梁国平, 李文芳, 陈佰鸿, 左存武, 马丽娟, 何红红, 万鹏, 安泽山, 毛娟^,甘肃农业大学园艺学院,兰州 730070

Effects of Different Sugar Sources on Protein Kinase Gene Expression in Grape Plantlets

LIANG GuoPing, LI WenFang, CHEN BaiHong, ZUO CunWu, MA LiJuan, HE HongHong, WAN Peng, AN ZeShan, MAO Juan^,College of Horticulture, Gansu Agricultural University, Lanzhou 730070

通讯作者: 毛娟,E-mail： maojuan@gsau.edu.cn

收稿日期:2018-07-25接受日期:2018-10-15网络出版日期:2019-04-01

基金资助:

国家自然科学基金.31460500 甘肃省科技重大专项计划.18ZD2NA006-4 甘肃省现代水果产业体系岗位专家项目.GARS-SG-3

Received:2018-07-25Accepted:2018-10-15Online:2019-04-01
作者简介 About authors
梁国平,Tel：18298344227;E-mail： 1143016341@qq.com。











摘要
【目的】探究不同外源糖对葡萄试管苗生长发育及蛋白激酶基因转录调控的影响,应用转录组测序挖掘蛋白质磷酸化过程中的基因,为葡萄蛋白激酶相关基因功能的验证奠定一定基础。【方法】在基本培养基中分别添加2%的蔗糖、葡萄糖和果糖,以无糖为对照,分别命名为S20、G20、F20和CK,经过37 d培养后,测定不同处理的地上和地下鲜重,并采用Illumina HiSeq ^TM 2000对各处理叶片进行转录组测序,通过综合生物信息学分析（参考基因组比对、差异基因（DEGs）筛选、COG（Cluster of Orthologous Groups of proteins）注释、GO（Gene Ontology）注释等）筛选出蛋白激酶相关基因,通过qRT-PCR分析该蛋白激酶相关基因的表达特性。【结果】F20、G20和S20处理下的葡萄（‘红地球’）试管苗与CK相比,地上鲜重具有明显差异,且F20最高,而G20地下鲜重最高。SNP统计发现,转换是主要的变异类型,颠换次之,且发生在基因间区的SNP数量最多,其次是下游;剪接位点供体和同义终止发生的基因数量最少且相等。4个样品中共获得了2 633个差异基因,3个处理与CK相比,共有差异基因180个且被聚类为3组,第一组中127个基因仅在CK中高表达,第二组19个基因仅在G20下高表达,而第三组34个基因在3个处理下表达模式不尽相同。这些共有的差异基因在COG中注释到了26个基因并分在11个功能类别中,且主要注释在一般的功能类别中。在GO分类中,共有的基因分别被注释在分子功能、生物学过程和细胞组分的14、22和13个功能类别中。共筛选出7种蛋白激酶,分别为葡萄糖激酶（Glucokinase,GK）、丝裂原活化蛋白激酶（Mitogen-activated protein kinases,MAPKs）、钙调蛋白激酶（Calcineurin protein kinase,CBL）、蛋白磷酸酶2（Protein phosphatase 2,PP2A）、己糖激酶（Hexokinase,HXK）、组氨酸蛋白激酶（Histidine protein kinase,HPK）和酪氨酸激酶（Tyrosine kinase,TK）,其不同激酶的基因在不同处理中具有各自的表达模式,经qRT-PCR验证,选择的20个差异基因中有17个基因表达与转录组测序结果相一致。【结论】在葡萄试管苗培养中,果糖较葡萄糖和蔗糖相比对生长较好。测序得出180个差异基因对3种不同糖均作出响应,这些基因在COG数据库中主要富集在膜酯转运和代谢、次级代谢物和碳水化合物的合成、转运和分解;GO中大多注释在蛋白激酶和氧化还原酶的活性中;筛选出了7种蛋白激酶,这些差异基因在数量、功能分类和代谢通路上对糖的响应各不相同。
关键词： RNA-Seq;外源糖;蛋白激酶;信号转导

Abstract
【Objective】 To explore the effects of different exogenous sugars on the growth and development of grape plantlets and the regulation of protein kinase gene transcription, the candidate genes were tapped in the process of protein phosphorylation by using transcription, which made a foundation for the verification of grape protein kinase-related gene function.【Method】 Sucrose (2%), glucose (2%) and fructose (2%) were added to the basic medium, and the free-sugar treatment was as control, which were named as S20, G20, F20 and CK, respectively. After 37 days of culture, the fresh weight of the leaf-stem and root under different treatments was determined. Transcriptome sequencing of each treated foliages was performed by using Illumina HiSeq ^TM 2000, and a series of protein kinases related genes were screened by integrated bioinformatics analysis, including reference genomic alignment, differentially expressed gene (DEGs) screening, COG (Cluster of Orthologous Groups of proteins) annotation, GO (Gene Ontology) annotation, etc., and the expression characteristic of these genes were further analyzed by qRT-PCR. 【Result】 Compared with CK, ‘Red Globe’ grape plantlets under F20, G20 and S20 treatments exhibited significant differences in the fresh weight of leaf-stem, and the highest was obtained by F20 treatment, while the weight of fresh root under G20 was the highest. The SNP statistics found that the Transition was the main type of mutation, the second was Transversion. The highest number of SNPs that occurred in the Intergenic, and the next was the Upstream. Splice_Site_Donor and Synonymous_Stop events occurred with the least number of genes and equal. A total of 2 633 deferentially expressed genes were obtained in the 4 samples. The Venn diagram showed that there were a total of 180 differential genes under the 3 treatments compared with CK, and these genes were clustered into 3 groups. In the first group, 127 genes were only highly expressed under CK. The 19 genes of the second group were only highly expressed under G20, while the expression patterns of the 34 genes in third group were different under three treatments. The common 180 differential genes were annotated with 26 genes in the COG database to 11 functional categories, and these DEGs were mainly enriched in general functional categories. In the annotation of GO, the common genes were annotated in 14, 22 and 13 functional categories of molecular function, biological process and cellular component, respectively. Seven kinds of protein kinases were screened by this sequencing, including Glucokinase (GK), Mitogen-activated protein kinases (MAPKs), Calcineurin protein kinase (CBL), Protein phosphatase 2 (PP2A), Hexokinase (HXK), Histidine protein kinase (HPK) and Tyrosine kinase (TK), and these different protein kinases genes showed their own expression patterns among different treatments. By qRT-PCR analysis, 17 out of 20 screened genes expression were consistent with the transcriptome sequencing results. 【Conclusion】 Compared with glucose and sucrose, fructose was the best sugar during grape culture process. The sequencing results showed that 180 DEGs all responded to three different sugars. In the COG annotation, these genes were mainly enriched in membrane ester transport and metabolism, the synthesis, transport and decomposition of secondary metabolites and carbohydrates. In the GO databases, the most of common DEGs were annotated in the activities of protein kinases and oxidoreductases. Seven protein kinases were identified, which were selectively in responses to different exogenous sugars in quantity, functional, category and metabolic pathways, and had their own choice of expression specificity.
Keywords：RNA-seq;exogenous sugar;protein kinase;signaling transduction


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本文引用格式
梁国平, 李文芳, 陈佰鸿, 左存武, 马丽娟, 何红红, 万鹏, 安泽山, 毛娟. 不同糖源对葡萄试管苗蛋白激酶相关基因表达的影响[J]. 中国农业科学, 2019, 52(7): 1119-1135 doi:10.3864/j.issn.0578-1752.2019.07.001
LIANG GuoPing, LI WenFang, CHEN BaiHong, ZUO CunWu, MA LiJuan, HE HongHong, WAN Peng, AN ZeShan, MAO Juan. Effects of Different Sugar Sources on Protein Kinase Gene Expression in Grape Plantlets[J]. Scientia Agricultura Sinica, 2019, 52(7): 1119-1135 doi:10.3864/j.issn.0578-1752.2019.07.001

0 引言

【研究意义】在植物组织培养过程中,糖不仅调节细胞内的渗透势,以维持水分平衡^[1],而且还是组培植物能量的供应者^[2]和信号分子^[3]。SOLAROVA等^[4]指出,糖是初期组培苗幼芽生长和叶片光合作用所必须的。在葡萄良种繁育和脱毒苗培育过程中,糖对试管苗的形态建成具有决定性作用^[5]。然而,在常规培养过程中,试管苗处在自养与异养的混合状态^[6],且不同外源糖的供应对试管苗的生长也具有不同的作用。蛋白激酶可将ATP分子上γ位的磷酸基团转移并共价结合到底物蛋白质氨基酸残基（丝氨酸、苏氨酸或络氨酸）上,从而实现蛋白质磷酸化并引起蛋白质构象的改变,进而调控蛋白质的活性和功能,最终对物质转运、能量代谢和信号转导等过程起到开关作用^[7]。因此,通过转录组测序深入分析不同外源糖对葡萄试管苗生长发育的调控,特别是对蛋白激酶基因表达的影响,为在组培条件下深入了解糖对植株的形态建成和进一步优化碳源具有重要意义。【前人研究进展】在组织培养诱导植物产生不定根的过程中,培养基的初始糖浓度对其生长有很大影响,低浓度糖难以满足植物生长的需要,而高浓度糖则导致细胞内渗透压过高,最终对不定根的生长产生抑制^[8]。邹英宁^[9]在培养基中分别添加蔗糖、葡萄糖、山梨醇、果糖4种糖源对中国李进行诱导增殖的研究中得出,葡萄糖浓度为30 g·L^-1和15 g·L^-1对促进增殖及生长和生根率及生根倍数最好。在诱导白桦愈伤组织的过程中发现,培养基中分别添加20—30 g·L^-1的蔗糖、葡萄糖和果糖后,其鲜重积累量均显著高于对照^[10]。王思瑶等^[11]研究了不同外源糖对柽柳丛生芽生长的影响,蔗糖浓度为10 g·L^-1时有助于其丛生芽的生长。然而,KOZAI等^[12]研究发现,将含有一片叶的单芽茎段葡萄试管苗继代在无糖培养基中也可生长。这些结果表明,不同外源糖对组培植物的生长发育具有不同的作用,且目的不同需求糖的种类也不尽相同。植物蛋白激酶在信号感知、传导、生长发育以及基因的表达调控中起重要作用。葡萄糖激酶（Glucokinase,GK）对葡萄糖具有特异性,在水稻^[13]、豌豆^[14]和马铃薯^[15]中研究发现,该酶可优先磷酸化葡萄糖,从而提前进入糖酵解途径。促分裂原活化蛋白激酶（Mitogen-activated protein kinases,MAPK）是一种丝氨酸/苏氨酸类蛋白激酶,它介导多种生物和非生物胁迫的相关信号通路,以磷酸化方式实现信号的级联放大,最后引起相应的生物学效应,对植物的生长发育起重要的调控作用^[16]。类钙调磷酸酶B蛋白作为（Calcineurin B-like protein,CBL）Ca²⁺传感蛋白之一^[17],以丝氨酸/苏氨酸蛋白激酶CIPK（CBL-interacting protein kinase）作为靶蛋白^[18,19],并形成蛋白激酶复合物CBL-CIPK,调节下游基因的表达来维持细胞离子平衡^[20]。在拟南芥突变体中研究发现,CBL-CIPK信号通路参与调控盐胁迫下Na⁺稳态分布和Na⁺、K⁺平衡^[21]。蛋白磷酸酶2A（Protein phosphatase 2,PP2A）包含一系列丝氨酸/苏氨酸磷酸酶^[22],该家族从酵母到哺乳动物在功能和结构上较为保守,并且在调节细胞的多种功能中起关键作用^[23]。己糖激酶（Hexokinase,HXK）催化己糖磷酸化并使之参与糖酵解^[24]。KARVE等^[25]用RT-PCR法研究拟南芥HXK基因中不同家族成员在6种器官（幼叶、成熟叶、根、花、角果和茎）的表达情况发现,AtHXK1在所有器官中都具有较高的丰度;AtHXK2在叶中高表达,但在库组织中表达略低。AtHXK3在根和角果中丰度高,但低于AtHXK1。AtHKL1和AtHKL2表达量低于AtHXK1,且这两者在不同器官中表达丰度相近,AtHKL3仅在花中有表达。组氨酸蛋白激酶（Histidine protein kinase,HPK）是一个磷酸化组氨酸保守残基的信号传导酶家族^[26],与它们的下游靶蛋白一起构成了双组分信号传导系统,响应各种生物和非生物胁迫^[27]。酪氨酸激酶（Tyrosinekinase,TK）是细胞信号转导过程中最为重要的物质之一,对细胞调节、通讯和发育生物学方面具有重要的意义。在动物中研究发现,该酶的活性过高会导致其下游信号途径激活,从而使细胞转化、增殖、对抗细胞凋亡促进细胞生存,形成非正常组织^[28],而植物中报道甚少。【本研究切入点】虽然前人已在组培中对碳源影响植物生长发育方面做了大量报道,目的是确定最适碳源和浓度,以达到商品化生产和降低组培成本,然而在分子水平研究不同外源糖对其生长的影响,特别是对蛋白磷酸化中基因表达的研究鲜有报道。【拟解决的关键问题】应用高通量测序手段,在分子层面深入分析在不同外源糖条件下‘红地球’葡萄（Vitis Vinifera L Red Globe）试管苗叶片中转录本的变化,特别是蛋白激酶相关基因如何响应不同的外源糖,为研究糖对植物生长发育的调控奠定一定的理论基础。

