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

摘要/Abstract

摘要： 【目的】分析稻曲病菌(Ustilaginoidea virens)致病力减弱的T-DNA插入突变菌株B1241的生物学性状和致病力，并结合分子生物学手段研究其T-DNA插入位点的侧翼基因，以解析突变基因在稻曲病菌生长和致病过程中的作用，从而为阐明稻曲病菌致病机制提供理论基础。【方法】以野生菌株P1作为对照，观察并检测突变菌株B1241的菌落形态、生长速率、孢子形态、产孢量等生物学性状；采用注射接种的方法将菌丝和孢子的混合液接种于水稻穗苞中，统计每穗发病的病粒数，分析B1241的致病性变化；突变菌株B1241在不含有潮霉素的PSA平板上转接5代之后，PCR检测T-DNA插入的稳定性并通过Southern杂交分析B1241中T-DNA插入的拷贝数；利用HiTail-PCR获得T-DNA插入位点的侧翼序列，经NCBI比对得到侧翼基因；运用RACE-PCR克隆插入位点侧翼基因全长；qRT-PCR分析侧翼基因的表达情况。【结果】经生物学特性观察及田间接种发现，与野生菌株P1相比，突变菌株B1241在固体培养基MM、PSA和TB3上的菌落和孢子形态以及生长速率无显著差异，其致病力、产孢能力均呈极显著下降。B1241在不含潮霉素的PSA平板上转接5代之后，仍能扩增到GFP和HPH基因，说明T-DNA已经稳定地插入到其基因组中。Southern杂交结果显示T-DNA在该突变菌株中以单拷贝的形式插入。经扩增并比对侧翼序列，T-DNA的插入位点处少了28 bp的稻曲序列，有37 bp在T-DNA及稻曲病菌基因组中都没有比对到。NCBI比对发现，侧翼基因Uvt-1241与UV-8b菌株的UV8b-7878基因同源，开放阅读框长2 317 bp，包含81和106 bp的2个内含子，编码709个氨基酸。经RACE-PCR获得基因全长2 650 bp，5'非编码区长度为14 bp，3'非编码区长度为319 bp。T-DNA插入在基因Uvt-1241的启动子区域，位于起始密码子之前516 bp处。qRT-PCR分析结果表明，Uvt-1241在该突变体中表达量下降。该基因编码一个糖基水解酶18家族的蛋白，同时含有一个保守结构域D××D×D×E。【结论】稻曲病菌突变菌株B1241中，T-DNA插入到基因Uvt-1241的启动子区域，从而导致该启动子功能部分缺失，基因表达量下降，使得突变菌株的生长、产孢等生物学特性及致病力发生改变，由此推测该基因可能在稻曲病菌生长及致病过程中起着重要的作用。

关键词: 稻曲病菌, T-DNA, 致病力, 糖基水解酶18家族, 基因克隆

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

薄惠文，俞咪娜，于俊杰，尹小乐，丁慧，王亚会，刘永锋. 稻曲病菌T-DNA插入突变菌株B1241侧翼基因克隆[J]. 中国农业科学, 2016, 49(9): 1685-1695.

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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