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作物学报 ›› 2022, Vol. 48 ›› Issue (9): 2265-2273.doi: 10.3724/SP.J.1006.2022.14109

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花GhIQM1基因克隆及抗黄萎病功能分析

李名江1(), 雷建峰2, 祖丽皮耶•托合尼亚孜2, 代培红1, 刘超1,*(), 刘晓东1,*()   

  1. 1.新疆农业大学生命科学学院, 新疆乌鲁木齐 830052
    2.新疆农业大学农学院, 新疆乌鲁木齐 830052
  • 收稿日期:2021-06-23 接受日期:2022-01-05 出版日期:2022-09-12 网络出版日期:2022-02-09
  • 通讯作者: 刘超,刘晓东
  • 作者简介:E-mail: 1091019753@qq.com
  • 基金资助:
    新疆农业大学生物学创新团队项目(ITB202108)

Cloning and functional verification of GhIQM1 gene of cotton in response to Verticillium wilt

LI Ming-Jiang1(), LEI Jian-Feng2, ZULIPIYE·Tuoheniyazi 2, DAI Pei-Hong1, LIU Chao1,*(), LIU Xiao-Dong1,*()   

  1. 1. College of Life Sciences, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China
    2. College of Agriculture, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China
  • Received:2021-06-23 Accepted:2022-01-05 Published:2022-09-12 Published online:2022-02-09
  • Contact: LIU Chao,LIU Xiao-Dong
  • Supported by:
    Innovative Team Foundation of Biology of Xinjiang Agricultural University(ITB202108)

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摘要：

棉花是我国重要的经济作物, 而黄萎病(Verticillium wilt)是棉花生产上的主要病害, 严重危害棉花产量和纤维品质。本研究通过转录组数据分析筛选出1个与棉花抗病相关、不依赖Ca2+的钙调素结合蛋白基因GhIQM1。该基因受黄萎病菌和水杨酸(SA)诱导表达。利用病毒诱导的基因沉默(virus-induced gene silencing, VIGS)技术研究其在棉花抗黄萎病中的功能发现, 抑制GhIQM1基因表达增强了植株对黄萎病的抗性, TRV:GhIQM1植株病情指数、维管束褐化程度、体内病原菌积累量都显著低于TRV:00。qRT-PCR分析表明, 抑制GhIQM1表达的植株接种黄萎病后, 作为参与水杨酸途径中的3个重要基因NPR1、NPR3和PR5的表达量显著高于对照。以上结果初步表明, GhIQM1基因可能通过抑制SA途径负调控了棉花的黄萎病抗性。

关键词: 棉花, 黄萎病, GhIQM1, 基因沉默

Abstract:

Cotton is an important economic crop in China, and Verticillium wilt is the main disease in cotton production, which seriously endangers cotton yield and fiber quality. In this project, GhIQM1, encode a Ca2+-independent calmodulin-binding protein in cotton, was screened out by transcriptome analysis. The gene was up-regulated by Verticillium dahliae and salicylic acid treatment in cotton. The function of GhIQM1 was explored in cotton in response to Verticillium wilt using virus-induced gene silencing (VIGS) technology. The results showed that disease index (DI), the symptoms of Verticillium wilt, the degree of browning of vascular tissue, and the relative fungal biomass pathogen accumulation in vivo in the GhIQM1-silenced plants (TRV:GhIQM1) were significantly lower than those in the TRV:00 cotton plant. The qRT-PCR results revealed that the gene expression level of NPR1, NPR3, and PR5, involved in the salicylic acid pathway, were significantly higher than the control after inoculation of Verticillium dahliae. The above results indicate that the GhIQM1 negatively regulates the resistance of cotton to Verticillium wilt through suppressing SA pathway.

