王晓阳¹, 王丽媛¹, 潘兆娥¹, 何守朴¹, 王骁¹, 龚文芳^,1,3,*, 杜雄明^,1,2,*1中国农业科学院棉花研究所 / 棉花生物学国家重点实验室, 河南安阳455000
²棉花生物学国家重点实验室郑州研究基地 / 郑州大学, 河南郑州 450001
³中南林业科技大学 / 经济林培育与保护教育部重点实验室, 湖南长沙 410004

Analysis of differentially expressed genes and fiber development in Gossypium arboreum fuzzless mutant

WANG Xiao-Yang¹, WANG Li-Yuan¹, PAN Zhao-E¹, HE Shou-Pu¹, WANG Xiao¹, GONG Wen-Fang^,1,3,*, DU Xiong-Ming^{,1,2,* 1}Institute of Cotton Research, Chinese Academy of Agricultural Sciences / State Key Laboratory of Cotton Biology, Anyang 455000, Henan, China
²Zhengzhou Research Base, State Key Laboratory of Cotton Biology / Zhengzhou University, Zhengzhou 450001, Henan, China
³Key Laboratory of Cultivation and Protection for Non-wood Forest, Ministry of Education / Central South University of Forestry and Technology, Changsha 410004, Hunan, China

通讯作者: *杜雄明, E-mail: dxm630723@163.com; 龚文芳, E-mail: gwf018@126.com

收稿日期:2019-09-2接受日期:2019-12-26网络出版日期:2020-01-15

基金资助:

本研究由国家自然科学基金项目.31601353 国家重点研发计划项目资助.2017YFD0101601-03

Received:2019-09-2Accepted:2019-12-26Online:2020-01-15

Fund supported:

This study was supported by the National Natural Science Foundation of China.31601353 the National Key Technology R&D Program.2017YFD0101601-03

作者简介 About authors
E-mail:wangxiaoyang198806@126.com。









摘要
棉花纤维是重要的天然纺织材料, 是最长的单细胞, 是研究纤维发育的良好材料。本研究以亚洲棉短绒突变体(FZ)及其野生型(fz)为材料, 结合扫描电镜、石蜡切片、RNA-seq技术, 解析棉花短绒起始的可能机制。与野生型(fz)的0DPA时期胚珠相比, 突变体的胚珠在该时期仅有少量的纤维起始。在+3DPA时, 突变体没有短绒细胞起始, 仅有长纤维细胞, 而野生型有大量的短纤维细胞和长纤维细胞。对这2个材料的0DPA、+3DPA、+5DPA和+8DPA胚珠差异基因分析结果显示, 在短绒突变体(FZ)和野生型(fz)的4个纤维发育时期共挖掘出3780个差异表达基因, 其中0DPA时差异基因数目最少, 随着胚珠发育时间的延长, 差异基因的数目逐渐增加。KEGG分析发现这些基因主要参与蜡质、角质生物合成, 以及苯丙烷代谢和植物信号传导过程。共表达趋势分析显示, 在突变体+3DPA上调的差异基因中, 参与离子结合、MAPK级联反应、氧化还原活性和转录调控的基因表达受到正影响(表达水平提高), 造成突变体短绒纤维不能正常起始。这些结果描述了二倍体亚洲棉短绒起始的动态变化, 可为进一步研究棉纤维发育提供参考。
关键词： 亚洲棉;短绒突变体;差异表达基因;共表达趋势

Abstract
Cotton fiber, one of main nature textile and longest single cell, is a good material for researching the cotton fiber development. In this study, fuzz mutant (FZ) and wild-type (fz) which belong to diploid Gossypium arboreum species were used for unraveling molecular mechanism of fuzz fiber initiation by scanning electron microscopy (SEM), paraffin section and RNA sequencing technology. It was found that any fibers scarcely initiated on the ovule surface of the mutant at 0DPA, compared with that of the wild-type. Only lint fiber cells emerged on the ovule surface of fuzz mutant, while abundant fuzz and lint fiber cells appeared in wild-type at +3DPA stage. The transcriptome analysis of FZ and fz across the four fiber development stages (0DPA, +3DPA, +5DPA, and +8DPA) indicated that the number of differentially expressed gene was 3780. There were litter differentially expressed genes at 0DPA in FZ and fz. In the proceed of ovule development, the number of differentially expressed genes was increased gradually. The KEGG enriched pathway analysis illustrated that these differentially expressed genes were involved in cutin, suberine and wax biosynthesis, phenylalanine metabolism pathways and plant hormone signal transduction pathways. The analysis of co-expressed gene trend lines indicated that the up-regulated genes in mutant at +3DPA were all involved in metal ion binding, MAPK cascade, oxidoreductase activity and transcription regulation, resulting in failed fuzz fiber cells initiation in mutant. These findings elaborated a dynamic variation about fuzz fiber initiation in diploid G. arboreum, which provides a reference for further research in the cotton fiber development.
Keywords：Gossypium arboreum;fuzzless mutant;differentially expressed gene;co-expressed trend


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本文引用格式
王晓阳, 王丽媛, 潘兆娥, 何守朴, 王骁, 龚文芳, 杜雄明. 亚洲棉短绒突变体纤维发育及其差异基因表达分析[J]. 作物学报, 2020, 46(5): 645-660. doi:10.3724/SP.J.1006.2020.94133
WANG Xiao-Yang, WANG Li-Yuan, PAN Zhao-E, HE Shou-Pu, WANG Xiao, GONG Wen-Fang, DU Xiong-Ming. Analysis of differentially expressed genes and fiber development in Gossypium arboreum fuzzless mutant[J]. Acta Agronomica Sinica, 2020, 46(5): 645-660. doi:10.3724/SP.J.1006.2020.94133


棉花是一种重要的经济作物, 种子是棉籽油的主要原料, 纤维更是一种重要的天然纺织资源。棉花纤维是高等植物中最长的单细胞突起, 由棉花胚珠表皮细胞产生。棉纤维的长度一般为30~40 mm ^[1]。棉花纤维根据形态可以分为长纤维和短绒2种不同的类型。具有重要经济价值的长纤维在开花前0DPA (day post anthesis)发育, 经过快速伸长, 纤维素生物合成和成熟, 最终形成成熟的长纤维, 通过轧花从种皮上收获并用于纺织产品^[2,3]。短绒纤维起始于+3 ~ +5DPA, 长度不到5 mm, 轧花后不能从种皮上分离下来, 紧紧附着于种皮上, 对种子传播起重要作用^[4]。胚珠上短纤维细胞和长纤维细胞的共存现象长期以来一直受到研究者的关注, 为研究植物细胞的分化提供了独特的模式体系。

棉纤维的发育受一系列基因调控, 这些基因与植物激素信号转导、细胞能量代谢、脂肪酸代谢、次生代谢等途径有关。目前关于调控纤维发育的基因和调控网络的信息还不太清楚。在棉花基因组测序之前, 许多研究者主要通过基因芯片技术筛选与棉花纤维发育有关的基因, 在分子水平上了解纤维发育的机制。如Shi等^[5]通过cDNAs芯片分析技术发现了778个cDNAs优先在发育的纤维中表达。同年, Yang等^[6]通过分析陆地棉及其光子突变体转录组, 鉴定出了受激素诱导的转录因子MIXTA、MYB5、GL2在纤维起始过程中高表达。随着棉花基因组测序的完成, 逐步完善了大量的棉花转基因技术, 使得通过转录组分析与纤维发育相关的基因与代谢通路成为可能^[7,8,9]。鉴定出一些与纤维发育及调控细胞壁沉积与强度的候选基因^[10]。通过分析0DPA和+10DPA的二倍体亚洲棉近等基因系有短绒无长纤维材料(Fl)和既有短绒又有长纤维材料(FL)的转录组发现, 一些参与植物激素信号转导、细胞能量代谢, 脂肪酸代谢、刺激代谢的转录因子如AP2- EREBP、AUX/IAA、bHLH、C2H2、C3H、HB、MYB、NAC、Orphans、PLATZ和WRKY在长纤维突变体+ 10DPA纤维中明显下调, 植物受体样激酶(plant receptor-like kinases, RLKs)、富含亮氨酸(Leucine Rich Repeats)的LRR家族蛋白和参与信号转导的编码丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)在突变体中也下调, 导致纤维生长减慢^[11]。

虽然上述转录组分析的研究为棉纤维的起始过程提供了一些研究基础, 但纤维发育的关键基因的研究仍处于初级阶段, 长纤维和短绒纤维发育及生理过程仍然不清楚, 并且目前这些转录组研究主要集中在纤维伸长阶段, 关于短绒纤维起始的动态过程研究较少。

突变体特有的性状是研究基因表达的有利资源, 分析植物突变体加速了特定基因功能的发现。本研究所用的材料为二倍体亚洲棉短绒突变体GA0149有长纤维没有短绒(其光籽性状受单基因显性控制, 基因型为FZ)及其野生型GA0146既有长纤维又有短绒(基因型为fz), 经过连续十代的自花授粉, 突变体植株光籽性状稳定, 突变体和野生型材料在形态和发育上区别微乎其微, 除了光籽突变体种皮上没有短绒^[8]。利用转录组测序技术分析突变体和野生型0DPA、+3DPA、+5DPA和+8DPA 4个时期的差异基因表达情况, 旨在探究棉花短绒起始的动态过程及其关键基因, 为棉花短绒起始的分子机制提供坚实基础。

1 材料与方法

1.1 试验材料

亚洲棉野生型毛子材料为中美棉DPL971 (fz), 短绒突变体材料为中美棉DPL972 (FZ), 均来自棉花种质资源中期库近10年的自交材料。2017年在中国农业科学院棉花研究所实验基地, 用标准的田间管理措施, 按3个重复种植每个材料。

