类钙调磷酸酶B亚基蛋白GhCBL3-A01调控棉花黄萎病抗性的功能分析  

Functional analysis of cotton calcineurin B-like protein GhCBL3-A01 in regulating the resistance to Verticillium wilt

作　　者：高升旗[1,2] 邵武奎 赵准 邵盘霞 胡文冉 黄全生[1,2] Gao Shengqi;Shao Wukui;Zhao Zhun;Shao Panxia;Hu Wenran;Huang Quansheng(Institute of Nuclear Technology and Biotechnology,Xinjiang Academy of Agricultural Sciences/Xinjiang Key Laboratory of Crop Biotechnology,Urumqi 830091,China;National Key Laboratory of Crop Genetic Improvement and Germplasm Innovation in Arid Desert Areas(Preparation),Urumqi 830091,China)

机构地区：[1]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 [2]省部共建干旱荒漠区作物遗传改良与种质创新国家重点实验室(筹备),乌鲁木齐830091

出　　处：《棉花学报》2023年第6期447-458,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】解析类钙调磷酸酶B亚基蛋白(calcium B-like protein,CBL)基因GhCBL3-A01在棉花抗黄萎病反应中的功能,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GhCBL3-A01基因的同源序列,进行生物信息学分析。采用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测大丽轮枝菌(Verticillium dahliae)、茉莉酸甲酯(methyl jasminate,MeJA)和H2O2处理的棉株中GhCBL3-A01基因的表达量变化。利用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术研究GhCBL3-A01在棉花抗黄萎病中的功能。通过检测活性氧积累以及相关基因的表达量,初步分析GhCBL3-A01的作用机制。【结果】陆地棉中GhCBL3-A01及其3个同源蛋白均含有3个CBL家族典型的EF-hand结构域。大丽轮枝菌、MeJA和H2O2处理后,GhCBL3-A01的表达量显著升高。VIGS沉默GhCBL3-A01导致病株率和病情指数显著降低,茎秆维管束褐变程度减轻,有病菌繁殖的茎段数量明显减少,增强了棉株对黄萎病的抗性。GhCBL3-A01基因沉默棉株叶片中活性氧积累增多,防御相关基因PR1、NPR1、PR4和PDF1.2以及茉莉酸信号通路关键基因AOS1、OPR3、MYC2和LOX2的表达量增加。【结论】GhCBL3-A01通过调控防御相关基因、茉莉酸信号通路基因的表达和活性氧积累负调控棉花对黄萎病的抗性,是提高棉花黄萎病抗性的候选基因。[Objective]This research aims to investigate the role of calcium B-like protein(CBL)gene GhCBL3-A01 in resistance to Verticillium wilt in cotton,so as to provide genetic resource for cotton disease resistance breeding.[Methods]The homologous sequences of GhCBL3-A01 gene were obtained from cotton genomic database for bioinformatic analysis.The relative expression levels of GhCBL3-A01 under Verticillium dahliae,methyl jasminate(MeJA)and H2O2 treatments were detected by quantitative real-time polymerase chain reaction(qRT-PCR).The function of GhCBL3-A01 in cotton resistance to Verticillium wilt was analysed by virus-induced gene silencing(VIGS)technique.The functional mechanisms of GhCBL3-A01 were analyzed by detecting the accumulation of reactive oxygen species(ROS)and the expression levels of related genes.[Results]GhCBL3-A01 and its three homologous proteins in upland cotton all contain three typical EF-hand domains of the CBL family.The expression level of GhCBL3-A01 was significantly increased under the treatment of Verticillium dahliae,MeJA,and H2O2.Silencing GhCBL3-A01 by VIGS resulted in a significant decrease in rate of diseased plants and disease index,a reduction in the browning degree of vascular bundles,and a decline in the number of hyphae in stem segments,which enhanced the resistance of cotton plants to Verticillium wilt.The expression levels of disease resistance-related genes(PR1,NPR1,PR4,and PDF1.2)and jasmonic acid(JA)signaling pathway genes(AOS1,OPR3,MYC2,and LOX2)were increased,and ROS accumulation was enriched in GhCBL3-A01 silenced cotton plants.[Conclusion]GhCBL3-A01 negatively regulates the resistance of cotton to Verticillium wilt by regulating the expression levels of defense-related genes and JA signaling pathway genes as well as the accumulation of ROS,which is a candidate gene for improving the resistance of cotton to Verticillium wilt.

关 键 词：棉花 黄萎病 类钙调磷酸酶B亚基蛋白 GhCBL3-A01 抗病基因 病毒诱导的基因沉默 

分 类 号：S435.621[农业科学—农业昆虫与害虫防治]