禾谷镰孢碳源代谢调控因子FgCreA的功能

doi:10.3864/j.issn.0578-1752.2018.02.006

摘要/Abstract

摘要： 【目的】敲除禾谷镰孢（Fusarium graminearum）中碳源代谢调控因子FgCreA，并对其营养生长、有性生殖和致病力等方面进行研究，为研究禾谷镰孢中碳源代谢机制提供依据。【方法】根据酵母数据库SGD和NCBI数据库中的序列，利用酿酒酵母中碳源代谢调控因子Mig1确定禾谷镰孢中的碳源调控因子FgCreA；在NCBI数据库中检索其他物种中碳源代谢调控蛋白，使用ClustalW2软件进行多重序列比对，并用MEGA5软件构建系统进化树，同时在InterProScan网站上预测其蛋白结构域；在NCBI数据库中检索与禾谷镰孢碳源吸收相关的结构基因和真菌毒素DON生物合成的相关基因，调取起始密码子上游1 000 bp的片段，预测FgCREA基因结合位点位置；用Primer5软件设计引物，利用Split-PCR和PEG介导原生质体转化的方法进行基因敲除，并通过PCR和Southern blot验证获得FgCREA基因敲除突变体Fgcrea。根据突变体Fgcrea营养生长、有性生殖和致病力等方面的变化分析FgCREA的功能。【结果】通过生物信息学方法，明确禾谷镰孢中只有一个碳源代谢调控因子基因FgCREA(FGSG_09715)，氨基酸序列416 aa，共包含两个保守的C2H2锌指结构区域。FgCreA同源蛋白在各类真菌中均存在一定程度的同源性，具有较高的保守性。禾谷镰孢碳源吸收相关结构基因（XYL2、ARA1、ICL1、PG1、SUC2）和真菌毒素DON生物合成相关基因（TRI1、TRI3、TRI4、TRI5、TRI6、TRI7、TRI8、TRI10、TRI12、TRI101）启动子区均含有FgCREA DNA结合位点。采用Split-PCR技术和PEG介导的原生质体转化，通过PCR和Southern blot获得并验证了两个FgCREA基因敲除突变体。表型观察发现，突变体Fgcrea的生长速度与野生型相比下降90%；分生孢子形态正常，但产孢量与野生型相比降低88%；有性生殖阶段，突变体Fgcrea可以产生正常的子囊壳、子囊和子囊孢子，但需要比野生型长20—28 d；突变体Fgcrea对钠盐较敏感，侵染小麦穗不发病。【结论】获得禾谷镰孢碳代谢调控因子FgCreA敲除突变体，明确FgCreA参与营养生长和有性生殖，并能够影响致病过程。但是否影响相关结构基因的转录水平和DON生物合成仍需进一步验证。

关键词: 禾谷镰孢, FgCREA, 基因敲除, 表型, 碳源代谢

Abstract: 【Objective】The objective of this study is to knock out the FgCreA of Fusarium graminearum, which is the carbon source metabolic regulation factor, to research the vegetative growth, sexual reproduction and pathogenicity of the knock out mutant, and to provide a basis for carbon source metabolic mechanism of F. graminearum. 【Method】 According to the budding yeast carbon source regulation factorMig1gene sequence on SGD and NCBI databases, the carbon source regulation factor FgCreA in F. graminearum was determined. the sequences of FgCreA orthologs of other species were retrieved from NCBI database. multiple sequence alignment of CreA orthologs was performed with ClustalW2 software, and phylogenetic tree was constructed by MEGA5 software to determine their evolutionary relationships. The protein properties of FgCreA were predicted via InterProScan. The promotor regions of the carbon absorption structure genes and DON biosynthesis genes were obtained from the NCBI database, then the potential FgCreA binding sites in these promotor regions were analyzed. Primer5 software was used to design primers, the FgCREA gene knockout construct was generated by Split-PCR and then introduced into the wild type strain PH-1 by PEG-mediated protoplast transformation. PCR and Southern blot were used to confirm the FgCREA gene deletion mutants (Fgcrea). The function of FgCREA was analyzed according to the vegetative growth, sexual reproduction and pathogenicity of the Fgcrea mutants. 【Result】There is only one carbon metabolism regulator gene FgCREA (FGSG_09715) in F. graminearum. The 416-amino acid protein encoded by FgCREA has two conserved C2H2 zinc finger regions. FgCreA homologue proteins have a certain degree of homology and are highly conserved in fungi. The promoter regions of the carbon absorption structure genes (XYL2, ARA1, ICL1, PG1, SUC2) and DON biosynthesis genes (TRI1, TRI3, TRI4, TRI5, TRI6, TRI7, TRI8, TRI10, TRI12, TRI101) contained FgCreA DNA binding sites. Two FgCREA gene knockout mutants were obtained and screened by PCR and Southern blot. The growth rate of Fgcrea mutants was 90% lower than that of wild type. The conidia morphology of mutants was normal, but the conidiation decreased by 88% compared with the wild type. In the sexual reproduction stage, the Fgcrea mutants could produce normal perithecia, ascus and ascospore, but it delayed 20-28 d longer than the wild type. The Fgcrea mutants were sensitive to salt stress, and were almost non-pathogenic on wheat. 【Conclusion】The carbon metabolic regulator FgCreA plays important roles in vegetative growth and sexual reproduction, and is essential for plant infection in F. graminearum. However, whether FgCreA affects transcription of the carbon absorption structure genes and DON biosynthesis genes remains to be verified.

Key words: Fusarium graminearum, FgCREA, gene knockout, phenotype, carbon metabolism

侯瑞，王晨芳. 禾谷镰孢碳源代谢调控因子FgCreA的功能[J]. 中国农业科学, 2018, 51(2): 257-267.

HOU Rui, WANG ChenFang. The Function of the Carbon Metabolism Regulator FgCreA in Fusarium graminearum[J]. Scientia Agricultura Sinica, 2018, 51(2): 257-267.

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