1.2 扫描电镜

将2个材料(fz和FZ) 0DPA、+3DPA的胚珠固定于含有2.5%戊二醛的0.1 mol L^-1磷酸缓冲液中, 用真空泵抽真空15 min, 使胚珠完全浸没于固定液中, 4℃过夜后用0.1 mol L^-1 pH 7.2磷酸缓冲液洗3次, 每次10 min; 接着, 用ddH₂O清洗2次, 每次30 min; 用30% (v/v)、50% (v/v)、70% (v/v)、80% (v/v)、95% (v/v)乙醇对样品梯度脱水, 每个梯度脱水20 min; 用无水乙醇对样品进行3次脱水; 再用等体积的乙醇: 乙酸异戊酯置换15 min; 最后用100% 的乙酸异戊酯置换15 min; 将样品放于样品笼中用CO₂干燥, 然后喷金, 用JSM-6390/LV扫描电镜观察。

1.3 石蜡切片

将新鲜的0DPA、+3DPA的胚珠固定于含有4%的FAA (甲醛-冰乙酸-无水乙醇)固定液中, 抽真空, 保存于4℃冰箱中过夜, 用系列梯度浓度的乙醇(50%、70%、85%、95%和100%)对样品进行脱水处理, 每梯度30 min; 将脱水处理过的胚珠包埋于处理过的石蜡(Sigma-Aldrich)中, 60℃透蜡2 h; 然后将胚珠放于预热的包埋框中, 迅速倒入融化的石蜡, 过夜使石蜡凝固。用石蜡切片机, 切成8 μm 厚的连续切片, 移至42℃的水浴中, 使样品展平, 将切片放在载玻片上, 37℃干燥过夜。用番红固绿染色, 用带有数码相机的显微镜照相。

1.4 文库构建与转录组测序

用北京天根公司的多糖多酚植物总RNA试剂盒提取FZ和fz 0DPA、+3DPA和+5DPA 的胚珠及+8DPA的纤维的RNA。将检测质量合格的RNA送至北京百迈客生物公司进行cDNA文库的构建, 借助 Illumina HiSeq 2000测序仪进行双末端测序, 获得原始数据。

1.5 转录组原始数据的评估以及分析

将得到的原始数据转化为FASTQ形式进行保存, 通过fastp软件过滤掉低质量数据和接头序列, 得到高质量的reads, 即clean data.利用TopHat2/Bowtie2软件将 clean reads 比对到亚洲棉三代基因组上^[8,12-13]。同时对比对到参考基因组的序列进行注释, 在比对过程中, 每个错配最多允许2个碱基。采用FPKM值(Fragments Per Kilobase of transcript per Million fragments mapped)来衡量基因表达^[13]。根据FPKM值, 以错误发现率FDR<0.01, |log₂ fold change| ≥ 1, P-value<0.01作为筛选突变体和野生型差异表达基因的标准。将获得的差异基因进行GO功能富集分析(WEGO: http://wego.genomics.org.cn/cgi-bin/wego/index.pl)和KEGG生物通路分析^[14,15]。

1.6 荧光定量Q-PCR验证

通过分析RNA-seq数据, 验证转录组数据的准确性, 随机挑选22个差异表达的基因, 利用qRT-PCR进行验证。利用在线软件NCBI-Primer (http://www.ncbi. nlm.nih.gov/tools/primer-blast/)设计引物(表1)。分别取不同组织样品的总RNA 1 μg, 利用cDNA反转录试剂盒PrimeScript RT reagent Kit (Perfect real time TaKaRa生物)合成cDNA。利用北京全世金生物公司荧光定量试剂盒(TransStart Top Green qPCR SuperMix), 以UBQ作为内参基因, 利用ABI7500fast Real-time PCR System (美国, ABI公司)开展荧光定量PCR验证。每个基因设计3次技术重复和3次生物学重复。以fz材料0DPA样品的基因表达量作为1, 分析突变体和野生型各个时期的基因相对表达量, 按2^-ΔΔCT法计算相对表达量^[16]。

Table 1
表1
表1用于荧光定量PCR验证的基因和引物
Table 1Primers used for real-time quantitative PCR verify of cotton differentially expressed genes (DEGs)

基因编号 Gene	正向引物 Forward primer (5'-3')	反向引物 Reverse primer (5'-3')	log2RT-QPCR	log2FPKM	功能注释 Functional annotations
Ga02G0173	GTGCTCGGAAGAAAACGCTC	CGCACCTGCCAAAGTTGTTA	1.31	1.10	谷胱甘肽转移酶 Glutathione S-transferase
Ga03G0324	ACAGCAAAAGGCAATCGAGAAC	CGTGGGAAGCTATCGGCTTT	2.85	1.65	谷胱甘肽转移酶 Glutathione S-transferase
Ga04G0014	CTTTCGGGTCAGGTCGAAGG	GTTGTGTTACTCGGGGGACC	3.52	2.89	细胞色素氧化酶 Cytochrome P450
Ga05G3679	GAGCCATCGCTAAGTTCTGGGC	TCCAACAATTTCAGGCACGCAA	-1.19	-1.40	谷胱甘肽转移酶 Glutathione S-transferase
Ga05G3965	CCTGCTATTGCCATCAACACTT	TTGTGCTGGACTTGTTCTTGCT	-2.00	-1.24	分子伴侣 Molecular chaperone
Ga07G0568	CAACGGCGAAAACAACACGGAA	TGCACTGGTCTCCTTCTGTGGA	1.31	1.18	甲基转移酶Methyltransferase
Ga07G1530	GGTGGTAACCCTAGCTTGTGGC	GCGCGTTATGTGTAACTTGCGA	1.26	1.11	转录因子WD40
Ga07G1742	GCCGTGCTAACTTATTCCCTTGC	AGCACCCCAAGTAGTAACCAAGC	-1.54	-1.20	钙联蛋白 Calnexin 1
Ga07G2088	TTGCATCAGAGCAGCCTGCATA	TCTGCTGCCCAAGATTTGCCAT	-1.54	-1.32	转录因子 MYB108
Ga07G2611	GCAGAGGCAGCTTTAAGTTTGGG	GCATCCCACCAGGCCTAAGTTC	-1.61	-1.55	网状蛋白 Reticulon-like protein
Ga08G0121	TGTCTTCGGAACCCGGAGATGA	GGCATCCCACTAGCAGCATTGA	4.47	6.69	蛋白受体激酶 Receptor-like protein kinase
Ga09G0409	TACCGGTGCACTGATCGACCTA	TTCTGAGTCGACTGTTCGTCGC	2.03	1.55	功能未知 Unknown
Ga09G0435	TGCAGGGTTGTTAAGGTGTGGG	TGATTTCGTTATCGGTCCGCCC	1.14	1.05	假定蛋白 Predicted protein
Ga09G0661	CGCCATGCTTGCCATCTTCATC	CACTGAAGGCAGCTTGATCCGA	1.28	1.31	还原酶 Dihydroflavonol-4-reductase
Ga10G0810	ATGAGCCGCCCTATTGACTGCC	TGGTAGATGCCTTCGTTGCTGA	2.24	2.51	RNA加工 RNA processing
Ga10G1419	CCAACAAGGGCCCAAGTGCTAT	TCTTGTTCCCTTGGCCGACTTG	-1.22	-1.20	NADH脱氢酶 NADH dehydrogenase
Ga11G2832	TAAGATCGGTTCGGCTTCGG	ATGCTCCACCCTAACTTGCC	2.01	2.45	假定蛋白 Hypothetical protein
Ga11G2833	CACTTCTCGTGTGATGACCTGT	CATCTTCTAACGCTGGAATCTG	3.70	5.20	谷胱甘肽转移酶 Glutathione S-transferase
Ga11G3625	ATGCTTCTCAACTGCGCAGGAA	GCTGTTGTCGAACTTTATGGGGC	2.45	2.86	谷胱甘肽转移酶 Glutathione S-transferase
Ga11G3670	GACCGATGAAGAGGAACCCAGC	CCATGTGGTGTCCCGGTATGAC	-2.88	-1.78	NADH脱氢酶 NADH dehydrogenase
Ga12G0113	TCGACGTCGCCCTGTTTTTCAT	CTGACGTGGACGCCATGTTGAT	1.53	1.58	假定蛋白 Hypothetical protein
Ga10G0013	TACCAGTTGAAGCCGAAGGCTG	CCATTGTAAGGCCTGCGCTTTG	2.18	1.81	甲基转移酶N-methyltransferase

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2 结果与分析

2.1 二倍体亚洲棉短绒纤维起始的动态学观察

前人研究结果表明, 长纤维细胞起始于开花当天(0DPA), 在纤维发育早期阶段如在+3DPA时, 长纤维已经突破表皮细胞, 长度约为2 mm, 而短纤维细胞还未突破表皮, 但是胚珠表皮细胞开始出现排列不规则现象。为了鉴定野生型毛子fz和突变体FZ短绒纤维起始的差异, 取这2个材料的0和+3DPA的胚珠, 用扫描电镜观察发现, 在0DPA时野生型和突变体都有明显的纤维突起, 但是野生型纤维明显长于突变体, 而且纤维数目多于突变体; 当胚珠发育到+3DPA时, fz和FZ长纤维长度基本一致, 但纤维数目fz多于FZ, 由于胚珠表面长纤维的干扰, 无法观察到胚珠表面的短纤维(图1-A)。为了进一步验证短绒在细胞水平上的突起, 在电子显微镜下观察fz和FZ 0DPA和+3DPA的胚珠的石蜡切片, 如图1-B所示, 在野生型 fz 胚珠表面能观察到长纤维和短纤维细胞, 其中红色箭头所指位置为长纤维, 而黑色箭头所指位置为短纤维细胞。在0DPA时野生型纤维细胞开始隆起, 而突变体只有少量突起; 随着胚珠的发育 fz 和 FZ 突起细胞数目都有所增加, 但是在+3DPA时, 短绒纤维开始在 fz 胚珠表面出现, 而 FZ 胚珠在相同的发育阶段没有短绒细胞突起, 表明短纤维细胞在FZ中起始失败。

图1

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图1野生型fz和短绒突变体FZ材料中短纤维发育过程的形态学观察

A: 野生型、短绒突变体的种子以及扫描电镜下看到的短绒纤维起始; B: 石蜡切片所示短绒纤维起始。红色箭头所示长纤维, 黑色箭头所示短纤维细胞。
Fig. 1Morphology analysis of fuzz-fiber development in normal wilt-type fz (upper) and fuzz mutant FZ (lower)

A: the seeds of wild type, natural fuzz mutant and fuzz fiber initiation under scanning electron microscope; B: paraffin section showing fuzz fiber initiation. Red arrow indicates lint fiber, and black arrow indicates fuzz fiber cells.

