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
遇文婧;刘志华;王志英;古丽吉米拉米吉提;黄颖;刁桂萍;
摘要(Abstract):
[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。
关键词(KeyWords): 深绿木霉ACCC30153;刺激植物响应蛋白;毕赤酵母
基金项目(Foundation): 哈尔滨市科技局青年科技创新人才项目(2013RFQXJ02)资助;; 中央高校基本科研业务费专项资金项目(2572014BA08)
作者(Author): 遇文婧;刘志华;王志英;古丽吉米拉米吉提;黄颖;刁桂萍;
Email:
DOI: 10.13989/j.cnki.0517-6611.2014.19.015
参考文献(References):
- [1]DJONOVI C'S,VARGAS W A,KOLOMIETS M V,et al.A proteinaceous elicitor Sm1 from the beneficial fungus Trichoderma virens is required for induced systemic resistance in maize[J].Plant Physiology,2007,145:875
- [2]SEIDL V,MARCHETTI M,SCHANDL R,et al.Epl1,the major secreted protein of Hypocrea atroviridis on glucose,is a member of a strongly conserved protein family comprising plant defense response elicitors[J].FEBS J,2006,273:4346-4359.
- [3]WANG Y,SONG J,WU Y,et al.Eplt4 proteinaceous elicitor produced in Pichia pastoris has a protective effect against Cercosporidium sofinum infections of soybean leaves[J].Appl Biochem Biotechnol,2013,169:722-737.
- [4]DJONOVI C'S,POZO M J,DANGOTT L J,et al.Sm1,a Proteinaceous elicitor secreted by the biocontrol fungus Trichoderma virens induces plant defense responses and systemic resistance[J].Mol Plant Microbe Interact,2006,19:838-853.
- [5]FREITAS R S,STEINDORFF A S,RAMADA M H,et al.Cloning and characterization of a protein elicitor Sm1 gene frome Trichoderma harzianum[J].Biotechnol Lett,2014,36:783-788.
- [6]胡砷.重组chi46在毕赤酵母中的表达及抗病原菌初步研究[C].哈尔滨:东北林业大学,2007.
- [7]BOOR H,SIMOLIN H,COTTAZ S,et al.Heterologous expression and sitedirected mutagenesis studies of tow Trichoderma harzianum chitinases,Chit33 and Chit42,in Escherichia coli[J].Protein Expression and Purification,2007,51:216-226.
- [8]KLEMASDAL S S,CLARKE J L,HOELL I A,et al.Molecular cloning,characterization,and expression studies of a novel chitinase gene(ech30)from the mycoparasite Trichoderma atroviride strain P1[J].FEMS Microbiol Lett,2006,256:282-289.
- [9]VITERBO A,LANDAU U,KIM S,et al.Characterization of ACC deaminase from the biocontrol and plant growth-promoting agent Trichoderma asperellum T203[J].FEMS Microbiol Lett,2010,305:42-48.
- [10]JUN H,BING Y,KEYING Z,et al.Expression of a Trichoderma reesi betaxylanase gene in Escherichia coli and activity of the enzyme on fiberbound substrates[J].Proteinv Expr Purify,2009,67:1-6.
- [11]BUENSANTEAI N,MUKHERJEE P K,HORWITZ B A,et al.Expression and purification of biologically active Trichoderma virens proteinaceous elicitor SM1 in Pichia pastoris[J].Protein Expr Purify,2010,72:131-138.
- [12]RUBIO M B,CARDOZA R E,HERMOSA R,et al.Cloning and characterization of the Thcut1 gene encoding a cutinase of Trichoderma harzianum T34[J].Curr Genet,2008,54:301-312.