陆地棉锌指蛋白基因GhZFP8黄萎病的抗性功能验证

doi:10.6048/j.issn.1001-4330.2025.01.017

新疆农业科学 ›› 2025, Vol. 62 ›› Issue (1): 137-145.DOI: 10.6048/j.issn.1001-4330.2025.01.017

• 耕作栽培·生理生化·种质资源·分子遗传学·土壤肥料 • 上一篇下一篇

陆地棉锌指蛋白基因GhZFP8黄萎病的抗性功能验证

程贯富(), 卢国强, 崔永祥, 侯奥成, 张国帅, 梁春燕, 雷建峰, 路伟, 代培红, 李月()

新疆农业大学生命科学学院/新疆极端环境生物生态适应与进化重点实验室,乌鲁木齐 830052

收稿日期:2024-08-11 出版日期:2025-01-20 发布日期:2025-03-11
通信作者: 李月(1984-),女,河南许昌人,副教授,博士,研究方向为棉花逆境分子生物学,(E-mail)liyue6905@126.com
作者简介:程贯富(1998-),男,山东济南人,硕士研究生,研究方向为棉花逆境分子生物学,(E-mail)cgfyouxi@163.com
基金资助:
国家自然科学基金项目(32160494);新疆维吾尔自治区自然科学基金项目(2023D01E03);国家级大学生创新创业训练计划项目(202210758001);新疆维吾尔自治区重点研发专项(2022B02033-1)

Functional analysis of a C2H2 zinc protein GhZFP8 gene in Gossypium hirsutum resistance to Verticillium wilt

CHENG Guanfu(), LU Guoqiang, CUI Yongxiang, HOU Aocheng, ZHANG Guoshuai, LIANG Chunyan, LEI Jianfeng, LU Wei, DAI Peihong, LI yue()

Xinjiang Key Laboratory for Ecological Adaptation and Evolution of Extreme Environment Biology/College of Life Sciences, Xinjiang Agricultural University, Urumqi 830052, China

Received:2024-08-11 Published:2025-01-20 Online:2025-03-11
Supported by:
National Natural Science Foundation of China(32160494);Natural Science Foundation of Xinjiang Uygur Autonomous Region(2023D01E03);National Innovation and Entrepreneurship Training Program for College Students(202210758001);Key Scientific R & D Program Project of Xinjiang Uygur Autonomous Region(2022B02033-1)

摘要/Abstract

摘要：

【目的】 探究陆地棉C2H2型锌指蛋白基因GhZFP8在棉花抗黄萎病反应中的功能,为挖掘棉花抗病基因奠定理论基础。【方法】 通过同源克隆方法得到一个陆地棉C2H2型锌指蛋白基因GhZFP8,利用生物信息学方法分析该基因的理化性质,构建该基因病毒诱导的基因沉默(VIGS)载体,通过农杆菌介导法转化棉花,检验GhZFP8的抗病功能。【结果】 GhZFP8开放阅读框(ORF)为789 bp、编码为262个氨基酸,相对分子量为28.12 kD、等电点为8.16、脂肪指数为59.31和平均疏水性为-0.718,为亲水性、碱性的非跨膜蛋白。GhZFP8蛋白具有2个ZnF-C2H2结构域,含有6个α-螺旋和4个β-折叠,属于C2H2型锌指蛋白。GhZFP8基因沉默植株对黄萎病菌的抗性相对减弱。【结论】 GhZFP8基因在棉花抗黄萎病过程中发挥正调控作用。

关键词: 黄萎病; GhZAT8; 基因克隆; 病毒诱导的基因沉默

Abstract:

【Objective】 To explore the function of the terrestrial cotton C2H2-type zinc finger protein gene GhZFP8 in the response to Verticillium wilt in cotton in the hope of laying a theoretical foundation for mining cotton disease resistance genes. 【Methods】 The cotton gene GhZFP8 was obtained by homologous cloning and its biological function explored. Bioinformatics methods were used to analyze the physicochemical properties of the gene, while the VIGS vector for the gene was constructed and transformed into cotton by Agrobacterium-mediated method to preliminarily test the disease resistance function of GhZFP8. 【Results】 The open reading frame (ORF) of GhZFP8 was 789 bp, encoded as a hydrophilic protein containing 262 amino acids, a relative molecular weight was 28.12 kD, a basic amino acid with an isoelectric point of 8.16, a lipid index of 59.31, and an average hydrophobicity of-0.718. The absence of a signal peptide in the GhZFP8 protein predicted that GhZFP8 was a non-transmembrane protein. GhZFP8 protein revealed that it had 2 ZnF-C2H2 structural domains. Its secondary structure prediction revealed that the GhZFP8 protein contained 6 α-helices and 4 β-folds. GhZFP8 gene silenced plants showed relatively reduced resistance to Verticillium wilt. 【Conclusion】 The GhZFP8 gene plays a positive regulatory role in cotton resistance to verticillium wilt.

