棉花抗枯萎病相关基因GhERF5-4D的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1065

摘要/Abstract

摘要：

研究陆地棉ERF（ethylene-responsive factor）转录因子基因GhERF5-4D在棉花抗枯萎病中的作用,为棉花抗病分子机制及棉花抗病育种研究奠定基础。以枯萎病菌诱导棉花根部基因表达谱中的差异表达基因为探针,结合棉花全基因组数据库获得1个ERF转录因子基因GhERF5-4D,并进一步利用实时荧光定量（qRT-PCR）技术和病毒诱导基因沉默（virus induced gene silencing,VIGS）技术研究枯萎病菌胁迫和激素处理后基因GhERF5-4D的表达特征及其在棉花抗病过程中的作用。结果表明,GhERF5-4D（GenBank：MF093148）位于陆地棉D07染色体上,开放阅读框（open reading frame,ORF）为663 bp,编码220个氨基酸,仅含1个AP2结构域且无内含子,属于ERF亚族。该基因的表达特征在棉花抗感枯萎病品种及不同激素胁迫处理中存在显著差异,其在抗病品种中为上调表达,在感病品种中为下调表达;乙烯、茉莉酸甲酯处理后,该基因上调表达,而水杨酸处理后则下调表达。GhERF5-4D基因沉默后并进行枯萎病菌接种实验,结果表明,与对照相比,沉默株系更感病。对下游抗病相关防卫基因表达特征进行分析,发现PR2、PR4、PR5、NPR1、ERF1相对表达量均低于对照,而WRKY70相对表达量高于对照。GhERF5-4D能够响应枯萎病菌胁迫,并通过参与乙烯、茉莉酸途径调控下游相关抗病防卫基因提高棉花对枯萎病抗性。

关键词: 棉花, 枯萎病菌, ERF转录因子, GhERF5-4D, 表达分析, 功能研究

Abstract:

To study the role of ERF (ethylene responsive factor) transcription factor gene GhERF5-4D in cotton Fusarium oxysporum resistance may lay a foundation for the study of cotton disease resistance molecular mechanism and cotton disease resistance breeding.Firstly,the differentially expressed genes obtained from the gene expression profile in cotton root tissues induced by F. oxysporum were employed as probes,based on the whole genome database of upland cotton,the ERF transcription factor gene GhERF5-4D was selected as a candidate gene. Then,the expression characteristics of GhERF5-4D under F. oxysporum and multiple hormones treatment,and its roles in cotton disease resistance were investigated via qRT-PCR and virus induced gene silencing （VIGS）techniques. GhERF5-4D gene（GenBank accession number ：MF093148）,located on the chromosome D07 of upland cotton,its open reading frame（ORF）was 663 bp encoding 220 amino acids. There was only one AP2 domain in its coding regions without any introns and belonged to the ERF subfamily. Its expression patterns in resistant and susceptible cotton cultivars and inoculation by hormones were significantly distinct,which was up-regulated in resistant varieties and down-regulated in susceptible cultivars,up-regulated after ethylene and methyl jasmonate treatment and down-regulated after salicylic acid tratment. When GhERF5-4D gene was silenced by VIGS and then inoculated with F. oxysporum, the silenced strains showed more susceptible to disease than the control. Moreover,the relative expressions of PR2, PR4, PR5, NPR1 and ERF1 genes were lower than those of control groups,while,the relative expression of WRKY70 gene was higher than that of the control. Thus,GhERF5-4D gene was confirmed to respond to F. oxysporum stress and improve cotton resistance to F. oxysporum by regulating downstream defense genes through ethylene and jasmonic acid pathway.

Key words: cotton, Fusarium oxysporum, ERF transcription factor, GhERF5-4D, expression analysis, functional study

赵曾强, 郭文婷, 张析, 李潇玲, 张薇. 棉花抗枯萎病相关基因GhERF5-4D的克隆及功能分析[J]. 生物技术通报, 2022, 38(4): 193-201.

ZHAO Zeng-qiang, GUO Wen-ting, ZHANG Xi, LI Xiao-ling, ZHANG Wei. Cloning and Functional Analysis of GhERF5-4D Gene Related to Fusarium oxysporum Resistance in Cotton[J]. Biotechnology Bulletin, 2022, 38(4): 193-201.

