摘要
运用选择性培养基LBCMC从环境土样中分离得到一株能利用羧甲基纤维素的芽孢杆菌Bacillus sp.NW-2004 a,并运用“鸟枪”克隆法构建了其基因组文库,且从此基因组文库中筛选得到两个阳性克隆.对其中一阳性克隆中插入的DNA片段(命名为GLUD)进行序列测定,发现了一长度为2 502 bp的开放阅读框(ORF),可编码834个氨基酸.BLAST分析结果表明,该基因与Bacillus sp.KSM-S237来源的纤维素酶基因AB018420具86%相似性,所编码的多肽与Bacillus sp.KSM-64来源的β-1,4-内切葡聚糖酶具89%的相似性,故该基因是一新发现的β-1,4-内切葡聚糖酶基因.该序列已收录于Genebank,登录号AY663839.另外,以CpoI andNotI限制性内切酶双酶切衍生于质粒pGEX的大肠杆菌表达载体pHBM625,然后将经T4 DNA polym erase处理后的β-1,4-内切葡聚糖酶基因克隆至载体pHBM625中得到重组质粒pH-BM625G lu,最后通过平板检测及SDS-PAGE凝胶电泳,均检测到该基因在大肠杆菌XL10-G lod中的表达.
Bacillus sp. NW - 2004a, which can hydrolyze carboxymethyl cellulose （CMC） , was obtained from soil sample through selective medium LBCMC, and its genomic library was constructed using “ shotgun” cloning strategy . Two positive clones were obtained from the genomic libray, one of which was chosen to sequence the inserted DNA fragment. An open - reading frame of 2 502 bp（ORF） , coding for 834 amino acids , was found . BLAST analysis of the sequence in NCBI database showed that it has 86 % similarity with the glucanase gene of Bacillus sp. KSM - S237, and the edcoded polypeptide shows 87 % similarity with the β- 1,4 -endo- glucanase of Bacillus sp. KSM -64 at amino acid level. It has been accepted by GeneBank with accession number AY663839 . In addition , treated with T4 polymerase, the/3 - 1,4 - endo glucanase gene was cloned into the plasmid pHBM625, which was derived from vector pGEX, through restriction endonuclease CpoI and NotI, and expressed in Escherichia coli XL10 - Gold.
出处
《湖北大学学报（自然科学版）》
CAS
北大核心
2005年第3期275-279,共5页
Journal of Hubei University：Natural Science
基金
农业微生物国家重点实验室开放课题(AML-0303)资助
武汉市科技攻关计划(20026002087)
