Construction and Preliminary Analysis of Verticillim dahliae Mutant Library

doi:10.13560/j.cnki.biotech.bull.1985.2022-1247

Abstract

Abstract:

In order to rapidly screen the key pathogenic genes of Verticillim dahliae, the random insertion mutant library was constructed by polyethylene glycol-mediated protoplast transformation. The growth phenotype and pathogenicity of partial positive transformants were analyzed to screen the growth, development and pathogenic defect mutants, and the target gene was located. The results showed that more than 13 030 positive transformants were obtained in this study, and five positive transformants were randomly selected. The colony diameter and spore production on PDA and different carbon source medium as well as pathogenicity of one mutant significantly decreased compared with V991 wild type strain. The results of flanking sequence sequencing and Blast alignment analysis demonstrated that the hygromycin resistance gene expression cassette in the defective mutant was located at 1 528 782 bp of chromosome 3 of V. dahliae, belonging to endoglucanase 1 gene（VDAG_04017）. Taken together, this study can preliminarily identify pathogenicity-related genes through a series of molecular biological technique, including the construction of insertion mutant library of V. dahliae, identification of the growth phenotypic indices and pathogenicity, and mapping of target genes so on, which lays a foundation for investigating the pathogenic mechanism of V. dahliae at the whole genome level.

Key words: Verticillium dahliae, polyethylene glycol mediated protoplast transformation, pathogenicity-related genes, pathogenicity, flanking sequence analysis

PAN Guo-qiang, WU Si-yuan, LIU Lu, GUO Hui-ming, CHENG Hong-mei, SU Xiao-feng. Construction and Preliminary Analysis of Verticillim dahliae Mutant Library[J]. Biotechnology Bulletin, 2023, 39(5): 112-119.

Figures/Tables 7

Table 1 Primer sequences for detection of biomass of V. dahliae in Nicotiana benthamiana root by qPCR

引物名称Primer name	序列 Sequence（5'-3'）
VdrDNA-qPCR-F	CCGCCGGTCCATCAGTCTCTCTGTTTATAC
VdrDNA-qPCR-R	CGCCTGCGGGACTCCGATGCGAGCTGTAAC
NbActin-qPCR-F	GGCTTCCTCAAGGTCGGCTATG
NbActin-qPCR-R	GCTGCATGTCATCCCACTTCTTC

Table 2 Primer sequences for the amplification of flanking sequences at insertion site

引物名称 Primer name	序列 Sequence（5'-3'）
AP1	Provided by Takara Genome Walking Kit, unknown sequence
SP1	ATCGTTGGTGTCGATGTCAGCTCC
SP2	GCGTTTCGGGTTTACCTCTTCCAG
SP3	CGAGATCAAGCAGATCAACGGTCG

Fig. 1 Schematic diagram and PCR amplification of hygromycin resistance gene expression cassette in pUC-Hyg A: Schematic diagram of hygromycin resistance gene expression cassette in pUC-Hyg. B: PCR amplification of hygromycin resistance gene expression cassette fragment. M: DNA marker. 1-4: Hygromycin resistance gene expression cassette fragment. N: Negative control（ddH2O）

Fig. 2 PCR identification of randomly inserted mutants of V. dahlia M: DNA marker. 1-5: PCR identification products of transformant 1-5 genomic DNA. WT: PCR identification products of V991 genomic DNA. N: Negative control（ddH2O）

Fig. 3 Colony growth phenotype of randomly inserted mutants on PDA and different carbon source medium A: Colony morphology of V. dahliae V991 and 5 randomly inserted mutants were cultured on PDA and different carbon source medium for 12 d. B: Colony diameters of V. dahliae V991 and 5 randomly inserted mutants were cultured on PDA and different carbon source medium for 12 d. C: Spore production of V. dahliae V991 and 5 randomly inserted mutants cultured on PDA and different carbon source medium for 12 d. Error bars indicate the standard deviations and different letters indicate significant differences at P<0.05

Fig. 4 Pathogenicity of randomly inserted mutants of V. dahliae to N. benthamiana A: Disease incidence of N. benthamiana plants after inoculation with V. dahliae V991 and 5 randomly inserted mutants for 12 d. Mock: N. benthamiana plants that were not inoculated with V. dahliae. B: Disease index of N. benthamiana plants after inoculation with V. dahliae V991 and 5 randomly inserted mutants for 12 d. C: Fungal biomass in the root tissues of N. benthamiana after inoculation with V. dahliae V991 and 5 randomly inserted mutants for 12 d. Error bars indicate the standard deviations and different letters indicate significant differences at P<0.05

Fig. 5 Flanking sequence analysis of insertion site for randomly inserted mutant M5 A: Flanking sequence detection of the insertion site of hygromycin resistance gene expression cassette fragment（M: DNA marker. 1-9: three rounds of PCR reaction amplification products）. B: Schematic diagram of insertion site of hygromycin resistance gene expression cassette fragment

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