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

摘要/Abstract

摘要：

【目的】明确棉花黄萎病菌（大丽轮枝菌Verticillium dahliae）中一个新基因（VdHP1）的功能,为解析棉花黄萎病菌的致病机制以及棉花黄萎病的防治提供依据。【方法】以大丽轮枝菌野生型菌株V592的基因组DNA和cDNA为模板,对VdHP1全长进行克隆并测序;利用逆转录实时荧光定量PCR（RT-qPCR）分别对棉花根系诱导不同时间VdHP1的表达量及V592菌株不同组织中VdHP1的表达量进行测定;构建针对VdHP1的敲除载体、互补载体和过表达载体,通过农杆菌介导的遗传转化筛选VdHP1基因敲除突变体、互补菌株和过表达菌株;以野生型菌株V592为对照,对VdHP1基因敲除突变体及互补菌株的菌落及菌丝形态进行观察,并对微菌核量、产孢量及致病力进行测定;通过RT-qPCR测定其他致病力相关的基因在VdHP1基因敲除突变体及过表达体菌株中的表达情况。【结果】VdHP1全长为862bp,预测编码蛋白含268个氨基酸,与GenBank中已注释的基因没有任何的序列相似性。野生型菌株V592受棉花根系诱导6—12 h时VdHP1表达水平显著上调,表明VdHP1在大丽轮枝菌侵染早期发挥作用。VdHP1在分生孢子中的表达量显著高于在菌丝和微菌核中的表达量,表明VdHP1在大丽轮枝菌不同组织中的表达具有差异性。与野生型菌株V592相比,VdHP1基因敲除突变体产孢量和产孢梗显著减少,菌丝分支呈螺旋状,对棉花的致病力明显下降。与侵染钉形成相关基因（VdCrz1、VdNoxB、VdPls1）、分泌蛋白释放相关基因（VdSep5）及分生孢子产生相关基因（Vdpf、VdSge1、VGB、VdPLP、VdCYC8、VdNLP1、VdNLP2）在VdHP1基因敲除体中的相对表达量显著下调,在过表达菌株中上调;而与黑色素合成相关基因（VdCmr1、VdSho1、VdLAC、VdPKS1）在VdHP1基因敲除突变体中则显著上调,在过表达菌株中下调。【结论】VdHP1与大丽轮枝菌分生孢子和产孢梗的产生有关,参与大丽轮枝菌致病;VdHP1对与侵染钉形成、分泌蛋白释放及分生孢子产生相关基因的表达具有正调控作用,对黑色素合成相关基因的表达具有负调控作用。

关键词: 棉花黄萎病, 大丽轮枝菌, VdHP1, 产孢, 致病力, 转录表达

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

孙琦,何芳,邵胜楠,刘政,黄家风. 棉花黄萎病菌VdHP1的克隆及功能分析[J]. 中国农业科学, 2020, 53(14): 2872-2884.

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.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2020.14.011

https://www.chinaagrisci.com/CN/Y2020/V53/I14/2872

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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