2.2 转录组数据质量评估

为了进一步理解短绒发育的动态过程和鉴定与短绒发育相关的基因, 选取突变体FZ和野生型材料fz 4个发育时期的胚珠和纤维(0DPA、+3DPA、+5 DPA、+8DPA)的RNA, 每个材料3个生物学重复, 构建了24个RNA文库。

24个测序文库获得的约3.2亿条原始数据经去接头和过滤后, 产生出约1.6亿条clean reads。每个文库平均得到65,841,318个高质量的reads (表2)。大约有88%的clean reads可成功比对到已发表的亚洲棉三代基因组上。比对到参考基因组上唯一位置和多个位置的比例分别约为76%~85%、3.53%~6.00%。比对到基因组正链的数目与比对到负链的数目几乎相当。另外每个样本的Q30比例均大于86%, GC含量在35%~65%衡量指标之间。

Table 2
表2
表2样品测序数据与参考基因组序列比对结果分析
Table 2Analysis of transcriptome data and comparison with reference genome

编号 No	过滤后的序列数 Clean reads	GC GC (%)	Q30 Q30 (%)	原始数 Total reads	比对到参考基因组的总数 Mapped reads	比对到参考基因组唯一位置的数目 Uniq mapped reads	比对到参考基因组多个位置的数目 Multiple mapped reads	比对到正链数 Reads map to “+”	比对到负链数 Reads map to “-“
L01	6E+07	42.75	88.41	1.13E+08	100098347(88.79%)	95615963 (84.82%)	4482384 (3.98%)	49356770 (43.78%)	49542917 (43.95%)
L02	7E+07	42.89	87.89	1.49E+08	132564763(88.84%)	126665191 (84.88%)	5899572 (3.95%)	65411403 (43.84%)	65613688 (43.97%)
L03	6E+07	43.73	88.21	1.29E+08	114781600(89.23%)	109876069 (85.42%)	4905531 (3.81%)	56731466 (44.10%)	56901283 (44.24%)
L04	7E+07	43.32	87.93	1.31E+08	115706679(88.26%)	109946001 (83.86%)	5760678 (4.39%)	57037850 (43.51%)	57182987 (43.62%)
L05	6E+07	43.45	88.18	1.15E+08	101447417(88.47%)	96871049 (84.47%)	4576368 (3.99%)	50115129 (43.70%)	50235739 (43.81%)
L06	7E+07	43.65	88.41	1.36E+08	119977298(88.23%)	114195412 (83.98%)	5781886 (4.25%)	59276583 (43.59%)	59413894 (43.69%)
L07	9E+07	43.47	88.41	1.85E+08	164355512(88.86%)	156168989 (84.43%)	8186523 (4.43%)	81092741 (43.84%)	81289251 (43.95%)
L08	6E+07	43.21	87.12	1.24E+08	108363137(87.68%)	102760244 (83.15%)	5602893 (4.53%)	53406006 (43.21%)	53552268 (43.33%)
L09	6E+07	43.53	88.53	1.25E+08	110741117(88.61%)	105077371 (84.08%)	5663746 (4.53%)	54559214 (43.66%)	54711263 (43.78%)
L10	6E+07	43.93	88.53	1.12E+08	95982638(85.64%)	89277314 (79.66%)	6705324 (5.98%)	47006407 (41.94%)	47142478 (42.06%)
L11	6E+07	43.62	87.74	1.24E+08	106630817(85.76%)	99506038 (80.03%)	7124779 (5.73%)	52250173 (42.03%)	52415958 (42.16%)
L12	7E+07	43.65	88.90	1.45E+08	125095168(86.41%)	116813022 (80.69%)	8282146 (5.72%)	61391019 (42.41%)	61584013 (42.54%)
L13	6E+07	43.78	89.76	1.28E+08	109195486(85.27%)	104512429 (81.62%)	4683057 (3.66%)	53869063 (42.07%)	54027240 (42.19%)
L14	7E+07	43.40	90.20	1.31E+08	115537224(88.07%)	110898150 (84.53%)	4639074 (3.54%)	57057026 (43.49%)	57220578 (43.61%)
L15	8E+07	43.20	90.60	1.63E+08	144883755(88.62%)	139117996 (85.09%)	5765759 (3.53%)	71628619 (43.81%)	71848062 (43.94%)
L16	7E+07	44.19	89.70	1.44E+08	122786455(85.34%)	116535579 (80.99%)	6250876 (4.34%)	60495477 (42.04%)	60621983 (42.13%)
L17	8E+07	44.14	90.20	1.56E+08	130032853(83.13%)	123871138 (79.19%)	6161715 (3.94%)	64115657 (40.99%)	64295623 (41.10%)
L18	7E+07	43.38	90.49	1.44E+08	127237888(88.61%)	121687992 (84.75%)	5549896 (3.87%)	62876344 (43.79%)	63029624 (43.90%)
L19	6E+07	43.32	91.03	1.19E+08	106495539(89.28%)	101489480 (85.08%)	5006059 (4.20%)	52555236 (44.06%)	52649155 (44.14%)
L20	6E+07	43.38	91.29	1.16E+08	101384931(87.60%)	96799488 (83.64%)	4585443 (3.96%)	50050156 (43.24%)	50157927 (43.34%)
L21	6E+07	43.63	91.17	1.12E+08	98144438(87.78%)	93630910 (83.74%)	4513528 (4.04%)	48444159 (43.33%)	48541604 (43.42%)
L22	6E+07	44.24	87.94	1.22E+08	100479675(82.20%)	92865206 (75.98%)	7614469 (6.23%)	49048631 (40.13%)	49206041 (40.26%)
L23	6E+07	44.06	87.32	1.23E+08	102829882(83.46%)	95769710 (77.73%)	7060172 (5.73%)	50222205 (40.76%)	50359879 (40.87%)
L24	6E+07	44.39	86.86	1.14E+08	96749028(84.55%)	90393952 (79.00%)	6355076 (5.55%)	47365502 (41.39%)	47485322 (41.50%)

L01, L02, L03 represent the three repeats of fz 0DPA; L04, L05, L06 represent the three repeats of fz +3DPA; L07, L08, L09 represent the three repeats of fz +5DPA; L10, L11, L12 represent the three repeats of fz +8DPA; L13, L14, L15 represent the three repeats of FZ 0DPA; L16, L17, L18 represent the three repeats of FZ +3DPA; L19, L20, L21 represent the three repeats of FZ +5DPA; L22, L23, L24 represent the three repeats of FZ +8DPA.
L01、L02、L03代表fz 0DPA的3个重复;L04、L05、L06 代表fz +3DPA的3个重复;L07、L08、L09代表fz +5DPA的3个重复;L10、L11、L12代表fz +8DPA的3个重复;L13、L14、L15代表FZ 0DPA的3个重复;L16、L17、L18代表FZ +3DPA的3个重复;L19、L20、L21代表FZ +5DPA的3个重复;L22、L23、L24代表FZ +8DPA的3个重复。

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为了鉴定24个样品间的生物学重复关系, 对各样本间生物学重复数据进行皮尔逊相关系数(Pearson Correlation Coefficient, PCC)分析(图2), 结果发现, fz材料3个生物学重复间的皮尔逊相关系数均大于0.98 (图2-A), FZ 材料3个生物学重复间的皮尔逊相关系数均大于0.94 (图2-B), 说明这2个材料不同样本的3个生物学重复较好, 可进行差异基因表达分析。

图2

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图2所有样本间皮尔逊相关系数分析

A和B分别代表fz和FZ
Fig. 2Pearson correlation coefficients of all samples

A and B indicate fz and FZ, respectively.

2.3 差异基因表达分析

计算FPKM值后, 选择P-value<0.05和|log₂(ratio)| ≥1作为筛选差异表达基因的标准。通过比较突变体 FZ和野生型fz材料0、+3、+5、+8DPA的FPKM值, 共鉴定出3784个差异基因(图3-A)。其中在 fz vs. FZ 0DPA时期特异表达的基因有201个, fz vs. FZ +3DPA时期特异表达的基因有313个, fz vs. FZ +5DPA特异表达的基因有1108个, fz vs. FZ +8DPA时期特异表达的基因有1303个。突变体和野生型4个纤维发育时期发现91个共有的差异基因, 这些基因的差异表达情况见图3-B和表3, 52个基因明显上调, 39个基因明显的下调, 对差异基因功能分析发现一些未知蛋白、转录因子、乙烯受体和抗病基因都参与了短绒纤维的发育。

图3

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图3fz及其短绒突变体FZ不同纤维发育时期共有的差异基因

A: 在fz和FZ材料中不同纤维发育时期差异基因韦恩图; B: 在fz和FZ两个材料中4个纤维发育时期91个共有的差异基因热图。
Fig. 3Common DEG at different fiber development stages in fz and fuzz-mutant FZ

A: Venn diagram of significant DEGs found in fz and FZ across various fiber development stages; B: Heatmap showing 91 common significant DEGs in fz and FZ across fiber development stages.


