卡地腐霉Pr1蛋白酶cDNA片段的分离和克隆

Isolating and Cloning of a cDNA Fragment of Pr1 Gene from Pythium carolinianum

目的 :分离和克隆灭蚊真菌卡地腐霉 (Pythiumcarolinianum)Pr1蛋白酶 (subtilisin likeprotease)的cDNA片段。方法 :用 1%的几丁质悬液诱导培养卡地腐霉菌丝 30h ,然后按下述流程操作 :抽提菌丝的总RNA ;逆转录合成cDNA第一链 ;根据Pr1的保守氨基酸序列设计合成一对简并引物 ,以cDNA第一链为模板 ,用MOPAC方法 (MixedoligonucleotidesprimedamplificationofcDNA)扩增cDNA ;将扩增产物纯化、连入pGEM T载体 ,转化大肠杆菌DH5α用于测序。结果 :所获片段中 ,1个 5 30bp的cDNA片段与枯草杆菌类蛋白酶基因有较高的相似性 ,尤其与真菌Aphanomycesastaci (卵菌纲、水霉目 )的Pr1在核苷酸序列及翻译得到的氨基酸序列水平有较高的相似性 ,分别为 5 9%和 4 9%。此序列已登录Genbank数据库 ,检索号 :AF4 990 0 6。P .car olinianum与A .astaciPr1部分cDNA序列的比对亦登录Genbank数据库 ,检索号 :AY0 94 976 ,AY0 94 977。结论 :这一DNA片段可能是卡地腐霉Pr1蛋白酶的一条cDNA片段。更进一步的工作是设法获得全长cDNA片段 ,并加以证实。

Objective: To isolate and clone a cDNA fragment of Pr1 from Pythium carolinianum encoding a subtilisin like protease. Methods: Mycelia of the fungus were cultured in 1% chitin suspension for 30 hours, and then processed as the follows: isolating the total RNA; synthesizing the first strand cDNA; amplifying cDNA (MOPAC) with a pair of degenerate primers based on the highly conservative aa regions of Pr1; purifying the amplification product, and inserting it into the pGEM T vector, transforming the E.coli DH5α for cloning and sequencing.Results:One of the obtained fragments, a 530bp cDNA, shows high homology to the Pr1 genes of other organisms, especially to that of a fungus Aphanomyces astaci (Oomycetes: Saprolegniales ) at both nt (59%) and aa (49%) levels. This fragment and the alignment of partial Pr1 cDNA between P. carolinianum and A. astaci have been submitted to Genbank database. The accession numbers are AF499006,AY094976,AY094977 respectively. Conclusion: This fragment might be a part of a novel Pr1 cDNA of P. carolinianum, and further work should be done to identify the full length of Pr1 cDNA and prove it.