深圳水库区双脐螺rDNA基因片段克隆与序列分析

Cloning and sequence analysis of partial rDNA fragment from a Biomphalaria snail collected in Shenzhen reservoir

目的克隆深圳水库区双脐螺rDNA基因片段并进行序列分析。方法采集深圳水库排洪渠水体的双脐螺,提取基因组DNA,PCR方法扩增r DNA基因片段,纯化后与p MD18-T质粒连接,转化大肠埃希氏菌JM109,筛选阳性克隆;重组质粒经双酶切鉴定后,进行序列测定,并以BLAST和MEGA4软件分析序列特点。结果深圳水库区双脐螺r DNA基因扩增片段大小约为959 bp;重组质粒经双酶切鉴定与预期结果相符;克隆的rDNA序列含有959个碱基,BLAST比对结果显示,rDNA克隆序列与Gen Bank中3株库恩双脐螺（B.kuhniana）序列的同源性为99%,与2株藁杆双脐螺（B.straminea）的同源性为98%,与1株中介双脐螺（B.intermedia）及2株亚马逊双脐螺（B.amazonica）的同源性介于96%~97%。应用邻位连接法（Neighbor-jioning,NJ）和最小进化法（Minimum Evolution,ME）2种方法构建系统发生树,深圳水库区双脐螺与3株B.kuhniana的遗传距离小,同属一个分支。结论成功克隆了深圳水库区双脐螺r DNA基因片段,其基因遗传特征与B.kuhniana双脐螺接近。

Objective To clone and analyze the sequences of rDNA fragment of a Biomphalaria snail collected atShenzhen reservoir. Methods The r DNA fragment extracted from a Biomphalaria snail collected in Shenzhen reservoir wasamplified by PCR and ligated with plasmid p MD-18 T to construct recombinant plasmids, and transformed into E.coli JM109.Positive bacteria clones were identified by double enzymes digestion methods. The sequence of inserted r DNA fragments wasdetermined and analyzed with BLAST and MEGA4 software. Results The amplified r DNA fragment of the Biomphalariasnail was about 959 bp in length. As aligned with the corresponding sequences of 8 isolates from Biomphalaria snails in theGen Bank database, the nucleotides had a homogeneity of 99% with 3 isolates of B. kuhniana, 98% with 2 isolates B. straminea,and 96%~97% with 2 isolates of B. amazonica and 1 isolate of B. intermedia,respectively. Phylogenetic analysis based onneighbor-joining（NJ） and minimum evolution（ME） methods indicated the snail have close genetic association with B.kuhnianaisolates. Conclusion The r DNA fragments of the Biomphalaria snail was cloned, whose characteristics was close to that ofB.kuhniana snails.