锯缘青蟹卵巢均一化cDNA文库的构建

Normalized cDNA Library Construction of Ovary of Scylla serrata

为探讨锯缘青蟹(Scylla serrata)性腺发育的分子机制,本研究提取锯缘青蟹卵巢的总RNA,经oligotex试剂分离mRNA,利用SMART(Switch mechanisms at the 5 end of RNAtranscript)技术及双链特异核酸内切酶DSN(duplex-specific nuclease)的特性构建均一化cDNA文库。经检测文库约含有4.7×107重组子,扩增后的文库含有1011以上重组子。从文库中随机挑取24个克隆进行PCR检测,文库的插入cDNA长度为500bp-2.5kb,重组率为95.8%。该文库的成功构建,为采用基因芯片技术开展锯缘青蟹性腺发育相关功能基因的研究奠定了基础。

Total RNA was isolated from ovary of Scylla serrata.Oligotex(QIAGEN) was used to isolate the mRNA from the total RNA.The first strand cDNA was synthesized by transcription of mRNA with the SMART technique.The LD-PCR was performed using a modified SMART primer as the primer set,and first-strand cDNA as the template to synthesize double strand cDNA.After treatment with DSN,the normalized cDNAs were digested with Sfi I enzyme and purified with gel purification kit to remove the small PCR products(<500bp).These normalized cDNAs were ligated into the Sfi I-digested pDNR-LIB Vector.E.coli(TOP10) were transformed with the ligation mixture to generate a normalized cDNA library.The titer of unamplified cDNA libraries was 4.7×104cfu/ml.The titer of amplified cDNA libraries was above 108cfu/ml.The cDNA inserts sizes ranged between 0.5-2.5kb.These results indicate that the normalized cDNA library is suitable quality to be used for further cloning and analysis of genes related to gonad development in Scylla serrata.