摘要
【目的】探究ghr-miR394-GhFBX6模块在棉花黄萎病抗性中的功能,为棉花抗病育种提供候选基因。【方法】利用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)分析ghr-miR394a和GhFBX6的表达模式。在棉花中瞬时超表达ghr-miR394a,利用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术在棉花中分别瞬时沉默ghr-miR394a和GhFBX6,研究二者在抗黄萎病中的功能。利用5'端RNA连接酶介导的cDNA末端快速扩增(5'-RNA ligase-mediated rapid amplification of cDNA ends,5'RLM-RACE)技术并结合荧光素酶(luciferase,LUC)报告系统,分析ghr-miR394a对GhFBX6的调控作用。【结果】qRT-PCR表明,ghr-miR394a在棉花叶片中的表达量较高,其次为茎和根。与清水处理相比,大丽轮枝菌侵染可显著降低根中ghr-miR394a的表达量,显著提高GhFBX6的表达量。与对照植株相比,在棉花中瞬时沉默ghr-miR394a导致病情指数、病株率显著降低,茎段恢复培养后菌丝积累量较少,棉花对黄萎病的抗性增强;而瞬时过表达ghr-miR394a或瞬时沉默GhFBX6则会降低棉花对大丽轮枝菌的抗性,具体表型为二者的病情指数和病株率均显著升高,过表达ghr-miR394a的茎段恢复培养后菌丝积累量较多,GhFBX6沉默棉株茎秆的维管束褐变程度加重。5'RLM-RACE和LUC报告系统结果表明,ghr-miR394a在转录后水平抑制GhFBX6表达。【结论】ghr-miR394a可靶向抑制GhFBX6的表达,且ghr-miR394a和GhFBX6在棉花黄萎病抗性响应中分别发挥负调控和正调控功能。
[Objective]This research aimed to investigate the function of the ghr-miR394-GhFBX6 module in cotton resistance to Verticillium wilt and to provide candidate genes for cotton disease-resistant breeding.[Methods]The expression patterns of ghr-miR394a and GhFBX6 were analyzed using quantitative real-time polymerase chain reaction(qRT-PCR).Virus-induced gene silencing(VIGS)was employed to transiently silence ghr-miR394a and GhFBX6 individually,and in combination with the transient overexpression of ghr-miR394a in cotton,to investigate their roles in resistance to Verticillium wilt.The regulatory effect of ghr-miR394a on GhFBX6 was analyzed using the 5'-RNA ligase-mediated rapid amplification of cDNA ends(5'RLM-RACE)technique combined with a luciferase(LUC)reporter system.[Results]qRT-PCR analysis revealed that ghr-miR394a was highly expressed in cotton leaves,followed by stems and roots.Compared with the water treatment,inoculation with Verticillium dahliae significantly downregulated the expression level of ghr-miR394a in roots,while significantly upregulated the expression level of GhFBX6.Compared with control plants,transient silencing of ghr-miR394a in cotton resulted in a significantly reduced disease index and rate of diseased plants,and less fungal biomass accumulation in stem segments during recovery culture,indicating enhanced resistance to Verticillium wilt.In contrast,transient overexpression of ghr-miR394a or silencing of GhFBX6 led to decreased resistance to V.dahliae,as evidenced by a significantly higher disease index and rate of diseased plants,increased fungal biomass accumulation in stem segments of ghr-miR394a-overexpressing plants,and more severe browning of vascular bundles in GhFBX6-silenced plants.The results from 5'RLM-RACE and the LUC reporter system demonstrated that ghr-miR394a inhibits the expression of GhFBX6 at the post-transcriptional level.[Conclusion]ghr-miR394a targets and inhibits the expression of GhFBX6.ghr-miR394a and GhFBX6 play negative and positive regulatory roles,respectively,in the cotton response to Verticillium wilt.
作者
廖婵娟
李丹
赵文军
吴琪
尹秀娟
钟贵买
王智
胡广
翟俊峰
Liao Chanjuan;Li Dan;Zhao Wenjun;Wu Qi;Yin Xiujuan;Zhong Guimai;Wang Zhi;Hu Guang;Zhai Junfeng(Shaoyang Polytechnic,Shaoyang,Hunan 422004,China;Technical Innovation Center for Development and Industrial Application of Melon Germplasm Resources,Shaoyang,Hunan 422004,China;Chinese Academy of Quality and Inspection&Testing,Beijing 100176,China)
出处
《棉花学报》
北大核心
2025年第3期165-174,I0001,I0002,共12页
Cotton Science
基金
湖南省教育科学“十四五”规划课题(XJK23CZY054,XJK24QZY004)
湖南省社会科学成果评审委员会课题(XSP25YBC202)
重庆社科项目管理中心职业教育改革课题(SSPMC22145)。
