黄孢原毛平革菌酵母双杂交cDNA文库的构建及应用  

Construction and Application of Yeast Two Hybrid cDNA Libraries of Phanerochaete chrysosporium

作　　者：胡国库[1] 颜永红[1] 姜权[1] 吴波[1] 冯红[1] 张义正[1] 

机构地区：[1]四川大学生命科学学院/四川省分子生物学及生物技术重点实验室,成都610064

出　　处：《应用与环境生物学报》2006年第5期678-682,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金资助项目(No.30470984);教育部博士点基金(No.20040610013)~~

摘　　要：作为研究木质素降解系统的模式微生物黄孢原毛平革菌,迄今还没有蛋白-蛋白相互作用方面的报道.本研究利用酵母双杂交技术构建了低氮培养基中培养2d和3d的酵母双杂交cDNA文库,分别得到2.5×105和3.0×105个重组子.以14-3-3蛋白为钓饵筛选3d cDNA文库,在SD/-Ade/-His/-Leu/-Trp缺陷培养基平板上共获得了约600个单克隆,进一步分析发现,有30个克隆可激活3个报告基因.分离自这些克隆中的质粒DNA能转化大肠杆菌.利用HaeⅢ和Sau3AⅠ限制酶切分析可将它们分为4类.序列测定和同源分析结果表明,其中一类为14-3-3蛋白基因,提示14-3-3蛋白可形成二聚体,另外3类在GenBank没有明显同源的基因.通过SBASE网站预测,编号为Y2H333的克隆含有一个OB-fold核酸结合结构域(OB-fold nucleic acid binding domain).这些研究结果表明,所构建的黄孢原毛平革菌cDNA酵母杂交文库是成功的,14-3-3蛋白在黄孢原毛平革菌中能以蛋白-蛋白相互作用的形式发挥功能.There has been no report on protein - protein interaction in Phanerochaete chrysosporium, a model microorganism for studying lignin degradation. In this study, two yeast two-hybrid libraries containing 2.5 × 10^5 and 3.0 × 10^5 recombinants, were constructed using 2 d and 3 d mRNAs derived from P. chrysosporium cultures growing in low-nitrogen medium. 14-3-3 gene was used to screen the 3-day library by yeast two-hybrid technique and more than 600 clones were obtained on the SD/-Ade/-His/-Leu/-Trp plates. Thirty positive recombinants could activate three reporter genes. Plasmids isolated from yeast transformants were successfully transformed into Escherichia coll. The thirty clones could be classified into 4 types on the basis of the digestion patterns by HaeⅢ and Sau3AⅠ , as well as their sequence analyses. Results showed that one type of clones encoded 14-3-3 protein, implying that homodimer could be formed between two molecules of 14-3-3 protein. The other three types of clones encoded unknown proteins. However, the Y2H333 contained an OB-fold nucleic acid binding domain on the basis of analysis in SBASE. These results demonstrated that the yeast two-hybrid cDNA libraries were qualified for studying protein - protein interactions and 14-3-3 protein may play important roles in P. chrysosporium.

关 键 词：黄孢原毛平革菌 酵母双杂交 CDNA文库 14-3-3蛋白 

分 类 号：Q78[生物学—分子生物学]