刺激植物响应蛋白基因Epl1的载体构建及酵母转化

Vector Construction of the Eliciting Plant Response Protein Gene Epl1 and Yeast Transformation

[目的]构建刺激植物响应蛋白Epl1基因的真核表达载体,筛选多拷贝酵母转化子。[方法]以深绿木霉(Trichoderma atroviride)ACCC30153的cDNA为模板进行PCR获得Epl1基因片段,并将目的片段插入表达载体pPIC9K的相应位置,获得重组表达载体,并将pPIC9K-Epl1转入毕赤酵母(Pichia pastoris)GS115中,并用不同中终浓度的遗传霉素G418筛选多拷贝重组酵母菌株。[结果]对重组载体进行PCR及双酶切检测为阳性;在含有终浓度为2 mg/ml遗传霉素G418的YPD平板上筛选得到7株多拷贝的转化子GS115-Epl1,经PCR鉴定1株转化子呈阳性。[结论]Epl1基因的载体构建及多拷贝酵母转化子筛选为以后大量分离纯化Epl1蛋白并研究其功能奠定基础。

[Objective] To construct eukaryon expression yector of the eliciting plant response protein gene Epl1 and screen multicopy yeast transformant.[Method] Based on the cDNA from biocontrol agent T.atroviride strain ACCC30153,the Epl1 gene segment was obtained by PCR and constructed into vector pPIC9K to obtain the recombinant vector pPIC9K-Epl1.The recombinant vector was transformed into P.pastoris strain GS115,and the multicopy yeast transformants was screened by Geneticin 418 with different final concentration.[Result] PCR and double digest detection identified that pPIC9K-Epl1 was obtained; the seven multicopy yeast transformants were obtained on the YPD medium containing with final concentration of 2 mg/ml,and one strain was positive by PCR detection.[Conclusion] The vector construction of Epl1 gene and screening of high expressing yeast transformant provide the high-efficiency expression strain for separating and purifying Epl1 and establish a basis for the further functional research of eliciting plant response protein Epl1.