Key words: Verticillium wilt; GhZFP8; gene cloning; VIGS

中图分类号:

S562

程贯富, 卢国强, 崔永祥, 侯奥成, 张国帅, 梁春燕, 雷建峰, 路伟, 代培红, 李月. 陆地棉锌指蛋白基因GhZFP8黄萎病的抗性功能验证[J]. 新疆农业科学, 2025, 62(1): 137-145.

CHENG Guanfu, LU Guoqiang, CUI Yongxiang, HOU Aocheng, ZHANG Guoshuai, LIANG Chunyan, LEI Jianfeng, LU Wei, DAI Peihong, LI yue. Functional analysis of a C2H2 zinc protein GhZFP8 gene in Gossypium hirsutum resistance to Verticillium wilt[J]. Xinjiang Agricultural Sciences, 2025, 62(1): 137-145.

图/表 9

表1 生物信息学分析工具

Tab.1 Bioinformatics analysis tools

分析工具 Analysis tools	功能 Function	网址 URL
ExPASy-ProtScale	蛋白理化性质分析	http://web.expasy.org/protparam/
SignaIP	信号肽预测	http://www.cbs.dtu.dk/services/SignalP
TMHMM	跨膜结构域预测	http://www.cbs.dtu.dk/services/TMHMM/
SMART	蛋白结构域预测	http://smart.embl-heidelberg.de/
PSIPRED	蛋白二级结构预测	http://bioinf.cs.ucl.ac.uk/psipred/
SWISS-MODEL	蛋白三级结构预测	https://swissmodel.expasy.org/

表2 引物及用途

Tab.2 Primer and application

基因 Gemne	序列 Sequence(5'-3')	用途 Application
GhZFP8	F:TCGGAAGTATTTGATATGGCGCTTG R:GCTTTGGACCTCTTCTTCATCTGGG	基因克隆
VIGS- GhZFP8	F: GAATTCACAACGTGGCGACTCTCCT R: GGTACCAGCGTTTATGACCACCCAAG	VIGS载体构建
qGhZFP8	F:ATTCGTCGGTCTTCCCCTTC R:TTTGCGGTGGCTGGCTTTAT	qRT-PCR
qGhUBQ7	F:GAAGGCATTCCACCTGACCAAC R:CTTGACCTTCTTCTTCTTGTGCTTG	qRT-PCR

图1 GhZFP8基因的克隆

Fig.1 Cloning of GhZFP8 gene M:Trans 2K Plus Ⅱ DNA Marker

图2 GhZFP8蛋白信号肽预测

Fig.2 The prediction signal peptide of GhZFP8 protein

图3 GhZFP8蛋白结构域预测

Fig.3 The prediction protein of GhZFP8

图4 GhZFP8蛋白的二、三级结构预测注:A:GhZFP8 蛋白的二级结构预测;B: GhZFP8 蛋白的三级结构预测

Fig.4 The prediction secondary and tertiary structure of GhZFP8 protein Notes: A: The prediction secondary structure of GhZFP8 protein; B: The prediction tertiary structure of GhZFP8protein

图5 GhZFP8的VIGS载体构建注:A:GhZFP8 目标片段的PCR扩增;B:TRV : GhZFP8 VIGS 载体的酶切鉴定;M:Trans 2K Plus Ⅱ DNA Marker

Fig.5 Construction of VIGS vector of GhZFP8 Notes: A:PCR amplification of target fragment of GhZFP8 ;B :identification of TRV: GhZFP8 VIGS vectors by restriction enzyme digestion.M :Trans 2K Plus Ⅱ DNA marker

图6 GhZFP8的沉默效率检测注:A:阳性对照植株表型;B:GhCLA1基因沉默效率检测;C:GhZFP8 基因沉默效率检测;相同小写字母表示差异不显著(P>0.05),不同小写字母之间表示差异显著(P<0.05),下同

Fig.6 Detection results of silencing efficiency of GhZFP8 gene Notes:A:Phenotype of positive control plant;B:Silencing efficiency of GhCLA1 gene;C:Silencing efficiency of GhZFP8 gene; The same lowercase letter indicates no significant difference(P>0.05),and different lowercase letters indicate significant difference (P<0.05),the same as below

图7 沉默植株接种黄萎病菌后表型及病情指数注:A:TRV:GhZFP8沉默植株表型与剖杆鉴定; B:病情指数统计; C:木质部染色切片

Fig.7 The Phenotype and disease index of silenced plants inoculated with Verticillium Notes: A: Phenotype analysis of plants after GhZFP8gene silencing and Identification by stem anatomy.B: Disease index statistics.C: the stained section of Xylem

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陆地棉锌指蛋白基因GhZFP8黄萎病的抗性功能验证

Functional analysis of a C2H2 zinc protein GhZFP8 gene in Gossypium hirsutum resistance to Verticillium wilt

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