采用酵母双杂交法筛选PSRPK相关蛋白基因

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

本课题组在前期研究中,从多头绒泡菌中分离到一个SR蛋白激酶基因psrpk,并将其编码蛋白命名为PSRPK(SR protein kinase ofPhysarum 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/-H is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500 bp的67个克隆测序获得35个cDNA片段,其中编码Plasm in C、branched-chain am inoacid am inotransferase(BCAT)类似蛋白、MSF1类似蛋白、m ixed-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.

A novel SR protein-specific kinases （SRPKs） gene psrpk, was recently isolated from Physarum polycephalum and the coding protein was named as PSRPK （ SR protein kinase of Physarum Polycephalum）. To investigate the biological function of PSRPK and screen for the PSRPK-related proteins, a 2 × 10^6 transformants/3μg pGADT7-Rec of transformation efficiency, 5.35 × 10^8 cells/mL of cell density and 2.34 × 10^9 cfu/mL titre of P. polycephalum two-hybrid AD library was constructed. When screening the AD library with psrpk, 1476 diploid yeast clones on the SD/-Leu/-Trp/-Ade/-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 （≥500 bp） , 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, FCYlp like protein and dOS ribosomal protein S2 like protein et al, the rest were presented in single clone. 15 of the cDNA fragments were found encoding homology proteins of other species. Most of the coding proteins contain more than 6% serine, showing a character of the substrates of the kinases.