根癌农杆菌介导的泡盛曲霉遗传转化系统的构建

目的]建立根癌农杆菌介导的泡盛曲霉遗传转化系统,探讨利用泡盛曲霉表达异源蛋白的可行性。方法]从国内主要菌种库中购买泡盛曲霉菌株,根据分泌蛋白图谱分析确定合适的宿主菌,在此基础上通过药物敏感性分析确定遗传转化的选择标记,并利用构建的泡盛曲霉遗传转化体系对米黑根毛霉脂肪酶基因RML进行转化、表达研究,通过转化子鉴定及性质分析考察泡盛曲霉作为异源蛋白表达系统的可行性。结果]通过分泌蛋白图谱分析确定泡盛曲霉CBS115.52和CICC2257作为异源蛋白表达的宿主菌;药物敏感性试验确定潮霉素抗性基因(HygBr)作为有效的遗传选择标记;在此基础上,利用根癌农杆菌介导的转化方法(agrobacterium tumefaciensmediated transformation,ATMT),将带有HygBr的质粒pHGW-amdS成功导入泡盛曲霉宿主菌CBS115.52,建立以HygBr为选择标记的泡盛曲霉遗传转化体系。利用该体系,介导米黑根毛霉脂肪酶(Rhizomucor miehei lipase,RML)的表达载体转化泡盛曲霉,通过底物水解试验、SDS-PAGE及Western blot确定RML基因已在泡盛曲霉中表达。结论]该研究证明了根癌农杆菌介导转化泡盛曲霉作为异源蛋白的表达体系具有潜在的可行性。

Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System for Aspergillus awamori

Objective] This study aimed to establish Agrobacterium tumefaciens-mediated genetic transformation system for Aspergillus awamori and investigate the feasibility of expressing heterologous proteins in A.awamori.Method] Appropriate A.awamori host strains were determined according to the secretory protein profile.Selectable marker was selected for genetic transformation by drug sensitivity analysis.The established A.awamori genetic transformation system was used for transformation and expression analysis of Rhizomucor miehei lipase(RML).The feasibility of using A.awamori to express heterologous proteins was investigated by identification of transformants and property analysis.Result] Based on the analysis of secretory protein profile,A.awamori strains CBS115.52 and CICC2257 were determined as the host strains for heterologous protein expression;drug sensitivity analysis shows that hygromycin B resistance gene(HygBr) is an effective genetic selectable marker;by using Agrobacterium tumefaciens-mediated transformation(ATMT) method,the plasmid pHGW-amdS containing HygBr was successfully transformed into A.awamori strain CBS115.52 to establish the genetic transformation system of A.awamori with HygBr as selectable marker.RML was transformed into A.awamori and its expression was validated by substrate hydrolysis test,SDS-PAGE and Western blot.Conclusion] This study demonstrates that the genetic transformation system of A.awamori mediated by Agrobacterium tumefaciens has potential feasibility for expression of heterologous proteins.