GhROP6通过调控茉莉酸合成与木质素代谢参与棉花抗黄萎病反应

doi:10.11963/cs20210047

摘要/Abstract

摘要：

【目的】克隆抗病相关基因GhROP6,解析其作用机制,为开展棉花抗病分子育种提供理论基础。【方法】利用生物信息学方法系统分析了陆地棉Rho鸟苷三磷酸酶基因（Rho-related guanosine triphosphatase from plants, ROP）家族成员在染色体上的分布及组织表达模式。克隆了GhROP6 （Gh_A01G1392.1）基因,通过实时荧光定量聚合酶链式反应、病毒诱导的基因沉默（virus-induced gene silencing, VIGS）技术、拟南芥遗传转化技术及代谢物测定等对该基因进行功能分析。【结果】在陆地棉中鉴定到28个ROP,其编码的多肽均含有ROP结构,包括4个GTP/GDP结合域、与下游靶蛋白结合的效应结构域和C末端的可变区域。染色体定位分析发现陆地棉ROP家族中24个基因对称分布在A亚基因组和D亚基因组中,另有3个基因分布在D亚基因组。实时荧光定量聚合酶链式反应分析发现,GhROP6在棉花不同器官表达量不同,在花瓣、柱头、开花后10 d的纤维中表达量较高,并受茉莉酸甲酯诱导上调表达。抑制GhROP6表达会降低茉莉酸生物合成相关基因GhLOX1、GhOPR3-1、GhOPR3-3、GhAOC1、GhAOS和茉莉酸信号通路相关基因GhMYC2的表达水平,削弱木质素合成相关基因GhCCR-1、GhF5H-1、GhCCoAOMT-2和GhCCoAOMT-3的表达,从而降低棉花对黄萎病的抗性;超表达组成型激活的GhROP6能增加转基因拟南芥茉莉酸-异亮氨酸含量和木质素含量,增强其对黄萎病菌抗性。【结论】 GhROP6可能通过茉莉酸合成和信号通路以及木质素合成代谢参与棉花抗黄萎病反应。

关键词: 棉花; 黄萎病; GhROP6; 抗病性; 茉莉酸; 木质素; 信号通路

Abstract:

[Objective] This study aims to characterize the GhROP6 and study its roles of resistance to Verticillium wiltin upland cotton (Gossypium hirsutum L.). [Method] The bioinformatics analysis was used to identify Rho-related guanosine triphosphatase from plants (ROP) genes in upland cotton. The chromosome distributions, expression pattern analysis of GhROP genes were investigated. The function of GhROP6 gene was studied by quantitative real-time polymerase chain reaction (qRT-PCR), virus-induced gene silencing (VIGS), plant genetic transformation and metabolism analysis. [Result] Totally, 28 ROP genes were identified in upland cotton. And the corresponding amino acid sequence contained the ROP protein specific structures, including four GTP/GDP binding domains, effector domain binding to downstream target proteins and variable C-terminal regions. Chromosomal mapping analysis showed that 24 ROP genes were symmetrically distributed in subgenome A and subgenome D, and 3 genes specifically distributed in subgenome D. qRT-PCR analysis showed that the transcript levels of GhROP6 varied in different organs, and showed higher expression level in petals, stigma, fiber of 10 days post anthesis. Meanwhile, the transcript level of GhROP6 was upregulated in cotton by methyl jasmonate (MeJA). Knock-down of GhROP6 through VIGS weakened the cotton resistance to Verticillium wilt, and reduced the expression of GhLOX1, GhOPR3-1, GhOPR3-3, GhAOC1, GhAOS involved in jasmonic acid (JA) synthesis, and the expression of GhMYC2 involved in JA signaling pathway, and the expression of GhCCR-1, GhF5H-1, GhCCoAOMT-2, and GhCCoAOMT-3 genes involved in lignin synthesis. However, constitutively activated GhROP6 in Arabidopsis enhanced the plants resistantce to V. dahliae. Further analysis showed that the contents of JA-isoleucine and lignin in transgenic Arabidopsis were higher than those of wild type. [Conclusion] GhROP6 may involve in the resistance of cotton to Verticillium wilt through JA synthesis and signaling pathway and lignin synthesis.

Key words: cotton; Verticillium wilt; GhROP6; disease resistance; jasmonic acid; lignin; signaling pathway

周雪慧, 高二林, 王钰静, 李焱龙, 袁道军, 朱龙付. GhROP6通过调控茉莉酸合成与木质素代谢参与棉花抗黄萎病反应[J]. 棉花学报, 2022, 34(2): 79-92.

Zhou Xuehui, Gao Erlin, Wang Yujing, Li Yanlong, Yuan Daojun, Zhu Longfu. GhROP6 involved in cotton resistance to Verticillium wilt through regulating jasmonic acid synthesis and lignin metabolism[J]. Cotton Science, 2022, 34(2): 79-92.