Table 3
表3
表3在fz和FZ两个材料中4个纤维发育时期91个共有的差异基因及其表达情况
Table 3Ninety-one common significant DEGs found in fz and FZ across fiber development stages and their expression

基因编号 Gene ID	基因注释Gene annotation	0DPA	+3DPA	+5DPA	+8DPA
Ga11G2831	含有WD40结构的蛋白 WD40 repeat-like superfamily protein	2.00	2.70	3.00	1.72
Cotton_newGene_517935	假定蛋白 Hypothetical protein	2.55	2.94	1.88	1.84
Ga03G0324	谷胱甘肽S-转移酶 Glutathione S-transferase	1.33	1.65	1.63	1.05
Ga11G0115	假定蛋白 Hypothetical protein	-1.25	-1.28	-1.42	-1.05
Ga07G1809	硫代硫酸盐ATP结合蛋白 Sulfate/thiosulfate ATP-binding protein	2.35	2.34	2.40	0.95
Cotton_newGene_190500	未鉴定蛋白 Uncharacterized protein	2.37	2.67	0.97	1.16
Cotton_newGene_217366	未鉴定蛋白 Uncharacterized protein	2.03	2.44	1.33	1.65
Ga11G3311	葡萄糖基转移酶 UDP-glycosyltransferase 83A1	1.25	1.98	1.50	1.85
Ga09G2202	乙烯响应因子 AP2/ERF	1.95	1.31	2.13	-1.42
Ga09G0608	亮氨酸拉链蛋白 Leucine-rich repeat protein kinase	-1.90	-1.49	-0.70	-0.20
Cotton_newGene_190685	假定蛋白 Hypothetical protein	1.39	1.60	0.94	0.89
Cotton_newGene_184413	假定蛋白 Hypothetical protein	0.86	0.82	0.68	0.20
Cotton_newGene_216530	假定蛋白 Hypothetical protein	1.42	1.92	0.70	1.48
Ga06G0475	泛素连接酶 E3 ligase	-0.74	-0.80	-0.53	-0.61
Cotton_newGene_206595	假定蛋白 Hypothetical protein	0.62	0.51	0.22	0.08
Cotton_newGene_188033	假定蛋白 Hypothetical protein	-3.18	-2.89	-2.89	-1.50
Ga14G2119	谷氨酸受体 Glutamate receptor	3.94	3.27	3.11	1.92
Cotton_newGene_206588	假定蛋白 Hypothetical protein	2.13	2.00	1.73	0.92
Ga11G2832	自噬相关蛋白 Autophagy-related protein	2.42	2.45	2.66	1.38
Ga06G1674	锌依赖的包外多肽酶蛋白 Zn-dependent exopeptidases protein	-1.22	-0.78	-0.75	-0.57
Cotton_newGene_313214	TMV抗性蛋白 TMV resistance protein	0.42	1.14	1.27	0.73
Ga04G1219	乙酰基葡萄糖转移酶 UDP-GlcNAc	1.21	1.37	1.40	0.78
Cotton_newGene_383086	ATP合成酶 ATP synthase	1.29	1.00	1.15	0.57
Cotton_newGene_336363	核酸内切酶 Endonuclease	1.47	1.00	1.30	0.46
Cotton_newGene_521387	功能未知 Unknown	1.87	1.81	1.73	1.36
Ga04G0915	功能未知 Unknown	1.43	1.30	1.23	0.32
Cotton_newGene_190683	细胞色素氧化酶 P450 Cytochrome P450	2.78	3.25	2.09	1.73
Cotton_newGene_94861	核酸酶 Putative nuclease	-1.00	-0.86	-0.93	-0.65
Cotton_newGene_195900	功能未知 Unknown	-3.47	-3.50	-3.45	-2.85
Ga10G0013	甲基转移酶 Histone-lysine N-methyltransferase	1.78	1.81	2.10	1.35
Ga11G0121	木糖梅 Alpha-xylosidase 1	-1.64	-0.77	-0.92	-1.26
Ga11G0187	假定蛋白 Hypothetical protein	-0.85	-0.74	-0.81	-0.48
Ga11G0110	假定蛋白 Hypothetical protein	0.82	1.21	0.71	0.84
Ga06G1755	脯氨酸受体蛋白激酶Proline-rich receptor-like protein kinase	-1.00	-0.52	-0.98	-0.95
Ga05G2954	天冬氨酸酶 Asparticase	0.65	0.58	1.25	0.24
Cotton_newGene_572274	假定蛋白 Hypothetical protein	2.24	2.62	1.48	1.69
Cotton_newGene_353692	假定蛋白 Hypothetical protein	-0.56	-0.63	-0.71	-0.49
Cotton_newGene_483966	假定蛋白 Hypothetical protein	2.77	2.77	2.71	2.30
Ga11G0111	微丝蛋白 Fb17	-0.31	-0.75	-1.21	-2.11
Cotton_newGene_99176	丝氨酸/苏氨酸蛋白磷酸酶 Serine/threonine protein phosphatase	1.08	0.85	0.97	1.10
Ga12G1137	乙烯受体蛋白 AP2/ERF	1.25	1.53	1.81	-1.25
Ga11G2354	假定蛋白 Hypothetical protein	1.73	1.58	1.68	0.31
Cotton_newGene_517938	细胞色素氧化酶 P450 Cytochrome P450	4.11	4.65	3.60	3.14
Cotton_newGene_300782	核酸内切酶亚基 Endonuclease subunit	1.47	1.44	1.06	1.14
Cotton_newGene_252500	Copia型多蛋白 Copia-type polyprotein	2.74	2.88	2.52	2.00
Cotton_newGene_517934	功能未知 Unknown	3.19	3.84	2.65	1.73
Cotton_newGene_353671	缬氨酸-tRNA连接酶 Valine-tRNA ligase	-0.39	-0.95	-0.65	-0.94
Ga05G3965	功能未知 Unknown	-1.55	-1.24	-1.95	-1.34
Ga09G1652	丝氨酸/苏氨酸蛋白磷酸酶 Serine/threonine protein phosphatase	-1.61	-1.34	-1.01	-1.25
Ga04G1773	转录因子 NAC1	1.20	0.98	2.02	-1.43
Ga09G1364	乙酰辅酶A氧化酶 Acyl-coenzyme A oxidase	2.66	2.52	2.53	2.06
Cotton_newGene_213765	假定蛋白 Hypothetical protein	-1.28	-1.29	-1.89	-1.22
Ga04G0280	富含脯氨酸蛋白 Proline-rich protein	0.71	0.77	1.46	1.21
Cotton_newGene_217374	假定蛋白 Hypothetical protein	1.56	1.89	0.72	1.26
Ga10G0798	细胞壁受体激酶 Wall-associated receptor kinase	0.44	0.41	0.30	0.13
Ga03G0640	功能未知 Unknown	1.88	1.62	1.14	1.16
Cotton_newGene_577041	功能未知 Unknown	3.08	1.66	3.58	0.40
Ga07G1742	含有CBS结构域的蛋白 CBS domain-containing protein	-1.45	-1.20	-1.17	-0.69
Ga07G2703	丝氨酸/苏氨酸蛋白磷酸酶 Serine/threonine-protein kinase	-0.81	-0.59	-0.39	-0.26
Ga07G0566	转录因子 bHLH137	1.05	1.03	3.13	-0.50
Cotton_newGene_190686	假定蛋白 Hypothetical protein	1.42	1.61	0.84	0.86
Ga04G1342	未鉴定蛋白 Uncharacterized protein	0.80	0.30	0.17	0.37
Cotton_newGene_563611	未鉴定蛋白 Uncharacterized protein	2.35	2.52	1.44	1.83
Cotton_newGene_592029	丝裂原活化蛋白激酶 Mitogen-activated protein kinase MMK1	-1.65	-1.23	-0.87	-0.46
Ga01G1687	富含半胱氨酸/组氨酸蛋白 Cysteine/histidine-rich protein	0.74	0.58	0.54	0.48
Cotton_newGene_54250	功能未知 Unknown	2.88	2.87	2.67	1.88
Cotton_newGene_353710	假定蛋白 Hypothetical protein	1.50	1.25	1.10	1.44
Cotton_newGene_474685	未鉴定蛋白 Uncharacterized protein	2.45	2.75	1.40	1.79
Cotton_newGene_67170	假定蛋白 Hypothetical protein	3.06	2.61	2.59	1.44
Cotton_newGene_353674	芳香类多肽 Pentafunctional arom polypeptide	1.41	1.31	1.33	1.02
Ga03G1055	木聚糖糖基化酶 Xyloglucan endotransglucosylase	0.83	2.34	-1.71	0.96
Ga11G0123	甲硫氨酸tRNA转移酶 Methionine-tRNA ligase	-2.16	-1.98	-1.73	-1.21
Ga07G0272	咖啡酸盐酯酶 Caffeoylshikimate esterase	-1.59	-1.52	-1.82	-1.20
Cotton_newGene_57872	假定蛋白 Hypothetical protein	0.51	0.71	0.36	0.36
Ga03G2383	受体蛋白 Receptor	-0.81	-1.43	-2.23	1.21
Ga02G0173	假定蛋白 Hypothetical protein	1.44	1.10	1.19	1.04
Cotton_newGene_509534	假定蛋白 Hypothetical protein	1.82	2.28	1.03	1.35
Ga11G0124	未鉴定蛋白 Uncharacterized protein	-2.92	-4.04	-3.64	-3.84
Ga13G1311	丝氨酸/苏氨酸蛋白磷酸酶 Serine/threonine-protein phosphatase	1.80	1.60	1.61	1.42
Ga10G1419	NADH脱氢酶 NADH dehydrogenase	-0.99	-1.20	-1.37	-1.31
Cotton_newGene_190499	未鉴定蛋白 Uncharacterized protein	1.30	1.60	0.80	0.91
Cotton_newGene_217363	假定蛋白 Hypothetical protein	1.69	2.16	0.97	1.75
Ga07G2215	未鉴定蛋白 Uncharacterized protein	1.38	2.23	-2.23	0.98
Ga11G2833	谷胱甘肽S-转移酶 Glutathione S-transferase	4.06	5.20	5.45	1.92
Cotton_newGene_473974	功能未知 Unknown	1.76	1.56	1.46	0.69
Ga07G0353	富含脯氨酸蛋白 Proline-rich protein	1.48	1.61	1.06	0.36
Cotton_newGene_99000	假定蛋白 Hypothetical protein	0.73	0.61	0.46	0.92
Ga07G1792	CBL9	-1.33	-1.33	-1.07	-0.68
Cotton_newGene_190497	假定蛋白 Hypothetical protein	2.55	2.89	2.28	1.86
Ga09G1540	蛋白YLS9 YLS9 protein	0.88	1.12	-0.82	0.66
Ga09G1216	川芎嗪-3 Tetraspanin-3	-1.02	-1.53	-0.94	-2.15

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通过比较同一材料不同纤维发育时期的差异基因发现(图4-A)在fz材料中, 开花当天至+3DPA时共有2227个基因上调, 1113个基因下调; +3DPA至+5DPA时期, 601个基因上调, 194个基因下调; +5DPA至+8DPA时期2993个基因上调, 3703个基因下调。+3DPA至+5DPA时期差异基因数目较少。在FZ材料中, 0到+3DPA时期, 2420个基因发生了上调, 下调基因的数目是1201; +3DPA到+5DPA时期, 611个基因上调, 753个基因下调; +5DPA到+8DPA时期, 5465个基因上调, 6686个基因下调。

图4

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图4fz及其短绒突变体FZ不同纤维发育时期差异基因概况

A: 在fz和FZ材料中不同纤维发育时期差异基因数目; 红色代表上调的基因, 绿色代表下调基因。B: 1783个差异基因在fz和FZ材料4个短绒纤维发育时期的表达趋势。C: fz和FZ材料4个短绒纤维发育时期差异基因KEGG 富集通路。
Fig. 4Overview of DEGS at different fiber development stages in fz and fuzz-mutant FZ

A: number of genes differentially expressed during fiber development within and between fz and FZ; Red represent up-regulated, green represent down-regulated gene. B: the expression trends of 1783 DEGs in fz and FZ across fiber development stages; C: the DEGs KEGG pathway in fz and FZ across fiber development stages.