1 材料与方法

1.1 试验材料及处理

本试验于2016—2017年在甘肃农业大学园艺学院果树生理与生物技术实验室进行,试验材料为本实验室保存的‘红地球’试管苗。

选择继代健壮、长势一致且无污染的试管苗,剪成单芽茎段（2—3 cm）并留一片叶,接种在不含糖以及含2%不同外源糖的50 mL MS培养基中,培养基分装在150 mL的三角瓶中,每瓶接1个外植体,外源糖分别为蔗糖、葡萄糖和果糖,以无糖为对照,基本培养基组分为：4.43 g·L^-1 MS+6 g·L^-1琼脂+0.2 mg·L^-1 IAA,pH为5.8—6.0,分别命名为CK、S20、G20和F20,每个处理接种30瓶,培养条件为（25±1）℃,白炽光36 mmol·m^-2·s^-1光照16 h,黑暗8 h。经过37 d培养后,每个处理随机挑选9瓶长势一致的试管苗,测定其生物量,并迅速收集叶片液氮速冻,-80℃保存用于RNA的提取。每个处理进行3次生物学重复。

1.2 生物量测定

将培养37 d后的试管苗从培养基中取出,不损伤根系,从基部第一个芽处剪断,测定其地上鲜重。用蒸馏水将根系表面的培养基洗干净,后将根系在滤纸上轻轻滚动一圈,吸干表面多余水分,用于测定其地下鲜重,每个处理重复3次。

1.3 RNA提取和质检

RNA提取采用植物提取试剂盒（中科泰瑞生物技术有限公司,北京）并按操作说明书进行。用Nanodrop和Agilent 2100（安捷伦有限责任公司,美国）检测RNA的纯度、浓度及完整性。

1.4 cDNA文库的构建

cDNA文库构建参照本课题组前期的试验方案进行^[29]。样品检测合格后,用带有Oligo（dT）的磁珠,通过A-T互补配对与mRNA的ployA尾结合的方式富集mRNA。随后加入Fragmentation Buffer将mRNA打断成短的片段。以打断的mRNA为模板,用六碱基随机引物合成第一条cDNA链,然后加入缓冲液、dNTPs和DNA polymerase I合成第二条cDNA链,随后利用AMPure XP beads纯化双链cDNA。纯化的双链cDNA再进行末端修复、加A尾并连接测序接头,然后用AMPure XP beads进行片段大小选择,最后通过PCR富集得到cDNA文库。

1.5 cDNA库检及测序

文库构建完成后,先使用Qubit 2.0进行初步定量,稀释文库至1 ng·μL^-1,随后使用Agilent 2100对文库插入片段的长度进行检测,插入片段大小符合预期后,使用qPCR方法对文库的有效浓度进行准确定量（文库有效浓度>2 nmol·L^-1）。

库检合格后,采用Illumina Hiseq^TM 2000进行测序,测序读长为PE100。

1.6 测序数据过滤

去除带测序接头的reads、无法确定碱基信息的比例大于10%的reads及低质量reads（Q_phred≤20的碱基数占整个read长度50%以上的reads）。

1.7 参考序列比对分析

利用TopHat将Clean Reads与指定的参考基因组进行序列比对^[30],获取在参考基因组或基因上的位置信息以及测序样品特有的序列特征信息。

1.8 差异基因筛选

通过设定FDR（False discovery rate）<0.01和∣Log₂（FC）∣≥2作为筛选差异基因的阈值进行差异基因的筛选^[31]。

1.9 qPCR验证

用差异基因的Gene ID对葡萄数据库（http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/）检索并获得相应的CDS区,通过在线引物设计软件Primer 3 Input（http://primer3.ut.ee/）进行引物设计,引物见表1。对1.3的RAN使用Primer Script^TM RT regent Kit with gDNA Eeaser（TaKaRa）试剂盒并按照说明书进行反转录获得cDNA,并用SYBR Primer Ex Taq^TM II（TaKaRa）试剂盒说明书进行定量,定量PCR仪为Light Cycler? 96 Real-Time PCR System（Roche,瑞士）。内参基因为GAPDH（GenBank accession no. CB973647）。引物由生工生物工程股份有限公司合成。反应程序为：95℃预变性30 s,95℃变性10 s,60℃退火30 s,72℃ 延伸30 s,40个循环,重复3次。反应结束后分析荧光值变化曲线及熔解曲线。基因的相对表达量采用2^-ΔΔCT计算^[32]。

Table 1
表1
表1qRT-PCR引物
Table 1qRT-PCR primer

基因号 Gene ID	引物序列（5′-3′） Primer sequence (5′-3′)
GSVIVG01014744001	F-TCAAGGACATCACCACCACA	R-TAGGCCCTTTACGACACACA
GSVIVG01015297001	F-ATAGAAGACGCGGTTGGACA	R-TACCCAAGATCACTGCAGCA
GSVIVG01014081001	F-AAGACAAGCAGCCATCTCCT	R-TGTGCCTGCAACAGCTTTAG
GSVIVG01019739001	F-AGGATTGGTTGAAGGCTCCA	R-AAGGCGTGACTCAGATGGAT
GSVIVG01026984001	F-TTGAAGGGGCACTGTCTCAT	R-ACTGGTGGGTCTGGATTGAA
GSVIVG01038192001	F-AGCCTCCAATCATGCCCATA	R-ATGATCCATGTGCCGCAAAA
GSVIVG01038760001	F-ACGTTAGTGGAGGGAATGCT	R-TGTCCCAATGTTCTCCCCAT
GSVIVG01001347001	F-TGGCCCTGAAAGTCCGTTAT	R-TTCAACCAAGGCCGTCAATG
GSVIVG01020128001	F-GGAAGCAGCAATAACGTGGT	R-TCTGCCAAGAGACAACCCAA
GSVIVG01025420001	F-GGGTTGGGTGCTGTTTTCAA	R-TGCCTCCTTATGCCGAAACT
GSVIVG01038559001	F-GGGGTCCAAATTCTTTCGCA	R-TTCCTCGCCATCATCATCCA
GSVIVG01000919001	F-TTCGGGCAAGTTTTGGAAGG	R-AAAAGGGAGAAGGAGGTGGA
GSVIVG01011043001	F-GGGTTGGACGGCTGAATTTT	R-GGAGGAACGAAACAATGGCA
GSVIVG01020071001	F-ATGAGGTTGGCTGCTACTGA	R-AAGGCAAGAACACTGTCCCT
GSVIVG01000561001	F-TCTTGGGCTTGGATGGAGTT	R-AGCATGTCGGTCCACTCTTT
GSVIVG01007646001	F-AGGCATGGAGAGAAGAAGCA	R-TCTGGGAGGTTTTCAGCACT
GSVIVG01009192001	F-AGCGAGGGTGTAAGGTTTGT	R-TCAGCAGTCACATCCTTGGT
GSVIVG01009685001	F-ACGGAACTCAGCGTATCAGT	R-TCGTCATCATCGGGTTTCCA
GSVIVG01013564001	F-GTTCCGGCCAAATATGAGCA	R-TTGACCGGAAGAAATTCGCC
GSVIVG01014110001	F-TGTAGCTGCACCAACAACAC	R-TGGAGGGATTGAACCGTTGA
GSVIVG01014128001	F-AGGCTGCCACAAACAACTTT	R-TGCGGATGTTGTAGAGCAGA
GSVIVG01023376001	F-TGGACTGTGAGGCTAAACGT	R-TTTAAAAGCCTAGCCGTGCC
GSVIVG01023676001	F-TCATCATCCTCATCGCCATCA	R-AACTTGTCGCATGGTTGGTC
GSVIVG01026000001	F-AGCGTTTGGGGACTTGAGTA	R-TCCCAGAGCAAGCAAGTACA
GSVIVG01026487001	F-TGACGGGAAAGCAGGAAGAA	R-AAACATGCCCAAAAGTCCCC
GSVIVG01029835001	F-TGGCTTACGGAGGTGAACTT	R-AGCACCCAGAGCAATCATCA
GSVIVG01032611001	F-TTCCCCACCCATCATTCACA	R-TACGGTCATGGTGTAGGCAA
GAPDH	F-TTCTCGTTGAGGGCTATTCCA	R-CCACAGACTTCATCGGTGACA

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2.0 统计分析

试验结果数据经Excel 2010整理,统计分析采用单因素ANOVO的Duncan’s法,显著性水平为P<0.05,用Origin 9.0作图。

2 结果

2.1 不同外源糖对葡萄试管苗生长量的影响

以CK为对照,在2%的不同外源糖处理下,葡萄试管苗经37 d培养后,地上和地下生长具有明显差异。其中地上鲜重表现出显著差异,且在F20下生物量最大,而S20和G20处理下无显著变化。地下的鲜重在S20和G20下没有显著差异,而F20与两个处理均表现出显著差异（图1-A、B）。

图1

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图1不同外源糖对葡萄试管苗生物量的影响

不同小写字母表示不同糖处理水平上差异显著（P<0.05）。下同
Fig. 1Effect of different exogenous sugars on the biomass of grape plantlets

Different small letters means significant difference between treatments (P<0.05). The same as below

2.2 测序数据统计

对4个样品的转录组测序共获得21.53 Gb Clean Data,各样品Clean Data均达到4.69 Gb,不同处理获得的Clean reads分别是：CK为19 840 139条,S20为19 619 720条,G20为24 923 975条和F20为20 579 635条。Q30碱基百分比均在90.26%以上（表2）。表明测序质量较好,可以用于后期的数据分析。

Table 2
表2
表2转录组测序数据
Table 2Transcriptome sequencing data

样品 Sample	Raw reads	Clean reads	Clean Data	GC含量 GC content (%)	%≥Q30
CK	20 632 503	19 840 139	4 998 523 222	45.43	90.26
S20	24 367 990	19 619 720	4 696 568 225	45.44	92.01
G20	25 739 838	24 923 975	6 279 049 182	45.44	90.38
F20	22 890 967	20 579 635	5 553 171 109	44.43	92.41

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2.3 SNP/InDel分析

SNP是个体基因组DNA同一位置单个核苷酸变异（转换、颠换、插入和缺失）所引起的多态性。对不同外源糖处理葡萄测序结果的单个核苷酸变异位点进行统计（表3）,4个样品的SNP数量分别为：403 332、489 805、358 014和331 527个,且主要发生的变异类型是转换,其频率在65.89%—67.18%,而颠换频率在32.82%—34.11%。进一步对不同的SNP事件数量进行统计,结果表明发生在基因间区的SNP数量最多,为238 351个;其次是发生在下游的,为42 382个;而剪接位点供体和同义终止发生最少,且相等。（图2）。

