哈维氏弧菌感染的杂色鲍全组织均一化cDNA文库的构建

Construction of whole body normalized cDNA library of Haliotis diversicolor Reeve challenged by Vibro harveryi

提取了哈维氏弧菌（Vibrio harveryi）感染的杂色鲍（Haliotis diversicolorReeve）总RNA,采用SMART方法合成双链cDNA,并用双链特异核酸酶进行均一化处理。割取0.5~1.0和1.0~3.0kb的片段分别连接pDNR-LIB并转化大肠杆菌（Esherichia coli）,最终构建2种片段大小的杂色鲍全组织均一化cDNA文库。2文库库容均在2.5×105cfu.mL-1左右,PCR检测阳性重组率为100%。随机挑取200个克隆测序,获得高质量EST序列174条。组装后得到149条Unigenes,冗余率为14.37%。序列注释结果表明,有39条Unigene序列与已知基因高度相似。综上所述,文章所建cDNA文库质量良好,可以满足后续研究工作的需要。

We extracted the total RNA of Vibrio harveyi-challenged abalone（Haliotis diversicolor Reeve）.First and second strand cDNA were synthetized by using Creator SMART cDNA Construction Kit and further homogenization of cDNA library was carried out with duplex-specific nuclease（DSN）treatment.Fragments at length of 0.5~1.0 kb and 1.0~3.0 kb were ligated to pDNR-LIB and transformed into Esherichia coli strain DH10B.Both two cDNA libraries have a capacity of 2.5×105 cfu·mL-1,and all of the 30 randomly picked colonies are identified as positive recombinant plasmids by PCR.Furthermore,200 randomly selected clones were sequenced and 174 high quality ESTs were acquired,which belong to 149 unigenes including 12 contigs and 137 singlets after assembling with a redundancy rate of 14.37%.The result indicates that 39 unigenes get high similarity with known genes.In conclusion,the cDNA library we have constructed has good quality and can facilitate further research work.