黄曲霉基因敲除体系的构建及其RNA依赖的RNA聚合酶1基因在生长发育中的作用

Construction of gene knockout system of Aspergillus flavus and its effect of RNA-dependent RNA polymerase 1 gene in the growth and development

目的探讨黄曲霉(A.flavus)基因敲除体系的构建和RNA依赖的RNA聚合酶1(RDRP1)基因在A.flavus生长发育中的作用。方法通过National Center for Biotechnology Information网址查找RDRP1基因,设计上下游序列引物,引入融合片段20 bp,采用重叠PCR(overlap PCR)法融合RDRP1基因上下游片段和嘧啶磺胺抗性基因(ptrA);采用聚乙二醇(PEG)介导方法将该融合片段导入A.flavus的原生质体中获得RDRP1阳性转化子(ptrA抗性),采用Sourthern blot鉴定筛选RDRP1基因突变菌株;对RDRP1基因突变菌株,采用十字交叉法测定生长速率、血细胞计数板统计产孢量,手动计数Wickerham Medium(WKM)+尿嘧啶尿苷(UU)培养基上产生的菌核数量。结果获得ptrA抗性转化子13个;Sourthern blot鉴定4个为RDRP1基因缺失菌株,效率30.8%;与CA14相比,RDRP1基因突变菌株在表型、生长率、产孢量及菌核发育上差异无统计学意义(P>0.05)。结论overlap PCR结合PEG介导转化的方式可短时间内获得A.flavus基因敲除突变菌株,RDRP1基因不参与A.flavus表型、生长率、产孢量及菌核发育的调控作用。

Objective To investigate the construction of Aspergillus flavus(A.flavus)knockout system and the role of the RNA-dependent RNA polymerase 1(RDRP1)gene in the growth and development of A.flavus.Methods The RDRP1 gene was searched through the National Center for Biotechnology Information,and primers were designed for upstream and downstream sequences,and a 20 bp fusion fragment was also designed for linking.The upstream and downstream fragments of RDRP1 gene were fused with pyrimidine resistance gene(ptrA)by overlap PCR.Then the fusion fragments were introduced into the protoplasts of Aspergillus flavus by Polyethylene glycol(PEG)mediated method and the positive transformants(ptrA resistance)were obtained.RDRP1 gene mutant strains were identified and screened strains by Sourthern blot;Phenotypic functional analysis of the obtained RDRP1 gene mutant strains:determination of growth rate by the crossover method,counting of spore production by hemocytometer plates,and manual counting of the number of nuclei.Results Thirteen ptrA resistant transformants were obtained;Sourthern blot identified 4 RDRP1 gene deletion strains,with 30.8%efficiency.Compared with CA14,the RDRP1 gene mutant strains did not show statistically difference in phenotype,growth rate,spore production,and nucleus development.Conclusion The gene knockout mutant could obtain by overlap PCR combined with PEG-mediated transformation in a short time.And the RDRP1 gene was not involved in the regulation of A.flavus phenotype,growth rate,sporulation,and sclerotia development.