分裂泛素化酵母双杂交系统研究信息素因子STE2蛋白相互作用
|
摘要
真菌的进化、分化及有性发育趋势受若干性别基因的调控,如交配型、信息素因子及异源三聚体G-蛋白ɑ-亚基基因等;同时,信息素因子调控真菌的有性生殖在酿酒酵母中也得到充分研究。匍柄霉属(Stemphylium)是丝状子囊真菌中的一类重要真菌类群,许多丝状子囊真菌属、种中含有信息素因子。在自然界中,匍柄霉属真菌大多数是以无性态的形式存在,但其中也有一小部分存在有性生殖阶段。为了深入研究信息素受体STE2及其相关基因对匍柄霉属典型种有性生殖的影响,本研究选取含有信息素因子STE2的匍柄霉属典型种Stemphylium eturmiunum为试验材料,克隆鉴定了信息素受体STE2,然后将其构建至诱饵蛋白表达载体上,并在cDNA文库初筛得到数个疑似互作蛋白片段基础上,克隆其基因全长构建至靶蛋白表达载体上,并利用分裂泛素化的膜酵母双杂交系统,进行一对一验证筛选STE2的相关互作蛋白。该研究筛选出的互作蛋白结果为后期的CO-IP、Poll-Down实验提供了基础。
The evolution, differentiation and sexual development of fungi are regulated by several sex genes, such as mating type, pheromone factors and heterotrimeric G-protein ɑ-subunit genes. At the same time, regulatory factor pheromone sexual reproduction of fungi are also well studied in Stemphylium cerevisiae. Stemphylium is an important group of fungi in filamentous ascomy fungi, and many filamentous ascospora genus and species contain pheromone factors. In nature, Most of the Stemphylium fungi are in the form of asexuality, but a small part of them also have sexual reproduction. In order to further study the effect of pheromone receptor STE2 and its related genes on the sexual reproduction of typical species of the Stemphylium, this study selected Stemphylium eturmiunum, a typical species of the genus Stemphylium, containing the pheromone factor STE2, and cloned and identified the information receptor STE2, and then constructed on the bait protein expression vector, the cDNA library is screened to obtain several suspected interaction protein fragments, and the full length of the gene is cloned into the prey protein expression vector, and the split ubiquitinated membrane yeast two-hybrid system was used to perform one-to-one validation screening of STE2 related interaction protein. The results of the interaction protein screened by this study provide the basic premise for the later CO-IP and Poll-Down experiments.
The evolution, differentiation and sexual development of fungi are regulated by several sex genes, such as mating type, pheromone factors and heterotrimeric G-protein ɑ-subunit genes. At the same time, regulatory factor pheromone sexual reproduction of fungi are also well studied in Stemphylium cerevisiae. Stemphylium is an important group of fungi in filamentous ascomy fungi, and many filamentous ascospora genus and species contain pheromone factors. In nature, Most of the Stemphylium fungi are in the form of asexuality, but a small part of them also have sexual reproduction. In order to further study the effect of pheromone receptor STE2 and its related genes on the sexual reproduction of typical species of the Stemphylium, this study selected Stemphylium eturmiunum, a typical species of the genus Stemphylium, containing the pheromone factor STE2, and cloned and identified the information receptor STE2, and then constructed on the bait protein expression vector, the cDNA library is screened to obtain several suspected interaction protein fragments, and the full length of the gene is cloned into the prey protein expression vector, and the split ubiquitinated membrane yeast two-hybrid system was used to perform one-to-one validation screening of STE2 related interaction protein. The results of the interaction protein screened by this study provide the basic premise for the later CO-IP and Poll-Down experiments.
