GhEIN3基因对棉花枯萎病胁迫响应的功能分析

doi:10.11963/cs20210046

摘要/Abstract

摘要：

【目的】乙烯（ethylene, ET）不敏感蛋白3（ethylene-insensitive 3, EIN3）/EIN3-like（EIL）家族基因是乙烯通路中的关键基因,参与植物生物胁迫响应。探究其功能可为解析陆地棉对枯萎病菌的响应机理提供依据。【方法】从枯萎病菌诱导棉花根部基因表达谱数据中筛选并从TM-1基因组数据库中鉴定获得EIN3/EIL基因家族的GhEIN3基因,利用生物信息学、实时荧光定量聚合酶链式反应技术和病毒诱导的基因沉默（virus-induced gene silencing, VIGS）技术分别分析基因序列、不同处理下的表达特征及GhEIN3基因在棉花抗病过程中的作用。【结果】GhEIN3基因（Genbank登录号：KY744279）开放阅读框为1 842 bp,编码的氨基酸序列中含有典型的EIN3蛋白结构域。枯萎病菌胁迫和不同外源激素ET、水杨酸（salicylic acid, SA）和茉莉酸（jasmonic acid, JA）处理后,该基因受枯萎病菌胁迫和ET诱导上调表达,而受SA和JA诱导下调表达;当GhEIN3基因沉默后,对沉默植株进行枯萎病菌接菌试验,结果显示,与对照相比,沉默GhEIN3基因的株系更感病;基因表达量测定结果表明,与对照比较,沉默株系中病程相关基因PR1、PR2、PR4的表达量均下降,乙烯响应因子基因ERF1和1-氨基环丙烷-1-羧酸氧化酶基因ACO的表达量下降,而PR5基因的表达量上升。【结论】从陆地棉中鉴定出的EIN3/EIL家族基因GhEIN3能够响应枯萎病菌和外源激素ET、SA、JA诱导,结合枯萎病菌、ET、SA与JA诱导后GhEIN3基因表达情况,以及枯萎病接种试验和VIGS结果认为,GhEIN3基因在棉花抗枯萎病的过程中起积极作用。

关键词: GhEIN3; 陆地棉; 枯萎病; 转录因子; 基因功能; 病毒诱导的基因沉默; 表达分析

Abstract:

[Objective] Ethylene (ET) insensitivity protein 3 (EIN3)/EIN3-like (EIL) family genes are key genes in ethylene pathway, which are involved in the biological stress response of plants. Its function was studied to provide a basis for analyzing the response mechanism of upland cotton to Fusarium wilt. [Method] GhEIN3 of EIN3/EIL gene family were selected from TM-1 genome database based on the data of gene expression profile from root infected with cotton Fusarium wilt in our laboratory. The gene coding protein and related sequence information were analyzed by bioinformatics methods, and the expression pattern of GhEIN3 in Fusarium wilt and different hormone treatments were analyzed by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The role of GhEIN3 in disease resistance was preliminarily analyzed by virus induced gene silencing (VIGS) technique. [Result] A new gene GhEIN3 was isolated from upland cotton (Genbank accession: KY744279), which was from EIN3/EIL family. Bioinformatic analysis showed that the open reading frame of this gene was 1 842 base pairs. The encoded amino acids sequence contains a typical EIN3 domain. After Fusarium oxysporum f. sp. vasinfectum (FOV) and different exogenous hormones ET, salicylic acid (SA) and jasmonic acid (JA) treatment, the expression of GhEIN3 was up-regulated by FOV and ET induction, but down-regulated by SA and JA induction. The VIGS results showed that the GhEIN3 gene silenced plants were more susceptible to FOV than the control. The results of gene expression analysis showed that compared with the control, the expression levels of pathogenesis-related genes PR1, PR2, and PR4, ethylene response factor gene ERF1 and 1-aminocyclopropane-1-carboxylate oxidase gene ACO were decreased, while the expression level of PR5 gene was increased after GhEIN3 gene silencing. [Conclusion] A EIN3/EIL family gene GhEIN3 identified from G. hirsutum responds to Fusarium wilt and hormones ET, SA and JA induction. Combined the GhEIN3 gene expression analysis of the cotton plants under ET, SA, JA and FOV treatments with VIGS results, this study showed that the GhEIN3 gene plays a positive role in the process of cotton to resist Fusarium wilt.

Key words: GhEIN3; Gossypium hirsutum; Fusarium wilt; transcription factor; gene function; virus-induced gene silencing (VIGS); expression analysis

赵曾强,张析,李潇玲,张薇. GhEIN3基因对棉花枯萎病胁迫响应的功能分析[J]. 棉花学报, 2022, 34(3): 173-186.

Zhao Zengqiang,Zhang Xi,Li Xiaoling,Zhang Wei. Functional analysis of GhEIN3 gene in response to cotton Fusarium wilt stress[J]. Cotton Science, 2022, 34(3): 173-186.

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基因登录号 Gene accession number	基因名称 Gene name	物种 Species
KAA3468812.1	EIN3-like	澳洲棉 Gossypium australe
XP_039018867.1	EIN3-like	木槿Hibiscus syriacus
EOY34301.1	EIN3-like	可可 Theobroma cacao
XP_021277894.1	EIN3-like	锦葵 Herrania umbratica
XP_022729333.1	EIN3-like	榴莲 Durio zibethinus
KHG09663.1	EIN3-like	亚洲棉 G. arboreum
XP_021611396.1	EIN3-like	木薯 M. esculenta
KAA3485749.1	EIN3-like	澳洲棉 G. australe
XP_016718322.1	EIN3	陆地棉 G. hirsutum
XP_002530192.1	EIN3	蓖麻 Ricinus communis

引物名称 Primer name	引物序列（5'→3'） Primer sequences (5' to 3')
GhERF1-F	CTTTAGATCACAACTCGCTTC
GhERF1-R	TTTTCTTTCGTCGGGCTT
GhACO-F	TGACCTCAAGTTCCAAGCCAA
GhACO-R	AGAATTTATCCATCAGCCGACT
GhPR1-F	TTTAGTAAGGTTTTTGACCGACGAA
GhPR1-R	GACTCTGTCCATCTTGGTTGTGCTA
GhPR2-F	TTCAAGGTTTGCACTCGGAAGA
GhPR2-R	CCACCAGCAGCAGAAGTTATCG
GhPR4-F	CTTGATATAAGATTGGTTAGCCC
GhPR4-R	TTTGCTTTTGTTGTATGTAGTCG
GhPR5-F	TTGGCTCTTACTTCCGACCATCT
GhPR5-R	GCCGTGATTCATACAGTTATCCTCA