绿僵菌蝗虫菌株Pr1蛋白酶基因的克隆及序列分析

Cloning and Sequence of pr1 Gene from a Locust-infecting Isolate of Metarhizium anisopliae

从绿僵菌蝗虫菌株M2189克隆了昆虫表皮降解酶Pr1的基因。以特异性引物,分别进行了DNA-PCR和mRNA-RT-PCR反应,得到了预期扩增片段,回收纯化后通过pGEM-T载体转化大肠杆菌JM109,得到重组质粒,EcoRⅠ酶切鉴定表明目的片段已插入质粒。对重组质粒中插入的DNA片段进行了核苷酸序列测定,表明该序列全长1369bp,其中有3个内含子,分别为76、59、67bp;编码序列长度为1167bp,含有起始密码子和终止密码子,是一个完整的蛋白读码框。与菌株Me1得到的Pr1编码序列(GenBank/M73795)相比,二者的核苷酸数目相同,同源性达到92.2%,编码的389个氨基酸中48个不同,占12.3%。

The pr1 gene, a potential virulence factor by virtue of its activity against insect cuticles, was cloned from isolate M2189 of Metarhizium anisopliae, highly virulent to locusts. Expected DNA fragments were amplified by DNA-PCR and mRNA-RT-PCR respectively with a pair of specific primers. The fragments were introduced into isolate JM109 of Escherichia coli by pGEM-T easy vector. Integrated DNA from recombinant plasmid was identified by electrophoresis after restriction enzyme digestion. Nucleotide of the expected DNA fragment was sequenced. Result showed that the fragment was 1369 bp in full length, in which there were 3 introns which were 76, 59 and 67 bp, respectively. Protein-encoding part was 1167 bp in length containing a full protein reading frame with an initiation codon and a termination codon at the two ends. The nucleotide number of the protein-encoding sequence was the same as that of prl from isolate Mel of M. anisopliae published by St. Leger, and identity of their nucleotide get to 92.2% . The two sequences predicted a protein of 389 amino acids respectively, and there are 48 different amino acids between them amounting to 12.3 % of the total.