt)
MGG_02804	-1.215728691	-1.092397138	Down	DNA repair protein rad16 (2883 nt)
MGG_09955	-1.969723867	-1.089511202	Down	hypothetical protein (345 nt)
MGG_12317	-12.93479659	-1.089306541	Down	hypothetical protein (864 nt)
MGG_07993	-7.271589209	-1.08604406	Down	predicted protein (417 nt)
MGG_14639	-1.974319842	-1.081068424	Down	predicted protein (1437 nt)
MGG_02259	-1.403222136	-1.077846845	Down	predicted protein (1590 nt)
MGG_12466	-1.067343468	-1.074085046	Down	predicted protein (174 nt)
MGG_08453	-2.110264831	-1.071790683	Down	conserved hypothetical protein (1473 nt)
MGG_03351	-1.431244789	-1.065406996	Down	glycosyl hydrolase (2391 nt)
MGG_08774	-3.790912355	-1.064388508	Down	chitin deacetylase (1107 nt)
MGG_01208	-1.560037961	-1.062644511	Down	conserved hypothetical protein (639 nt)
MGG_01653	-1.978306305	-1.056904511	Down	nuclear transcription factor Y subunit B-3 (615 nt)
MGG_13944	-3.269625202	-1.054479897	Down	hypothetical protein (288 nt)
MGG_07734	-2.383553079	-1.053901938	Down	M-phase inducer phosphatase (1710 nt)
MGG_09949	-2.024300402	-1.046379935	Down	oxysterol-binding protein (1572 nt)
MGG_03649	-1.215690451	-1.043931126	Down	protein phosphatase 1 regulatory subunit SDS22 (1146 nt)
MGG_09199	-1.672048117	-1.042885813	Down	repressible acid phosphatase (1374 nt)
MGG_02423	-1.010896389	-1.03922131	Down	ER lumen protein retaining receptor 2 (645 nt)
MGG_03219	-1.467366269	-1.038280996	Down	conidiophore development protein hymA (963 nt)
MGG_00203	-1.832486716	-1.037786759	Down	conserved hypothetical protein (1881 nt)
MGG_01390	-1.164289467	-1.031395196	Down	MFS hexose transporter (1533 nt)
MGG_00593	-3.36099766	-1.029202535	Down	liver carboxylesterase (2088 nt)
MGG_07569	-1.330902164	-1.022779869	Down	cellobiose dehydrogenase (1728 nt)
MGG_08201	-4.122816385	-1.018223292	Down	DUF636 domain-containing protein (411 nt)
MGG_07872	-1.06287969	-1.017921908	Down	conserved hypothetical protein (477 nt)
MGG_12671	-1.353471223	-1.016753905	Down	DUF718 domain-containing protein (447 nt)
MGG_02982	-1.055602066	-1.011866587	Down	averantin oxidoreductase (1569 nt)
MGG_08550	-1.226015216	-1.005932956	Down	lipolytic enzyme (1083 nt)
MGG_00631	-1.991577141	-1.005719087	Down	conserved hypothetical protein (1851 nt)
MGG_09816	-2.674503854	-1.004576686	Down	conserved hypothetical protein (933 nt)

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2.5 qRT-PCR验证

为了验证RNA-seq数据的准确性,随机选择了10个DEGs进行qRT-PCR验证（表4）。结果显示,与RNA-Seq数据一致,与在野生菌株Guy11中相比,MGG_01391、MGG_03825、MGG_09000等基因表达水平在Δznf1 和Δpmk1中显著下调,而MGG_08944、MGG_11084在Δznf1 中表达水平显著升高。MGG_02246在Δznf1中下调表达,但在Δpmk1中上调表达26倍（图4）。
Table 4
表4
表4用于qRT-PCR验证的基因及其RNA-Seq测序结果
Table 4The RNA-Seq result of genes selected for qRT-PCR analysis

