采用酵母双杂交法筛选PSRPK相关蛋白基因-《深圳大学学报理工版》

文章信息/Info

Title:: Screening of the psrpk一related protein genes with yeast two-hybrid method

Author(s):: OUYANG Qiu-ling ; LIU Shi-de; ZHANG Jian-hua; WANG Shao-wen TIAN Sheng-li; and XING Miao; College of Life Science Shenzhen Key Laboratory of Microbial Gene Engineering Shenzhen University

关键词:: 多头绒胞菌; 酵母双杂交; PSRPK; Plasmin C ; BCAT类似蛋白; MSF1类似蛋白; mixedlinked glucanase precursor类似蛋白; FCY1p类似蛋白; 40S ribosomal protein S2 类似蛋白

Keywords:: Pltysarum polycephalum; Two-hybrid system; PSRPK; Plasmin C; BCAT like protein; MSF1-like protein mixed-linked glucanase precursor like protein; FCY1p like protein; 4OS ribosomal protein S2 like protein

摘要:: 本课题组在前期研究中，从多头绒胞菌中分离到一个SR蛋白激酶基因psrpk，并将其编码蛋白命名为PSRPK(SR protein kinase of Physarum Polycephalum).为分离PSRPK相关蛋白基因以了解PSRPK的功能，构建了转化率为2×10⁶转化子/3μg pGADT7-Rec、密度为5.35×10⁸cells/mL、滴度为2.34× 10⁹cfu/mL的多头绒泡菌酵母双杂交AD库.以PSRPK为饵蛋白筛选该文库得到接合率为41.18%的杂交酵母在SD/-Leu/-Trp/-Ade/-His培养板上筛选获得1476个杂交克隆，其中，X-gal滤纸显色呈强蓝色的克隆有342个。对大于500bp的67个克隆测序获得35个cDNA 片段，其中编码plasmin C, branched-chain amino acid aminotransferase (BCAT)类似蛋白、MSFI类似蛋白、mixed-linked glucanase precursor类似蛋白、FCY1p类似蛋白和40S ribosomal protein S2类似蛋白等7个cDNA片段在阳性杂交酵母克隆中出现多次，其余仅出现一次。在35个cDNA片段的编码序列中有15个具有同源蛋白，31个编码序列的丝氨酸含量大于或等于6%，符合激酶底物的组成特征。

Abstract:: A novel SR protein-specific kinases(SRPKs) gene psrpk,，was recently isolated from (SR protein kinase of Physarum polycephalum).To investigate the biological function of PSRPK and screen for the PSRPK-related proteins, a and the coding protein was the biological function of PSRPK and screen for the PSRPK-related proteins, a 2 x106 transformants/3μg pGADT7-Rec of transformation efficiency,5. 35 x 10R cells/ml, of cell density and 2. 34× 109 cfu/mL titre of P. Pnlycephalum two-hybrid AD library was constructed.When screening the AD library with psrpk, 1476 diploid yeast clones on the SD/-Leu/-Trp/-Adc/一His plates were obtained with a mating efficiency of 41.8%. After screening on X-gal filter, 342 positive clones were confirmed with blue color,67 of them were sequenced basing on the inserts size(≥500bp),35 cDNA fragments were obtained. Among the cDNA fragments, 7 of them were presented in multiclones, which encode the plasmin C, branched-chain amino acid aminotransferase (BCAT)like protein，MSF1-like protein，mixed-linked glucanase precursor like protein, FCY1p like protein and 4OS ribosomal protein S2 like proteins of other species. Most of the coding proteins contain more than 6% serine, showing a character of the substrates of the kinases.