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链接本文: https://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/cs20210047

https://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2022/V34/I2/79

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本研究所用的引物序列"

引物名称Primer name	序列Sequence	用途Purpose
qGhROP6-F	GCTCATCTCCTACACCAGCAATAC	检测相应基因的表达水平 detect gene expression by qRT-PCR
qGhROP6-R	CAGCAGTATCCCACAATCCAAG
GhLOX1-F	TAGAGAGGACATTTTGCCCTGG
GhLOX1-R	GGTCAAGGTCGTCCAGAGATTTTA
GhAOS1-F	CGGATTAGAGCCTCAGTGTCGG
GhAOS1-R	ATCTTGAGAAATGAAAGGACCAGG
GhAOS2-F	TGCCACCTGGTCCTTTCATTTC
GhAOS2-R	GCGTGTTTGGGCTCGGAAGGGTCG
GhAOC1-F	CAACCCCTTCACTACCACTGCC
GhAOC1-R	AGGGCTGCTTCTGTCTCTCTCG
GhOPR3-1-F	ATGCTGTTCATGCCAAAGGAGG
GhOPR3-1-R	TTTCTGATGTTTCCAGGGGTCG
GhMYC2-F	GCTCCGCCACTACCGTGCTC
GhMYC2-R	CTCGAAGCACTTTTTTACGGTGTTC
GhCCR-1-F	ATTGTTATGGGAAGGCAGTGGC
GhCCR-1-R	ACGAGAAGGTGTGCTAATGCG
GhF5H-1-F	TTGGAGGCAGATGCGAAAGATT
GhF5H-1-R	TCCTCTTGCCCGTGTTTGTTAC
GhCCoAOMT-2-F	TACGACAACACGCTGTGGAATGGG
GhCCoAOMT-2-R	CATCGCCGACAGGAAACATACAGA
GhCCoAOMT-3-F	GAGACCAGTGTGTATCCGAGGG
GhCCoAOMT-3-R	CAAGGGCAGTGGCTAAGAGAGA
GhUB7-F	GAAGGCATTCCACCTGACCAAC
GhUB7-R	CTTGACCTTCTTCTTCTTGTGCTTG
AtPAL1-F	CTTGTCAGGAGCAACACCATCA
AtPAL1-R	AACGAGCAAGGCATATTTGAAGAG
AtPAL2-F	CTACCCCATCAACCTGAACCCA
AtPAL2-R	TATGGTCCAGAAGCGGATGTGT
AtC4H-F	GCTTAGCAACAATGGTGGAATG
AtC4H-R	CATCCTTGGTATTACTTTGGGTCG
AtF5H-F	TCCTCTTGCCCGTGTTTGTTAC
AtF5H-R	TTGGAGGCAGATGCGAAAGATT
AtAOS1-F	TTTTATCGCCGAGAATCCAC
AtAOS1-R	CCTCCGCTAATCGGTTATGA
AtAOC1-F	AACTGAGCGTGTACGAAATCAAT
AtAOC1-R	CAAACATACTGCATTCACAAGGA
AtOPR3-F	CGGCGTTGGCAGAGTATTAT
AtOPR3-R	GCGAGCTTTGAGCCATTAAC
AtMYC2-F	ACGACTGAAACAACTCCGACG
AtMYC2-R	AACCGTCGTATGATTTCTCCG
RT-GhROP6-F	TCCTACACCAGCAATACTTTC	RT-PCR检测GhROP6表达水平 Detect the expression of GhROP6 by RT-PCR
RT-GhROP6-R	GGAGGAATTAAGAAAGCTGAT
VIGS-GhROP6-F	CAGTGCCCATTACCACAGCC	构建GhROP6沉默载体Construction of GhROP6 silencing vector
VIGS-GhROP6-R	AGGAAAGTGTGAGAACACAAAGGG	构建GhROP6沉默载体Construction of GhROP6 silencing vector
GhROP6-F	ATGAGTGCATCAAGGTTCATCA	扩增 GhROP6 基因 Amplification of full-length GhROP6 gene
GhROP6-R	TCACAATATCGAGCAGGCCTT	扩增 GhROP6 基因 Amplification of full-length GhROP6 gene
CA1-GhROP6-F	ACAATAATTGAAGCAAGAG	构建组成型激活的GhROP6载体 Construction of constitutively active GhROP6 vector
CA1-GhROP6-R	GTGACGTGTGCCGTCGGCAAGA
CA2-GhROP6-F	TCACTGTTGGTGACGGTGCA
CA2-GhROP6-R	ATTCCGGAGTTGAGTCATACTT
DN1-GhROP6-F	ATTACTTTCACTTTTGCAGCAT	构建组成型失活的GhROP6载体Construction of dominant negative GhROP6 vector
DN1-GhROP6-R	CGGCAAGAGAATGCATGCT
DN2-GhROP6-F	AAGGAGTGCATGCTCATCTC
DN2-GhROP6-R	ACCCTGGAGCAGTGCCCATT

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基因ID Gene ID	长度 length/bp	染色体位置 Chromosome localization	基因ID Gene ID	长度 length/bp	染色体位置 Chromosome localization
Gh_A01G1392.1	597	A01	Gh_D03G0072	591	D03
Gh_A02G0857	588	A02	Gh_D05G0243	603	D05
Gh_A05G0179	630	A05	Gh_D05G1765	588	D05
Gh_A05G1588	588	A05	Gh_D05G2437	591	D05
Gh_A06G0026	591	A06	Gh_D06G0618	714	D06
Gh_A06G0551	630	A06	Gh_D06G1409	645	D06
Gh_A06G2039	594	A06	Gh_D06G2288	591	D06
Gh_A08G0520	639	A08	Gh_D08G0612	630	D08
Gh_A08G1258	627	A08	Gh_D08G1547	627	D08
Gh_A11G1595	594	A11	Gh_D10G0271	603	D10
Gh_A12G0343	597	A12	Gh_D11G1753	564	D11
Gh_A12G2499	636	A12	Gh_D12G0319	597	D12
Gh_D01G1636	552	D01	Gh_D12G2627	666	D12
Gh_D02G0984	588	D02	Gh_Sca006742G01	591