陆地棉CRK基因家族的鉴定及其表达分析

doi:10.3864/j.issn.0578-1752.2018.13.002

摘要/Abstract

摘要： 【目的】富含半胱氨酸类受体激酶（CRK）是植物中最大的类受体激酶家族之一，在植物生长发育、激素信号传导和抗逆境胁迫中发挥重要作用。从全基因组水平鉴定陆地棉CRK基因家族并进行生物信息学和表达模式分析，为研究和利用陆地棉CRK基因家族奠定基础。【方法】从Pfam数据库下载stress-antifung结构域氨基酸序列，应用BLASTp程序搜索棉花基因组数据库，鉴定棉花CRK基因家族；利用Compute pI/Mw tool、SignalP、TMHMM Server V2.0、WoLF POSRT等在线工具预测陆地棉CRK家族蛋白的分子量、信号肽、跨膜结构域和亚细胞定位等；用ClustalX1.8软件对棉花和拟南芥CRK蛋白质进行氨基酸序列比对，MEGA5.0分析棉花和拟南芥CRK蛋白的系统进化关系；使用TBtools制作陆地棉CRK基因家族的染色体定位、基因结构和蛋白质结构域示意图；应用植物顺式调控元件数据库PlantCARE分析棉花启动子序列；通过植物磷酸化位点数据库PlantPhos预测陆地棉CRK家族蛋白的磷酸化位点；从NCBI数据库下载RNA-Seq数据，利用转录组定量工具Kallisto计算TPM值，通过在线工具Morpheus绘制陆地棉CRK家族基因表达热图。【结果】陆地棉基因组中有70个CRK基因，分布于14条染色体，其中52个基因（74.3%）集中串联成簇分布于A6/D6、A9/D9、A10/D10染色体，且在A/D染色体组之间呈现高度共线性关系。编码302—901个氨基酸，58个蛋白质（82.9%）具有跨膜结构域，主要定位于叶绿体、质膜和胞外。磷酸化位点预测结果表明，陆地棉和拟南芥CRK有5个相同的磷酸化位点基序，包括3种丝氨酸磷酸化位点基序和2种苏氨酸磷酸化位点基序。65个陆地棉CRK基因的启动子区（92.9%）至少含有一种逆境激素响应元件，69个基因启动子区（98.6%）至少含有一种生物或非生物胁迫响应元件。根据RNA-Seq数据分析结果，陆地棉CRK基因可分为3种不同的组织表达特征类型；盐、干旱、冷、热胁迫以及接种大丽轮枝菌均可以导致部分陆地棉CRK基因表达水平的改变。GhCRK25在根、茎、叶和胚珠中优势表达，在纤维中几乎不表达，ABA、GA3、SA、PEG-6000、氯化钠和大丽轮枝菌Vd991处理均能刺激GhCRK25迅速上调表达。应用病毒诱导的基因沉默技术（VIGS）沉默GhCRK25可导致棉花对大丽轮枝菌Vd991更为敏感。【结论】陆地棉CRK基因家族有70个成员，具有保守的基因结构和功能结构域，多样化的组织表达特征，大多数基因受激素和逆境调控。

关键词: 陆地棉, 富含半胱氨酸蛋白激酶, 基因家族, 抗逆性, GhCRK25

Abstract: 【Objective】 The cysteine-rich receptor kinase (CRK) is one of the largest family of receptor-like kinases in plants, and plays important roles in plant growth and development, hormone signal transduction and stress tolerance. So far, few cotton CRK genes have been reported. In silico identification, bioinformatics and expression analysis of CRK family genes in upland cotton on whole genome level lays the foundation for in-depth study and utilization of CRK family genes in cotton. 【Method】 The conserved stress-antifung domain sequence downloaded from the Pfam database was used as query sequence to search the Gossypium hirsutum (cv. TM-1) genome database to identify cotton CRKs by using BLASTp program; the theoretical isoelectric point and molecular weight, signal peptide, transmembrane domain, subcellular localization of cotton CRK proteins was predicted applying Compute pI/Mw, SignalP, TMHMM Server V2.0, WoLF POSRT online program respectively; amino acid sequence alignment of CRK proteins in cotton and Arabidopsis thaliana was performed using ClustalX1.8 software. phylogenetic relationships of cotton and Arabidopsis CRK proteins were analyzed with MEGA5.0; the chromosome location, gene structure and conserved domain were visualized with TBtools. The promoter sequences of cotton CRK genes were In silico analyzed by searching PlantCARE database; phosphorylation sites were predicted with PlantPhos. RNA-Seq data were download the from the NCBI database, TPM values were calculated using transcriptome quantification tool Kallisto, the heatmap of CRK gene expression was drawn with online tools Morpheus. 【Result】 There are 70 CRK genes in upland cotton genome distributed on 14 chromosomes, 52 genes (74.3% of the total) are intensively distributed in clusters on A6/D6, A9/D9 and A10/D10 chromosomes characterized by collinear relationships between A/D chromosomes. These CRK genes encode proteins containing 302-901 amino acids, 58 proteins (82.9%) have a transmembrane domain, mainly located in the chloroplasts, plasmalemma and extracellular. Phosphorylation site prediction results showed that cotton and Arabidopsis CRK share 5 consensus phosphorylation sites, including three serine phosphorylation motifs and two threonine phosphorylation motifs. The promoter regions of 65 cotton CRK genes (account for 92.9%) contain at least one stress hormone response element, and 69 (98.6%) genes contain at least one biotic or abiotic stress response element. RNA-Seq data analyses showed that the tissue expression patterns of CRKs could be divided into three types, and that the expression of some CRK genes were altered in response to salt, drought, cold, heat stress and inoculation with Verticillium dahliae Kleb. GhCRK25 was predominantly expressed in roots, stems, leaves, and ovules, but barely accumulated in fibers. ABA, GA3, SA, PEG-6000, NaCl, and Verticillium dahliae Vd991 can stimulate rapid up-regulation of GhCRK25 expression. GhCRK25-silenced cottonby using virus induced gene silencing technology (VIGS) showed increased susceptibility to Verticillium dahliae Vd991. 【Conclusion】 There are 70 members of CRK family gene in the upland cotton genome. They have conserved gene structure and functional domain, diverse tissue expression characteristics; most of cotton CRK genes are responsive to hormone and stress stimulus.

Key words: upland cotton, cysteine-rich protein kinase, gene family, stress tolerance, GhCRK25

张中起，王娇，靳炜，葛冬冬，刘康，吕芬妮，孙敬. 陆地棉CRK基因家族的鉴定及其表达分析 [J]. 中国农业科学, 2018, 51(13): 2442-2461.

ZHANG ZhongQi, WANG Jiao, JIN Wei, GE DongDong, LIU Kang, Lü FenNi, SUN Jing. Identification and Expression Analysis of CRK Gene Family in Upland Cotton[J]. Scientia Agricultura Sinica, 2018, 51(13): 2442-2461.

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