棉花抗黄萎病相关基因GhERF020功能的初步分析

doi:10.13304/j.nykjdb.2023.0407

中国农业科技导报 ›› 2024, Vol. 26 ›› Issue (9): 112-121.DOI: 10.13304/j.nykjdb.2023.0407

• 动植物健康 • 上一篇

棉花抗黄萎病相关基因GhERF020功能的初步分析

翁慧婷¹^,²(), 刘海洋³, 郭惠明⁴, 程红梅⁴, 李君¹^,², 张超¹^,²(), 苏晓峰⁴()

^1.河北农业大学生命科学学院，河北保定 071001
^2.河北省农业微生物生物信息利用技术创新中心，河北保定 071001
^3.新疆农业科学院植物保护研究所，乌鲁木齐 830091
^4.中国农业科学院生物技术研究所，农业微生物资源发掘与利用全国重点实验室，北京 100081

收稿日期:2023-05-25 接受日期:2023-07-26 出版日期:2024-09-15 发布日期:2024-09-13
通讯作者: 张超,苏晓峰
作者简介:翁慧婷 E-mail：wht-bio@163.com
基金资助:
河北省重点研发计划项目(22322912D);国家自然科学基金项目(32160624)

Preliminary Function Analysis of GhERF020 Gene in Response to Verticillium Wilt in Cotton

Huiting WENG¹^,²(), Haiyang LIU³, Huiming GUO⁴, Hongmei CHENG⁴, Jun LI¹^,², Chao ZHANG¹^,²(), Xiaofeng SU⁴()

^1.College of Life Science，Hebei Agricultural University，Hebei Baoding 071001，China
^2.Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes，Hebei Baoding 071001，China
^3.Institute of Plant Protection，Xinjiang Academy of Agricultural Sciences，Urumqi 830091，China
^4.National Key Laboratory of Agriculture Microbiology，Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2023-05-25 Accepted:2023-07-26 Online:2024-09-15 Published:2024-09-13
Contact: Chao ZHANG,Xiaofeng SU

摘要/Abstract

摘要：

黄萎病（Verticillium wilt）是影响我国棉花产量和品质的重要病害，其病原菌是大丽轮枝菌（Verticillium dahliae）。AP2/ERF类转录因子在植物生长发育和胁迫适应性响应中的作用日益突显。?为挖掘棉花抗黄萎病相关基因并探究其生物学功能，利用拟南芥抗大丽轮枝菌相关基因AT1G22810.1，同源比对获得棉花抗黄萎病候选基因GhERF020（XM_016842745.1）。生物信息学分析结果表明，GhERF020开放阅读框的长度为441 bp，编码147个氨基酸；其蛋白含有1个保守的AP2结构域，相对分子质量为16.16 kD，无跨膜结构，属于亲水蛋白；预测其启动子区域含有水杨酸、茉莉酸甲酯和脱落酸等响应元件。基因表达模式分析发现，GhERF020受大丽轮枝菌侵染诱导表达。亚细胞定位分析表明，GhERF020蛋白定位于细胞核中。利用病毒诱导的基因沉默（virus-induced gene silencing， VIGS）技术下调GhERF020表达后，棉花植株对大丽轮枝菌的敏感性显著增强，病情指数和病原菌生物量明显升高。由此表明，GhERF020是棉花抗黄萎病的正向调控因子。

关键词: 棉花黄萎病, 大丽轮枝菌, 转录因子, GhERF020, VIGS

Abstract:

Verticillium wilt （VW） is a disruptive symptom caused by Verticillium dahliae， which affects fiber yield and quality of cotton in China. AP2/ERF transcription factors play prominent role in plant growth， development and stress response. In order to excavate the genes related to resistance to VW in cotton and explore their biological functions， the candidate gene GhERF020 （XM_016842745.1） was obtained by homologous alignment of AT1G22810.1 related gene of Arabidopsis thaliana against V. dahliae. Bioinformatic analysis showed that the open reading frame length of GhERF020 was 441 bp， encoded 147 amino acids. GhERF020 contained a conserved AP2 domain， the relative molecular weight was 16.16 kD without transmembrane structure， belonging to the hydrophilic protein. It was predicted that the promoter region contained response elements of salicylic acid （SA）， methyl jasmonate （MEJA）， abscisic acid （ABA） and other response elements. The expression of GhERF020 was induced by V. dahliae infection. Subcellular localization analysis showed that GhERF020 protein was localized in the nucleus. As the expression of GhERF020 was downregulated by virus-induced gene silencing （VIGS） technique， the susceptibility of cotton plants to V. dahliae was significantly enhanced， and the disease index and pathogen biomass were distinctly increased. Above results indicated that GhERF020 was a positive regulator in resistance against V. dahliae in cotton.

