Crops ›› 2023, Vol. 39 ›› Issue (3): 246-253.doi: 10.16035/j.issn.1001-7283.2023.03.034

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Functional Analysis of SRRM1-Like Transcription Factor of Magnaporthe grisea

Qiu Kaihua(), Fang Shumei, Liang Xilong()   

  1. Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang, China
  • Received:2021-11-24 Revised:2022-02-17 Online:2023-06-15 Published:2023-06-16

Abstract:

Transcription factors played an important role in the growth and development, adversity adaptation and pathogenicity of Magnaporthe grisea. To clarify the relationship between pre-mRNA splicing regulated by transcription factor SRRM1-like and the growth and adversity adaptation of M.grisea, the SRRM1-like gene- deficient strains were obtained by gene-specific knockout technology based on the principle of gene recombination, and analyzed for vegetative growth and adversity adaptation. The results showed that the knockout vector was successfully constructed and the SRRM1-like gene in the rice blast fungus Y34 strain was knocked out, and three gene deletion mutants were obtained. The deletion of the SRRM1-like gene did not significantly affect the biotrophic growth of M.grisea, but it was sensitive to stress by Congo-Red, SDS, sorbitol, NaCl and the fungicide pyraclostrobin, and showed weakened stress resistance. SRRM1-like played an important role in the adversity adaptation of M.grisea.

Key words: Magnaporthe grisea, Splicing, PWI domain, Bioinformatics analysis, Gene knockout

Table 1

Primer information for knockout vector construction and mutant verification"

引物名称
Primer name
引物序列
Primer sequence (5′→3′)
产物长度
Product length (bp)
酶切位点
Restriction site
M-0-L-S GAATTCTATCCGAAACAAACCTGAG 1152 EcoR I
M-0-L-A GAGCTCGGAGATCGGGTTGTTGTAG Sac I
M-0-R-S GGATCCCTACAACATCCGCCCTTAC 1305 BamH I
M-0-R-A AAGCTTGTCTGAGCCTTCGGAACAT Hind III
M-0-G-S AGGCTGGATGTCACGCTAA 653 -
M-0-G-S ATTTCGACGATGCAGGAGA -
Hyg-S GCCCTTCCTCCCTTTATT 753 -
Hyg-A TGTTGGCGACCTCGTATT -
M-0-L-H-S TACATCAGCACCCAAGGC 2044 -
M-0-L-H-A GCTATTTACCCGCAGGAC -
M-O-H-R-S CCGTGGTTGGCTTGTATG 2193 -
M-O-H-R-A CGGGCTCTAACCTCCAGTA -

Fig.1

The spatial structure of SRRM1-like protein (a) the secondary structure of SRRM1-like protein; (b) the domain of SRRM1-like protein; (c) the tertiary structure of SRRM1-like protein"

Fig.2

Evolutionary relationship and structural analysis of SRRM1-like protein"

Fig.3

PCR amplification of left and right arms of M-0 gene"

Fig.4

Electropherogram of pXEH20, P-L, P-LR plasmid P is the knockout vector pXEH20; P-L is the recombinant vector connecting the left arm of the gene; P-LR is the recombinant vector connecting the left and right arms of the gene"

Fig.5

Schematic diagram and verification of construction of knockout vector of M-0 gene (a): knockout vector pXEH20-LR construction and recombination diagram; (b): knockout vector verification electropherogram, track one and three are water controls, track two and four are the left and right arms of the M-0 gene, respectively"

Fig.6

Electropherogram of the verified M-0 transformant target genes Track one is the wild-type control; track two is the water control; track three, four, and five are the Hyg in mutants M9, M11, and M17; track six, seven, and eight are the gene in the mutants M9, M11, and M17; track nine is water control; track ten is wild-type control"

Fig.7

Electropherogram of the verified M-0 transformant flank sequences Track one is the wild-type control; track two is the water control; track three, four, and five are the M-0-L-H in mutants M9, M11, and M17; track six, seven, and eight are the M-0-H-R in the mutants M9, M11, and M17; track nine is water control; track ten is wild-type control"

Fig.8

Effects of gene knockout on the vegetative growth and stress adaptation of Magnaporthe grisea Different lowercase letters indicate significant difference at 0.05 level"

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