索式桃花水母全长cDNA文库的构建

目的]构建索式桃花水母的全长cDNA文库,为桃花水母的生长、发育、衰老过程功能基因的进一步筛选、克隆和表达奠定基础。方法]利用RNAiso试剂盒提取桃花水母的总RNA,采用SMART技术合成全长cDNA,经SfⅠi酶切消化后,将cDNA克隆到质粒载体pDNR-LIB,并对cDNA文库进行质量评价。结果]经鉴定,原始文库的滴度为7.9×10^5cfu/ml,库容量约为1.0×10^6cfu。随机挑取28个克隆,经PCR快速鉴定,插入片段大小在0.5～2.5kb,平均大小在1.0kb左右,重组率达94%,表明获得了高质量的桃花水母cDNA文库。结论]该试验成功构建了桃花水母cDNA文库,其cDNA原始文库的库容,根据真核生物能够满足建库的要求,同时文库的cD-NA插入片段序列是完整的,从而证实构建的文库质量较好,可以用于桃花水母目的基因的筛选。

Construction of Full-length cDNA Library of Craspedacusta sowerbyi

Objective] The study aimed to construct the full-length cDNA library of Craspedacusta sowerbyi and laid the foundation for further screening,cloning and expression of the function genes involving the growth and development process of C.sowerbyi.Method] Total RNA was extracted from C.sowerbyi using RNAiso Plus kit.Double-stranded cDNAs were synthesized with SMART technique and were digested by the enzyme SfiⅠ,and then the cDNAs were cloned into the vector pDNR-LIB.Result] The identification showed that the titer of primary library was 7.9×105 cfu/ml,with the library volume of about 1.0×106 cfu.The results from the random sampling of 28 clones indicated that the inserted fragments ranged from 0.5 kb to 2.5 kb by PCR amplification,with an average size of 1.0 kb,and the recombination rate was 94%,which showed that a full-length cDNA library with high quality on C.sowerbyi was well constructed.Conclusion] The experiment constructed the full-length cDNA library of C.sowerbyi successfully.Its library volume of cDNAs primary library could meet the demand of building the library according to eukaryote and the inserted fragments from the cDNAs primary library was intact,which proved that the constructed library had good quality and it could be used to screen the objective gene.