弧菌科质粒pJV的全序列测定、基因预测及其复制区的鉴定

Full-sequence determination,gene prediction and identification of the replication region of plasmid pJV from Vibrionaceae

为了从条件致病菌鳗弧菌中提取到天然质粒,获得该质粒的全序列,并得到在弧菌中起复制作用的区域,试验采用向上引物和向下引物共9条,双向测通该质粒,经拼接得到质粒p JV的全序列,利用Glimmer3基因预测软件对该质粒可能包含基因模型进行预测和基因注释,通过在线工具OriFinder预测分析该质粒的复制区域,并将该区域克隆到T载体中,验证重组质粒在弧菌科菌种中的复制效果。结果表明:该质粒p JV全长5 982 bp(Gen Bank登录号为JQ782192),有4个含有核糖体结合位点的基因(UQY-2、UQY-3、UQY-4、UQY-6);质粒p JV的复制元件是一段长313 bp的序列(含Dna A boxes)。说明经过试验验证,该片段使T载体具有在维氏气单胞菌中复制的能力,即证明该片段为鳗弧菌质粒p JV的复制区,可用于新弧菌科载体的构建和疫苗研制等。

To extract a natural plasmid from Vibrio anguillarum, and obtain the complete sequence of the plasmid to get the region which played a role in replication in Vibrio anguillarum, a total of nine up and down primers were used in the test. The complete sequence of plasmid pJV was obtained through slicing based on bidirectional sequencing. Glimmer3 software for gene prediction was used for the prediction and gene annotation on the gene models that might be contained in the plasmid. The replication region of pJV was predicted using an on - line software, Ori - Finder. The replication region was cloned into T vector to verify the replication effect of recombinant plasmid in a strain from Vibrionaceae. The results showed that the full length of plasmid pJV （GenBank No. JQ782192）was 5 982 bp, and there were 4 genes containing a ribosome binding site on pJV, including the UQY - 2, UQY - 3, UQY - 4, and UQY - 6. The replication element from the plasmid was a sequence of 313 bp and contained DnaA boxes. The results indicate that the T vector has the ability to replicate in Aeromonas veronii due to the fragment, which proves that this fragment is a replication region of plasmid pJV from Vibrio anguillarum. Therefore, these results provide a theoretical basis for the construction of novel vector in Vibrionaceae and for vaccine development of V. anguillarum.