Screening and bioinformatics analysis of accase from a bac library of dairy cow rumen microbiota | 奶牛瘤胃微生物BAC文库中ACCase基因的筛选与生物信息学分析

Chinese. 【目的】从奶牛瘤胃微生物BAC文库中筛选含乙酰CoA羧化酶(ACCase)基因的克隆子,并对其全长测序和生物信息学分析。【方法】利用PCR序列驱动筛选法,从瘤胃微生物BAC文库中筛选ACCase基因,鸟枪法测定基因序列后,进行基因注释、比对和系统发育分析。【结果】筛选得到了含ACCase基因的克隆(U12),其插入片段序列(URE12)全长43 358 bp,GC含量为43.75%,经GeneMark程序预测含有38个基因,经Signature程序预测属于变形菌门。ACCase基因(Acc-32)编码159个氨基酸,与Elusimicrobium minutum的ACCase基因具有41%的相似度,编码蛋白分子量为17.89 kD,等电点为5.27,在距离Acc-32上游的33 kb区域,发现一段能编码ACCase连接酶的基因。Acc-32的结合位点氨基酸残基为GQVICVIEAMKVFNELKA,系统发育分析表明Acc-32属于ε-变形菌纲。【结论】得到了含有ACCase基因的序列片段,并且ACCase基因具有新的结构特征。

English. 【Objective】 The objective of the experiment is to screen, sequence and bioinformatical analyze the ACCase clone from a BAC library of the dairy cow rumen microbiota. 【Method】 ACCase gene was screened from BAC library of the rumen microbiota by PCR sequence-driven analysis. Whole insert sequence of screened clone was sequenced by shotgun method. Then it was analyzed by gene annotation, blast and phylogenetic tree analysis. 【Result】 One ACCase clone (U12) was obtained , and its insert fragment (URE12) had the length of 43 358 bp and GC content of 43.75%. URE12 contained 38 CDSs predicted by GeneMark software, and it was probably originated from Proteobacteria by Signature analysis. ACCase encoded by Acc-32 was predicted to be 159 amino acids with a molecular weight of 17.89 kD and pI of 5.27. Acc-32 had the similarity of 41% with that from Elusimicrobium minutum. The combining site AA residue of Acc-32 was GQVICVIEAMKVFNELKA, and Acc-32 belonged to ε-Proteobacteria by phylogenetic tree analysis. 【Conclusion】 The fragment containing ACCase was screened, and ACCase had some novel gene characteristics.