摘要
根据铜结合区的保守氨基酸序列设计简并引物,扩增栓菌420(Trametessp.420)基因组,结合长距离反向PCR(LD-IPCR)技术,克隆得到新型漆酶同工酶B基因(lacB),包括结构基因(2255bp)及5′-和3′-非编码序列。lacB含12个内含子,其cDNA序列长1560bp,编码495aa成熟多肽和24aa信号肽。lacB与其它不同来源的真菌漆酶基因具有较高的同源性,而与植物、细菌、昆虫的漆酶基因同源性低于25%。将不含信号序列的lacBcDNA通过质粒pPIC9克隆到表达载体pPIC9K上,电击转化毕赤酵母GS115细胞,经BMM-ABTS平板筛选得到漆酶分泌阳性转化子。
A novel laccase gene （lacB） was cloned from the genomic DNA isolated from Trametes sp. 420, using the degenerate primers based on the conserved copper-binding regions in fungal laccases. Long distance-inverse PCR （LD-IPCR） was used to amplify the flanking sequences, generating the entire lacB DNA sequence including the 2255-bp structure gene and 5＇ -and 3＇-noncoding sequences. The lacB gene is interrupted by 12 introns. The lacB cDNA is 1560 bp long, encoding a 519 amino acid polypeptide composed of a 24 amino acid sig-nal sequence and a 495 amino acid mature protein. In addition, lacB displays a relatively high homology with lac-case genes from other different fungi but a low homology of less than 25% with those from plants, bacteria and in-sects. Furthermore, the lacB cDNA without its signal sequence was cloned into the expression vector pPIC9K through the pPIC9 plasmid and transformed into the Pichia pastoris strain GS115. The laccase-producing transfor-mants were screened out from the BMM plate containing substrate ABTS （2,2＇-azino-bis-（3-ethylbenztbiozo-line6-sulfonic acid）.
出处
《生物学杂志》
CAS
CSCD
2007年第3期25-28,共4页
Journal of Biology
基金
国家自然科学基金(30370045
30470056
30670069)
安徽省优秀青年基金(04043048)
安徽省教育厅自然基金重点(2006kj049A)
安徽大学211工程学术创新团队基金(02203109)
关键词
栓菌420
毕赤酵母
漆酶
基因克隆
异源表达
Trametes sp.420
Pichia pastoris
laccase
gene cloning
heterologous expression