Table 3
表3
表3SNP位点统计
Table 3Statistical SNP sites

样品 Sample	SNP Number	Genic SNP	Intergenic SNP	转换 Transition (%)	颠换 Transversion (%)	杂合型 Heterozygosity (%)
CK	403 332	269 783	133 549	66.55	33.45	35.50
S20	489 805	313 349	176 456	67.18	32.82	37.19
G20	358 014	263 791	94 223	65.95	34.05	36.42
F20	331 527	252 625	78 902	65.89	34.11	35.16

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图2

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图2SNP注释分析

Fig. 2SNP annotation analysis



通过InDel注释分类,结果如图3所示。2%的不同外源糖处理‘红地球’测序结果与参考基因组比对后,共获得15种InDel类型,有21 479个差异表达基因的InDel类型为其他类型,而发生在基因间区的数量最多,为16 824个,其次是在下游,为3 343个;密码子改变与缺失、终止获得及剪接位点受体这3类事件的数量最少,都为1个。

图3

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图3InDel注释分析

Fig. 3InDel annotation analysis

2.4 可变剪接事件分析

对每个样品存在的可变剪接类型统计,结果如图4所示。不同外源糖处理下的葡萄试管苗中,其第一个外显子可变剪接和最后一个外显子可变剪接事件数量最多,其次是单内含子滞留,而多外显子跳跃（模糊边界）和单外显子跳跃（模糊边界）事件数量最少。

图4

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图4可变剪接分析

Fig. 4Alternative splice analysis

2.5 差异基因表达分析

通过设定FDR和Log₂（FC）两个阈值对不同外源糖处理的葡萄试管苗测序结果进行差异基因筛选,共获得2 633个差异基因,其中共有的差异基因为180个（图5-A）。对共有的差异基因以Log₂（FPKM）值进行表达量分析（图5-B）,所有基因被聚类为3组,第一组包括127个基因,仅CK中高表达,而其余处理下均下调表达;第二组包括19个基因,仅在G20下高表达,其余处理中均下调;第三组的34个基因,与CK相比表达模式不尽一致,且表达在处理之间具有明显差异。

图5

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图5差异基因的Venn图和共有差异基因的表达分析

Fig. 5Venn diagram of DEGs and common DEGs analysis

2.6 差异基因的COG分析

将共有的180个差异基因进行COG注释,结果仅注释到了26个,包括6个上调基因和20个下调基因（图6）。其中,注释到一般功能预测的差异基因数量最多,为10个,其次是次级代谢物的合成、转运和分解,包括4个上调和3个下调差异基因。在转录,能量产生和转运,氨基酸转运和代谢、翻译后修饰、蛋白质周转、分子伴侣和防御机制4个功能分类中各注释到1个基因。

图6

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图6差异基因COG分析

Fig. 6COG analysis of DEGs

2.7 差异基因的GO富集分析

通过对共有的差异基因进行GO功能注释,发现这些差异基因主要被注释在分子功能（14）、生物学过程（22）和细胞组分（13）功能类别中（图7）。在分子功能中,注释在ATP结合（ATP binding (GO: 0005524)）、丝氨酸/苏氨酸蛋白激酶活性（protein serine/threonine kinase activity (GO: 0004674)）和氧化还原酶活性（oxidoreductase activity (GO: 0016491)）的基因数量最多;生物学过程中,注释在蛋白质磷酸化（protein phosphorylation (GO: 0006468)）、氧化还原过程（oxidation-reduction process (GO: 0055114)）和碳水化合物代谢过程（carbohydrate metabolic process (GO: 0005975)）的基因个数分别为23、20和10个,而在细胞组分中,细胞外区域（extracellular region (GO: 0005576)）、质膜（plasma membrane (GO: 0005886)）占比最高。在分子功能中也注释到了蛋白激酶活性（protein kinase activity (GO: 0004672)）、6-磷酸果糖激酶活性（6-phosphofructokinase activity (GO: 0003872)）和生长因子激活（growth factor activity (GO: 0008083)）等。生物学过程中也注释到了负调控细胞程序性死亡（negative regulation of programmed cell death (GO: 0043069)）、果糖6-磷酸代谢过程（fructose 6-phosphate metabolic process (GO: 0006002)）及生长发育等（developmental growth (GO: 0048589)）功能类别。

图7

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图7差异基因GO富集分析

Fig. 7GO enrichment analysis of DEGs

2.8 7个蛋白激酶差异基因表达

蛋白激酶对植物的生长具有重要的作用。通过对本次测序结果进行筛选,获得了7种与植物生长发育相关的蛋白激酶基因,包括GK的2个基因、MAPKs的34个基因、CBL的6个基因、PP2A的6个基因、HXK的6个基因、HPK的14个基因和TK的164个基因（图8-A—G,G图基因名称见电子版附件）。与CK相比,2个GK基因HXK2在S20处理中上调表达（图8-A）。MAPKs中的NTF3、MKK6、NPK1、MPK9、M2K2和MEKK1在CK中上调表达,而在其余处理中均下调表达,S20下的4个基因包括NPK1、MKKA、MKKA和ANP较CK、G20和F20表达量高（图8-B）。CBL中的CBL1分别在CK和S20中出现下调,而CBL7在S20和F20中均上调表达（图8-C）。有5个PP2A,包括PP2A、PP2A2、TAP46、PP2A3及PP2A仅在G20中上调表达,其余处理下均出现下调（图8-D）。在HXK中,与CK相比在S20中仅有HXKL2和HXK1为下调表达,其余的4个基因为上调表达（图8-E）。在HPK中,仅Vitisvinifer_newGene_6370在CK中高表达,而在S20、G20和F20中出现低表达,BETTA-AD、AHK3、WNK5、AMPD 和Vitisvinifer_newGene_6370只在S20中上调,而AHP1、GSVIVG01019546001、AHK2、CYP20-2和AHP1只在G20中上调（图8-F）。TK的164个基因在不同的处理中,表达具有明显差异（图8-G）。

图8

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图87个蛋白激酶的表达分析

A: GK; B: MAPKs; C: CBL; D: PP2A; E: HXK; F: HPK; G: TK
Fig. 8Analysis of the expression of seven protein kinases

2.9 qRT-PCR验证

选择了20个差异基因包括MAPK（5个）、HXK（1个）、PP2A（4个）、HPK（2个）和TK（8个）进行qRT-PCR分析,结果如图9所示。有3个基因分别为GSVIVG01015297001（HXK1）GSVIVG01009192001（CTR1）和GSVIVG01014110001（LRR-RLK）在不同的外源糖处理下,表达趋势与转录组测序结不一致,其余的17个基因基本一致,表明测序结果可靠。

图9

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图9差异基因的qRT-PCR分析

Fig. 9qRT-PCR analysis of DEGs

3 讨论

糖对植物的形态建成和基因表达调控是其代谢产生的效应。近年来研究表明,糖作为信号分子在胞内形成不同信号转导途径,将信号转导到细胞核或细胞器中目标基因的转录起始位点上,控制相关基因的转录,引起相应的生理生化变化,从而调控植物的生长和发育^[33]。本次测序共获得21.53 Gb Clean Data,各样品Clean Data均达到4.69 Gb。对SNP统计发现,获得转换和颠换的SNP覆盖率分别为65.89%—67.18%和32.82%—34.11%不等。在注释到的15种InDel类型中,除21 479个差异基因的InDel类型未知外,最多的是发生在基因间区和下游中,获得终止、密码子改变与缺失和剪接位点受体这三类事件的数量最少,均为1个差异基因。可变剪接能够使一个转录单位形成多个转录本和蛋白同系物,从而增加转录本和蛋白质的复杂性,这是导致真核生物基因和蛋白质数量较大差异的重要原因^[34]。对本次测序结果进行可变剪接分析得出,12种可变剪接事件中TTS和TSS数量最多,其次是IR,而MSKIP和XMSKIP事件最少。

进行COG注释,可从基因组水平上寻找直系同源体,预测未知的ORF生物学功能,并提高基因注释的准确性和完整性^[35]。对筛选出的180个共有的差异基因通过COG注释,共获得了26个基因,包括6个下调和20个上调,且注释到最多的是一般功能预测,而转录相关和信号转导机制功能类别次之,最少的则是核苷酸转运与代谢和染色体结构与活力,在细胞核结构、细胞运动和胞外结构中均没有注释到差异基因,这一结果与张雪等^[36]研究山葡萄果皮着色的转录组注释结果基本一致,表明转录和信号转导对植物生长发育和果实着色的调控具有重要作用。GO注释分类发现这些差异基因主要被注释在分子功能、生物学过程和细胞组分的12、22和13个功能类别中。在分子功能中,注释到丝氨酸/苏氨酸蛋白激酶活性的基因数量较多,其次是氧化还原酶活性;在生物学过程中,注释到蛋白质磷酸化、氧化还原过程和碳水化合物代谢过程的基因个数分别为21、20和7个,而在细胞组分中,细胞外区域和质膜占比最高,达到11.2%和5.6%,这一结果与魏利斌等^[37]在研究芝麻发育转录组分析结果一致。

大量研究表明蛋白质的磷酸化与去磷酸化过程在细胞的信号识别与转导中起重要作用,它是生物体中普遍存在的一种调节机制,几乎涉及所有的生理和病理过程,如糖代谢、细胞的生长发育、光合作用、基因表达等^[38]。本研究通过设定差异基因阈值,筛选出了与植物生长发育相关的7种蛋白激酶,包括GKs、MAPKs、CBLs、PP2A、HXK、HPK和TK,这些蛋白激酶基因在不同外源糖处理下表达各不相同。

GKs能催化葡萄糖和其他己糖的磷酸化,包括果糖、甘露醇和半乳糖,也在大肠杆菌、酵母、哺乳动物和植物中充当信号感受器^[39,40]。在拟南芥中研究发现,与线粒体胞膜相关的HXK1感知葡萄糖存在后,会特异性地下调编码与光合作用过程中相关蛋白质基因的表达,例如Chl a/b结合蛋白（CAB）和Rubisco小亚基（RBCS）等^[41,42]。在本研究中,GK中的两个基因HXK2在S20处理中上调表达,在G20处理中下调,这一结果与CHO等^[43]的研究一致。HXK作为双功能酶存在于所有的有机体中,它不仅磷酸化己糖转变为己糖6磷酸,而且对糖信号的感知具有重要作用^[44]。对测序结果进行HXK筛选得出,G20下分别有两个HXKL2和HXK2与其余3个处理相比均上调表达,表明这几个基因较其余的处理更能快速感应葡萄糖的存在。

PP2A在植物中具有多重作用,例如对细胞周期性进程的调控,根皮质细胞伸长,组织发育和植物对生物和非生物胁迫的反应等^[45]。RAZAVIZADEH等^[46]研究发现,PP2A3在不同程度的干旱胁迫和4 μmol·L^-1 的ABA处理下均表现出显著上调。本研究中对PP2A进行差异基因筛选,获得6个差异表达基因,其中PP2A、PP2A2、TAP46、PP2A3及PP2A仅在G20中上调表达,其余处理均出现下调,且与对照相比,PP2A3在果糖中出现上调趋势。拟南芥中研究发现,CIPK9与CBL3相互作用有助于在低K⁺胁迫下细胞K⁺的动态平衡,且CBL2和CBL3的过表达提高了对低K⁺的敏感性^[47],而本研究筛选到的6个CBL中,CBL1、CBL10和CBL7在G20下低表达,另一个CBL7、CBL3和CBL9却表现出上调,表明这些基因在响应不同的外源糖时表达模式不尽相同。