基因编号 Gene ID	描述 Description	Log₂ ratio (Δznf1/Guy11)	Log₂ ratio (Δpmk1/Guy11)
MGG_10422	C₆转录因子OefC C₆ transcription factor OefC	-1.6	-6.6
MGG_01391	贝壳杉烯氧化酶Ent-kaurene oxidase	-3.0	-5.3
MGG_03825	Isotrichodermin C-15羟化酶 Isotrichodermin C-15 hydroxylase	-3.3	-9.8
MGG_09000	蛋白激酶结构域 Protein kinase domain-containing protein	-2.2	-11.7
MGG_09994	脂质磷酸磷酸酶1 Lipid phosphate phosphatase 1	-3.2	-2.9
MGG_05719	30 kD热激蛋白 30 kD heat shock protein	-2.1	-3.4
MGG_03329	小分子热激蛋白 Small heat shock protein	-3.2	-2.6
MGG_02246	保守假定蛋白Conserved hypothetical protein	-1.7	3.9
MGG_08944	预测蛋白 Predicted protein	6.1	0.10
MGG_11084	TPR结构域蛋白 TPR domain-containing protein	5.1	-0.64

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图410个差异表达基因的qRT-PCR分析纵轴表示与在野生菌株Guy11中相比（定为1）,10个DEGs在Δznf1 或Δpmk1中相对表达水平,“**”代表与Guy11相比,差异极显著（P<0.01）,误差线表示标准差
-->Fig. 4qRT-PCR analysis on 10 DEGsThe vertical axis represented the relative expression levels of the 10 DEGs in Δznf1 or Δpmk1 when compared with the wild-type strain Guy11 (arbitrarily set to 1). Asterisks indicated significant differences (**, P<0.01). Error bars represented standard deviation
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3 讨论