比较突变体FZ和野生型fz同一纤维时期的差异基因(图4-A, B)表明, 在开花当天与野生型材料相比, 突变体中341个基因上调, 174个基因下调; 在+3DPA时, 突变体中612个基因上调, 下调基因数目170; 在+5DPA时期, 突变体中发生了1018个基因上调, 下调基因数目为802个; 在+8DPA时期, 1230个基因下调, 641个基因上调; 突变体中在短绒起始期上调基因的数目多于下调基因的数目, 这些结果证明大部分基因负调控短绒纤维的起始。

2.4 与短绒起始相关的差异基因KEGG通路分析

通过对2个材料4个纤维发育时期的差异基因KEGG (Kyoto Encyclopedia of Genes and Genomes)通路富集分析(图4-C)发现, 40个基因参与到植物-病原菌互作过程; 20个基因参与到精氨酸和脯氨酸代谢过程; 52个基因参与到内质网上蛋白加工过程; 30个基因涉及到苯丙烷代谢过程; 17个基因涉及到角质、木质素和蜡质生物合成过程; 44个基因参与到苯丙烷生物合成过程; 92个基因汇集到植物激素信号传导过程。对KEGG途径富集因子较高的植物激素信号传导途径中的苯丙烷生物合成途径和角质木质素以及蜡质生物合成途径的差异基因进一步分析发现(表4和表5), 26个差异基因与生长素响应以及生物合成有关, 与野生型(fz)相比, 突变体(FZ)大部分基因在+5DPA时上调, 在+8DPA时下调, 在+3DPA时基因表达差异不明显, 表明生长素(IAA)对短绒的起始没有影响, 对短绒纤维的伸长起正调控作用; 12个基因参与脱落酸(ABA)和乙烯信号转导过程, 如乙烯响应因子和脱落酸受体, 这些基因在短绒纤维起始时期+5DPA突变体中显著上调, Ga11G2673基因差异倍数达到15以上, 而这些基因在纤维伸长时期+8DPA突变体中下调, 这些结果说明棉花体内过量的ABA和乙烯抑制纤维的发育, 而少量的乙烯能促进纤维伸长。参与角质、软木质、蜡质生物合成途径的基因有8个, 主要是细胞色素氧化酶P450和脂肪酸羟化酶(表4); 参与苯丙烷代谢和生物合成途径有关的基因有23个, 大部分为过氧化物酶以及2个苯丙氨酸裂合酶、3个甲基转移酶和1个花青素葡萄糖基转移酶, 说明这些基因与短绒纤维的起始和发育有关。

Table 4
表4
表4角质、软木质、蜡质生物合成途径以及苯丙烷代谢途径的差异基因及其表达情况
Table 4DEGs functioning in cutin, suberine and wax biosynthesis, and phenylalanine metabolism pathways and their expression

基因编号Gene ID	基因功能 Gene function	0DPA	+3DPA	+5DPA	+8DPA	KEGG_pathway_annotation
Ga01G2133	ω-羟基棕榈酸酯-乙酰酰转移酶 Omega-hydroxypalmitate-feruloyl transferase	0.05	0.52	-0.11	1.07	Cutin, suberine, and wax biosynthesis
Ga12G0500	细胞色素氧化酶 P450 Cytochrome P450	-0.06	0.18	-1.22	-1.24	Cutin, suberine, and wax biosynthesis
Ga07G2444	细胞色素氧化酶 P450 Cytochrome P450	0.06	1.06	-0.18	-0.40	Cutin, suberine, and wax biosynthesis
Ga08G1573	细胞色素氧化酶 P450 Cytochrome P450	-0.81	-0.68	-0.63	1.02	Cutin, suberine, and wax biosynthesis
Ga08G2220	细胞色素氧化酶 P450 Cytochrome P450	-1.95	-2.11	-2.22	0.14	Cutin, suberine, and wax biosynthesis
Ga10G2228	ω-羟基棕榈酸酯-乙酰酰转移酶 Omega-hydroxypalmitate-feruloyl transferase	0.59	-1.55	0.32	0.59	Cutin, suberine, and wax biosynthesis
Ga04G1297	脂肪酸羟化酶 Fatty acid hydroxylase	-0.95	-0.15	0.31	2.10	Cutin, suberine, and wax biosynthesis
Ga07G2443	细胞色素氧化酶 P450 Cytochrome P450	0.05	1.16	-0.09	-0.37	Cutin, suberine, and wax biosynthesis
Ga05G2018	过氧化物酶Peroxidase	0.34	0.88	1.20	-0.25	Phenylalanine metabolism
Ga14G0143	肉桂醇脱氢酶 Cinnamyl alcohol dehydrogenase	-0.06	0.47	0.77	-1.41	Phenylpropanoid biosynthesis
Ga10G0561	甲基转移酶 Caffeic acid-methyltransferase	0.08	-0.02	-0.19	-1.30	Phenylalanine metabolism
Ga11G3625	过氧化物酶 Peroxidase	0.65	2.86	1.37	1.11	Phenylalanine metabolism
Ga12G1737	甲基转移酶 Caffeic acid-methyltransferase	-1.18	-0.54	-0.96	1.15	Phenylpropanoid biosynthesis
Ga05G1817	过氧化物酶 Peroxidase	-0.05	0.33	-0.17	-2.71	Phenylalanine metabolism
Ga12G2843	过氧化物酶 Peroxidase	-1.71	-1.17	-0.29	-0.02	Phenylalanine metabolism
Ga12G0336	甲基转移酶 Caffeic acid-methyltransferase	0.35	0.44	-0.05	-1.12	Phenylpropanoid biosynthesis
Ga03G0366	花青素葡萄糖基转移酶 Anthocyanidin-glucosyltransferase	0.25	0.72	-0.22	1.45	Phenylpropanoid biosynthesis
Ga05G0239	乙烯脱氢酶 Aldehyde dehydrogenase	-0.01	0.02	0.37	-3.41	Phenylpropanoid biosynthesis
Ga09G0681	葡萄糖苷酶 Glucosidase	-0.18	0.30	-0.15	-1.20	Phenylpropanoid biosynthesis
Ga04G1983	过氧化物酶 Peroxidase	0.08	0.33	0.23	-1.16	Phenylalanine metabolism
Ga09G1700	苯丙氨酸氨裂合酶 Phenylalanine ammonia-lyase	-0.19	0.37	-1.21	-0.84	Phenylalanine metabolism
Ga01G2475	过氧化物酶 Peroxidase 68	0.42	2.69	0.60	-0.49	Phenylalanine metabolism
Ga10G0968	肉桂酸4-羟化酶 Cinnamate 4-hydroxylase	0.24	-0.26	-1.28	-0.01	Phenylalanine metabolism
Ga11G1671	过氧化物酶 Peroxidase 17	0.13	0.46	0.05	-2.06	Phenylalanine metabolism
Ga10G1786	过氧化物酶 Cationic peroxidase 1	0.46	1.38	0.50	-0.25	Phenylalanine metabolism
Ga10G1772	莽草酸O-羟基肉桂酰基转移酶 Shikimate O-hydroxycinnamoyltransferase	0.04	1.14	1.78	1.38	Phenylpropanoid biosynthesis
Ga11G2088	过氧化物酶 Peroxidase 72	0.42	0.22	0.79	-2.38	Phenylalanine metabolism
Ga13G0640	细胞色素氧化酶 P450 Cytochrome P450	-0.09	0.15	0.12	-1.35	Phenylpropanoid biosynthesis
Ga12G1739	氨基甲酸N-甲基转移酶 Anthranilate N-methyltransferase	-1.30	-0.86	-0.68	0.34	Phenylpropanoid biosynthesis
Ga04G0842	苯丙氨酸裂解酶 Phenylalanine ammonia-lyase	-0.12	0.06	-1.08	-0.88	Phenylalanine metabolism
Ga04G1242	过氧化物酶 Peroxidase 51	0.44	1.08	-0.02	0.17	Phenylalanine metabolism

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Table 5
表5
表5植物激素转导途径中的差异基因及其表达情况
Table 5DEGs in plant hormone signal transduction pathways and their expression