在植物中,由环境条件或激素改变产生的信号均是通过MAPKs所介导的级联反应进行传递的^[48]。通过遗传和生化方法研究发现,MAPKs不仅参与生物和非生物胁迫的信号转导,而且还参与激素对生长发育信号的转导过程^[49]。如今,MAPKs已在大量植物物种中被发现,表明MAPKs级联信号转导机制在高等植物中也非常保守^[50]。通过转录组测序,本研究筛选到了MAPKs的34个差异表达基因,其中NTF3、MKK6、NPK1、MPK9、M2K2和MEKK1在CK中上调表达,而在其余处理下均下调表达。HPKs是一个大的信号转导酶家族,能够在保守的组氨酸残基上自磷酸,并与其下游的靶蛋白形成双组分信号系统响应生物和非生物胁迫^[51]。本研究筛选到了10个组氨酸蛋白激酶基因,通过聚类分析发现,BETTA-AD、AHK3、WNK5、AMPD 和Vitisvinifer_newGene_6370只在S20中上调,而AHP1、GSVIVG01019546001、AHK2、CYP20-2和AHP1只在G20中上调,表明这些基因对响应不同的糖具有明显选择性。酪氨酸（Tyr）磷酸化是动物体内受体酪氨酸激酶（RTKs）介导信号转导的关键分子开关^[52],在植物中虽然缺乏典型的RTKs,但是拥有数百个与动物具有相似结构域结构的RTKs^[53]。最近研究表明,在植物中酪氨酸磷酸化对于调控诱导免疫过程（PTI）的信号传递具有重要作用^[54]。本研究筛选到酪氨酸蛋白激酶的164个差异基因,这些基因在响应不同的外源糖时具有各自的表达模式。

应用qRT-PCR对测序结果挑选了20个基因进行验证,发现有17个基因的表达趋势与测序结果一致,表明转录组测序结果可靠,这为通过基因表达研究蛋白激酶响应不同外源糖,并深入分析糖在植物生长发育中的作用提供了理论依据。

4 结论

本研究应用RNA-Seq技术对不同外源糖处理下的葡萄试管苗进行测序分析,共获得了2 633个差异基因,有180个差异基因对3种不同外源糖都作出响应,且共有的这些基因表达对糖源具有选择性。筛选到了7种蛋白质磷酸化相关的基因,这些差异基因在数量、功能分类和代谢通路上对糖的响应各不相同,且有各自的选择表达特异性,为丰富糖在调节植物生长发育及进一步探究蛋白质磷酸化响应外源糖提供了参考。

（责任编辑 赵伶俐）

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Sugar plays an essential role in plant cold acclimation (CA), but the interaction between CA and sugar remains unclear in tea plants. In this study, during the whole winter season, we investigated...

[4] SOLAROVA J, POSPHILSILOVA J, CATSKY J, SANTRUCEK J . Photosynthesis and growth of tobacco plantlets independence on carbon supply
Photosynthetica, 1989,23(4):629-637.
[本文引用: 1]

[5] SMEEKENS S, MA J K, HANSON J, ROLLAND F . Sugar signals and molecular networks controlling plant growth
Current Opinion in Plant Biology, 2010,13(3):273-278.
DOI:10.1016/j.pbi.2009.12.002URLPMID:20056477 [本文引用: 1]
In recent years, several regulatory systems that link carbon nutrient status to plant growth and development have emerged. In this paper, we discuss the growth promoting functions of the hexokinase (HXK) glucose sensor, the trehalose 6-phosphate (T6P) signal and the Target of Rapamycin (TOR) kinase pathway, and the growth inhibitory function of the SNF1-related Protein Kinase1 (SnRK1) and the C/S1 bZIP transcription factor network. It is crucial that these systems interact closely in regulating growth and in several cases crosstalk has been demonstrated. Importantly, these nutrient controlled systems must interact with other growth regulatory pathways.

[6] NGUYEN Q T, KOZAI T, NIU G, NGUYEN U V . Photosynthetic characteristics of coffee (Coffea arabusta) plantlets in vitro in response to different CO₂ concentrations and light intensities.
Plant Cell Tissue & Organ Culture, 1999,55:133-139.
DOI:10.1023/A:1006224026639URL [本文引用: 1]
The photosynthetic characteristics of coffee ( Coffea arabusta) plantlets cultured in vitro in response to different CO 2 concentrations inside the culture vessel and photosynthetic photon flux (PPF) were investigated preliminarily. The estimation of net photosynthetic rate (P n ) of coffee plantlets involved three methods: (1) estimating time courses of actual P n in situ based on measuring CO 2 concentrations inside and outside the vessel during a 45-day period, (2) estimating P n in situ at different CO 2 concentrations and PPFs using the above measuring approach for 10-day and 30-day old in vitro plantlets, and (3) estimating P n of a single leaf at different CO 2 concentrations and PPFs by using a portable photosynthesis measurement system for 45-day old in vitro coffee plantlets. The results showed that coffee plantlets in vitro had relatively high photosynthetic ability and that the P n increased with the increase in CO 2 concentration inside the vessel. The CO 2 saturation point of in vitro coffee plantlets was high (4500鈥5000 渭mol mol -1 ); on the other hand, the PPF saturation point was not so high as compared to some other species, though it increased with increasing CO 2 concentration inside the vessel.

[7] 张曦, 林金星, 单晓昳 . 拟南芥无机氮素转运蛋白及其磷酸化调控研究进展
植物学报, 2016,51(1):120-129.
DOI:10.11983/CBB14222URLMagsci [本文引用: 1]
<p>氮元素是植物必需的营养元素之一, 氮素供需失衡会严重影响植物的生长发育。无机氮(硝酸根NO₃^&ndash;和铵根NH4⁺)是植物体内氮素的主要来源, 对其有效吸收和利用依赖于多种类型转运蛋白的协同作用。其中, 部分无机氮素转运蛋白的活性受到可逆磷酸化作用的精准调控。该文将对模式植物拟南芥(<em>Arabidopsis thaliana</em>)中硝酸根和铵根转运蛋白的分类、结构、定位和功能特点等进行总结, 并重点对可逆磷酸化调控转运蛋白的分子机制加以阐述。</p>
ZHANG X, LIN J X, SHAN X Y . Progress in inorganic nitrogen transport proteins and their phosphorylation regulatory mechanism in Arabidopsis.
Chinese Bulletin of Botany, 2016,51(1):120-129. (in Chinese)
DOI:10.11983/CBB14222URLMagsci [本文引用: 1]
<p>氮元素是植物必需的营养元素之一, 氮素供需失衡会严重影响植物的生长发育。无机氮(硝酸根NO₃^&ndash;和铵根NH4⁺)是植物体内氮素的主要来源, 对其有效吸收和利用依赖于多种类型转运蛋白的协同作用。其中, 部分无机氮素转运蛋白的活性受到可逆磷酸化作用的精准调控。该文将对模式植物拟南芥(<em>Arabidopsis thaliana</em>)中硝酸根和铵根转运蛋白的分类、结构、定位和功能特点等进行总结, 并重点对可逆磷酸化调控转运蛋白的分子机制加以阐述。</p>

[8] 尹双双, 高文远, 王娟, 刘辉, 左北梅 . 药用植物不定根培养的影响因素
中国中药杂志, 2012,37(24):3691-3694.
DOI:10.4268/cjcmm20122403 [本文引用: 1]
随着中药现代化,药用植物资源 远远不能满足中药产业的需求,因此亟需为中药资源的开发开辟新的途径,药用植物不定根大规模培养是实现中药产业化的一个重要途径,而如何建立良好的不定培 养体系是药用植物不定根培养的关键。该文主要从植物激素、外植体、蔗糖、接种量、无机盐等几个方面综述了药用植物不定根培养的影响因素。
YIN S S, GAO W Y, WAN J, LIU H, ZUO B M . Influencing factors on culture of medicinal plants adventitious roots
China Journal of Chinese Material Medcia, 2012,37(24):3691-3694. (in Chinese)
DOI:10.4268/cjcmm20122403 [本文引用: 1]
随着中药现代化,药用植物资源 远远不能满足中药产业的需求,因此亟需为中药资源的开发开辟新的途径,药用植物不定根大规模培养是实现中药产业化的一个重要途径,而如何建立良好的不定培 养体系是药用植物不定根培养的关键。该文主要从植物激素、外植体、蔗糖、接种量、无机盐等几个方面综述了药用植物不定根培养的影响因素。

[9] 邹英宁 . 不同碳源对中国李离体培养的影响
安徽农业科学, 2010, 38(4): 1720, 1776.
DOI:10.3969/j.issn.0517-6611.2010.04.025URL [本文引用: 1]
[目的]研究不同碳源种类对中国李"海湾红宝石"离体培养的影 响.[方法]以3年生"海湾红宝石"李新稍茎段为试材,在初代培养和生根培养中添加蔗糖、葡萄糖、山梨醇、果糖4种碳源进行外植体接种,30、60 d后分别统计增殖倍数、新梢率和生根率.[结果]在中国李增殖、诱导过程中,葡萄糖(30 g/L)作碳源明显促进增殖和生长.生根诱导过程中,采用蔗糖(15 g/L)为碳源,外植体生根率和生根倍数均最高.[结论]初代培养和生根培养中分别选用葡萄糖和蔗糖作碳源,中国李培养效果好.
ZOU Y N . Effect of different carbon sources on the culture of Chinese plum (Prunus salicina) in vitro
Journal of Anhui Agricultural Sciences, 2010, 38(4): 1720, 1776. (in Chinese)
DOI:10.3969/j.issn.0517-6611.2010.04.025URL [本文引用: 1]
[目的]研究不同碳源种类对中国李"海湾红宝石"离体培养的影 响.[方法]以3年生"海湾红宝石"李新稍茎段为试材,在初代培养和生根培养中添加蔗糖、葡萄糖、山梨醇、果糖4种碳源进行外植体接种,30、60 d后分别统计增殖倍数、新梢率和生根率.[结果]在中国李增殖、诱导过程中,葡萄糖(30 g/L)作碳源明显促进增殖和生长.生根诱导过程中,采用蔗糖(15 g/L)为碳源,外植体生根率和生根倍数均最高.[结论]初代培养和生根培养中分别选用葡萄糖和蔗糖作碳源,中国李培养效果好.

[10] 王博, 范桂枝, 詹亚光, 李康 . 不同碳源对白桦愈伤组织生长和三萜积累的影响
植物生理学通讯, 2008,44(1):97-99.
URL [本文引用: 1]
在一个继代周期中,培养基中添加10～50 g·L~(-1)的蔗糖、葡萄糖和果糖后,白桦愈伤组织的鲜重均上升,浓度为20～30 g·L~(-1)的三种糖,其鲜重积累量均较高。培养基中添加不同浓度葡萄糖和果糖的白桦愈伤组织中三萜物质含量先上升后下降,而添加蔗糖的则呈直线上升。其中,葡萄糖、果糖和蔗糖浓度为30 g·L~(-1)的三萜类物质积累量最高。
WANG B, FAN G Z, ZHAN Y G, LI K . Effects of different carbon sources on callus growth and accumulation of triterpenoids in Birch (Betula platyphylla suck.).
Plant Physiology Communications, 2008,44(1):97-99. (in Chinese)
URL [本文引用: 1]
在一个继代周期中,培养基中添加10～50 g·L~(-1)的蔗糖、葡萄糖和果糖后,白桦愈伤组织的鲜重均上升,浓度为20～30 g·L~(-1)的三种糖,其鲜重积累量均较高。培养基中添加不同浓度葡萄糖和果糖的白桦愈伤组织中三萜物质含量先上升后下降,而添加蔗糖的则呈直线上升。其中,葡萄糖、果糖和蔗糖浓度为30 g·L~(-1)的三萜类物质积累量最高。

