棉花黄萎病抗性相关基因GhTIFY9的克隆与功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1519

摘要/Abstract

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）

李秀青, 胡子曜, 雷建峰, 代培红, 刘超, 邓嘉辉, 刘敏, 孙玲, 刘晓东, 李月. 棉花黄萎病抗性相关基因GhTIFY9的克隆与功能分析[J]. 生物技术通报, 2022, 38(8): 127-134.

LI Xiu-qing, HU Zi-yao, LEI Jian-feng, DAI Pei-hong, LIU Chao, DENG Jia-hui, LIU Min, SUN Ling, LIU Xiao-dong, LI Yue. Cloning and Functional Analysis of Gene GhTIFY9 Related to Cotton Verticillium Wilt Resistance[J]. Biotechnology Bulletin, 2022, 38(8): 127-134.

图/表 7

图1 GhTIFY9的克隆

Fig. 1 Cloning of GhTIFY9 gene M：Trans 2K Plus Ⅱ DNA marker

表1 GhTIFY9的生物信息学分析

Table 1 Bioinformatics analysis of GhTIFY9

基因 Gene	开放阅读框ORF/bp	氨基酸 Amino acid	相对分子质量Relative molecular weight/kD	理论等电点Theoretical isoelectric point	平均疏水性Average hydrophobicity	信号肽 Signal peptide	亚细胞定位 Subcellular localization	脂肪系数Fat coefficient	跨膜结构域 Transmembrane domain
GhTIFY9	594	197	21.65	9.08	-0.477	无 None	细胞核 Nucleus	76.63	无 None

图2 GhTIFY9蛋白结构域预测

Fig.2 Protein domain prediction of GhTIFY9

图3 黄萎病诱导下GhTIFY9的表达模式分析 *：在0.05水平上存在显著差异,**：在0.01水平上存在显著差异。下同

Fig.3 Analysis of the expression patterns of GhTIFY9 under the treatment of Verticillium dahliae *：Significant difference at the 0.05 level. **：Significant difference at the 0.01 level. The same as below

图4 GhTIFY9的VIGS载体构建 A：GhTIFY9目标片段的PCR扩增;B：TRV﹕GhTIFY9 VIGS载体的酶切鉴定。M：Trans 2K Plus II DNA marker

Fig. 4 VIGS vector construction of GhTIFY9 A：PCR amplification of target fragment of GhTIFY9. B：Identification of TRV﹕GhTIFY9 vectors by restriction enzyme digestion. M：Trans 2K Plus II DNA marker

图5 GhTIFY9的沉默效率检测 A：在棉花中沉默GhCLA1的表型;B：GhCLA1的沉默效率检测;C：GhTIFY9的沉默效率检测

Fig. 5 Silencing efficiency detection of GhTIFY9 gene A：The phenotype of silenced GhCLA1 in cotton. B：Silencing efficiency detection of GhCLA1. C：Silencing efficiency detection of GhTIFY9

图6 GhTIFY9沉默植株的抗病性鉴定 A：GhTIFY9沉默后植株的表型分析;B：病情指数统计;C：剖杆鉴定;D：病原菌恢复培养

Fig. 6 Disease resistance identification of GhTIFY9 gene-silenced plants A：Phenotype analysis of plants after GhTIFY9 gene silencing. B：Disease index statistics. C：Identification by stem anatomy. D：Restoration culture of pathogenic bacteria

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