利用Site-Finding PCR克隆琼胶酶基因  

Cloning of An New Agarase Gene Using Site-finding PCR

作　　者：刘丹[1] 邢培川[1] 于文功[1] 路新枝[1] 

机构地区：[1]中国海洋大学医药学院海洋药物教育部重点实验室山东省糖科学与糖工程重点实验室,山东青岛266003

出　　处：《现代生物医学进展》2013年第1期5-10,共6页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(31070712;31000361);国家高技术研究发展计划(863计划)(2011AA090703);海洋公益性行业科研专项(201105027-3)

摘　　要：目的:新型琼胶酶基因的筛选。方法:根据α-琼胶酶基因序列的同源性,设计了兼并引物,利用兼并PCR对所筛选到的琼胶酶产生菌株进行筛选,阳性菌株进行16s rDNA序列测定并构建了进化树。利用染色体步移技术Site-finding PCR获得目的基因的上下游序列,经过拼接获得全长的目的基因序列,并利用Blast对其进行分析。将目的基因插入pET 24a(+)载体,转化大肠杆菌BL21(DE3),利用平板水解圈初步鉴定了重组酶的性质,并利用DNS法检测了重组酶发酵上清液的酶活。结果:获得了一株疑似α-琼胶酶产生菌株,16s rDNA序列鉴定显示为Thalassomonas sp.,命名为Thalassomonas sp.LD5。获得了一个新的基因,命名为agaD。agaD开放阅读框长4401 bp,编码1466个氨基酸,理论分子量为158.8kDa。序列分析表明,agaD编码的蛋白AgaD与已有的两种α-琼胶酶的相似性分别为89%和77%。重组AgaD经诱导后可以直接降解琼胶平板产生水解圈,其发酵上清液酶活为0.2 U.ml-1,说明该蛋白为琼胶酶。结论:采用分子克隆技术分离出新的琼胶酶基因,该基因的发现为活性寡糖的制备提供了新的工具。Objective： To clone new agarase gene. Methods： The degenerate primer was designed according to the known α-agarases and degenerate PCR was used for α-agarase gene screening. The 16s rDNA of positive strain was sequenced and phylogenetic tree was constructed. A chromosome walking technique, site-finding PCR, was used to obtain the upstream and downstream sequences. After being assembled, the full-length of agaD gene was inserted into expression plasmid pET24a （＋）, then transformed into E.coli BL21 （DE3）. Recombinant was screened on agar plate and the fermented culture supernatant activity was quantified by DNS （3,5-dinitrosali- cylic acid） method. Results： A possible ct-agarase producing strain was screened and named as Thalassomonas sp. LD5 according to the phylogenetic tree. A new gene agaD was cloned from Thalassomonas sp. LD5 genomic. The full length of agaD consists of a 4401 bp open reading frame, encoding 1466 amino acid residues, with a putative molecular mass of 158.8 kDa. Sequence analysis revealed that agarase AgaD shared 89% identity with the α-agarase of Alteromonas sp. GJ1B and 77% with that of Thalassomonas sp. JAMB-A33. Recombinant AgaD formed depressions on agar plate and the activity of culture supernatant was 0.2 U. ml＂1, which indicated that agaD was an agarase gene. Conclusions： A new agarase gene agaD was obtained, which provides a new tool for agarooligosaccharides preparation.

关 键 词：α-琼胶酶 单胞菌Thalassomonas SiteFinding PCR 兼并PCR 

分 类 号：Q78[生物学—分子生物学] Q936