[11] 王思瑶, 崔瞳肸, 翟睿, 林香雨, 李欣, 孙璐, 詹亚光, 尹静 . 不同碳源对柽柳丛生芽生长、三萜及黄酮物质积累的影响
植物生理学报, 2017,53(12):2189-2196.
URL [本文引用: 1]
本研究以内蒙古柽柳（Tamarix chinensis）嫩枝为外植体,分别在培养基中添加不同碳源（蔗糖、果糖、葡萄糖、麦芽糖、淀粉）及不同浓度（10、20、30、40 g·L~（-1））的蔗糖,研究柽柳丛生苗生长量和次生产物含量积累的变化。结果表明,不同碳源及蔗糖浓度均对柽柳丛生苗生长量和次生产物积累有显著影响,其中,适合柽柳丛生芽生长的碳源为蔗糖,最适蔗糖浓度为10 g·L~（-1）,培养35 d后其生长量显著高于其他处理。当以蔗糖为碳源时,柽柳组培苗一个生长周期（35 d）内的总黄酮及三萜组分（白桦脂酸、白桦脂醇、齐墩果酸）含量均较高。当蔗糖浓度为30 g·L~（-1）时有利于总黄酮的积累,含量可达16.65mg·g~（-1）（DW）;而10 g·L~（-1）蔗糖浓度对三萜组分积累更有利,白桦脂酸、白桦脂醇、齐墩果酸含量分别可达2.67、6.86、0.85mg·g~（-1）（DW）。当碳源浓度为30 g·L~（-1）时,总三萜含量以葡萄糖处理为最高,可达62.43 mg·g~（-1）（DW）,蔗糖次之,麦芽糖最低。该研究结果为通过生物发酵方法进行柽柳药用成分的规模化生产奠定了基础。
WANG S Y, CUI T X, ZHAI R, LIN X Y, LI X, SUN L, ZHAN Y G, YIN J . Effects of different carbon sources on the growth and accumulation of triterpenoids and flavonoids in tufted buds of Tamarix chinensis.
Plant Physiology Journal, 2017,53(12):2189-2196. (in Chinese)
URL [本文引用: 1]
本研究以内蒙古柽柳（Tamarix chinensis）嫩枝为外植体,分别在培养基中添加不同碳源（蔗糖、果糖、葡萄糖、麦芽糖、淀粉）及不同浓度（10、20、30、40 g·L~（-1））的蔗糖,研究柽柳丛生苗生长量和次生产物含量积累的变化。结果表明,不同碳源及蔗糖浓度均对柽柳丛生苗生长量和次生产物积累有显著影响,其中,适合柽柳丛生芽生长的碳源为蔗糖,最适蔗糖浓度为10 g·L~（-1）,培养35 d后其生长量显著高于其他处理。当以蔗糖为碳源时,柽柳组培苗一个生长周期（35 d）内的总黄酮及三萜组分（白桦脂酸、白桦脂醇、齐墩果酸）含量均较高。当蔗糖浓度为30 g·L~（-1）时有利于总黄酮的积累,含量可达16.65mg·g~（-1）（DW）;而10 g·L~（-1）蔗糖浓度对三萜组分积累更有利,白桦脂酸、白桦脂醇、齐墩果酸含量分别可达2.67、6.86、0.85mg·g~（-1）（DW）。当碳源浓度为30 g·L~（-1）时,总三萜含量以葡萄糖处理为最高,可达62.43 mg·g~（-1）（DW）,蔗糖次之,麦芽糖最低。该研究结果为通过生物发酵方法进行柽柳药用成分的规模化生产奠定了基础。
王爱民, 刘文, 傅中滇 . 不同碳源对红边朱蕉组培苗生长的影响
徐州师范大学学报 (自然科学版), 2003,21(3):76-78.
[本文引用: 1]

WANG A M, LIU W, FU Z D . A preliminary study on the reducing cost of tissue culture plantlet of cordyline terminalis
Journal of Xuzhou Normal University ((Nature Sciences Edition), 2003,21(3):76-78. (in Chinese)
[本文引用: 1]

[12] KOZAI T, KUBOTA C, JEONGB R . Environmental control for the large-scale production of plants through in vitro techniques
Plant Cell Tissue & Organ Culture, 1997,51:49-56.
DOI:10.1023/A:1005809518371URL [本文引用: 1]
Leafy or chlorophyllous explants of a number of plant species currently micropropagated have been found to have high photosynthetic ability. Their growth and development have been promoted on sugar-free medium rather than on sugar-containing medium, provided that the environmental factors, such as CO 2 concentration, light intensity and relative humidity, are controlled for promoting photosynthesis and transpiration of explants/shoots/plantlets in vitro. Thus, environmental control is essential for promoting photosynthetic growth and development of in vitro plantlets.Several types of sugar-free (photoautotrophic) culture systems for large-scale micropropagation of plants have been developed. Advantages of sugar-free over conventional (heterotrophic or photomixotrophic) micropropagation systems are as follows: growth and development of plantlets in vitro are faster and more uniform, plantlets in vitro have less physiological and morphological disorders, biological contamination in vitro is less, plantlets have a higher percentage of survival during acclimatization ex vitro, and larger culture vessels could be used because of less biological contamination. Hence, production costs could be reduced and plant quality could be improved significantly with photoautotrophic micropropagation. Methods for the measurement and control of in vitro environments and the beneficial effects of environmental control on photosynthetic growth, development, and morphogenesis in large-scale production of micropropagated plantlets are presented.

[13] GUGLIELMINETTI L, PERATA P, MORITA A, LORETI E, YAMAGUCHI J, ALPI A . Characterization of isoforms of hexose kinases in rice embryo
Phytochemistry, 2000,53(2):195-200.
DOI:10.1016/S0031-9422(99)00541-5URLPMID:10680171 [本文引用: 1]
Hexose kinases in rice embryos have been characterized. Six isoforms were detected: i.e. three glucokinases (GK1–3), two hexokinases (HK1 and HK2) and one fructokinase (FK1). Out of these, GK3, HK1 and HK2 were inhibited by mannoheptulose and glucosamine, known inhibitors of hexokinase activity. These inhibitors are also known to be modulators of sugar sensing processes. The results suggest that GK3, HK1 and HK2 may play a role in sensing the cellular sugar status in the rice embryo.

[14] TURNER J F , CHENSEE, HARISSON D D. Glucokinase of pea seeds
Biochimica et Biophysica Acta-Enzymology, 1997,480:367-375.
[本文引用: 1]

[15] MARTINEZ-BARAJAS E, RANGALL D D . Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruct.
Planta, 1998,205:567-573.
[本文引用: 1]

[16] 赵书平, 谈宏斌, 鹿丹, 裴丽丽, 崔喜艳, 马有志, 陈明, 徐兆师, 张小红 . 植物蛋白激酶介导的非生物胁迫和激素信号转导途径的研究进展
植物遗传资源学报, 2017,18(2):358-366.
DOI:10.13430/j.cnki.jpgr.2017.02.022URL [本文引用: 1]
干旱、盐渍、低温和高温等非生物胁迫严重影响植物的生长发育和作物的产量.在长期的进化过程中,植物逐渐形成了对外部刺激快速感知和主动适应的能力,其中植物体内逆境信号的传递在植物快速感知外部刺激和主动适应非生物胁迫过程中起着非常重要的作用.蛋白激酶和蛋白磷酸酶催化的蛋白质磷酸化和去磷酸化是植物体内存在的最普遍且最重要的信号转导调节方式.其中,蛋白激酶的主要作用是将ATP或GTP上的γ磷酸基团转移到特定的底物蛋白上,使蛋白磷酸化,被磷酸化的蛋白发挥相应的生理功能.近年来,利用生物技术和基因工程等手段从细胞、分子水平上研究有关蛋白激酶的抗逆机理,通过基因沉默、基因过表达等策略提高植物的抗逆性成为国内外抗逆分子生物学与分子育种学研究的热点.本文主要对植物蛋白激酶在介导非生物胁迫和激素信号通路中的作用进行综述,为进一步研究植物蛋白激酶功能提供有价值的信息.
ZHAO S P, TAN H B, LU D, PEI L L, CUI X Y, MA Y Z, CHEN M, XU Z S, ZHANG X H . Research progress of plant protein kinase mediated abiotic stress and hormone signal transduction pathway
Journal of Plant Genetic Resources, 2017,18(2):358-366. (in Chinese)
DOI:10.13430/j.cnki.jpgr.2017.02.022URL [本文引用: 1]
干旱、盐渍、低温和高温等非生物胁迫严重影响植物的生长发育和作物的产量.在长期的进化过程中,植物逐渐形成了对外部刺激快速感知和主动适应的能力,其中植物体内逆境信号的传递在植物快速感知外部刺激和主动适应非生物胁迫过程中起着非常重要的作用.蛋白激酶和蛋白磷酸酶催化的蛋白质磷酸化和去磷酸化是植物体内存在的最普遍且最重要的信号转导调节方式.其中,蛋白激酶的主要作用是将ATP或GTP上的γ磷酸基团转移到特定的底物蛋白上,使蛋白磷酸化,被磷酸化的蛋白发挥相应的生理功能.近年来,利用生物技术和基因工程等手段从细胞、分子水平上研究有关蛋白激酶的抗逆机理,通过基因沉默、基因过表达等策略提高植物的抗逆性成为国内外抗逆分子生物学与分子育种学研究的热点.本文主要对植物蛋白激酶在介导非生物胁迫和激素信号通路中的作用进行综述,为进一步研究植物蛋白激酶功能提供有价值的信息.

[17] RANTY B, ALDON D, GALAUD J P . Plant calmodulins and calmodulin-related proteins: Multifaceted relays to decode calcium signals
Plant Signaling & Behavior, 2006,1(3):96-104.
DOI:10.4161/psb.1.3.2998URLPMID:19521489 [本文引用: 1]
The calmodulin (CaM) family is a major class of calcium sensor proteins which collectively play a crucial role in cellular signaling cascades through the regulation of numerous target proteins. Although CaM is one of the most conserved proteins in all eukaryotes, several features of CaM and its downstream effector proteins are unique to plants. The continuously growing repertoire of CaM-binding proteins includes several plant-specific proteins. Plants also possess a particular set of CaM isoforms and CaM-like proteins (CMLs) whose functions have just begun to be elucidated. This review summarizes recent insights that help to understand the role of this multigene family in plant development and adaptation to environmental stimuli.

[18] BATISTIC O, KUDLA J . Integration and channeling of calcium signaling through the CBL calciumsensor/CIPK protein kinase network
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[21] YU Q Y, AN L J, LI W L . The CBL-CIPK network mediates different signaling pathways in plants
Plant Cell Reports, 2014,33:203-214.
DOI:10.1007/s00299-013-1507-1URL [本文引用: 1]

[22] KENTARO F, TOMOYUKI F, SHUN-ICHI Y, TETSU S, YUSUKE K, YUTAKA Y, HIROMI K, HITOSHI N, TOMOTAKE K . The PP2A-like protein phosphatase ppg1 and the far complex cooperatively counteract CK2-mediated phosphorylation of Atg32 to inhibit mitophagy
Cell Reports, 2018,23:3579-3590.
DOI:10.1016/j.celrep.2018.05.064URL [本文引用: 1]
Mitophagy plays an important role in mitochondrial quality control. In yeast, phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 (CK2) upon induction of mitophagy is a prerequisite for interaction of Atg32 with Atg11 (an adaptor protein for selective autophagy) and following delivery of mitochondria to the vacuole for degradation. Because CK2 is constitutively active, Atg32 phosphorylation must be precisely regulated to prevent unrequired mitophagy. We found that the PP2A (protein phosphatase 2A)-like protein phosphatase Ppg1 was essential for dephosphorylation of Atg32 and inhibited mitophagy. We identified the Far complex proteins, Far3, Far7, Far8, Far9, Far10, and Far11, as Ppg1-binding proteins. Deletion of Ppg1 or Far proteins accelerated mitophagy. Deletion of a cytoplasmic region (amino acid residues 151&ndash;200) of Atg32 caused the same phenotypes as incells, which suggested that dephosphorylation of Atg32 by Ppg1 required this region. Therefore, Ppg1 and the Far complex cooperatively dephosphorylate Atg32 to prevent excessive mitophagy.