图/表 8

表1 引物列表

Table 1 Table of primers

引物名称Primer name	引物序列Primer sequence（5'-3'）
KpnI-GhERF5-4D-F	GGGGTACCATGGCTTCTTTCGGAGA
BamHI-GhERF5-4D-R	CGGGATCCCATAACGGCGAGTCCTG
qGhERF5-4D-F	AATCCACCCTAAGCCAACGTC
qGhERF5-4D-R	CTTTTCTCGTCATGTTCCTCGAT
GhUBQ7-F	GAAGACCTACACCAAGCCCAA
GhUBQ7-R	CGGACTCTACTCAATCCCCACC
XbaI-GhERF5-4D-CO-F	GCTCTAGATCCCACCCACAAGTTTTAACAT
KpnI-GhERF5-4D-CO-R	CGGGTACCCTCCTTTTCCCCGACTCTG
GhERF1-F	CTTTAGATCACAACTCGCTTC
GhERF1-R	TTTTCTTTCGTCGGGCTT
GhPR1-F	TTTAGTAAGGTTTTTGACCGACGAA
GhPR1-R	GACTCTGTCCATCTTGGTTGTGCTA
GhPR2-F	TTCAAGGTTTGCACTCGGAAGA
GhPR2-R	CCACCAGCAGCAGAAGTTATCG
GhPR4-F	CTTGATATAAGATTGGTTAGCCC
GhPR4-R	TTTGCTTTTGTTGTATGTAGTCG
GhPR5-F	TTGGCTCTTACTTCCGACCATCT
GhPR5-R	GCCGTGATTCATACAGTTATCCTCA
GhWRKY70-F	TGGCAACATACGAAGGACAACACA
GhWRKY70-R	CTTCGCTCGTCCCGGAAAATATCGC
GhNPR1-F	CACGTGGTGCTGTTGTTGTTACTG
GhNPR1-R	GCGAATCGTTGCTTTCTTCTTCA

图1 GhERF5-4D的PCR扩增及序列分析 A：GhERF5-4D的cDNA序列及推定氨基酸序列（方框代表起始密码子ATG和终止密码子TAA,*为终止密码子,红色框为保守域结构域）;B：GhERF5-4D的PCR扩增产物;C：GhERF5-4D酶切验证;D：GhERF5-4D蛋白结构域分析;E：GhERF5-4D启动子序列分析

Fig.1 PCR amplification and sequence analysis of GhERF5-4 D gene A：The cDNA sequence and the putative amino acid sequence of the GhERF5-4D（The square frame refers to initiation codon ATG and termination codon TAA,* indicates termination codon,and red square frame indicates conserved domain）. B：PCR amplification result of GhERF5-4D. C：Enzyme digesting verification of GhERF5-4D. D：Analysis of structure domain of GhERF5-4D protein. E：Sequence analysis of GhERF5-4D gene promoter

图2 棉花中枯萎病菌诱导下GhERF5-4D的表达模式 A：中棉所12;B：新陆早7。不同字母表示P=0.05水平时差异显著,下同

Fig.2 Expression pattern of GhERF5-4 D gene after treat by Fusarium oxysporum in cotton A：Zhongmiansuo 12. B：Xinluzao 7. Different letters indicate significant difference at P = 0.05 level. The same below

图3 不同激素处理下GhERF5-4D的表达模式

Fig.3 Expression pattern of GhERF5-4 D gene after the treatment of different hormones

图4 pTRV2-GhERF5-4D-CO载体的构建 A：GhERF5-4D-CO片段的扩增;B：GhERF5-4D-CO片段的酶切验证; C：pTRV2-GhERF5-4D-CO双酶切验证;D：pTRV2-GhERF5-4D-CO菌液PCR验证;M：DL2000

Fig.4 Vector construction of pTRV2-GhERF5-4D-CO A：PCR amplification of GhERF5-4D-CO. B：Enzyme digestion result of GhERF5-4D-CO. C：Enzyme digesting verification of pTRV2-GhERF5-4D-CO. D：PCR amplification result of pTRV2-GhERF5-4D-CD of agrobacterium. M：DL2000

图5 GhERF5-4D沉默效率与叶片漂白情况 A：注射GhCLA1后叶片漂白情况;B：GhERF5-4D沉默效率。**表示P=0.01水平差异显著

Fig.5 GhERF5-4D gene silencing efficiency and leaf bleaching A：Leaf bleaching after the injection of GhCLA1 gene. B：GhERF5-4D gene silencing efficiency. ** indicates significant difference at P=0.01

图6 GhERF5-4D沉默后接菌处理 A：沉默植株接菌12 d后的表型;B：沉默植株接菌20 d后的表型;C：接菌20 d后棉花茎段在PDA培养基培养

Fig.6 GhERF5-4 D gene silencing followed by bacterial treatment A：The incidence phenotype of silencing plants at 12 d. B：The incidence phenot-ype of silencing plants at 20 d. C：20 d stem sections were plated on PDA medium

图7 与抗病相关防卫基因表达特征分析

Fig.7 Expression characteristics of defense genes related to disease resistance

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