红曲菌cDNA消减文库的构建

应用抑制性消减杂交技术,构建红曲菌产桔霉素和不产桔霉素差异表达的cDNA消减文库.分别从产桔霉素和不产桔霉素的红曲菌丝体中提取mRNA,依次合成单链和双链cDNA,经酶切成大小为250～750bp的片断,将产桔霉素的cDNA分为两组,分别与两种不同的接头连接,再与不产桔霉素的红曲菌的cDNA进行两次消减杂交及两次抑制性PCR后,将产物与T/A载体连接构建成功cDNA消减文库,并转染大肠杆菌进行文库扩增,文库扩增后得到283个克隆,经PCR法快速测定,均得到250～750bp的插入片断.所构建的红曲菌cDNA消减文库为进一步筛选红曲菌中与产桔霉素性状相关的基因奠定了基础.

CONSTRUCTION OF A CDNA SUBTRACTIVE LIBRARY OF MONASCUS

A cDNA subtractive library of Monascus was constructed by using suppression subtractive hybridization technique. The library only contains the differently expressing cDNAs between Monascus producing citrinin and not producing citrinin. mRNA was isolated from mycelium of Monascus. Single-strand cDNAs and double-strand cDNAs were synthesized in turn. After enzyme restriction, cDNAs between 250-750bp were obtained. cDNAs from Monascus that producing citrinin then were divided into two groups and ligated to the specific adaptor1 and adaptor 2 respectively. After the cDNAs hybridized twice with cDNAs from Monascus that not producing citrinin and underwent two times of nested PCR, then linked with arms of T/A plasmid vectors, the subtractive library was set up. Amplification of the library was carried out with the E.coli strain JM109. The amplified library contains 283 positive clones. Rapid analysis by PCR shows that all plasmids in the clones contain 250-750bp inserts. The constructed cDNA subtractive library of Monascus lays solid foundation for screening key genes from citrinin biosynthetic pathway in species of Monascus.