[23] 刘钊, 贾霖, 贾盟, 关明俐, 曹英豪, 刘丽娟, 曹振伟, 李莉云, 刘国振 . 水稻PP2Ac类磷酸酶蛋白质在盐胁迫下的表达
中国农业科学, 2012,45(12):2339-2345.
DOI:10.3864/j.issn.0578-1752.2012.12.001URLMagsci [本文引用: 1]
【目的】了解重要蛋白质的表达模式进而探讨水稻耐盐的分子机理。【方法】采用基于抗体的蛋白质组学策略，用免疫印迹（western blotting）调查了5个PP2Ac类磷酸酶蛋白质在苗期盐胁迫条件下的表达。【结果】发现在耐盐水稻品种兰胜中，OsPP2Ac-4的表达上调，在盐敏感的水稻品种9311中，OsPP2Ac-2、OsPP2Ac-3和OsPP2Ac-5的表达也发生了上调，但OsPP2Ac-4的表达下调。比较2个品种间PP2Ac蛋白质的表达，发现在正常生长条件下，PP2Ac蛋白质的表达没有显著区别且基本保持恒定，其表达变化仅发生在盐胁迫条件下。分析水稻MPSS数据库提供的苗期盐胁迫的转录数据，发现OsPP2Ac-2、OsPP2Ac-3和OsPP2Ac-5在盐胁迫条件下转录水平下调。【结论】发现了4个盐胁迫条件下表达发生变化的PP2Ac蛋白质。
LIU Z, JIA S, JIA M, GUAN M L, CAO Y H, LIU L J, CAO Z W, LI L Y, LIU G Z . Expression on profiling of rice PP2Ac type phosphatase proteins in seedlings under salt-stressed conditions
Scientia Agricultura Sinica, 2012,45(12):2339-2345. (in Chinese)
DOI:10.3864/j.issn.0578-1752.2012.12.001URLMagsci [本文引用: 1]
【目的】了解重要蛋白质的表达模式进而探讨水稻耐盐的分子机理。【方法】采用基于抗体的蛋白质组学策略，用免疫印迹（western blotting）调查了5个PP2Ac类磷酸酶蛋白质在苗期盐胁迫条件下的表达。【结果】发现在耐盐水稻品种兰胜中，OsPP2Ac-4的表达上调，在盐敏感的水稻品种9311中，OsPP2Ac-2、OsPP2Ac-3和OsPP2Ac-5的表达也发生了上调，但OsPP2Ac-4的表达下调。比较2个品种间PP2Ac蛋白质的表达，发现在正常生长条件下，PP2Ac蛋白质的表达没有显著区别且基本保持恒定，其表达变化仅发生在盐胁迫条件下。分析水稻MPSS数据库提供的苗期盐胁迫的转录数据，发现OsPP2Ac-2、OsPP2Ac-3和OsPP2Ac-5在盐胁迫条件下转录水平下调。【结论】发现了4个盐胁迫条件下表达发生变化的PP2Ac蛋白质。

[24] 雍彬, 何兵, 徐攀, 虞传洋, 王东 . 甘薯HXK基因的克隆、组织表达及生物信息分析
四川大学学报(自然科学版), 2014,51(2):378-384.
DOI:10.3969/j.issn.0490-6756.2014.02.029URL [本文引用: 1]
通过RT-PCR技术从甘薯的总 RNA 中克隆得到了全长的己糖激酶基因,测序结果表明HXK(hexokinase)基因编码区长1491bp,编码496个氨基酸,其翻译的蛋白质分子量为53.4kD,其氨基酸序列与其它已知高等植物HXK基因具有很高的同源性.构建了原核表达载体pET-HXK,经IPTG诱导后可表达获得相对分子量约为71 kD的外源融合蛋白,与预测结果一致.并对HXK基因在不同组织中的转录水平进行了数字表达谱和荧光定量分析.
YONG B, HE B, XU P, YU C Y, WANG D . Cloning, organ-specific expression pattern and sequence analysis of HXK gene in sweet potato.
Journal of Sichuan University (Natural Science Edition), 2014,51(2):378-384. (in Chinese)
DOI:10.3969/j.issn.0490-6756.2014.02.029URL [本文引用: 1]
通过RT-PCR技术从甘薯的总 RNA 中克隆得到了全长的己糖激酶基因,测序结果表明HXK(hexokinase)基因编码区长1491bp,编码496个氨基酸,其翻译的蛋白质分子量为53.4kD,其氨基酸序列与其它已知高等植物HXK基因具有很高的同源性.构建了原核表达载体pET-HXK,经IPTG诱导后可表达获得相对分子量约为71 kD的外源融合蛋白,与预测结果一致.并对HXK基因在不同组织中的转录水平进行了数字表达谱和荧光定量分析.

[25] KARVE A, RAUH B L, XIA X . Expression and evolutionary features of the hexokinase gene family in Arabidopsis.
Planta, 2008,228(3):411-425.
[本文引用: 1]

[26] HERICOURT F, CHEFDOR F, DJEGHDIR I, LARCHER M, LAFONTAINE F, COURDAVAULT V, AUGUIN D, COSTE F, DEPIERREUX C, TANIGAWA M, MAEDA T, GLEVAREC G, CARPIN S . Functional divergence of poplar histidine-aspartate kinase hk1 paralogs in response to osmotic stress
International Journal of Molecular Sciences, 2016,17(12):2061.
DOI:10.3390/ijms17122061URLPMID:5187861 [本文引用: 1]
Previous works have shown the existence of protein partnerships belonging to a MultiStep Phosphorelay (MSP) inPopulusputatively involved in osmosensing. This study is focused on the identification of a histidine-aspartate kinase, HK1b, paralog of HK1a. The characterization of HK1b showed its ability to homo- and hetero-dimerize and to interact with a few Histidine-containing Phosphotransfer (HPt) proteins, suggesting a preferential partnership in poplar MSP linked to drought perception. Furthermore, determinants for interaction specificity between HK1a/1b and HPts were studied by mutagenesis analysis, identifying amino acids involved in this specificity. TheHK1bexpression analysis in different poplar organs revealed its co-expression with threeHPts, reinforcing the hypothesis of partnership participation in the MSP in planta. Moreover, HK1b was shown to act as an osmosensor with kinase activity in a functional complementation assay of an osmosensor deficient yeast strain. These results revealed that HK1b showed a different behaviour for canonical phosphorylation of histidine and aspartate residues. These phosphorylation modularities of canonical amino acids could explain the improved osmosensor performances observed in yeast. As conserved duplicates reflect the selective pressures imposed by the environmental requirements on the species, our results emphasize the importance ofHK1gene duplication in poplar adaptation to drought stress.

[27] WOLANIN P M, THOMASON P A, STOCK J B . Histidine protein kinases: Key signal transducers outside the animal kingdom
Genome Biology, 2002, 3(10): reviews3013. 1.
[本文引用: 1]

[28] HUPFELD T, CHAPUY B, SCHRADER V, BEUTLER M, VELTKAMP C, KOCH R, CAMERON S, AUNG T, HAASE D, LAROSEE P, TRUEMPER L, WULF G G . Tyrosinekinase inhibition facilitates cooperation of transcription factor SALL4 and ABC transporter A3 towards intrinsic CML cell drug resistance
British Journal of Haematology, 2013,161(2):204.
DOI:10.1111/bjh.12246URLPMID:23432194 [本文引用: 1]
Summary Top of page Summary Introduction Material and methods Results Discussion Acknowledgements Authorship and disclosures References Supporting Information Although BCR-ABL1 tyrosine kinase inhibitors reliably induce disease remission for patients with chronic myeloid leukaemia (CML), unlimited extension of therapy is necessary to prevent relapse from persistent leukaemic cells. Here, we analysed model cell lines and primary CML cells for the expression and functions of the ABC transporter A3 (ABCA3) as well as the embryonic stem cell-associated transcription factor SALL4. ABCA3 protected leukaemic cells from the cytotoxic effects of the tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib. In the surviving cells, exposure to tyrosine kinase inhibitors significantly enhanced ABCA3 expression in vivo and in vitro , and was associated with increased expression of SALL4, which binds the ABCA3 promoter. Inhibition of ABCA3 or SALL4 by genetic silencing or indomethacin, but not interferon gamma, interrupted SALL4-dependent regulation of ABCA3 and restored susceptibility of leukaemic cells to tyrosine kinase inhibition. Tyrosine kinase inhibitor exposure facilitates a protective loop of SALL4 and ABCA3 cooperation in persistent leukaemic cells.

[29] MAO J, LI W F, MI B Q, DAWUDA M M, CALDERON-URREA A, MA Z H, ZHANG Y M, CHEN B H . Different exogenous sugars affect the hormone signal pathway and sugar metabolism in ‘Red Globe’ (Vitis vinifera L.) plantlets grown in vitro as shown by transcriptomic analysis.
Planta, 2017,246:537-552.
[本文引用: 1]

[30] KIM D, PERTEA G, TRAPENLL C, PIMENTEL H, KELLEYR R, SALZBERG S L . TopHat2: Accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions
Genome Biology, 2013,14(4):R36.
DOI:10.1186/gb-2013-14-4-r36URLPMID:4053844 [本文引用: 1]
TopHat is a popular spliced aligner for RNA-sequence (RNA-seq) experiments. In this paper, we describe TopHat2, which incorporates many significant enhancements to TopHat. TopHat2 can align reads of various lengths produced by the latest sequencing technologies, while allowing for variable-length indels with respect to the reference genome. In addition to de novo spliced alignment, TopHat2 can align reads across fusion breaks, which can occur after genomic translocations. TopHat2 combines the ability to identify novel splice sites with direct mapping to known transcripts, producing sensitive and accurate alignments, even for highly repetitive genomes or in the presence of pseudogenes. TopHat2 is available at http://ccb.jhu.edu/software/tophat .

[31] ANDERS S, HUBER W . Differential expression analysis for sequence count data
Genome Biology, 2010,11(10):R106.
DOI:10.1186/gb-2010-11-10-r106URL [本文引用: 1]

[32] LIVAK K J, SCHMITTGEN T D . Analysis of relative gene expression data using real-time quantitative and the 2 ^-ΔΔCT method
Methods, 2001,25:402-408.
DOI:10.1006/meth.2001.1262URL [本文引用: 1]

[33] ROLLAND F, BAENA-GONZALEZ E, SHEEN J . Sugar sensing and signaling in plants: Conserved and novel mechanisms
Annual Review of Plant Biology, 2006,57:675-709.
DOI:10.1146/annurev.arplant.57.032905.105441URLPMID:16669778 [本文引用: 1]
Sugars not only fuel cellular carbon and energy metabolism but also play pivotal roles as signaling molecules. The experimental amenability of yeast as a unicellular model system has enabled the discovery of multiple sugar sensors and signaling pathways. In plants, different sugar signals are generated by photosynthesis and carbon metabolism in source and sink tissues to modulate growth, development, and stress responses. Genetic analyses have revealed extensive interactions between sugar and plant hormone signaling, and a central role for hexokinase (HXK) as a conserved glucose sensor. Diverse sugar signals activate multiple HXK-dependent and HXK-independent pathways and use different molecular mechanisms to control transcription, translation, protein stability and enzymatic activity. Important and complex roles for Snf1-related kinases (SnRKs), extracellular sugar sensors, and trehalose metabolism in plant sugar signaling are now also emerging.

[34] FILICHKIN S A, HAMILYON M, DHARMAWARDHANA P D, SINGH S K, SULLIVAN C, BEN-HUR A, REDDY A S N, JAISWAL P . Abiotic stresses modulate landscape of poplar transcriptome via alternative splicing, differential intron retention, and isoform ratio switching
Frontiers in Plant Science, 2018. doi: 10.3389/fpls.2018.00005.
DOI:10.3389/fpls.2018.00005URL [本文引用: 1]
Abiotic stresses affect plant physiology, development, growth, and alter pre-mRNA splicing. Western poplar is a model woody tree and a potential bioenergy feedstock. To investigate the extent of stress-regulated alternative splicing (AS), we conducted an in-depth survey of leaf, root, and stem xylem transcriptomes under drought, salt, or temperature stress. Analysis of approximately one billion of genome-aligned RNA-Seq reads from tissue- or stress-specific libraries revealed over fifteen millions of novel splice junctions. Transcript models supported by both RNA-Seq and single molecule isoform sequencing (Iso-Seq) data revealed a broad array of novel stress- and/or tissue-specific isoforms. Analysis of Iso-Seq data also resulted in the discovery of 15,087 novel transcribed regions of which 164 show AS. Our findings demonstrate that abiotic stresses profoundly perturb transcript isoform profiles and trigger widespread intron retention (IR) events. Stress treatments often increased or decreased retention of specific introns a phenomenon described here as differential intron retention (DIR). Many differentially retained introns were regulated in a stress- and/or tissue-specific manner. A subset of transcripts harboring super stress-responsive DIR events showed persisting fluctuations in the degree of IR across all treatments and tissue types. To investigate coordinated dynamics of intron-containing transcripts in the study we quantified absolute copy number of isoforms of two conserved transcription factors (TFs) using Droplet Digital PCR. This case study suggests that stress treatments can be associated with coordinated switches in relative ratios between fully spliced and intron-retaining isoforms and may play a role in adjusting transcriptome to abiotic stresses.