RNA-Seq是用来研究某一生物对象在特定生物过程中基因表达差异的技术^[39-40]。该技术结合了转录组建库实验方法与数字基因表达谱的信息分析手段,可广泛应用于生理调控、农业性状、环境改造等领域。该技术主要用来研究基因表达的情况,在基因表达量分析、基因表达差异分析以及差异基因模式和功能聚类等方面应用广泛^[41]。近年来,基于RNA-Seq的全基因表达谱分析技术已广泛应用于稻瘟病菌,为稻瘟病菌基因功能和结构的研究提供了许多有参考价值的数据。SOANES等^[42]通过RNA-Seq和HT-SuperSAGE技术研究了稻瘟病菌在不同营养生长条件以及附着胞形成各阶段中基因的差异表达情况,鉴定到了一组在附着胞形成阶段特异高表达的基因;LI等^[43]利用RNA-Seq初步明确了稻瘟病菌产孢相关转录因子Cos1的调控基因;PHAM等^[44]将Chip-Seq和RNA-Seq数据结合分析了组蛋白H₃K₄甲基转移酶MoSET1在营养生长阶段和附着胞发育阶段的调控网络。稻瘟病菌Znf1是与附着胞形成、侵染、致病过程相关的转录因子^[18],利用RNA-Seq技术,可为进一步鉴定Znf1下游调控基因,明确Znf1的调控网络提供参考数据。因此,本研究中,笔者对稻瘟病菌野生菌株Guy11和突变体Δznf1 进行RNA-Seq分析,获得了Δznf1在营养生长阶段的差异表达基因。同时,对稻瘟病菌附着胞形成调控因子PMK1的缺失突变体也进行了RNA-Seq测序,并筛选出了Δznf1和Δpmk1中共同的差异表达基因。测序质量评估分析显示测序质量较高,qRT-PCR验证表明测序结果可靠,测序所得数据可为后续研究提供依据。
Δznf1表达谱分析共筛选到709个差异表达基因,其中上调表达基因299个,下调表达基因410个（附表1）。本试验差异表达基因的筛选标准为∣log₂ratio (Δznf1/Guy11)∣≥1,且FDR≤0.001,因此可能还有部分受Znf1调控的基因,但不符合标准而未被筛选到。一些与稻瘟病菌致病相关的基因表达与Znf1有关。例如,HOX7表达水平在Δznf1 中有一定程度的下调（表3）,且Δhox7与Δznf1 的表型类似,均不能形成成熟的附着胞,仅在芽管或菌丝尖端形成无黑色素化的膨大结构^[16],因此,HOX7可能直接或间接受Znf1调控。之前的研究发现,与野生菌株Guy11相比,Δznf1对胞壁降解酶Glucanex敏感性升高,同等条件下Δznf1原生质体释放量增加^[18]。用等量的Glucanex对Guy11和Δznf1菌丝处理2.5 h,计数显示,Guy11原生质体释放量为（15.2±1.17）×10⁷个,而Δznf1原生质体释放量为（27.2±0.71）×10⁷个。本试验RNA-Seq数据显示几丁质合成相关基因CHS3以及α-1,3-葡聚糖编码基因AGS1在Δznf1中有一定程度的下调表达（表3）。因此,笔者推测Znf1可能通过调控CHS3和AGS1的表达水平从而影响稻瘟病菌细胞壁结构的完整性。在Δznf1差异表达基因中,有近60%的基因编码预测蛋白（hypothetical protein）或保守的假定蛋白（conserved hypothetical protein）。MGG_08944编码一个假定蛋白,在其他丝状真菌中不能找到其同源蛋白。测序数据显示log₂ratio（Δznf1/Guy11）为6.1,qRT-PCR进一步证明其在Δznf1中上调表达30倍。MGG_11005、MGG_14426等在Δznf1中显著下调（附表1）。在今后的研究中,可以通过基因敲除技术进一步明确这些受Znf1调控的假定蛋白基因的功能。最近,有研究以稻瘟病菌70-15菌株为背景,对47个C₂H₂转录因子基因进行了敲除,其中MGG_14931（本文的ZNF1）命名为VRF1,并对Δvrf1在饥饿诱导下的菌丝进行了表达谱分析^[25]。本研究以Δznf1营养菌丝为材料进行表达谱测序,结果与Δvrf1数据有一定的差异,可能是由于菌株的不同以及对菌丝样品的处理不同。
Pmk1是稻瘟病菌附着胞形成相关基因的重要调控因子^[42]。Δznf1和Δpmk1表型类似,都不能形成附着胞,对寄主致病性完全丧失,都能响应外源cAMP的诱导^[13,18]。在附着胞发育阶段,ZNF1表达水平在Δpmk1中显著下调^[18]。将Δznf1和Δpmk1中差异表达基因比较发现,Δznf1里709个差异表达基因中,有397个（约56%）基因同时也受Pmk1调控,其中,317个基因在Δznf1和Δpmk1 中上调/下调趋势相同（图3,附表3）。该结果可以进一步证明Znf1可能是位于Pmk1下游,参与调控附着胞的形成^[18]。3个在Δznf1和Δpmk1中均显著下调的基因,MGG_03825、MGG_02329和MGG_08498,都编码isotrichodermin C-15羟化酶,它们都含有细胞色素P450超家族的保守结构域,其中MGG_03825的表达经qRT-PCR验证（图4,附表3）。拟分枝镰孢（Fusarium sporotrichioides）和小麦赤霉病菌（Fusarium graminearum）中TRI11与这3个基因同源,其编码一个细胞色素P450单加氧酶。研究表明TRI11与单端孢霉烯毒素合成相关,主要负责C-15的羟基化反应^[45-46]。这3个基因是否与稻瘟病菌生长、产孢、附着胞形成、致病等过程相关尚不明确,有待进一步研究。此外,Δznf1和Δpmk1中的共同差异基因数仅占Δpmk1中差异基因数目（3 169个）的12.6%,表明Znf1和Pmk1在调控机制上存在很大差异,Pmk1下游还存在其他转录因子负责这些差异表达基因的转录。而且,有一些基因在Δznf1和Δpmk1中上调/下调趋势虽然相同,但上调/下调倍数有很大差异。疏水蛋白基因MPG1在Δpmk1中下调倍数达130倍,但在Δznf1中仅有轻微的下调表达。菌落疏水性测定表明Δpmk1 菌落疏水性丧失,但Δznf1菌落疏水性没有明显的改变（数据未显示）。可见,在这些基因的调控上,Znf1与Pmk1有很大的不同。
SOANES等^[42]通过RNA-Seq和HT-SuperSAGE技术对稻瘟病菌菌株Guy11在营养生长（CM培养基）阶段和附着胞形成阶段（分生孢子分别诱导4、6、8、14和 16 h）分别进行了表达谱分析,发现与在营养生长阶段相比,共有1 838个基因的表达水平在附着胞形成阶段显著上调。结合本试验数据分析发现,这些在附着胞发育阶段上调表达的基因中有48个基因在Δznf1 和Δpmk1中均显著下调表达（附表4）。比如,MGG_09000编码CMGC/CDK蛋白激酶,其log₂ratio（Δznf1/Guy11）及log₂ratio（Δpmk1/Guy11）分别为-2.23和-11.72,而在CM及附着胞形成阶段（T4、T6、T8、T14、T16）表达量分别为1.96、40.35、25.67、32.17、33.44、49.80^[42]（附表）。HOX7（MGG_12865）编码一个含Homeobox结构域的转录因子,参与稻瘟病菌附着胞形成、侵染和致病过程^[16],其在稻瘟病菌附着胞形成阶段,特别是在分生孢子诱导4 h后表达水平显著升高（附表）。然而,在突变体Δznf1和Δpmk1中,HOX7表达水平降低。这些与附着胞形成相关的基因同时受Znf1和Pmk1调控,表明与Pmk1类似,Znf1也参与这些附着胞相关基因的调控。
Table S4
附表4
附表4在附着胞形成阶段上调表达但在Δznf1和Δpmk1中下调表达的基因^[42]
Table S4Genes up-regulated during appressorium formation, but down-regulated in Δznf1 and Δpmkl

