罗尼氏弧菌Vibrio shilonii BY新琼胶酶基因的克隆、序列分析及表达  

Cloning, functional analysis and expression of a novel gene encoding agarase from Vibrio shilonii BY

作　　者：杨光[1] 叶秀云[1,2] 林娟[1,2] 李婧玒 韩尧跃 李仁宽[1,2] 

机构地区：[1]福州大学生物科学与工程学院,福建福州350116 [2]福建省海洋酶工程重点实验室,福建福州350116

出　　处：《微生物学通报》2015年第11期2133-2142,共10页Microbiology China

基　　金：国家海洋公益性行业科研专项项目(No.201305015)

摘　　要：【目的】从海洋来源的罗尼氏弧菌菌株BY中克隆得到一个具有琼胶酶活性的新基因,并对其进行重组表达。【方法】对实验室保藏的产琼胶酶菌株BY进行16S r RNA基因序列分析,并构建系统发育树。根据已报道的琼胶酶基因序列的同源性,设计简并引物,利用降落PCR(Touch-down PCR)及染色体步移技术扩增琼胶酶基因序列全长,对基因序列进行生物信息学分析。将目的基因插入p ET22a(+)载体,转化大肠杆菌BL21(DE3),对重组酶进行表达,利用DNS法测定了重组酶的酶活,对该重组琼胶酶酶学性质进行研究。【结果】克隆得到一条新的琼胶酶基因,命名为Vibrio sp.BY(Gen Bank登录号:AIW39921.1),Vibrio sp.BY基因序列全长2 232 bp,编码744个氨基酸,理论分子量为85 k D,Vibrio sp.BY的氨基酸序列基因库中与已知的琼胶酶氨基酸序列Vibrio sp.EJY3的相似度为86%。发酵液琼胶酶酶活力为71.73 U/m L,证明表达的蛋白为琼胶酶。酶学性质研究表明重组琼胶酶的最适温度及p H分别为50°C和7.0,并且具有较好的稳定性。【结论】利用染色体步移技术克隆得到一条新的琼胶酶基因,并在大肠杆菌BL21(DE3)中实现了重组表达,为琼胶酶的应用奠定了基础。[Objective] The novel gene encoding agarase in a strain of Vibrio shilonii screened from marine was cloned and expressed in E. coli BL21. [Methods] Isolate an agarase producing strain BY, 16 S r RNA gene sequence analysis and construct phylogenetic tree. The degenerate primer was designed according to the known agarase gene sequence, touch-down PCR and chromosome walking techniques were used to obtain agarase full-length gene sequence. After being assembled, the full-length of agarase gene was inserted into expression plasmid p ET22a（＋）, then transformed into E. coli BL21（DE3）. Activity of recombinant enzyme was determined utilizing DNS method, study the enzymatic properties of the recombinant enzyme. [Results] A new gene Vibrio sp. BY was cloned from strain BY genomic. The full length of Vibrio sp. BY consists of a 2 232 bp, encoding 744 amino acids, with a putative molecular mass of 85 k D, the amino acids sequence of agarase Vibrio sp. BY shared 86% identity with the agarase of Vibrio sp. EJY3. The activity of agarase was 71.73 U/m L, which indicated that Vibrio sp. BY was an agarase gene. The optimum temperature and p H for the recombinant activity was at 50 °C and 7.0, have a high agarase stability. [Conclusion] A new agarase gene was cloned by chromosome walking techniques, realized recombinant in E. coli BL21（DE3）, which lays a good foundation for the research on application of agarase.

关 键 词：琼胶酶 罗尼氏弧菌 染色体步移技术 表达 

分 类 号：Q93[生物学—微生物学] Q78