生物技术通报 ›› 2022, Vol. 38 ›› Issue (8): 127-134.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1519

• 研究报告 • 上一篇    下一篇

棉花黄萎病抗性相关基因GhTIFY9的克隆与功能分析

李秀青1(), 胡子曜2, 雷建峰1, 代培红2, 刘超2, 邓嘉辉2, 刘敏2, 孙玲1, 刘晓东2, 李月2()   

  1. 1.新疆农业大学农学院,乌鲁木齐 830052
    2.新疆农业大学生命科学学院,乌鲁木齐 830052
  • 收稿日期:2021-12-07 出版日期:2022-08-26 发布日期:2022-09-14
  • 作者简介:李秀青,男,硕士,研究方向:作物逆境分子生物学;E-mail: 549222451@qq.com
  • 基金资助:
    国家自然科学基金项目(32160494)

Cloning and Functional Analysis of Gene GhTIFY9 Related to Cotton Verticillium Wilt Resistance

LI Xiu-qing1(), HU Zi-yao2, LEI Jian-feng1, DAI Pei-hong2, LIU Chao2, DENG Jia-hui2, LIU Min2, SUN Ling1, LIU Xiao-dong2, LI Yue2()   

  1. 1. College of Agronomy,Xinjiang Agricultural University,Urumqi 830052
    2. College of Life Sciences,Xinjiang Agricultural University,Urumqi 830052
  • Received:2021-12-07 Published:2022-08-26 Online:2022-09-14

摘要:

初步探究陆地棉TIFY家族基因GhTIFY9在棉花黄萎病反应中的功能,为挖掘棉花抗病相关基因及棉花抗病育种奠定基础。通过转录组筛选、克隆获得一个TIFY家族基因GhTIFY9,利用生物信息学的方法分析该基因的理化性质,通过荧光定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)方法分析GhTIFY9在黄萎病诱导下的表达模式。同时构建该基因的VIGS载体,利用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术对其在棉花抗黄萎病中的功能进行了初步探究。结果表明,以陆地棉TM-1的cDNA为模板克隆获得GhTIFY9,其开放阅读框(ORF)为594 bp,编码一个含197个氨基酸的碱性亲水性蛋白,相对分子质量为21.65 kD,定位于细胞核,属于TIFY亚家族。RT-qPCR分析结果表明GhTIFY9响应黄萎病菌诱导,抑制该基因表达后沉默植株对黄萎病菌的敏感性显著增强。GhTIFY9是棉花抗黄萎病反应的一个正向调控因子。

关键词: 棉花黄萎病, GhTIFY9, 基因克隆, 病毒诱导的基因沉默(VIGS)

Abstract:

The function of TIFY family gene GhTIFY9 in cotton verticillium wilt response was preliminarily explored,which laid a foundation for mining cotton disease resistance related genes and cotton disease resistance breeding. A TIFY family gene GhTIFY9 was obtained by transcriptome screening and cloning. The physicochemical properties of the gene were analyzed by bioinformatics methods. The expression pattern of GhTIFY9 under verticillium wilt induction was analyzed by real-time quantitative polymerase chain reaction(RT-qPCR). Meanwhile,the VIGS vector of the gene was constructed,and its function in the cotton resistance to verticillium wilt was preliminarily explored by virus-induced gene silencing(VIGS)technology. GhTIFY9 was cloned using the cDNA of upland cotton TM-1 as the template. The open reading frame(ORF)of GhTIFY9 was 594 bp,encoding an alkaline hydrophilic protein containing 197 amino acids. The relative molecular weight was 21.65 kD,it was located in the nucleus and belonged to the TIFY subfamily. RT-qPCR analysis showed that GhTIFY9 responded to verticillium wilt induction,and the sensitivity of silent plants to verticillium wilt was significantly enhanced after inhibiting the expression of this gene. GhTIFY9 is a positive regulator of cotton resistance to verticillium wilt.

Key words: cotton verticillium wilt, GhTIFY9, gene cloning, virus-induced gene silencing(VIGS)