Key words: cotton, Verticillium wilt, GhIQM1, gene silencing

表1

本研究中使用的引物序列"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Application
GhIQM1-F GAATTCAGACCCGTTAGTGAGCTTG 片段扩增Gene fragment amplification
GhIQM1-R GGTACCCTCAAGATTTACTTCTTTCCC 基因片段扩增Gene fragment amplification
qGhIQM1-F ATCAGCCTGGCAACAAATTC 荧光定量Quantitative real time-PCR
qGhIQM1-R CAGCTTTGCCGTTTCTCAAA 荧光定量Quantitative real time-PCR
ITS-F AAAGTTTTAATGGTTCGCTAAGA 荧光定量Quantitative real time-PCR
VE1-R CTTGGTCATTTAGAGGAAGTAA 荧光定量Quantitative real time-PCR
SA-GhNPR1-F CTAGCTTGCGGAGGGATTGATACC 荧光定量Quantitative real time-PCR
SA-GhNPR1-R GAGATGGCTGACCTGTCAAACTGC 荧光定量Quantitative real time-PCR
SA-GhNPR3-F GCGGAGAGCATTCGGTTACT 荧光定量Quantitative real time-PCR
SA-GhNPR3-R GCTCAACATCGTCGGAGTCT 荧光定量Quantitative real time-PCR
SA-GhPR5-F TGAGTGCTCCTACACCGTCT 荧光定量Quantitative real time-PCR
SA-GhPR5-R CGACCCCAAATACGAGCCAT 荧光定量Quantitative real time-PCR
JA-GhPDF1.2-F CTGTGGTAGCGGATGGTGATAAG 荧光定量Quantitative real time-PCR
JA-GhPDF1.2-R GTGCAGACGCATTTGCGAAGGAA 荧光定量Quantitative real time-PCR
JA-GhAOC-F AATAGAGCATAAACCCGAAATGAAAG 荧光定量Quantitative real time-PCR
JA-GhAOC-R CAAAAATGCCAGACCCACCAGTA 荧光定量Quantitative real time-PCR
JA-GhAOS-F TGCCACCTGGTCCTTTCATTTC 荧光定量Quantitative real time-PCR
JA-GhAOS-R GCGTGTTTGGGCTCGGAAGGGTCG 荧光定量Quantitative real time-PCR
GhUBQ7-F GAAGGCATTCCACCTGACCAAC 内参基因Reference gene
GhUBQ7-R CTTGACCTTCTTCTTCTTGTGCTTG 内参基因Reference gene

图1

GhIQM1基因的表达分析 CK: 对照组; V991: 大丽轮枝菌处理组; SA: 水杨酸处理组。**和***分别表示在0.01、0.001水平显著差异。每组样品取3个生物学重复。"

图2

GhIQM1基因的VIGS载体构建及沉默效率检验 A: M: Trans2K Plus II DNA marker; a: GhIQM1片段的PCR产物扩增; b: pTRV2-GhIQM1载体的酶切验证。B: 沉默GhCLA1基因表型; C: GhCLA1和GhIQM1基因沉默效率检测。*、**分别表示在0.05和0.01水平显著差异。"

图3

沉默GhIQM1增强了棉花对黄萎病菌的抗性 A: 黄萎病菌侵染20 d后对照植株TRV:00和沉默植株TRV:GhIQM1植株的病症; B: 黄萎病菌侵染15 d后TRV:00和TRV:GhIQM1植株的病情指数; C: 接种黄萎病菌15 d后, TRV:00和TRV:GhIQM1植株维管束褐化情况; D: TRV:00和TRV:GhIQM1在接种黄萎病菌15 d后相对生物含量; E: 接种黄萎病菌20 d后TRV:00和TRV:GhIQM1棉株茎部的恢复培养试验。**表示TRV:00与TRV:GhIQM1在0.01水平显著性差异。"

图4

GhIQM1基因沉默后SA/JA信号通路相关基因的表达分析 TRV:00: 对照组; TRV:GhIQM1: 沉默组。*、**、***分别表示在0.05、0.01和0.001水平显著差异。"

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