微紫青霉菌(Penicillium janthinellum)P-type ATPase及金属硫蛋白相关基因的克隆

Coloning of P-type ATPase and Metallothionein Genes from Penicillium janthinellum

本研究以高抗多种重金属盐的微紫青霉菌(Penicillium janthinellum)菌株GXCR为材料构建基因组fosmid文库。其插入片段集中在36～50kb,含13348个克隆,重组率为100%,大约覆盖了GXCR基因组的14.83倍。基于序列特异性和简并引物,利用PCR扩增分析了与酿酒酵母(Saccharomyces cerevisiae)重金属盐抗性相关的CRS5和CUP2基因;基于兼并引物和序列特异性引物,利用PCR扩增分析了GXCR的P-type ATPase基因。通过菌落原位杂交和Southern blot鉴定了一个含铜转运P-type ATPase基因的阳性fosmid克隆,经亚克隆测序分析表明该基因与棒曲霉(Aspergillus clavatus菌株)NRRL1的P-type copper ATPase相似性达97%。没有筛选到与CRS5和CUP2基因同源的克隆,说明GXCR中可能不存在与酿酒酵母CUP2和CRS5高度同源的MT基因,同时也暗示酵母与丝状真菌的重金属盐的抗性机制有本质上的差异或者独特性。

The Penicillium janthinellum strain GXCR,briefly named GXCR,has multiple-heavy-metal tolerance. In this study,the GXCR genomic fosmid library was constructed,which consists of 13 348 clones containing inserts with the size of 36～50 kb. Total length of inserts in the library approximately covered GXCR genome,with 14.83-fold. Heavy-metal-resistant genes homologous to yeast （Saccharomyces cerevisiae） CRS5 and CUP2,as well as GXCR P-type ATPase gene were analyzed by PCR based on sequence-specific and degenerate primers,respectively. A fosmid clone containing P-type ATPase gene was identified through in situ colony hybridization followed by Southern blot,subcloned and then sequenced,showing 97% similarly with the P-type copper AT-Pase of Aspergillus clavatus NRRL1. However,no candidate clones containing genes homologus to CRS5 and CUP2 were obtained,suggesting that there not exist genes homologous to yeast CRS5 and CUP2 in the GXCR and further hinting that there are essential differences between yeasts and filamentous fungi in heavy-metal-resis-tant mechanisms.