基因编号 Gene ID	基因注释 Gene annotation	基因名称Gene name	激素Hormone	0DPA	+3DPA	+5DPA	+8DPA
Ga06G1125	乙烯响应因子 EIN3-binding F-box protein 1	EIN3	ETH	1.1	1.1	4.6	0.4
Ga01G1781	生长素响应因子Auxin response factor 9	ARF9	IAA	1.0	1.2	2.4	0.6
Ga09G2536	生长素诱导蛋白Auxin-induced protein 22D	AUX22D	IAA	1.6	1.0	2.6	1.6
Ga02G1198	脱落酸不敏感蛋白Abscisic acid-insensitive protein	ABI3	ABA	0.7	0.8	2.1	0.4
Ga01G2735	转录因子MYC4 Transcription factor MYC4	MYC4	JA	1.7	1.4	4.1	0.5
Ga13G1098	生长素诱导蛋白Auxin-induced protein X10A	AIP10A	IAA	1.0	1.0	0.6	2.2
Ga03G2153	吲哚乙酸合成酶Indole-3-acetic acid-amido synthetase	IAA	IAA	0.9	5.0	2.7	0.6
Ga05G2728	生长素诱导蛋白Auxin-induced protein 6B	AIP6B	IAA	1.2	0.9	3.2	1.0
Ga11G1087	生长素响应蛋白Auxin-responsive protein IAA16	IAA16	IAA	0.8	1.1	4.2	0.4
Ga07G0242	脱落酸受体Abscisic acid receptor	PYL2	ABA	1.2	1.7	1.5	0.7
Ga01G0771	吲哚乙酸合成酶 Indole-3-acetic acid-amido synthetase	IAA	IAA	1.4	1.3	3.0	0.5
Ga08G2804	乙烯响应因子 Ein3-binding F-box protein 4	EIN3	ETR	1.4	1.2	4.6	0.7
Ga06G2183	磷酸转移蛋白Histidine-containing phosphotransfer protein 4	AHP4	CK	1.0	1.0	0.6	0.4
Ga10G2395	生长素响应蛋白 Auxin-responsive protein IAA26	SRK2I	IAA	0.8	1.4	2.3	0.7
Ga10G3077	生长素诱导蛋白Auxin-induced protein X15	AIPX15	IAA	0.3	0.4	0.7	1.0
Ga08G2252	生长素响应蛋白Auxin-responsive protein IAA11	IAA11	IAA	1.2	0.9	2.5	0.6
Ga03G2608	生长素诱导蛋白Auxin-induced protein X10A	AIP10A	IAA	2.2	0.8	1.3	1.0
Ga03G0428	乙烯受体Ethylene receptor 2	ETR2	ETH	1.3	0.9	2.6	0.7
Ga11G1046	乙烯受体Ethylene receptor	EIN4	ETH	1.3	1.2	2.2	0.8
Ga07G0142	蛋白磷酸酶 Protein phosphatase 2C	PP2C	ABA	1.5	1.6	4.0	0.8
Ga05G0163	乙烯响应蛋白EIN3-binding F-box protein 1	EIN3	ETH	0.9	1.2	3.6	0.4
Ga09G2537	生长素响应蛋白 Auxin-responsive protein IAA14	IAA14	IAA	1.0	0.5	0.4	1.2
Ga12G2846	赤霉素受体 Gibberellin receptor GID1B	GID1B	GA	1.2	1.8	2.1	0.4
Ga08G2885	蛋白磷酸酶 Protein phosphatase 2C	PP2C77	ABA	1.4	1.3	3.9	0.4
Ga07G0455	生长素响应蛋白 Auxin-responsive protein IAA9	IAA9	IAA	1.6	0.8	2.6	0.3
Ga03G2632	生长素转运蛋白 Auxin transporter-like protein 5	ATP5	IAA	0.9	1.1	2.3	0.8
Ga11G3045	脱落酸受体 Abscisic acid receptor	PYL9	ABA	1.6	1.2	4.2	0.4
Ga11G1086	生长素诱导蛋白 Auxin-induced protein 22D	AUX22D	IAA	1.3	1.8	6.4	0.5
Ga03G2592	生长素诱导蛋白 Auxin-induced protein 15A	AUX15A	IAA	0.9	0.6	0.5	2.2
Ga05G1344	生长素响应蛋白 Auxin-responsive protein IAA16	IAA16	IAA	1.5	1.0	2.3	0.6
Ga05G2981	细胞分裂素响应蛋白 Cytokinin ARR17	ARR17	IAA	1.0	1.2	1.0	0.5
Ga11G2673	乙烯响应转录因子 Ethylene-responsive transcription factor	ERF10	ETH	1.8	1.0	15.7	0.1
Ga12G2331	ABA相关蛋白激酶 ABA-related protein kinase	SNF1	ABA	1.2	1.3	2.1	0.9
Ga05G0157	生长素响应蛋白IAA29 Auxin-responsive protein IAA29	IAA29	IAA	1.3	0.8	0.5	0.5
Ga02G1269	水杨酸相关蛋白 Salicylic acid-related protein	PRP1	SA	0.8	0.9	0.4	0.5
Ga06G2185	磷酸转移蛋白 Histidine phosphotransfer protein	AHP3	CK	1.0	0.9	0.7	0.5
Ga13G2114	生长素诱导蛋白Auxin-induced protein 22B	AUX15A	IAA	1.4	1.2	2.1	1.9
Ga05G3500	生长素诱导蛋白 Auxin-induced protein X15	AUX15	IAA	3.6	0.7	0.9	0.6
Ga07G1535	吲哚乙酸合成酶 Indole-3-acetic acid-amido synthetase	IAA	IAA	0.8	1.3	1.5	0.4
Ga09G2560	乙烯受体 Ethylene receptor 2	ETR2	ETH	1.3	1.4	6.3	0.3
Ga03G2582	生长素诱导蛋白 Auxin-induced protein 15A	AUX15A	IAA	1.0	0.9	2.1	0.8
Ga05G1383	生长素响应蛋白 Auxin-responsive protein IAA27	IAA27	IAA	1.1	1.1	1.3	0.2
Ga11G1865	生长素响应蛋白 Auxin-responsive protein	IAA	IAA	1.3	0.9	2.0	0.4
Ga05G1988	生长素诱导蛋白 Auxin-induced protein 6B	AUX6B	IAA	1.1	0.8	2.9	0.3

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2.5 差异基因共表达趋势分析

为了研究野生型(fz)和短绒突变体(FZ)的差异基因在胚珠和纤维中的表达情况, 对2个材料4个纤维发育时期的3784个差异基因绘制时空表达模式图(图5-A), 结果发现3784个基因在2个材料4个纤维发育时期可以划分为9组(cluster), 同一组的基因有相同的表达趋势。clusters 1和2的基因在突变体(FZ)的开花当天表达高, 主要参与应答胁迫反应以及能量传递过程; clusters 4、5和7的基因在突变体的+3DPA表达高, 主要参与转录调控和钙离子, 铁离子, 铜离子的结合; clusters 6 和8的基因在突变体材料开花后5 d表达高, 也主要涉及转录调控, 钙离子, 铁离子和铜离子的结合过程; clusters 3和9的基因主要在突变体+8DPA表达高, 主要参与水解酶活性, 转运活性, 核酸激活, 脂肪酸生物合成途径和ATP结合过程(图5-B)。

图5

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图5差异基因共表达趋势

A: 在fz 和FZ两个材料中4个纤维发育时期所有差异表达基因的热图分析。B: 差异基因功能注释; 黑色代表0DPA时期高表达的基因, 蓝色代表+3DPA时期高表达的基因, 橙色代表+5DPA时期高表达的基因, 绿色代表+8DPA时期高表达的基因。
Fig. 5Coexpression analysis of all significant DEGs

A: heatmap analysis of the expression of all significant DEGs between fz and FZ at four time points. B: functional annotations of significant DEGs; Black bars indicate genes highly expressed at 0DPA, blue bars indicate genes highly expressed at +3DPA, orange bars indicate genes highly expressed at +5DPA, and green bars indicate genes highly expressed at +8DPA.

2.6 陆地棉控制短绒基因在亚洲棉短绒突变体FZ及其野生型fz的表达分析

前人通过图位克隆, 在四倍体陆地棉中分别定位出了一个控制短绒发育和长纤维发育的基因, 即MYBMIXTA-like转录因子GhMML3_A12和GhMML4_D12; GhMML3_A12在TM-1短绒起始期表达量明显高于其在短绒突变体N1中的表达量; GhMML4_D12在仅有长纤维的短绒突变体n2NSM纤维起始期的表达量明显高于其在陆无絮XZ142FLM中的表达量^[17,18]。为了验证GhMML3_A12和GhMML4_D12在亚洲棉短绒突变体FZ和其野生型fz中的表达量, 将这2个基因的蛋白序列在亚洲棉中进行同源性比对(图6)表明, GhMML3_A12和GhMML4_D12在亚洲棉中为同一个基因Ga12G1199, 基因功能注释显示该基因属于MYB类转录因子, 其相似性达到97%。转录组数据分析表明, Ga12G1199在FZ材料短绒起始期表达量高于其在fz材料中的表达量(图7), 说明Ga12G1199可能负调控短绒发育。

图6

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图6MYBMIXTA-like基因在亚洲棉中的同源序列比对

Fig. 6Homologous sequence alignment of MYBMIXTA-like in G. arboreum

图7

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图7Ga12G1199在FZ和fz材料中的表达差异

* P < 0.05, ** P < 0.01, **** P < 0.0001.
Fig. 7Expression of Ga12G1199 in FZ and fz materials

2.7 差异基因实时荧光定量PCR验证

为了验证转录组数据的可靠性, 随机选择22个差异基因在2个材料+3DPA胚珠中进行荧光定量PCR验证。结果显示, RNA-seq结果与荧光定量PCR中的基因表达变化一致。通过单样本t检验, 野生型fz +3DPA胚珠中22个差异基因表达量与突变体FZ胚珠中表达量有显著差异。将qRT-PCR和RNA-seq中差异基因表达量经 log₂ 转化后做相关性分析, 所得的皮尔逊相关系数为0.89 (图8), 证明RNA-Seq结果可信。

图8

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图8RT-QPCR (X-轴)结果与RNA-seq (Y-轴)差异基因相关性分析

散点图代表RT-QPCR与RNA-seq的差异基因相对表达量的log₂值。
Fig. 8Comparison of expression ratios from RT-QPCR (Y-axis) and transcriptomic (X-axis) profiling

Scatter plots represent log₂ expression ratios calculated from RT- QPCR and RNA-seq.


