稻曲病菌分生孢子高产突变体A2588的T-DNA 插入位点侧翼基因分析

doi:10.3864/j.issn.0578-1752.2013.24.007

摘要/Abstract

摘要： 【目的】克隆稻曲病菌（Ustilaginoidea virens）分生孢子高产突变菌株A2588的T-DNA插入突变基因，解析该基因在稻曲病菌分生孢子形成中的功能，为进一步揭示稻曲病菌的分生孢子形成机制提供理论基础。【方法】测定A2588的产分生孢子能力、孢子萌发率、菌丝直线生长能力和热敏感性等生物学特性，利用hiTAIL-PCR、RACE和半定量PCR等方法克隆T-DNA插入突变基因，并结合生物信息学方法分析T-DNA插入导致分生孢子产量提高的分子机理。【结果】与稻曲病菌野生型菌株70-22相比，突变菌株A2588在PS培养液中产生数量为10倍以上的分生孢子，在MM培养液中产生数量约20倍的分生孢子，其在PS培养液中培养的菌球较为致密；在PSA和MM固体培养基上A2588产孢周期较短，孢子萌发率较低，虽然菌丝生长速度无明显差异，但热处理后菌丝不能恢复正常生长速率。hiTAIL-PCR和RACE法克隆了A2588中T-DNA侧翼基因，并利用RT-PCR检测侧翼基因表达水平，结果表明T-DNA侧翼基因spo76表达水平下降，进一步分析发现T-DNA可能插入至spo76的启动子区域，从而破坏了该基因启动子的部分功能。【结论】稻曲病菌突变菌株A2588中，T-DNA插入导致spo76启动子功能部分缺失，spo76表达水平下降，可能使A2588产孢周期缩短，并产生更多分生孢子。

关键词: 稻曲病菌 , 分生孢子 , ATMT转化 , spo76

Abstract: 【Objective】The objective of this study is to clone the mutated gene, which causes enhanced conidiation of the T-DNA insertional mutant A2588, and to shed light on the conidiation mechanism in Ustilaginoidea virens.【Method】The sporulation, conidial germination, mycelial growth rate and heat tolerance of the mutant A2588 were determined, and wild-type strain 70-22 was employed as the control. hiTAIL-PCR and RACE were used to identify the insertional T-DNA flanking genes in A2588, and RT-PCR were applied to analyzed the expression level of the T-DNA flanking genes as well. Additionally, the function of T-DNA insertion region in the genome of A2588 was bioinformatically predicted. 【Result】Compared to the wild-type U. virens strain 70-22, the mutant A2588 produced more than 10 folds of conidia in PS broth and approximately 20 folds of conidia in MM liquid medium, and formed more compact mycelial balls in PS. Despite of having an equivalent mycelial growth rate to 70-22, A2588 exhibited a shorter sporulation cycle, a lower conidia germination rate and slower recovery after heat treatment on the PSA and MM solid media. The T-DNA in mutant A2588 was found inserting into the promoter region of spo76 and reduced expression level of this gene. 【Conclusion】 Due to the partial damage of the spo76 promoter, the spo76 expression level in mutant A2588 was reduced and sporulation cycle of A2588 was accelerated. This may result in enhanced conidiation of U. virens mutant A2588.

Key words: Ustilaginoidea virens , conidia , ATMT transformation , spo76

于俊杰, 聂亚锋, 俞咪娜, 尹小乐, 胡建坤, 黄磊, 陈志谊, 刘永锋. 稻曲病菌分生孢子高产突变体A2588的T-DNA 插入位点侧翼基因分析[J]. 中国农业科学, 2013, 46(24): 5132-5141.

YU Jun-Jie, NIE Ya-Feng, YU Mi-Na, YIN Xiao-Le, HU Jian-Kun, HUANG Lei, CHEN Zhi-Yi, LIU Yong-Feng. Characterization of T-DNA Insertion Flanking Genes of Enhanced-Conidiation Ustilaginoidea virens Mutant A2588[J]. Scientia Agricultura Sinica, 2013, 46(24): 5132-5141.

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