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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (9): 2265-2273.doi: 10.3724/SP.J.1006.2022.14109

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2022-09-12 Published:2022-02-09
  • Contact: LIU Chao,LIU Xiao-Dong E-mail:1091019753@qq.com;xiaodongliu75@aliyun.com;liuch_86@126.com
  • Supported by:
    Innovative Team Foundation of Biology of Xinjiang Agricultural University(ITB202108)

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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

Table 1

Primer sequences used in this study"

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

Fig. 1

Relative expression pattern of GhIQM1 genes CK: control; V991: Verticillium dahliae treatment; SA: salicylic acid treatment. ** and *** mean significant difference at the 0.01 and 0.001 probability levels, respectively. Three biological replicates are for each set of samples."

Fig. 2

Construction of VIGS vector of GhIQM1 gene and its silencing efficiency test A: M: Trans2K Plus II DNA marker; a: PCR product amplification of GhIQM1 fragment; b: restriction digestion verification of pTRV2-GhIQM1 vector. B: silent GhCLA1 gene phenotype; C: GhCLA1 and GhIQM1 gene silencing efficiency detection. * and ** indicate significant difference at the 0.05 and 0.01 probability levels, respectively."

Fig. 3

Silencing GhIQM1 enhances cotton resistance to Verticillium dahliae A: control plant TRV:00 and silent plant TRV:GhIQM1 plant disease after 20 days of V991 infection; B: TRV:00 and TRV: disease index of GhIQM1 plant 15 days after V991 infection; C: 15 days after inoculation, TRV:00 and TRV:GhIQM1 plant vascular bundle browning situation; D: TRV:00 and TRV:GhIQM1 relative biological content of Verticillium dahliae 15 days after inoculation; E: twenty days after inoculation with Verticillium dahliae, TRV:00 and TRV:GhIQM1 cotton plant stem recovery culture experiment. ** means significant difference between TRV:00 and TRV: GhIQM1 at the 0.01 probability level."

Fig. 4

Relative expression patterns of SA/JA signal pathway related genes after GhIQM1 gene silencing TRV:00: control; TRV:GhIQM1: silent group. *, **, and *** mean significant difference at the 0.05, 0.01, and 0.001 probability levels, respectively."

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