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棉花学报 ›› 2022, Vol. 34 ›› Issue (2): 79-92.doi: 10.11963/cs20210047

• 研究与进展 •    下一篇

GhROP6通过调控茉莉酸合成与木质素代谢参与棉花抗黄萎病反应

周雪慧(),高二林,王钰静,李焱龙,袁道军,朱龙付*()   

  1. 华中农业大学/作物遗传改良国家重点实验室,武汉 430070
  • 收稿日期:2021-07-06 出版日期:2022-03-15 发布日期:2022-07-19
  • 通讯作者: 朱龙付 E-mail:lfzhu@mail.hzau.edu.cn
  • 作者简介:周雪慧(1994―),女,硕士研究生, 1272569018@qq.com
  • 基金资助:
    国家自然科学基金(31771844)

GhROP6 involved in cotton resistance to Verticillium wilt through regulating jasmonic acid synthesis and lignin metabolism

Zhou Xuehui(), Gao Erlin, Wang Yujing, Li Yanlong, Yuan Daojun, Zhu Longfu*()   

  1. Huazhong Agricultural University/National Key Laboratory of Crop Genetic Improvement, Wuhan 430070, China
  • Received:2021-07-06 Online:2022-03-15 Published:2022-07-19
  • Contact: Zhu Longfu E-mail:lfzhu@mail.hzau.edu.cn

摘要:

【目的】 克隆抗病相关基因GhROP6,解析其作用机制,为开展棉花抗病分子育种提供理论基础。【方法】 利用生物信息学方法系统分析了陆地棉Rho鸟苷三磷酸酶基因(Rho-related guanosine triphosphatase from plants, ROP)家族成员在染色体上的分布及组织表达模式。克隆了GhROP6 Gh_A01G1392.1)基因,通过实时荧光定量聚合酶链式反应、病毒诱导的基因沉默(virus-induced gene silencing, VIGS)技术、拟南芥遗传转化技术及代谢物测定等对该基因进行功能分析。【结果】 在陆地棉中鉴定到28个ROP,其编码的多肽均含有ROP结构,包括4个GTP/GDP结合域、与下游靶蛋白结合的效应结构域和C末端的可变区域。染色体定位分析发现陆地棉ROP家族中24个基因对称分布在A亚基因组和D亚基因组中,另有3个基因分布在D亚基因组。实时荧光定量聚合酶链式反应分析发现,GhROP6在棉花不同器官表达量不同,在花瓣、柱头、开花后10 d的纤维中表达量较高,并受茉莉酸甲酯诱导上调表达。抑制GhROP6表达会降低茉莉酸生物合成相关基因GhLOX1GhOPR3-1GhOPR3-3GhAOC1GhAOS和茉莉酸信号通路相关基因GhMYC2的表达水平,削弱木质素合成相关基因GhCCR-1GhF5H-1GhCCoAOMT-2GhCCoAOMT-3的表达,从而降低棉花对黄萎病的抗性;超表达组成型激活的GhROP6能增加转基因拟南芥茉莉酸-异亮氨酸含量和木质素含量,增强其对黄萎病菌抗性。【结论】 GhROP6可能通过茉莉酸合成和信号通路以及木质素合成代谢参与棉花抗黄萎病反应。

关键词: 棉花; 黄萎病; GhROP6; 抗病性; 茉莉酸; 木质素; 信号通路

Abstract:

[Objective] This study aims to characterize the GhROP6 and study its roles of resistance to Verticillium wiltin upland cotton (Gossypium hirsutum L.). [Method] The bioinformatics analysis was used to identify Rho-related guanosine triphosphatase from plants (ROP) genes in upland cotton. The chromosome distributions, expression pattern analysis of GhROP genes were investigated. The function of GhROP6 gene was studied by quantitative real-time polymerase chain reaction (qRT-PCR), virus-induced gene silencing (VIGS), plant genetic transformation and metabolism analysis. [Result] Totally, 28 ROP genes were identified in upland cotton. And the corresponding amino acid sequence contained the ROP protein specific structures, including four GTP/GDP binding domains, effector domain binding to downstream target proteins and variable C-terminal regions. Chromosomal mapping analysis showed that 24 ROP genes were symmetrically distributed in subgenome A and subgenome D, and 3 genes specifically distributed in subgenome D. qRT-PCR analysis showed that the transcript levels of GhROP6 varied in different organs, and showed higher expression level in petals, stigma, fiber of 10 days post anthesis. Meanwhile, the transcript level of GhROP6 was upregulated in cotton by methyl jasmonate (MeJA). Knock-down of GhROP6 through VIGS weakened the cotton resistance to Verticillium wilt, and reduced the expression of GhLOX1, GhOPR3-1, GhOPR3-3, GhAOC1, GhAOS involved in jasmonic acid (JA) synthesis, and the expression of GhMYC2 involved in JA signaling pathway, and the expression of GhCCR-1, GhF5H-1, GhCCoAOMT-2, and GhCCoAOMT-3 genes involved in lignin synthesis. However, constitutively activated GhROP6 in Arabidopsis enhanced the plants resistantce to V. dahliae. Further analysis showed that the contents of JA-isoleucine and lignin in transgenic Arabidopsis were higher than those of wild type. [Conclusion] GhROP6 may involve in the resistance of cotton to Verticillium wilt through JA synthesis and signaling pathway and lignin synthesis.

Key words: cotton; Verticillium wilt; GhROP6; disease resistance; jasmonic acid; lignin; signaling pathway