Key words: cotton Verticillium wilt, Verticillium dahliae, transcription factor, GhERF020, VIGS

中图分类号:

S562

翁慧婷, 刘海洋, 郭惠明, 程红梅, 李君, 张超, 苏晓峰. 棉花抗黄萎病相关基因GhERF020功能的初步分析[J]. 中国农业科技导报, 2024, 26(9): 112-121.

Huiting WENG, Haiyang LIU, Huiming GUO, Hongmei CHENG, Jun LI, Chao ZHANG, Xiaofeng SU. Preliminary Function Analysis of GhERF020 Gene in Response to Verticillium Wilt in Cotton[J]. Journal of Agricultural Science and Technology, 2024, 26(9): 112-121.

图/表 9

表1 本研究所用引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5’-3’）
GhERF020-qRT-F	TAAGCCTGAAGAATTGCCTGAAC
GhERF020-qRT-R	CATGCCGGATAGTAGTGGCAGAG
GhERF020-VIGS-F	TCTGTGAGTAAGGTTACCGAATTCTCAAAGGAAATACAAGGGAGTGC
GhERF020-VIGS-R	ACGCGTGAGCTCGGTACCGGATCCTTCAATGGATATGCTCAAAGTCTC
p1132-GhERF020-F	TCTGTGAGTAAGGTTACCGAATTCTCAAAGGAAATACAAGGGAGTGC
p1132-GhERF020-R	ACGCGTGAGCTCGGTACCGGATCCTTCAATGGATATGCTCAAAGTCTC
GhUBQ7-RT-F	GCAAGTGTTGGGTTCAAAGCTGGTG
GhUBQ7-RT-R	CCAGGTTGAGGAGTTACTCGGAATGCTG
Vd-ITS-F	TCCGTAGGTGAACCTGCGG
Vd-ITS-R	TCCTCCGCTTATTGATATGC

图1 棉花GhERF020蛋白的同源性及结构分析A：不同物种GhERF020同源蛋白系统发育树分析；B：GhERF020结构域分析

Fig. 1 Homology and structure analysis of GhERF020 in cottonA： Phylogenetic tree analysis of GhERF020 homologous protein in different species； B： GhERF020 domain analysis

表2 GhERF020蛋白理化性质

Table 2 Physical and chemical properties of GhERF020

指标Index	值Value
氨基酸数量Number of amino acid/aa	147
分子量Molecular weight/kD	16.16
分子式Formula	C₆₉₁H₁₀₉₅N₂₀₇O₂₂₁S₁₀
理论等电点Theoretical pI	7.79
脂肪族氨基酸指数Aliphatic index	60.48
不稳定性指数Instability index	73.88
N端N-terminal	Met
总平均亲水性 Grand average of hydropathicity	-0.586

图2 GhERF020蛋白理化性质分析A：GhERF020蛋白亲疏水性分析；B：GhERF020的氨基酸序列的跨膜区段预测；C：GhERF020蛋白二级结构；D：GhERF020蛋白三级结构

Fig. 2 Physicochemical properties of GhERF020A： Hydrophilic analysis of GhERF020； B： Transmembrane prediction of amino acid sequence of GhERF020； C： Secondary structure of GhERF020； D： Tertiary structure of GhERF020

图3 GhERF020基因启动子顺式元件预测

Fig. 3 Cis-element prediction of GhERF020 promoter

图4 GhERF020在棉花接种大丽轮枝菌中的表达

Fig. 4 Expression of GhERF020 in cotton post inoculation with V. dahliae

图5 GhERF020亚细胞定位

Fig. 5 Subcellular localization of GhERF020

图6 GhERF020基因沉默效果A：TRV：： CLA1植株；B：GhERF020在棉花沉默植株中的基因表达量；**表示在P<0.01水平差异显著

Fig. 6 GhERF020 silencing efficiencyA： TRV：： CLA1 plant； B： Gene expression of GhERF020 in cotton plants； ** indicates significant difference at P<0.01 level

图7 GhERF020基因沉默棉花植株黄萎病抗性鉴定A：V991侵染15 d后TRV：：00和TRV：：GhERF020植株的表型；B：接种10 d后，TRV：： 00和TRV：： GhERF020棉花植株病情指数；C：接种10 d后，TRV：： 00和TRV：： GhERF020棉花植株相对真菌生物量。**表示P < 0.01水平差异显著

Fig. 7 Identification of GhERF020 gene-silenced cotton in resistance to VWA： Phenotype of TRV：： 00 and TRV：： GhERF020 plants after infection 10 d with V991； B： Disease indices of TRV：： 00 and TRV：： GhERF020 cotton plants after 10 d post inoculation； C： Fungal biomass of TRV：： 00 and TRV：： GhERF020 plants after infection 10 d with V991. ** indicates significant difference at P<0.01 level

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棉花抗黄萎病相关基因GhERF020功能的初步分析

Preliminary Function Analysis of GhERF020 Gene in Response to Verticillium Wilt in Cotton

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