经qRT-PCR验证, 细胞色素氧化酶P450 (Ga04G0014)、甲基转移酶(Ga07G0568)、转录因子WD40(Ga07G1530)、蛋白受体激酶(Ga08G0121)、二氢黄酮醇-4-还原酶(Ga09G0661)和谷胱甘肽S-转移酶(Ga11G2833)在突变体FZ中上调; 分子伴侣(Ga05G3965)、钙联蛋白(Ga07G1742)、转录因子MYB108(Ga07G2088)、网状蛋白(Ga07G2611)和NADH脱氢酶(Ga11G3670)在突变体材料中下调。

3 讨论

棉纤维起始是决定纤维产量的关键过程。在过去的10年中, 许多参与棉纤维发育的基因已经被鉴定出来。相比之下, 对于野生型种皮上的短绒发育了解比较少。长纤维在生产上主要用于纺织, 短纤维在生产上主要用于火药、造纸和医药用品, 由于短纤维一些特有性质和价值, 吸引了一些科学家研究其发育机制^[19]。

植物激素例如赤霉素(GA)、生长素、细胞分裂素、油菜素内酯(BR)、脱落酸(ABA)、乙烯、茉莉酸甲酯(JA)和水杨酸是植物内源小信号分子^[20]。许多研究证明生长素在纤维发育过程中起重要作用。外源补充吲哚乙酸(IAA)能弥补纤维伸长的缺陷性, 并且能显著增加纤维的总体积^[21]。生长素响应蛋白GhARF2和GhARF18, 在纤维起始的细胞中表达高, 当把这2个生长素响应蛋白在拟南芥中超表达后, 能显著促进拟南芥表皮毛起始, 说明GhARF2和GhARF18正调控棉花纤维细胞起始^[22]。与GhARF2和GhARF18作用相反, INDOLEACETIC ACIDINDUCED PROTEIN 16 (GhIAA16)负调控纤维的起始和伸长。GhIAA16在野生型胚珠中表达非常低, 然而在无短绒无长纤维突变体中转录水平在开花后急剧升高^[23], 这与本研究结果一致。GaIAA16在短绒突变体+5DPA的表达量比野生型的高4倍。前人研究显示向体外离体培养的胚珠中加入ABA, 纤维细胞的起始受到抑制, 而且Ligon-lintless 1突变体0DPA的胚珠中积累ABA的量比野生型的高^[24]。这些结果说明ABA负调控棉花纤维起始。本研究结果显示, 与ABA信号传递相关的基因表达量在突变体中明显升高, 与纤维起始呈负相关的植物激素ABA响应因子(Ga11G3045、Ga12G2331)在突变体短绒起始期也显著的上调, 进而抑制了短绒纤维的起始。这些转录组分析结果需要进一步的试验验证。

最近研究显示, 植物体内信号分子, 如钙离子(Ca²⁺)和活性氧(ROS), 特别是超氧阴离子(O^2-)和过氧化氢(H₂O₂)参与棉花纤维发育。当用H₂O₂处理光子突变体(N1)和短绒突变体(XinFLM)时, 突变体纤维在0DPA时开始起始, 说明ROS对纤维起始具有促进作用^[25]。本研究显示参与活性氧清除的谷胱甘肽转移酶(GST)基因在突变体FZ短绒起始期明显升高。Ca²⁺也参与棉纤维细胞的起始, 与-1DPA的胚珠相比, 0DPA的胚珠中钙离子的积累明显升高, 基因芯片分析结果显示, 参与钙离子信号转导的基因钙结合蛋白在+1DPA胚珠中表达量明显上调^[26]。钙调蛋白(CBL)在Ca²⁺信号转导过程中起重要作用, 其表达量在短绒突变体胚珠中下调了2倍, 与前人结果一致。Ca²⁺正调控纤维起始^[27,28]。

前人研究结果显示, GhMML3 (GhMYB25-like)基因正调控陆地棉短绒纤维起始, 而GhMML4基因正调控陆地棉长纤维起始^[17,18]。而这2个基因在亚洲棉中的同源基因Ga12G1199在短绒突变体中的表达量明显高于其在野生型中的表达量, 该基因可能负调控短绒的发育。造成这种现象的原因可能是陆地棉和亚洲棉短绒发育机制不一样。

本文通过短绒突变体及其野生型4个纤维发育时期的RNA-Seq测序数据分析筛选到4988个差异表达的基因, 其中上调表达的2612个, 下调表达的2376个, 但仅21.5%的基因在棉花基因组数据库中找到注释信息, 有大量未知基因信息存在。参与KEGG排名前5富集通路的基因, 如参与角质、木质素和蜡质合成过程的基因(Ga08G2220, 细胞色素氧化酶)在突变体(FZ)中下调, 说明这些基因可能正调控短绒的发育; 参与苯丙烷代谢和生物合成过程的基因(Ga11G3625、Ga05G2018, 过氧化物酶)在FZ中明显上调, 说明这些基因可能负调控短绒发育; 参与植物激素信号转导途径中的乙烯响应因子相关基因(Ga06G1125、Ga08G2804、Ga05G0163和Ga11G2673)以及ABA合成和信号转导相关基因(Ga02G1198、Ga07G0242、Ga07G0142、Ga08G2885和Ga11G3045)在FZ短绒起始期+5DPA明显上调, 说明这些基因可能负调控短绒发育。在91个共有的差异基因中, 与转座现象相关的基因Cotton_newGene_252500 copia型多蛋白, 转录因子NAC1(Ga04G1773), 以及氧化还原相关基因谷胱甘肽S-转移酶(Ga11G2833)在FZ中明显上调, 说明这些基因可能负调控短绒发育; 而NADPH脱氢酶(Ga10G1419)基因, 与钙信号转导相关基因(Ga07G1792)在FZ中下调, 说明这些基因正调控短绒发育。

4 结论

短绒突变体(FZ)及其野生型(fz)的表型差异为阐明调控棉纤维发育的机制和品系间的形态差异提供了信息。短绒的起始在+3DPA。造成短绒纤维细胞在突变体中未起始的原因是, 一些促进纤维伸长的基因下调, 而另外一些抑制纤维起始的基因上调。例如参与蜡质合成的基因以及Ca²⁺信号转导的基因在突变体中都下调; 清除活性氧的基因以及参与ABA信号转导的基因在突变体中上调。该研究对于理解亚洲棉短绒纤维起始的机制具有一定的理论意义, 对于提高棉纤维产量和品质具有重要的实践意义。

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New Phytol, 2018,217:883-895.
DOI:10.1111/nph.14844URLPMID:29034968 [本文引用: 2]
Cotton, with cellulose-enriched mature fibers, is the largest source of natural textiles. Through a map-based cloning strategy, we isolated an industrially important lint fiber development gene (Li₃ ) that encodes an MYB-MIXTA-like transcription factor (MML) on chromosome D12 (GhMML4_D12). Virus-induced gene silencing or decreasing the expression of the GhMML4_D12 gene in n₂ NSM plants resulted in a significant reduction in epidermal cell prominence and lint fiber production. GhMML4_D12 is arranged in tandem with GhMML3, another MIXTA gene responsible for fuzz fiber development. These two very closely related MIXTA genes direct fiber initiation production in two specialized cell forms: lint and fuzz fibers. They may control the same metabolic pathways in different cell types. The MIXTAs expanded in Malvaceae during their evolution and produced a Malvaceae-specific family that regulates epidermal cell differentiation, different from the gene family that regulates leaf hair trichome development. Cotton has developed a unique transcriptional regulatory network for fiber development. Characterization of target genes regulating fiber production has provided insights into the molecular mechanisms underlying cotton fiber development and has allowed the use of genetic engineering to increase lint yield by inducing more epidermal cells to develop into lint rather than fuzz fibers.

[19] Du S J, Dong C J, Zhang B, Lai T F, Du X M, Liu J Y . Comparative proteomic analysis reveals differentially expressed proteins correlated with fuzz fiber initiation in diploid cotton (Gossypium arboreum L.)
J Proteomics, 2013,82:113-129.
DOI:10.1016/j.jprot.2013.02.020URLPMID:23474080 [本文引用: 1]
In this study, a comparative proteomic analysis was employed to identify fuzz fiber initiation-related proteins in wild-type diploid cotton (Gossypium arboreum L.) and its fuzzless mutant. Temporal changes in global proteomes were examined using 2-DE at five developmental time points for fuzz fiber initiation, and 71 differentially expressed protein species were identified by MS, 45 of which were preferentially accumulated in the wild-type. These proteins were assigned to several functional categories, mainly in cell response/signal transduction, redox homeostasis, protein metabolism and energy/carbohydrate metabolism. It was remarkable that more than ten key proteins with high-abundance were involved in gibberellic acid (GA) signaling and ROS scavenging, and increasing concentrations of active GAs and H2O2 were also detected approximately 5dpa in wild type ovules. Furthermore, in vivo GA and H2O2 treatments of ovules inside young bolls showed that these compounds can synergistically promote fuzz fiber initiation. Our findings not only described a dynamic protein network supporting fuzz initiation in diploid cotton fiber ovules, but also deepened our understanding of the molecular basis of cotton fiber initiation.

[20] Daviere J M, Achard P . A pivotal role of DELLAs in regulating multiple hormone signals
Mol Plant, 2016,9:10-20.
DOI:10.1016/j.molp.2015.09.011URLPMID:26415696 [本文引用: 1]
Plant phenotypic plasticity is controlled by diverse hormone pathways, which integrate and convey information from multiple developmental and environmental signals. Moreover, in plants many processes such as growth, development, and defense are regulated in similar ways by multiple hormones. Among them, gibberellins (GAs) are phytohormones with pleiotropic actions, regulating various growth processes throughout the plant life cycle. Previous work has revealed extensive interplay between GAs and other hormones, but the molecular mechanism became apparent only recently. Molecular and physiological studies have demonstrated that DELLA proteins, considered as master negative regulators of GA signaling, integrate multiple hormone signaling pathways through physical interactions with transcription factors or regulatory proteins from different families. In this review, we summarize the latest progress in GA signaling and its direct crosstalk with the main phytohormone signaling, emphasizing the multifaceted role of DELLA proteins with key components of major hormone signaling pathways.

