棉花黄萎病菌VdHP1的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2020.14.011

Abstract

Abstract:

【Objective】The objective of this study is to determine the function of a novel gene (VdHP1) in Verticillium dahliae causing cotton verticillium wilt, and to provide a basis for analyzing the pathogenic mechanism of V. dahliae and the prevention and treatment of cotton verticillium wilt.【Method】The full length of VdHP1 was cloned and sequenced from V. dahliae wild-type strain V592 genomic DNA and cDNA. The relative expression of VdHP1 in V592 strain induced by cotton roots for different times and from different tissues of V592 was measured by reverse transcription quantitative real-time PCR (RT-qPCR). VdHP1 gene knockout vector, complementary vector and overexpressed vector were constructed to produce VdHP1 gene knockout strains, complementary strains and overexpressed strains by Agrobacterium tumefaciens-mediated transformation, respectively. Taking wild-type strain V592 as the control, colony growth on PDA and hypha morphology were observed, microsclerotia production, conidial production and pathogenicity to cotton of VdHP1 gene knockout mutants and complementary strains were measured. The relative expression of other genes involved in pathogenicity in VdHP1 knockout mutants and overexpressed strains was measured by RT-qPCR.【Result】The full length of VdHP1 was determined to be 862 bp and deduced protein contained 268 amino acids, which shared no significant sequence similarity to any known annotated gene in GenBank. The transcriptional expression of VdHP1 was significantly up-regulated when V592 strain induced by cotton roots for 6-12 h, indicating that VdHP1 plays a role at the early stage of the infection. The transcriptional expression of VdHP1 in conidia was significantly higher than that in mycelia and microsclerotia, indicating that VdHP1 is differentially expressed in different tissues of V. dahliae. Compared with wild-type strain V592, VdHP1 gene knockout mutants showed significantly decreased conidia and conidiophores, branching hyphae were spirally shaped, and the pathogenicity to cotton was significantly decreased. The relative expression of genes involved in penetration peg formation (VdCrz1, VdNoxB, VdPls1), delivery of secretory protein (VdSep5) and conidial production (Vdpf, VdSge1, VGB, VdPLP, VdCYC8, VdNLP1, VdNLP2) was significantly down-regulated in VdHP1 knockout mutants, but was up-regulated in the overexpressed strains; whereas, the relative expression of genes involved in melanin synthesis (VdCmr1, VdSho1, VdLAC, VdPKS1) was significantly up-regulated in VdHP1 knockout mutants, and was down-regulated in the overexpressed strains. 【Conclusion】VdHP1 is participated in the production of conidia and conidiophores, and is involved in pathogenicity in V. dahliae. VdHP1 positively regulates the transcriptional expression of genes involved in penetration peg formation, delivery of secretory protein and conidial production, and negatively regulates the transcriptional expression of genes involved in melanin synthesis.

Key words: cotton verticillium wilt, Verticillium dahliae, VdHP1, conidiation, pathogenicity, transcriptional expression

SUN Qi,HE Fang,SHAO ShengNan,LIU Zheng,HUANG JiaFeng. Cloning and Functional Analysis of VdHP1 in Verticillium dahliae from Cotton[J].Scientia Agricultura Sinica, 2020, 53(14): 2872-2884.

Figures/Tables 9

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References 33

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引物Primer	引物序列Primer sequence (5′-3′)
VdHP1 full-F	ATGCGTTTCTTCGCCTTTTT
VdHP1 full-R	TAGTCCATTCTGATCCATGT
VdHP1 up-F	CTTGCTGAGGTCTTAATTAAACCTCCAAGGCATCGTTGC
VdHP1 up- R	AGTGCTGAGGCATTAATTAACAAACGAGACGCGAATGGTG
VdHP1 down-F	CCCGCTGAGGACTTAATTAA GGATCTTGCGTCTCGTAGGT
VdHP1 down- R	CTCGCTGAGGGTTTAATTAA AGGCCATTCATTACGATGCC
HygU	AACCACGGCCTCCAGAAGAA
HygL	AGCCTGACCTATTGCATCTCCC
ECVdHP1-F	CGGCCAGTGCCAAGCTT AAATATCGTGTGGTGCGAAA
ECVdHP1-R	GCAGCTTCTGCGAATTC TTAGTCCATTCTGATCCATG
OEVdHP1-F	AATGAATATAGGCCGTCGACATGCGTTTCTTCGCCTTTTT
OEVdHP1-R	CTGCATCCGAATTCACTAGTTTAGTCCATTCTGATCCATGC
VdHP1-qPCR-F	AGAGCCAGAGGGTTCGTGGA
VdHP1-qPCR-R	ATGCGTTTCTTCGCCTTTTTACAGA

