农杆菌介导pepA基因对黑曲霉菌丝体的遗传转化  

Genetic transformation of pepA gene to Aspergillus niger mycelium via Agrobacterium tumefaciens

作　　者：李杰[1] 李硕然[1] 张谷楠[1] 汪天虹[2] 钟耀华[2] 赵宁[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]山东大学生命科学学院,济南250100

出　　处：《东北农业大学学报》2013年第5期29-35,F0002,共8页Journal of Northeast Agricultural University

基　　金：山东大学微生物技术国家重点实验室开放项目(M2008-17)

摘　　要：通过重叠延伸PCR将宇佐米曲霉中1 347 bp的酸性蛋白酶基因pepA与黑曲霉糖化酶基因5'同源臂和3'同源臂进行拼接,构建pepA基因表达框。将此表达框插入载体pSZH中,构建黑曲霉同源重组表达载体pSZH-pepA。将载体pSZH-pepA通过冻融法转化农杆菌AGL1,进而通过农杆菌介导法转化高产糖化酶的黑曲霉菌丝体。以PDA作为共培养培养基,乙酰丁香酮(简称AS)浓度200μmol.mL-1的条件下,28℃恒温静置培养48 h,经潮霉素筛选和PCR鉴定获得3株重组菌株。以出发菌株作为对照,对3株重组菌株静置培养7 d后的发酵液上清进行酶活测定,结果发现酸性蛋白酶酶活最高达45.56 U.mL-1,而出发菌株的酸性蛋白酶酶活仅为3.72 U.mL-1,表明酸性蛋白酶基因pepA在高产糖化酶的黑曲霉中得到表达。研究建立了农杆菌介导的pepA基因导入黑曲霉菌丝体的转化体系,为实现酸性蛋白酶在黑曲霉菌丝体中高效表达奠定基础。A-1347bp pepA gene from Aspergillus usamii and glucoamylase homologous arms 5＇GLA and 3＇GLA are spliced by overlap extension PCR, constructed pepA expression cassette inset into the expression vector-pSZH. The resulting plasmid named with pSZH-pepA was transformed into Agrobactetium tumefaciens AGLlusing a freeze-thaw method, and then was transferred into AspergiUus niger mycelium through the mediation of Agrobacterium Tumefaciens. Four positive transformants were obtained on PDA medium plate with the concentration of AS 200 pmol . mL1 after the mixture of Aspergillus niger and Agrobacterium Tumefaciens were plated and staticly grown at 28 （3 for 48 h. Cation Aspergillus niger transformats were identified by PCR amplification, and results showed that the pepA gene was inserted into Aspergillus nigers through nonhomologous recombination. Three positive transformants were measured for the enzyme activity of acidic protease, and the highest enzyme activity was 45.56 U mL＂ when the transformants was grown to the seventh day, in contrast, the enzyme activity of original strain only was 3.72 U mL4. The establishment of Agrobactenum mediated transformation into Aspergillus niger mycelium lay a foundation on expression of acid protease in mycelia of Aspergillus niger.

关 键 词：酸性蛋白酶 黑曲霉 重叠延伸PCR ATMT 

分 类 号：S767.5[农业科学—森林保护学]