[21] Chen Z J, Guan X Y . Auxin boost for cotton
Nat Biotechnol, 2011, 29.
DOI:10.1038/nbt.1858URLPMID:21552241 [本文引用: 1]

[22] Xiao G H, He P, Zhao P, Liu H, Zhang L, Pang C, Yu J . Genome-wide identification of the GhARF gene family reveals that GhARF2 and GhARF18 are involved in cotton fibre cell initiation
J Exp Bot, 2018,69:4323-4337.
DOI:10.1093/jxb/ery219URLPMID:29897556 [本文引用: 1]
Auxin signalling plays an essential role in regulating plant development. Auxin response factors (ARFs), which are critical components of auxin signalling, modulate the expression of early auxin-responsive genes by binding to auxin response factor elements (AuxREs). However, there has been no comprehensive characterization of this gene family in cotton. Here, we identified 56 GhARF genes in the assembled Gossypium hirsutum genome. This gene family was divided into 17 subfamilies, and 44 members of them were distributed across 21 chromosomes. GhARF6 and GhARF11 subfamily genes were predominantly expressed in vegetative tissues, whereas GhARF2 and GhARF18 subfamily genes were highly expressed during seed fibre cell initiation. GhARF2-1 and GhARF18-1 were exclusively expressed in trichomes, organs similar to cotton seed fibre cells, and overexpression of these genes in Arabidopsis enhances trichome initiation. Comparative transcriptome analysis combined with AuxRE prediction revealed 11 transcription factors as potential target genes of GhARF2 and GhARF18. Six of these genes were significantly expressed during seed fibre cell initiation and were bound by GhARF2-1 and GhARF18-1 in yeast one-hybrid assays. Our results suggest that GhARF2 and GhARF18 genes may be key regulators of cotton seed fibre initiation by regulating the expression of several transcription factor genes. This study deepens our understanding of auxin-mediated initiation of cotton seed fibre cells and helps us in breeding better cotton varieties in the future.

[23] Han , X Cloning and expression analysis of novel Aux/IAA family genes in Gossypium hirsutum
Genes, 2012,503:83-91.
DOI:10.1016/j.gene.2012.03.069URLPMID:22575728 [本文引用: 1]
Members of the Aux/IAA gene family encode proteins that mediate the responses of auxin-regulated gene expression and regulate various aspects of plant morphological development. Here, we provide the first identification and characterization of nine cDNAs encoding the complete open reading frame (ORF) of the Aux/IAA family in cotton. These were designated GhAux1 to GhAux9 (Gossypiumhirsutum Aux/IAA). The proteins encoded by these nine genes had either whole or partially conserved domains of the Aux/IAA superfamily, with sequence identity ranging from 14% to 69%. A pair of homeologs exists for each Aux/IAA in G. hirsutum acc. TM-1 with high identity both in ORF sequences and amino acid level. Tissue- and organ-specific analysis showed that transcripts of GhAux1, GhAux2, and GhAux3 were abundant in vegetative organs, whereas GhAux4, GhAux5, GhAux6, and GhAux7 were preferentially expressed in ovules on the day of anthesis. GhAux8 and GhIAA16 (previously reported) were also preferentially expressed during fiber developmental stages, especially GhAux8 in fiber early elongation stages, and GhIAA16 in fiber initiation and secondary cell wall thickening stage. GhAux9 was specifically expressed in developing fibers. During the fiber initiation stage, except for GhAux3 and GhAux6, the expression of the other eight GhAuxs in various lintless-fuzzless and linted-fuzzless mutants demonstrated that they were significantly up-regulated compared with linted-fuzzy TM-1.

[24] Gilbert M K, Bland J M, Shockey J M, Cao H, Hinchliffe D J, Fang D D, Naoumkina M . A transcript profiling approach reveals an abscisic acid-specific glycosyltransferase (UGT73C14) induced in developing fiber of ligon lintless-2 mutant of cotton ( Gossypium hirsutum L.)
PLoS One, 2013,8.
[本文引用: 1]

[25] Zhang D Y, Zhang T Z, Guo W Z . Effect of H₂O₂ on fiber initiation using fiber retardation initiation mutants in cotton (Gossypium hirsutum)
J Plant Physiol, 2010,167:393-399.
DOI:10.1016/j.jplph.2009.10.005URLPMID:19931935 [本文引用: 1]
Single-celled fibers initiate at anthesis from cotton seed epidermal cells of normal developmental cotton cultivars; however, fiber initiation is retarded in some cotton fiber mutants. In this study, the relationship between genes associated with fiber initiation retardation and fiber initiation development was investigated using three cotton fiber developmental mutants: recessive naked seed n2; dominant naked seed N1; and Xinxiangxiaoji Linted-Fuzzless Mutant (XinFLM); with genetic standard line TM-1 (TM-1) as control. Retardation during fiber initiation development was observed in N1 and XinFLM by scanning electron microscope (SEM) analysis. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of genes related to the fiber initiation development showed that the expression of GhEXP1 and GhMYB25 was lower in N1 and XinFLM than in TM-1 and n2, however, the expression of GhTTG1 and GhTTG3 in XinFLM and n2 was higher than in TM-1 and N1. In vivo and in vitro treatments on ovules demonstrated that 30% hydrogen peroxide (H2O2) could prevent fiber initiation retardation in XinFLM, but no evident effect on N1. To further confirm the relationship between gene expression and the effects of H2O2 in XinFLM, qRT-PCR analysis of four differentially expressed genes was performed using -1d post-anthesis (DPA) ovules of XinFLM treated for 24 and 48h with 30% H2O2 and H2O, respectively, with 0 and 1 DPA untreated ovules from XinFLM and TM-1 as control. The results showed that the expression of GhMYB25 and GhEXP1 showed significant difference in XinFLM after -1 DPA ovule treated for 24h relative to the untreated or H2O-treated ovules, with the expression of GhMYB25 increased significantly and that of GhEXP1 decreased. This implied that H2O2 might be one of the upstream signal molecules affecting the expression of GhMYB25 and GhEXP1 genes. The fiber initiation retardation in XinFLM might be related to the production of reactive oxygen species (ROS).

[26] Taliercio E W, Boykin D . Analysis of gene expression in cotton fiber initials
BMC Plant Biol, 2007,7:22.
DOI:10.1186/1471-2229-7-22URLPMID:17506877 [本文引用: 1]
Cotton (Gossypium hirsutum L.) fibers are trichomes that initiate from the ovule epidermis. Little is known about the developmental pathway causing fiber to differentiate from ovular epidermal cells even though limits on the number of cells that differentiate into fiber will limit yield.

[27] Kudla J, Batistic O, Hashimoto K . Calcium signals: the lead currency of plant information processing
Plant Cell, 2010,22:541-563.
DOI:10.1105/tpc.109.072686URLPMID:20354197 [本文引用: 1]
Ca(2+) signals are core transducers and regulators in many adaptation and developmental processes of plants. Ca(2+) signals are represented by stimulus-specific signatures that result from the concerted action of channels, pumps, and carriers that shape temporally and spatially defined Ca(2+) elevations. Cellular Ca(2+) signals are decoded and transmitted by a toolkit of Ca(2+) binding proteins that relay this information into downstream responses. Major transduction routes of Ca(2+) signaling involve Ca(2+)-regulated kinases mediating phosphorylation events that orchestrate downstream responses or comprise regulation of gene expression via Ca(2+)-regulated transcription factors and Ca(2+)-responsive promoter elements. Here, we review some of the remarkable progress that has been made in recent years, especially in identifying critical components functioning in Ca(2+) signal transduction, both at the single-cell and multicellular level. Despite impressive progress in our understanding of the processing of Ca(2+) signals during the past years, the elucidation of the exact mechanistic principles that underlie the specific recognition and conversion of the cellular Ca(2+) currency into defined changes in protein-protein interaction, protein phosphorylation, and gene expression and thereby establish the specificity in stimulus response coupling remain to be explored.

[28] Steinhorst L, M?hs A, Ischebeck T, Zhang C X, Zhang X X, Arendt S, Schültke S, Heilmann I, Kudla J . Vacuolar CBL- CIPK12 Ca ²+-sensor-kinase complexes are required for polarized pollen tube growth
Curr Biol, 2015,25:1475-1482.
DOI:10.1016/j.cub.2015.03.053URLPMID:25936548 [本文引用: 1]
Polarized tip growth is a fundamental process of specialized eukaryotic cells like neuronal axons, fungal hyphae, and plant root hairs and pollen tubes. In pollen tubes, a tip-focused oscillating Ca(2+) gradient governs ions fluxes, vesicle transport, and cytoskeleton dynamics to ensure proper polarized cell growth [1, 2]. While a crucial role of vacuolar Ca(2+) signaling is established for cellular movements like guard cell dynamics [3-5], its contribution to polarized growth remains to be defined. Here we identified the two closely related tonoplast-localized Ca(2+)-sensor proteins CBL2 and CBL3 as crucial regulators of vacuolar dynamics and polarized pollen tube growth. Overexpression of CBL2 or CBL3 in Arabidopsis and tobacco pollen tubes affected vacuolar morphology, pollen germination, and tube growth, but did not alter actin organization, PI(4,5)P2 distribution, or?tip-focused Ca(2+) oscillations. Similarly, loss of function of each single Ca(2+) sensor and cbl2/cbl3 double mutants exhibited impaired pollen tube growth in?vitro and in?vivo. Both Ca(2+) sensors interacted with the kinase CIPK12, which translocated from the cytoplasm to the vacuolar membrane upon this interaction. Also, overexpression of CIPK12 induced severe vacuolar phenotypes, and loss of function of CIPK12 lead to impairment of polar growth. Remarkably, co-expression of CBL2 or CBL3 with CIPK12 resulted in a phosphorylation-dependent, massively enhanced vacuolar inflation and further disruption of polar growth. Together, these findings identify an essential role of the vacuole and vacuolar Ca(2+) signaling for polarized tip growth. We propose that a faithfully balanced activity of Ca(2+)-activated CBL2/3-CIPK12 complexes fulfills fundamental functions to enable the fast growth of pollen tubes in higher plants.