玉蜀黍尾孢菌VelB基因敲除及生物信息学分析  

Knockout and bioinformatics analysis of VelB gene in Cercospora zeae-maydis

作　　者：路媛媛 孙承龙 谭玉琴 魏文杰 史咸春 宋艳波[1] 李丽[1] 刘振宇[3] Lu Yuanyuan;Sun Chenglong;Tan Yuqin;Wei Wenjie;Shi Xianchun;Song Yanbo;Li Li;LiuZhenyu(College of Life Science,Shanxi Agricultural University,Taigu 030801,China;College of Horticulture,Shanxi Agricultural University,Taigu 030801,China;College of Information Science and Engineer,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学生命科学学院,山西太谷030801 [2]山西农业大学园艺学院,山西太谷030801 [3]山西农业大学信息科学与工程学院,山西太谷030801

出　　处：《山西农业大学学报（自然科学版）》2022年第2期56-64,共9页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：山西省高等学校科技创新项目(2020L0130);山西农业大学科技创新基金项目(2018YJ30)。

摘　　要：[目的]通过对玉蜀黍尾孢菌VelB基因进行生物信息学分析并构建敲除载体,为研究CzVelB调控玉蜀黍尾孢菌致病性的分子机制奠定基础。[方法]从JGI数据库中获得CzVelB基因序列,对其进行生物信息学分析,包括蛋白质的性质、保守区域、磷酸化位点、亚细胞定位、跨膜结构域和聚类分析,分析其基因功能。并采用同源互补原理构建CzVelB基因敲除载体,最终采用农杆菌介导遗传转化技术进行基因敲除。[结果]CzVelB基因编码385 AA,预测pI和MW分别为9.05和42.13645 kDa,是亲水性蛋白,无跨膜结构域。在亚细胞水平上,CzVelB定位在细胞核中。在氨基酸序列方面,具有多个丝氨酸、苏氨酸和酪氨酸激酶磷酸化位点;在功能上具有2个Velevt的保守结构域,并与Aspergillus flavus、A.niger亲缘关系较近,具有较高同源性。根据CzVelB基因序列设计特异性引物,扩增侧翼片段和标记基因,分别连入骨架载体pPZP100中,成功构建CzVelB基因的敲除载体,最终利用ATMT技术获得其敲除转化子。[结论]本文系统研究玉蜀黍尾孢菌VelB基因功能,并成功构建其敲除载体,获得敲除转化子,为后续研究CzVelB基因功能提供基础材料。[Objective]The study was aimed to analyze the bioinformation of VelB of Cercospora zeae-maydis and construct the deletion vector,to enrich the study on the molecular mechanism of CzVelB regulating pathogenesis.[Methods]CzVelB gene was found in JGI database and bioinformatics analysis was performed,including protein properties,conserved regions,phosphorylation sites,subcellular localization,transmembrane domain and cluster analysis to clarify the gene function.Then,CzVelB gene knockout vector was constructed by homologous complementation.Finally,ATMT method was used for gene knockout.[Results]Bioinformatics analysis showed that CzVelB encodes 385 AA,the theoretical isoelectric point pI value and molecular weight were 9.05 and 42.13645 kDa,respectively.CzVelB was a hydrophilic member protein with no transmembrane domain.At the subcellular level,CzVelB located in the nucleus.More than one of the serine,threonine and tymsine kinase phosphorvlation sites in CzVelB amino acids sequence were identified.In the functional structure,CzVelB protein contained two typical conserved Velvet functional domain,and was high homology to VelB of Aspergillus flavus and A.Niger.The specific primers were designed according to the CzVelB gene sequence;The flanking fragments and marker genes were amplified and were connected into the backbone vector Ppzp100.The knockout vector of CzVelB gene was successfully constructed,and the knockout transformant was obtained by ATMT technology.[Conclusion]In this study,the function of CzVelB gene was systematically investigated;its deletion vector was successfully constructed and the mutant strain were obtained.It is the basis for the subsequent study of CzVelB gene function.

关 键 词：玉蜀黍尾孢菌 VelB 生物信息学 敲除载体 敲除突变体 

分 类 号：S432.1[农业科学—植物病理学]