Cloning and Functional Analysis of GhERF5-4D Gene Related to Fusarium oxysporum Resistance in Cotton

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

Abstract

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

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.

Figures/Tables 8

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

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

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

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

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

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

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

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

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