稻曲病菌T-DNA插入突变菌株B1241侧翼基因克隆

doi:10.3864/j.issn.0578-1752.2016.09.005

Abstract

Abstract: 【Objective】The objective of this study is to explore the function of pathogenicity-relative gene basing on the phenotypes and T-DNA integration flanking sequence of a mutant strain B1241, and to provide a theory foundation for illustrating the pathogenic mechanism of Ustilaginoidea virens.【Method】 With the wild type strain P1 as the control, biological phenotypes of B1241 were analyzed. Artificial inoculation of mixtures of hyphae broken and conidia was made by injecting at the leaf sheath of the rice plant seven days before booting stage. Number of diseased grain per panicle was counted until false smut balls developed. After five generations of mutant strain B1241 inoculated on the PSA without hygromycin, the stability of T-DNA insertion was detected. Southern blot was used to identify the copy number of T-DNA in B1241. The flanking sequence of T-DNA was obtained by HiTail-PCR and the full length of the flanking gene was cloned by RACE-PCR. The gene expression was detected by quantitative RT-PCR.【Result】There was no significant difference in colony morphology and growth rate between B1241 and P1 culturing on MM, PSA and TB3. A field inoculation trials showed that the pathogenicity of B1241 was significantly declined compared with P1. After five generations of mutant strain B1241 inoculated on the PSA without hygromycin, gene GFP and HPH were still amplified, which indicated that T-DNA had been stably inserted into the genome of B1241. Genomic southern blot analysis confirmed that B1241 is a single T-DNA insertional event. Analysis of flanking sequence indicated that there were 28 bp sequences of U. virens loss in T-DNA insertion site and 37 base pairs were not found in T-DNA sequences and U. virens genome. Sequences analysis indicated that full length of flanking gene was 2 650 bp with a 14 bp of 5′ untranslated regions and a 319 bp of 3′ untranslated regions. The cloned flanking gene homologous with UV8b-7878 in strain UV-8b encodes a glycoside hydrolase family 18 protein which has a conserved domain D××D×D×E. The T-DNA insertion into the promoter region of Uvt-1241 led to lower transcription levels of gene Uvt-1241 in this mutant strain.【Conclusion】The mutant strain B1241 showed great changes on the conidia production and pathogenicity, probably due to inserted T-DNA in the promoter region of the gene. These results implied that gene Uvt-1241 might played an important role during the growth and pathogenic process of U. virens.

Key words: Ustilaginoidea virens, T-DNA, pathogenicity, glycoside hydrolase family 18 protein, gene clone

BO Hui-wen, YU Mi-na, YU Jun-jie, YIN Xiao-le, DING Hui, WANG Ya-hui, LIU Yong-feng. Molecular Cloning Flanking Sequences of T-DNA Insertion from the Ustilaginoidea virens Mutant Strain B1241[J].Scientia Agricultura Sinica, 2016, 49(9): 1685-1695.

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