基因Gene	引物Primer	引物序列Primer sequence (5′-3′)
VdPKS1 (VDAG_00190)	VdPKS1-F	ATGGTCGGCACCATGTCTTTTCTCC
VdPKS1 (VDAG_00190)	VdPKS1-R	GCCTGTTCGAGAAAGGTCTTGGCAA
VMK1 (VDAG_09461.1)	VMK1-F	CGCAGCAACGCCCCTAATC
VMK1 (VDAG_09461.1)	VMK1-R	GGCAGTGGTCATCGGAGAGGT
VdNLP1 (VDAG_04701.1)	VdNLP1-F	TCGGTCTTTGCCCTCGTC
VdNLP1 (VDAG_04701.1)	VdNLP1-R	GCCTGGTTTGCGTTGTTC
VdNLP2 (VDAG_01995.1)	VdNLP2-F	AAGCCGTACCTCAAGGTGTTCA
VdNLP2 (VDAG_01995.1)	VdNLP2-R	CCGACCCAAAGTCCGTGTTCT
VdCYC8 (VDAG_07052)	VdCYC8-F	GGATGCCCTCGATGCTTACT
VdCYC8 (VDAG_07052)	VdCYC8-R	CGTCGCTGATCTGGTTGTTG
VGB (JQ665433.1)	VGB-F	GCAATCTCCAAACGACGTGTCG
VGB (JQ665433.1)	VGB-R	GCGAACTGACGTGTGGTGTCGG
VdSge1 (VDAG_06298.1)	VdSge1-F	CATGGATCCTTCCGAGGCATCTAG
VdSge1 (VDAG_06298.1)	VdSge1-R	GATGATGCGGGACGCTTCTGAAC
VdHog1 (VDAG_08982)	VdHog1-F	CTTCCACGTGTCTACTGGCAGG
VdHog1 (VDAG_08982)	VdHog1-R	TGCTCCTTACCACGACCTTACCGA
VdNoxB (VDAG_09930)	VdNoxB-F	TGCGTGGCAAGCATAAGACATAC
VdNoxB (VDAG_09930)	VdNoxB-R	GACAGCACGAGTGAAATCACCAAC
VdPls1 (VDAG_01769)	VdPls1-F	ATGGTCAACAAGATCCTCGCGA
VdPls1 (VDAG_01769)	VdPls1-R	TCCGGCTGCTCAAACATGTTGT
VdSep5 (VDAG_04382)	VdSep5-F	AGCTCGACCTGGACGAGGA
VdSep5 (VDAG_04382)	VdSep5-R	GAGGCTTCGTTATCAATCTCGTCTC
VdSho1 (VDAG_01836)	VdSho1-F	GAGATAACCCAAAGGGCCATGGG
VdSho1 (VDAG_01836)	VdSho1-R	GAGAGCGTATCCAATCGCACC
VdCrz1 (VDAG_03208)	VdCrz1-F	ATGGATCAGCAAGCTCAACATCG
VdCrz1 (VDAG_03208)	VdCrz1-R	GATCCAGACCGAGACCGAGAC
VdLAC (VDAG_00189)	VdLAC-F	ATGCTCTTCTCGCGTTTCCTCA
VdLAC (VDAG_00189)	VdLAC-R	GCCACTGACCATTGATGCCAAT
VdCmr1 (VDAG_00195)	VdCmr1-F	GCGCCACAAGCTCTGCATCTTC
VdCmr1 (VDAG_00195)	VdCmr1-R	CAGAATCAAGGTGGCGCGATACAC
VdPLP (VDAG_00942)	VdPLP-F	GCTGACCAGTATCTGTCGGAGG
VdPLP (VDAG_00942)	VdPLP-R	ATGACGACTGGCTTCTCGGCCT
Vdpf (VDAG_08521.1)	Vdpf-F	ACCATTTTCAACAGTCGGGTACGCG
Vdpf (VDAG_08521.1)	Vdpf-R	GTGTGACGTACCAGCAACCGCTT
β-tubulin (DQ266153)	β-tubulin-F	TCACCAGCCGTGGCAAGGTTG
β-tubulin (DQ266153)	β-tubulin-R	AGCAAAGGGCGGTCTGGACGTTG

Cloning and Functional Analysis of VdHP1 in Verticillium dahliae from Cotton

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