利用Site-Finding PCR克隆琼胶酶基因

Cloning of An New Agarase Gene Using Site-finding PCR

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.