引文
[1]王群,王石,刘小缦,等.丝孢真菌无性与有性进化分析[J].菌物研究,2017,15(4):213-221
[2]Wallroth FG.Flora Cryptogamica Germaniae[J].Google Scholar,1833,2:182-184
[3]Inderbitzin P,Mehta YR,Berbee ML.Pleospora species with Stemphylium anamorphs:a four locus phylogeny resolves new lineages yet does not distinguish among species in the Pleospora herbarum clade[J].Mycologia,2009,101(3):329-339
[4]Jenness DD,Burholder AC,Hartwell LH.Binding of K-factor pheromone to yeast a cells:Chemical and genetic evidence for an K-factor receptor[J].Cell,1983,35(2 Pt 1):521-529
[5]Nakayama N,Miyajima A,Arai K.Nucleotide sequences of STE2 and STE3,cell type-specic sterile genes from Saccharomyces cerevisiae[J].The EMBO Journal,1985,4(10):2643-2648
[6]武明花,李桂源.分离的泛素系统——一种新型的膜蛋白酵母双杂交系统[J].国外医学遗传学分册,2005,28(2):88-90
[7]Fields S,Song OK.A novel genetic system to detect protein-protein interactions[J].Nature,1989,40(18):245-246
[8]Coates PJ,Hall PA.The yeast two-hybrid system for identifying protein-protein interactions[J].J Pathol,2003,199(l):4-7
[9]Brachmann RK,Boeke JD.Tag games in yeast:the two-hybrid system and beyond[J].Curr Opin Biotechnol,1997,8(5):561-568
[10]Reinders A,Schulze W,Thaminy S,et al.Intra-and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays[J].Structure,2002,10(6):763-772
[11]Stagljar I,Korostensky C,Johnsson N,et al.A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo[J].Proc Natl Acad Sci USA,1998,95(9):5187-5192
[12]Johnsson N,Varshavsky A.Split ubiquitin as a sensor of protein interactions in vivo[J].Proc.Nati.Acad.Sci.,1994,91:10340-10344
[13]蔺芳芳,杨旭,武小翠,等.利用分裂泛素酵母双杂交技术钓取小麦Ta SC互作蛋白质[J].作物学报,2013,39(3):423-430
[2]Wallroth FG.Flora Cryptogamica Germaniae[J].Google Scholar,1833,2:182-184
[3]Inderbitzin P,Mehta YR,Berbee ML.Pleospora species with Stemphylium anamorphs:a four locus phylogeny resolves new lineages yet does not distinguish among species in the Pleospora herbarum clade[J].Mycologia,2009,101(3):329-339
[4]Jenness DD,Burholder AC,Hartwell LH.Binding of K-factor pheromone to yeast a cells:Chemical and genetic evidence for an K-factor receptor[J].Cell,1983,35(2 Pt 1):521-529
[5]Nakayama N,Miyajima A,Arai K.Nucleotide sequences of STE2 and STE3,cell type-specic sterile genes from Saccharomyces cerevisiae[J].The EMBO Journal,1985,4(10):2643-2648
[6]武明花,李桂源.分离的泛素系统——一种新型的膜蛋白酵母双杂交系统[J].国外医学遗传学分册,2005,28(2):88-90
[7]Fields S,Song OK.A novel genetic system to detect protein-protein interactions[J].Nature,1989,40(18):245-246
[8]Coates PJ,Hall PA.The yeast two-hybrid system for identifying protein-protein interactions[J].J Pathol,2003,199(l):4-7
[9]Brachmann RK,Boeke JD.Tag games in yeast:the two-hybrid system and beyond[J].Curr Opin Biotechnol,1997,8(5):561-568
[10]Reinders A,Schulze W,Thaminy S,et al.Intra-and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays[J].Structure,2002,10(6):763-772
[11]Stagljar I,Korostensky C,Johnsson N,et al.A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo[J].Proc Natl Acad Sci USA,1998,95(9):5187-5192
[12]Johnsson N,Varshavsky A.Split ubiquitin as a sensor of protein interactions in vivo[J].Proc.Nati.Acad.Sci.,1994,91:10340-10344
[13]蔺芳芳,杨旭,武小翠,等.利用分裂泛素酵母双杂交技术钓取小麦Ta SC互作蛋白质[J].作物学报,2013,39(3):423-430