[35] 贾新平, 孙晓波, 邓衍明, 梁丽建, 叶晓青 . 鸟巢蕨转录组高通测序及分析
园艺学报, 2014,41(11):2329-2341.
URLMagsci [本文引用: 1]
采用新一代高通量测序技术Illumina HiSeq 2000对鸟巢蕨转录组（Asplenium nidus）进行测序，共获得29 254 595个序列读取片段（reads），包含了5 908 586 517个碱基序列（bp）信息。对reads进行序列组装，共获得42 907个单基因簇（Unigene），平均长度936 bp，序列信息达到了40.16 Mb。数据库中的序列同源性比较表明，24 993个Unigene与其他物种的已知基因具有不同程度的同源性。鸟巢蕨转录组中的Unigene根据GO功能大致可分为细胞组分、分子功能和生物学过程3大类51个分支，其中有大量的Unigene与代谢进程、结合活性、催化活性和细胞进程相关。将Unigene与COG数据库进行比对，根据其功能大致可分为24类。KEGG数据库作为参考，依据代谢途径可将Unigene定位到116个代谢途径分支。SSR位点查找发现，从42 907个Unigene中共找到6 067个SSR位点。SSR不同重复基序类型中，出现频率最高的为AG/CT，其次是AC/GT、A/T和AGG/CCT。针对这些序列，设计了20对引物进行了扩增效率和多态性检测，其中7对引物在不同蕨类材料中表现出多态性。
JIA X P, SUN X B, DENG Y M, LIANG L J, YE X Q . Sequencing and analysis of the transcriptome of Asplenium nidus.
Acta Horticulturae Sinica, 2014,41(11):2329-2341. (in Chinese)
URLMagsci [本文引用: 1]
采用新一代高通量测序技术Illumina HiSeq 2000对鸟巢蕨转录组（Asplenium nidus）进行测序，共获得29 254 595个序列读取片段（reads），包含了5 908 586 517个碱基序列（bp）信息。对reads进行序列组装，共获得42 907个单基因簇（Unigene），平均长度936 bp，序列信息达到了40.16 Mb。数据库中的序列同源性比较表明，24 993个Unigene与其他物种的已知基因具有不同程度的同源性。鸟巢蕨转录组中的Unigene根据GO功能大致可分为细胞组分、分子功能和生物学过程3大类51个分支，其中有大量的Unigene与代谢进程、结合活性、催化活性和细胞进程相关。将Unigene与COG数据库进行比对，根据其功能大致可分为24类。KEGG数据库作为参考，依据代谢途径可将Unigene定位到116个代谢途径分支。SSR位点查找发现，从42 907个Unigene中共找到6 067个SSR位点。SSR不同重复基序类型中，出现频率最高的为AG/CT，其次是AC/GT、A/T和AGG/CCT。针对这些序列，设计了20对引物进行了扩增效率和多态性检测，其中7对引物在不同蕨类材料中表现出多态性。

[36] 张雪, 唐铭浩, 陈蒙, 李春艳, 刘海峰 . 山葡萄不同着色时期果皮转录组测序分析
果树学报, 2017,34(7):781-789.
DOI:10.13925/j.cnki.gsxb.20160404URL [本文引用: 1]
【目的】探究山葡萄果皮转色过程中相关基因的表达。【方法】以不同成熟期的山葡萄果皮为材料,采用高通量测序技术进行转录组分析,探讨其转色过程中花色苷含量及其相关基因的变化。【结果】经过测序得到3个时期的clean reads分别为：转色前20 157 930条,50%着色期16 197 432条,完熟期16 410 824条;GC含量分别为48.41%、48.04%和49.54%;各时期的Q30大于等于94.73%。通过与参考基因组进行序列比对,各样品Read与参考基因组的比对效率为60.57%-67.77%,并且唯一比对位置的数量均超过58.99%,比对到基因组上的序列绝大部分分布在外显子区,均为96.3%以上。本试验将3个样品中转色前分别与50%成熟期和完熟期进行了基因差异表达统计分析,结果显示COG功能注释分别为1 305、1 602个。【结论】明确了山葡萄果皮转色过程中相关基因的变化,为进一步大量挖掘山葡萄果皮成熟过程中重要表达基因奠定一定的基础。
ZHANG X, TANG M H, CHEN M, LI C Y, LIU H F . Sequencing analysis of transcriptome of Vitis amurensis during different periods of coloration.
Journal of Fruit Science, 2017,34(7):781-789. (in Chinese)
DOI:10.13925/j.cnki.gsxb.20160404URL [本文引用: 1]
【目的】探究山葡萄果皮转色过程中相关基因的表达。【方法】以不同成熟期的山葡萄果皮为材料,采用高通量测序技术进行转录组分析,探讨其转色过程中花色苷含量及其相关基因的变化。【结果】经过测序得到3个时期的clean reads分别为：转色前20 157 930条,50%着色期16 197 432条,完熟期16 410 824条;GC含量分别为48.41%、48.04%和49.54%;各时期的Q30大于等于94.73%。通过与参考基因组进行序列比对,各样品Read与参考基因组的比对效率为60.57%-67.77%,并且唯一比对位置的数量均超过58.99%,比对到基因组上的序列绝大部分分布在外显子区,均为96.3%以上。本试验将3个样品中转色前分别与50%成熟期和完熟期进行了基因差异表达统计分析,结果显示COG功能注释分别为1 305、1 602个。【结论】明确了山葡萄果皮转色过程中相关基因的变化,为进一步大量挖掘山葡萄果皮成熟过程中重要表达基因奠定一定的基础。

[37] 魏利斌, 苗红梅, 张海洋 . 芝麻发育转录组分析
中国农业科学, 2012,45(7):1246-1256.
DOI:10.3864/j.issn.0578-1752.2012.07.002URLMagsci [本文引用: 1]
【目的】系统了解芝麻发育及种子形成转录组特征，丰富芝麻转录组数据信息。【方法】选用6份芝麻样品（5个不同芝麻品种的完整植株、1份不同发育阶段的芝麻籽粒），构建转录组测序文库并进行Illumina RNA-seq双端测序及生物信息学分析。【结果】共获得原始数据12.69 Gb，有效数据8.80 Gb。通过de novo拼接获得了长度大于100 bp的转录物26 837条（<a href="http://www">http://www</a>. ncbi.nlm.nih.gov/genbank/TSA.html，登录号：JP631635&mdash;JP668414）；转录物总长度18.35 Mb，平均长度683 bp，N50长度1 006 bp。转录物注释结果显示，25 331条转录物序列具有同源比对信息；1 506条转录物序列无匹配（no hits）序列信息，可能为芝麻特有的基因序列。采用COG、GO功能分类工具可将已注释转录物序列划分为24个或42个功能类别，共涉及物质及能量代谢、信号传导、转录调控及防卫反应等诸多生理生化过程。通过比较不同材料间转录物序列及表达水平，初步确定1 277条序列在种子形成过程中表达量下调10倍以上，990条序列仅在芝麻植株中表达而未在籽粒中表达；660条序列在种子形成过程中表达量上调10倍以上，296条序列可能与种子形成特异相关。【结论】利用高通量测序技术对芝麻野生种和不同栽培种现蕾期植株以及种子形成过程的转录组进行研究，揭示了芝麻发育转录组的整体表达特征，在得到大量芝麻转录组unigene序列的同时，获得了一批在芝麻生长发育及籽粒形成过程中有重要功能的基因序列。为深入开展芝麻生长发育、籽粒发育相关基因功能及调控以及芝麻分子标记开发等研究提供了丰富的数据资源。
WEI L B, MIAO H M, ZHANG H Y . Transcriptomic analysis of sesame development
Scientia Agricultura Sinica, 2012,45(7):1246-1256. (in Chinese)
DOI:10.3864/j.issn.0578-1752.2012.07.002URLMagsci [本文引用: 1]
【目的】系统了解芝麻发育及种子形成转录组特征，丰富芝麻转录组数据信息。【方法】选用6份芝麻样品（5个不同芝麻品种的完整植株、1份不同发育阶段的芝麻籽粒），构建转录组测序文库并进行Illumina RNA-seq双端测序及生物信息学分析。【结果】共获得原始数据12.69 Gb，有效数据8.80 Gb。通过de novo拼接获得了长度大于100 bp的转录物26 837条（<a href="http://www">http://www</a>. ncbi.nlm.nih.gov/genbank/TSA.html，登录号：JP631635&mdash;JP668414）；转录物总长度18.35 Mb，平均长度683 bp，N50长度1 006 bp。转录物注释结果显示，25 331条转录物序列具有同源比对信息；1 506条转录物序列无匹配（no hits）序列信息，可能为芝麻特有的基因序列。采用COG、GO功能分类工具可将已注释转录物序列划分为24个或42个功能类别，共涉及物质及能量代谢、信号传导、转录调控及防卫反应等诸多生理生化过程。通过比较不同材料间转录物序列及表达水平，初步确定1 277条序列在种子形成过程中表达量下调10倍以上，990条序列仅在芝麻植株中表达而未在籽粒中表达；660条序列在种子形成过程中表达量上调10倍以上，296条序列可能与种子形成特异相关。【结论】利用高通量测序技术对芝麻野生种和不同栽培种现蕾期植株以及种子形成过程的转录组进行研究，揭示了芝麻发育转录组的整体表达特征，在得到大量芝麻转录组unigene序列的同时，获得了一批在芝麻生长发育及籽粒形成过程中有重要功能的基因序列。为深入开展芝麻生长发育、籽粒发育相关基因功能及调控以及芝麻分子标记开发等研究提供了丰富的数据资源。

[38] PROUD C G . Phosphorylation and signal transduction pathways in translational control
Cold Spring Harb Perspect Biology, 2018. doi: 10.1101/cshperspect.a033050.
DOI:10.1101/cshperspect.a033050URL [本文引用: 1]

[39] IRWIN D M, TAN H . Evolution of glucose utilization: Glucokinase and glucokinase regulator protein
Molecular Phylogenetics & Evolution, 2014,70(1):195-203.
DOI:10.1016/j.ympev.2013.09.016URLPMID:3897444 [本文引用: 1]
Glucose is an essential nutrient that must be distributed throughout the body to provide energy to sustain physiological functions. Glucose is delivered to distant tissues via be blood stream, and complex systems have evolved to maintain the levels of glucose within a narrow physiological range. Phosphorylation of glucose, by glucokinase, is an essential component of glucose homeostasis, both from the regulatory and metabolic point-of-view. Here we review the evolution of glucose utilization from the perspective of glucokinase. We discuss the origin of glucokinase, its evolution within the hexokinase gene family, and the evolution of its interacting regulatory partner, glucokinase regulatory protein (GCKR). Evolution of the structure and sequence of both glucokinase and GCKR have been necessary to optimize glucokinase in its role in glucose metabolism.

[40] GUTIERREZNOGUES A, GARCIAHERRERO C M, ORIOLA J, VINCENT O, NAVAS M A . Functional characterization of MODY2 mutations in the nuclear export signal of glucokinase
Biochimica et Biophysica Acta (BBA)-Molecular Bassis of Disease, 2018,1864(7):2385-2394.
DOI:10.1016/j.bbadis.2018.04.020URLPMID:29704611 [本文引用: 1]
In conclusion, our results have provided functional and structural insights regarding the GCK-NES and contributed to a better knowledge of the molecular mechanisms involved in the nucleo-cytoplasmic shuttling of glucokinase. Impairment of this regulatory mechanism by some MODY2 mutations might contribute to the hyperglycaemia in the patients.

