陆地棉钙依赖蛋白激酶GhCDPK28-A10参与抗黄萎病的功能分析  

Functional analysis of cotton calcium-dependent protein kinase GhCDPK28-A10 involved in resistance to Verticillium wilt

作　　者：邵武奎 赵准 胡文冉[2] 郝晓燕[2] 高升旗[2] 李建平[2] 陈果[2] 刘晓东[1] 黄全生[2] Shao Wukui;Zhao Zhun;Hu Wenran;Hao Xiaoyan;Gao Shengqi;Li Jianping;Chen Guo;Liu Xiaodong;Huang Quansheng(College of Life Sciences,Xinjiang Agricultural University,Urumq 830052,China;Institute of Nuclear Technology and Biotechnology,Xinjiang Academy of Agricultural Sciences/Xinjiang Key Laboratory of Crop Biotechnology,Urumqi 830091,China)

机构地区：[1]新疆农业大学生命科学学院,乌鲁木齐830052 [2]新疆农业科学院核技术生物技术研究所/新疆农作物生物技术重点实验室,乌鲁木齐830091

出　　处：《棉花学报》2023年第1期17-28,共12页Cotton Science

基　　金：国家自然科学基金-新疆联合基金(U1703114);中央引导地方科技发展专项资金项目(ZYYD2022B07)。

摘　　要：【目的】解析GhCDPK28-A10在棉花黄萎病反应中的作用机制,并为棉花抗病育种提供基因资源。【方法】从棉花基因组数据库获得Gh CDPK28-A10基因同源序列,进行生物信息学分析。采用实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,qRT-PCR)方法检测在经大丽轮枝菌(Verticillium dahliae)、茉莉酸甲酯(methyl jasminate,MeJA)、水杨酸(salicylic acid,SA)或H_(2)O_(2)处理的棉株中GhCDPK28-A10基因的表达量变化。通过病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术验证GhCDPK28-A10在棉花抗黄萎病中的作用。【结果】进化树和基因结构分析表明,陆地棉中GhCDPK28-A10的6个同源蛋白具有相似数量的基序和CDPK家族典型的丝氨酸/苏氨酸蛋白激酶结构域;启动子区域的序列分析发现GhCDPK28-A10包含响应茉莉酸(jasmonic acid,JA)的顺式作用元件。组织特异性表达分析表明GhCDPK28-A10在根、茎和真叶中均表达,且在真叶中表达水平最高。在大丽轮枝菌、MeJA和H_(2)O_(2)胁迫下,GhCDPK28-A10的表达量显著升高。VIGS沉默Gh CDPK28-A10的棉株中抗病相关基因PR1、NPR1、PR4表达增强;JA合成负调控基因JAZ1表达量降低,说明随着CDPK28-A10表达水平的降低,JAZ1对JA合成的抑制作用减弱,活性氧富集,进而增强棉株的黄萎病抗性。【结论】GhCDPK28-A10通过调控防御相关基因的表达,负向调控棉花对黄萎病的抗性反应,是提高棉花黄萎病抗性的候选基因。[Objective]This study aimed to analyze the mechanism of GhCDPK28-A10 in Verticillium wilt response in cotton,and to provide genetic resources for disease resistance breeding in cotton.[Methods]The homologous sequence of the GhCDPK28-A10 gene was obtained from the cotton genome database and bioinformatic analysis was performed.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the effect of GhCDPK28-A10 gene on the resistance to Verticillium dahliae,methyl jasminate(MeJA),salicylic acid(SA),or H_(2)O_(2)-treated cotton plants.The role of GhCDPK28-A10 in cotton resistance to Verticillium wilt was verified by virus-induced gene silencing(VIGS)technique.[Results]Phylogenetic tree and gene structure analysis showed that the six GhCDPK28-A10 homologs in upland cotton shared similar number of motifs and contained the typical serine/threonine protein kinase structural domain of the CDPK family.Analysis of the promoter sequence revealed that GhCDPK28-A10 contains a cis-acting element in response to jasmonic acid(JA).Tissue-specific expression analysis showed that GhCDPK28-A10 was expressed in root,stem and true leaves.And the highest expression level was found in true leaves.The expression of GhCDPK28-A10 was significantly increased under stress induction by V.dahliae,MeJA, and H_(2)O_(2). The expression of disease resistance-related genes PR1, NPR1, and PR4 was enhanced and the negative regulator of JA synthesis gene JAZ1 was decreased in GhCDPK28-A10 silenced cotton plants. This indicated that the JAZ1 regulated JA synthesis was inhibited and reactive oxygen species was enriched when the CDPK28-A10 expression level was decreased, which in turn enhanced the resistance to Verticillium wilt of cotton plants. [Conclusion] GhCDPK28-A10 negatively regulates the resistance response of cotton to Verticillium wilt by regulating the expression of defense-related genes, and is a candidate gene for improving the resistance of cotton to Verticillium wilt.

关 键 词：棉花 黄萎病 钙依赖性蛋白激酶 抗病基因 病毒诱导的基因沉默 

分 类 号：S435.621.2[农业科学—农业昆虫与害虫防治]