苜蓿根瘤菌聚羟丁酸解聚酶基因(phbD)突变体的构建及其特性  

Construction and Characteristics of Poly-3-hydroxybutyrate Depolymerase Encoding Gene (phbD ) Mutant in Sinorhizobium meliloti

作　　者：戴美学[1] 武波[2] 柏学亮[2] 张成刚[1] 马庆生[2] 

机构地区：[1]中国科学院沈阳应用生态研究所,沈阳110015 [2]广西大学农业分子遗传农业部重点实验室,南宁530005

出　　处：《农业生物技术学报》2003年第2期115-120,共6页Journal of Agricultural Biotechnology

摘　　要：根据苜蓿根瘤菌(Sinorhizobiummeliloti)Rm1021基因组中与Ralstonia eutropha phaz基因同源部分序列设计1对引物,从S.meliloti基因组中用PCR扩增出835 bp phbD基因片段并克隆到载体pGEM(R)-TEasy上;通过在phbD基因内插入ΩSmSp和基因置换构建了phbD突变体.该突变体可积累比野生型菌株多1.0～2.6倍的聚羟丁酶(PHB).在YMA和TY平板上形成非粘液型菌落,而在以乙酰乙酸或3-羟丁酸为唯一碳源的M9基本培养基(M9-AA,M9-HB)上形成粘液型菌落.碱性磷酸酶测定结果表明,通过接合引入phbD突变体菌株中的exoF-phoA融合基因在YMB培养基中低量表达,而在M9-AA和M9-HB中高量表达.Rhizobia, as so many other bacteria, can synthesize the carbon storage compound, polyhydroxyalkanoates (PHAs), when non-carbon nutrient , such as N, P or O2, is limited to grow and excessive carbon is available. When subsequent conditions make carbon limit, the internal PHA stores may serve as sources of carbon and energy. Poly-3-hydroxybutyrate (PHB) is a common member of PHAs family.The ability to degrade PHAs depends on the production PHA depolymerases, which release dimers or monomers from the PHA polymer. Bacterial PHA depolymerases are classified as intracellular or extracellular and widely distributed among microorganisms. So far, intracellular PHA or PHB depolymerase mutants have only been described in two strains of bacteria: Pseudomonas oleovorans and Ralstonia eutropha. All Sinorhizobium meliloti PHB pathway mutants other than phbD mutant have been characterized. Creation of S. meliloti phbD mutant would be useful to study the role of phbD gene in the PHB pathway and to understand the PHB cycle in more detail. The S. meliloti phbD gene sequence was obtained from the S.meliloti 1021 genome sequencing project. A homologue of the Ralstonia eutropha phaZ gene, encoding intracellular PHB depolymerase, was identified and designated to be phbD. Using a pair of primers designed by the sequence of S. meliloti Rm1021 genome which is homologous to phaZ gene of R. eutropha , an 835 bp fragment of phbD gene was amplified by PCR from S. meliloti genome and cloned onto vector pGEM-T Easy and generated the recombinant plasmid pDC45. The insert was sequenced to ensure that the cloned fragment does contain the desired sequence. A unique Kpn21 site was identified to be located within the coding region at 299 bp from the translation start codon. Interposon ΩSmSp was inserted into phbD gene at the Kpn21 site and generated the plasmid pDC48. phbD :: ΩSmSp fragment was excised from pDC48 as an Eco RⅠfragment and ligated with Eco RⅠdigested plasmid pK19mobsacB to get pDC50. Plasmid pDC50 was introduced into

关 键 词：苜蓿根瘤菌 聚羟丁酸 解聚酶 基因 突变体 

分 类 号：S154.381[农业科学—土壤学] S54[农业科学—农业基础科学]