[41] KAWAI S, MUKAI T, MORI S, MIKAMI B, MURATA K . Hypothesis: structures, evolution, and ancestor of glucose kinases in the hexokinase family
Journal of Bioscience & Bioengineering, 2005,99(4):320-330.
DOI:10.1263/jbb.99.320URLPMID:16233797 [本文引用: 1]
Glucose kinase, which we tentatively use in this review, represents the enzymes catalyzing the phosphorylation of glucose and other hexoses by means of phosphoryl donors (ATP, ADP, and inorganic polyphosphate [poly(P)]). Except for glucose kinases utilizing ADP, all other glucose kinases belong to the hexokinase (HK) family and are classified into three groups based on primary structural information, i.e., groups HK, A, and B. The structural and evolutionary relationships of glucose kinases belonging to the above three groups have been controversial due to the lack of tertiary structural information on those in groups A and B. However, recent studies on the tertiary structures of poly(P)/ATP-glucomannokinase (GMK: a glucose kinase in group B) from Arthrobacter sp. strain KM and glucokinase (GK) (ecoGK: a glucose kinase in group A) from Escherichia coli have shed light on this problem. A comparison of the tertiary structures of GMK and ecoGK with those of glucose kinases in group HK demonstrated that both GMK and ecoGK are structurally homologous with glucose kinases in group HK, and that glucose kinases belonging to groups HK, A, and B in the HK family evolved divergently from a common ancestor. Based on the simple structure of GMK compared to those of ecoGK and glucose kinases in group HK, and the putative poly(P)-binding site in GMK, we propose that the ancestor of glucose kinases in the HK family was similar to GMK and used poly(P). We also discuss the ancestor and evolutionary process of ROK proteins, whose primary structures are homologous with those of glucose kinases in group B, in connection with the ancestor and evolutionary process of glucose kinases in the HK family.

[42] MOORE B, SHEEN J . Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling.
Science, 2003,300(5617):332-336.
DOI:10.1126/science.1080585URLPMID:12690200 [本文引用: 1]
Glucose modulates many vital processes in photosynthetic plants. Analyses of Arabidopsis glucose insensitive2 (gin2) mutants define the physiological functions of a specific hexokinase (HXK1) in the plant glucose-signaling network. HXK1 coordinates intrinsic signals with extrinsic light intensity. HXK1 mutants lacking catalytic activity still support various signaling functions in gene expression, cell proliferation, root and inflorescence growth, and leaf expansion and senescence, thus demonstrating the uncoupling of glucose signaling from glucose metabolism. The gin2 mutants are also insensitive to auxin and hypersensitive to cytokinin. Plants use HXK as a glucose sensor to interrelate nutrient, light, and hormone signaling networks for controlling growth and development in response to the changing environment.

[43] CHO Y H, YOO S D, SHEEN J . Regulatory functions of nuclear hexokinase1 complex in glucose signaling
Cell, 2007,127(2):579-589.
DOI:10.4161/psb.2.2.3894URLPMID:17081979 [本文引用: 1]
Arabidopsis hexokinase1 (HXK1) is a glucose sensor that regulates gene expression and plant growth and development. We have previously developed a high glucose (6%) assay based on the seedling developmental arrest to isolate and characterize the glucose-insensitive (gin) mutants. The analysis of gin2 as a null HXK1 mutant has revealed that the regulatory functions of HXK1 are distinct from its conventional role in glycolysis. In the Nov 3rd issue of Cell, we presented a new insight into the mechanism of HXK1-dependent glucose signaling. By combining proteomic and binary interaction screens, we discover two HXK1 unconventional partners (HUPs). HXK1 and HUPs form a core complex in the nucleus and directly regulate glucose-responsive gene expression and plant growth. As the 6% glucose assay is complicated by additional osmotic stress and nitrate signals, we have tested the gin2 and hup mutants using the 2% glucose assay. We believe that the new and more physiological glucose assay could help us better dissect the molecular mechanisms that link glucose regulation to diverse plant signaling pathways. Further functional analysis of gin mutants and the components in the novel nuclear HXK1 complex will provide more comprehensive mechanistic understanding of glucose sensing and signaling in plants.

[44] KELLY G, SADE N, DORON-FAIGENBOIM A, LERNER S, SHATIL-COHEN A, YESELSON Y, EGBARIA A, KOTTAPALLI J, SCHAFFER A A, MOSHELION M, GRANOT D . Sugar and hexokinase suppress expression of PIP aquaporins and reduce leaf hydraulics that preserves leaf water potential
The Plant Journal, 2017,91:325-339.
DOI:10.1111/tpj.13568URLPMID:28390076 [本文引用: 1]
Abstract Sugars affect central aspects of plant physiology, including photosynthesis, stomatal behavior and the loss of water through the stomata. Yet, the potential effects of sugars on plant aquaporins (AQPs) and water conductance have not been examined. We used database and transcriptional analyses, as well as cellular and whole-plant functional techniques to examine the link between sugar-related genes and AQPs. Database analyses revealed a high level of correlation between the expression of AQPs and that of sugar-related genes, including the Arabidopsis hexokinases 1 (AtHXK1). Increased expression of AtHXK1, as well as the addition of its primary substrate, glucose (Glc), repressed the expression of 10 AQPs from the plasma membrane-intrinsic proteins (PIP) subfamily (PIP-AQPs) and induced the expression of two stress-related PIP-AQPs. The osmotic water permeability of mesophyll protoplasts of AtHXK1-expressing plants and the leaf hydraulic conductance of those plants were significantly reduced, in line with the decreased expression of PIP-AQPs. Conversely, hxk1 mutants demonstrated a higher level of hydraulic conductance, with increased water potential in their leaves. In addition, the presence of Glc reduced leaf water potential, as compared with an osmotic control, indicating that Glc reduces the movement of water from the xylem into the mesophyll. The production of sugars entails a significant loss of water and these results suggest that sugars and AtHXK1 affect the expression of AQP genes and reduce leaf water conductance, to coordinate sugar levels with the loss of water through transpiration. 2017 The Authors The Plant Journal 2017 John Wiley & Sons Ltd.

[45] HU R B, ZHU Y F, SHEN G X, ZHANG H . Overexpression of the PP2A-C5gene confers increased salt tolerance in Arabidopsis thaliana.
Plant Signaling & Behavior, 2017,12(2):e1276687.
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[46] RAZAVIZADEH R, SHOJAIE B, KOMATSU S . Characterization ofPP2A-A3, mRNA expression and growth patterns in Arabidopsis thaliana, under drought stress and abscisic acid
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[47] LIU L L, REN H M, CHEN L Q, WANG Y, WU W H . A protein kinase, calcineurin B-like protein-interacting protein Kinase9, interacts with calcium sensor calcineurin B-like Protein3 and regulates potassium homeostasis under low-potassium stress in Arabidopsis.
Plant Physiology, 2013,161(1):266-277.
DOI:10.1104/pp.112.206896URLPMID:23109687 [本文引用: 1]
Potassium (K+) is an essential macronutrient for plant growth and development. Previous studies have demonstrated that Calcineurin B-Like Protein1 (CBL1) or CBL9 and CBL-Interacting Protein Kinase23 (CIPK23) regulate K+ uptake in Arabidopsis (Arabidopsis thaliana) roots by modulating K+ channel Arabidopsis K+ Transporter1. In this study, we show that the protein kinase CIPK9 interacts with the calcium sensor CBL3 and plays crucial roles in K+ homeostasis under low-K+ stress in Arabidopsis. Arabidopsis wild-type plants showed leaf chlorotic symptoms when grown for 10 d on low-K+ (100 mu M) medium. Here, we show that plants lacking CIPK9 displayed a tolerant phenotype to low-K+ stress, which still maintained green leaves when the wild-type plants showed typical K+-deficient symptoms. Overexpressing lines of CIPK9 resulted in a low-K+-sensitive phenotype compared with wild-type plants. Furthermore, CBL2 and CBL3 were identified as upstream regulators of CIPK9. Both CBL2- and CBL3-overexpressing lines displayed similar low-K+-sensitive phenotypes and K+ contents to CIPK9-overexpressing lines. However, only cbl3 mutant plants, but not cbl2 mutant plants, showed the low-K+-tolerant phenotype similar to cipk9 mutants. Taken together, these results demonstrate that CIPK9 and CBL3 work together and function in K+ homeostasis under low-K+ stress in Arabidopsis.

[48] COLCOMBET J, HIRT H . Arabidopsis MAPKs: A complex signalling network involved in multiple biological processes
Biochemical Journal, 2008,413(2):217-226.
DOI:10.1042/BJ20080625URLPMID:18570633 [本文引用: 1]
Abstract Many changes in environmental conditions and hormones are mediated by MAPK (mitogen-activated protein kinase) cascades in all eukaryotes, including plants. Studies of MAPK pathways in genetic model organisms are especially informative in revealing the molecular mechanisms by means of which MAPK cascades are controlled and modulate cellular processes. The present review highlights recent insights into MAPK-based signalling in Arabidopsis thaliana (thale cress), revealing the complexity and future challenges to understanding signal-transduction networks on a global scale.

[49] ANDREASSON E, ELLIS B . Convergence and specificity in the Arabidopsis MAPK nexus.
Trends in Plant Science, 2010,15(2):106-113.
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http://linkinghub.elsevier.com/retrieve/pii/S1360138509002994

[50] SINGHA H S, CHAKRABORTY S, DEKA H . Stress induced MAPK genes show distinct pattern of codon usage in Arabidopsis thaliana, Glycine max and Oryza sativa.
Bioinformation, 2014,10(7):436-442.
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[51] WOLANIN P M, THOMASON P A, STOCE J B . Histidine protein kinases: Key signal transducers outside the animal kingdom
Genome Biology, 2002, 3(10): reviews3013. 1.
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[52] LIM W A, PAWSON T . Phosphotyrosine signaling: Evolving a new cellular communication system
Cell, 2010,142:661-667.
DOI:10.1016/j.cell.2010.08.023URLPMID:20813250 [本文引用: 1]
Tyrosine phosphorylation controls many cellular functions. Yet the three-part toolkit that regulates phosphotyrosine signaling—tyrosine kinases, phosphotyrosine phosphatases, and Src Homology 2 (SH2) domains—is a relatively new innovation. Genomic analyses reveal how this revolutionary signaling system may have originated and why it rapidly became critical to metazoans.

[53] LIN W W, LI B, LU D P, CHEN S X, ZHU N, HE P, SHAN L B . Tyrosine phosphorylation of protein kinase complex BAK1/BIK1 mediates Arabidopsis innate immunity.
Proceedings of the National Academy of Sciences of the United States of America, 2014,111(9):3632-3637.
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[54] REDDY M M, RAJASEKHARAN R . Serine/threonine/tyrosine protein kinase from Arabidopsis thaliana is dependent on serine residues for its activity.
Archives of Biochemistry & Biophysics, 2007,460(1):122-128.
DOI:10.1016/j.abb.2007.01.003URLPMID:17291444 [本文引用: 1]
Genome-wide analysis of Arabidopsis thaliana with tyrosine kinase motif from animals predicted that tyrosine phosphorylation could be brought about only by dual-specificity protein kinases in plants. However, their regulation is poorly understood. In the present study, we have investigated the role of serines required for the activity of Arabidopsis thaliana serine/threonine/tyrosine protein kinase (AtSTYPK). There are eight serines in the kinase catalytic domain. The role of each serine residue was studied individually by substituting them with alanine. Serines at positions 215, 259, 269 and 315 are required for the kinase activity both in terms of auto and substrate phosphorylations of myelin basic protein. The mutant S265A showed slight increase in auto and substrate phosphorylations. Other serines at positions 165, 181 and 360 did not show any change in the phosphorylation status as compared to wild-type. In conclusion, these results suggest the importance of serine residues required for dual-specificity protein kinase activity.