基因编号 Gene ID	Log₂ratio (Δpmk1/Guy11)	Log₂ratio (Δznf1/Guy11)	描述 Description
MGG_02942	-1.468094002	-4.161723188	conserved hypothetical protein (1749 nt)
MGG_05250	-1.389007222	-3.245205238	conserved hypothetical protein (2520 nt)
MGG_02290	-3.545532356	-3.141142101	conserved hypothetical protein (858 nt)
MGG_01391	-5.276496666	-3.070166018	ent-kaurene oxidase (1569 nt)
MGG_10322	-1.926158373	-2.87570589	cortical patch protein (765 nt)
MGG_12865	-1.990940661	-2.412767326	conserved hypothetical protein (1344 nt)
MGG_01368	-2.284535475	-2.345597001	conserved hypothetical protein (714 nt)
MGG_10429	-1.969063833	-2.277318632	conserved hypothetical protein (963 nt)
MGG_07088	-1.983069604	-2.257691984	hypothetical protein (315 nt)
MGG_09000	-11.72280753	-2.230954435	protein kinase domain-containing protein (2160 nt)
MGG_01542	-1.740912779	-2.072756342	endo-1,4-beta-xylanase (996 nt)
MGG_15428	-1.279201609	-2.012748493	para-nitrobenzyl esterase (1674 nt)
MGG_03764	-1.207232614	-2	salicylate hydroxylase (1395 nt)
MGG_09001	-5.224001674	-1.915662644	hypothetical protein (474 nt)
MGG_00993	-3.513254318	-1.89630787	vacuolar iron transporter Ccc1 (1014 nt)
MGG_09828	-1.149050919	-1.763810581	vacuolar conductance protein (1959 nt)
MGG_05358	-1.222148207	-1.714428705	LYR family protein (267 nt)
MGG_09323	-2.623386485	-1.703160095	phenylacetone monooxygenase (1944 nt)
MGG_02929	-1.30712216	-1.675273272	phosphotransferase enzyme family protein (789 nt)
MGG_10359	-2.127489735	-1.673771768	conserved hypothetical protein (1248 nt)
MGG_10422	-6.573787425	-1.646850887	C6 transcription factor OefC (2112 nt)
MGG_01798	-1.143330494	-1.587742596	conserved hypothetical protein (906 nt)
MGG_15228	-2.91083778	-1.538419915	predicted protein (354 nt)
MGG_05897	-1.372405891	-1.535135392	predicted protein (462 nt)
MGG_15174	-1.676243008	-1.476305437	predicted protein (342 nt)
MGG_04594	-1.06210985	-1.422470257	conserved hypothetical protein (1410 nt)
MGG_06260	-3.62058641	-1.418952549	conserved hypothetical protein (2727 nt)
MGG_07211	-1.990331352	-1.418645279	conserved hypothetical protein (1482 nt)
MGG_05871	-2.888578717	-1.397592365	integral membrane protein (1896 nt)
MGG_05051	-1.568568062	-1.385121921	predicted protein (864 nt)
MGG_05114	-1.957295549	-1.380370367	hypothetical protein (1662 nt)
MGG_00899	-1.038913905	-1.372926068	tubulin-specific chaperone C (1056 nt)
MGG_04835	-1.101212251	-1.367605669	conserved hypothetical protein (1008 nt)
MGG_03392	-3.572023445	-1.357010554	cytochrome P450 11B1, mitochondrial precursor (1527 nt)
MGG_01612	-2.97012947	-1.320419788	hypothetical protein (1818 nt)
MGG_07455	-1.525919085	-1.252739522	sorbitol utilization protein SOU2 (879 nt)
MGG_00819	-1.540981307	-1.252476214	niemann-Pick C1 protein precursor (3588 nt)
MGG_10059	-2.754979047	-1.236333192	Gelsolin repeat-containing protein (1281 nt)
MGG_10058	-1.574549094	-1.195351422	predicted protein (813 nt)
MGG_05976	-1.212222528	-1.190497206	transesterase (1218 nt)
MGG_10525	-1.593134174	-1.170024378	conserved hypothetical protein (2520 nt)
MGG_09371	-1.111678983	-1.145365428	phosphatidate cytidylyltransferase (1143 nt)
MGG_05521	-1.41214922	-1.142717043	conserved hypothetical protein (1707 nt)
MGG_08983	-2.207727034	-1.121015401	catechol 1,2-dioxygenase 1 (960 nt)
MGG_01653	-1.978306305	-1.056904511	nuclear transcription factor Y subunit B-3 (615 nt)
MGG_09199	-1.672048117	-1.042885813	repressible acid phosphatase (1374 nt)
MGG_01390	-1.164289467	-1.031395196	MFS hexose transporter (1533 nt)
MGG_12671	-1.353471223	-1.016753905	DUF718 domain-containing protein (447 nt)

