米曲霉尿嘧啶营养缺陷型突变体筛选及转化体系建立  

Screening of Uracil Auxotrophic Mutants of Aspergillus oryzae and Establishment of Transformation System

作　　者：秦坤海 刘琪聪 黄慧 曾斌 胡志宏[1,2] QIN Kun-hai;LIU Qi-cong;HUANG Hui;ZENG Bin;HU Zhi-hong(Coll.of Life Sci.Jiangxi Sci.&Tech Nor.Uni.,Nanchang 330013;Jiangxi Prov.Key Lab.of Bioprocess,Nanchang 330013;Coll.of Pharm.,Shenzhen Tech.Uni.,Shenzhen 518118)

机构地区：[1]江西科技师范大学生命科学学院,江西南昌330013 [2]江西科技师范大学江西省生物加工过程重点实验室,江西南昌330013 [3]深圳技术大学药学院,广东深圳518118

出　　处：《微生物学杂志》2023年第1期51-56,共6页Journal of Microbiology

基　　金：国家自然科学基金项目(31700068);江西省科技厅青年重点项目(20192ACBL20012);江西省教育厅自然科学基金项;目(GJJ201141);江西科技师范大学青年拔尖人才项目(2019QNBJRC004);。

摘　　要：米曲霉(Aspergillus oryzae)是一种十分重要的工业微生物,但由于其菌丝体和孢子均含有多个细胞核,并且对多种抗性药物具有抗性,导致其遗传转化和分子生物学研究较其他模式丝状真菌困难。目前米曲霉遗传转化主要采用营养缺陷型作为筛选标记。尿嘧啶营养缺陷型是米曲霉转化中最常用的一种营养缺陷型标记,其筛选标记基因pyrG编码乳清酸核苷-5′-磷酸脱羧酶为尿嘧啶的前体尿苷合成所必需。本研究以米曲霉3.042为背景,利用紫外线诱变和5-氟乳清酸(5-FOA)胁迫筛选获得5株尿嘧啶营养缺陷型菌株,经DNA测序5株菌株均为pyrG基因不同位点突变,确定为尿嘧啶营养缺陷型。以筛选到的尿嘧啶营养缺陷型菌株为背景,利用农杆菌介导的米曲霉转化系统,成功构建了米曲霉尿嘧啶营养缺陷型为筛选标记的农杆菌转化体系。Aspergillus oryzae is a very important industrial microorganism.However,because of its mycelium and spore contain multiple nucleis and having resistance to various resistant drugs that result in difficult to study its genetic transformation and molecular biology as compare with other model of filamentous fungi.At present,the genetic transformation of A.oryzae mainly uses nutritional defects as screening markers.Uracil auxotroph is the most commonly used auxotroph marker in A.oryzae transformation,its screening marker gene pyrG encodes nucleoside-5′-phosphate decarboxylase necessary for the synthesis of uracil precursor uridine.Based on the background of Huniang 3.042,five uracil auxotrophic strains were screened and obtained by UV mutation and 5-fluorowhey acid(5-FOA)coercion in this study.DNA sequencing showed that the five strains were all mutations at different sites of pyrG gene and confirmed as uracil auxotroph.The selected auxotroph strains as background an A.oryzae Agrobacterium mediated transformation system was used and successfully constructed Agrobacterium transformation system with uracil auxotroph A.oryzae as screening marker transformation system.

关 键 词：米曲霉3.042 尿嘧啶营养缺陷型 农杆菌转化 紫外线诱变 5-氟乳清酸 

分 类 号：Q933[生物学—微生物学]