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本研究获得的Δznf1在菌丝生长阶段的差异表达基因数目相对较少,而且表达差异倍数在10倍以上的基因不多,可能是由于Δznf1营养生长与野生型Guy11相比没有明显的差异。与野生型菌株相比,Δznf1产孢量略微增多,不能形成附着胞,不能穿透寄主表皮和进行侵染生长,说明病菌的这些发育阶段受Znf1调控,但调控的基因可能未被筛选到。此外,有小部分基因无论是在Guy11还是Δznf1或Δpmk1中表达丰度都较低,这可能与这些基因呈阶段特异性表达有关。因此,在后续的研究中可对Δznf1在分生孢子、附着胞形成的不同阶段、侵染阶段的样品进行RNA-Seq测序,获得差异表达基因,并与本试验的数据比较分析,获得阶段性受Znf1调控的基因,为更系统深入的研究稻瘟病菌致病分子机理提供参考数据。

4 结论

利用RNA-Seq技术获得了Δznf1在营养生长阶段709个差异表达基因的信息,其中一些稻瘟病菌致病相关基因以及附着胞阶段表达水平升高的基因受Znf1调控;397个基因受Znf1和Pmk1共同调控。研究结果为进一步明确稻瘟病菌Znf1转录调控机制以及挖掘新的稻瘟病菌致病相关基因提供了数据资源。
The authors have declared that no competing interests exist.