日本对虾精巢和卵巢全长cDNA文库的构建

The Construction of Full-length Testis and Ovary cDNA Libraries of Marsupenaeus japonicus

采用RDP试剂提取日本对虾精巢和卵巢的总RNA ,经Oligotex试剂盒纯化得到mRNA。根据SMART(switchingmechanismat5′endofRNAtranscript)技术 ,利用含SfiⅠB酶切位点的oligo(dT)引物合成cDNA第一条链 ,利用含SfiⅠA酶切位点的SMART核苷酸作为cDNA第一条链在mRNA 5′端延伸出去的模板 ,采用LDPCR引物合成双链cDNA ,双链cDNA用SfiⅠ酶切和过柱分级分离后 ,与λTriplEx2两臂进行连接 ,随后进行λ噬菌体体外包装反应 ,构建成精巢和卵巢之全长cDNA文库。构建好的文库中 ,精巢的文库含有约 1 0 4× 1 0 6的重组子 ,卵巢的文库含有约为 1 2× 1 0 6的重组子。文库扩增后 ,滴度分别达7 3× 1 0 8(Pfu ml)和 9 1× 1 0 8(Pfu ml) ,插入cDNA长度为 4 0 0bp～ 3kb ,说明两文库均具有良好的质量 ,为进一步筛选、克隆精巢与卵巢特异表达基因奠定了基础。

To clone the testis and ovary specific expression genes of Marsupenaeus japonicus, we constructed two full-length cDNA libraries using the SMART cDNA library construction kit (Clontech). Total RNAs were isolated from tests and ovaries using RDP, a reagent made by us and modified from that used by Chomczynski and Sacchi (1987) for the simultaneous isolation of RNA, DNA, and protein. Oligotex (QIAGEN) was used to separate the mRNA of testes and ovaries from total RNA. The “anchor first-strand cDNA' containing asymmetrical SfiⅠ restriction enzyme sites (A & B) was synthesized by transcription of mRNA with the SMART technique. The LDPCR was performed using a modified oligo(dT) primer and an anchor primer as the primer set, and anchor first-strand cDNA as the template to enrich the cDNA population for full-length sequences. After digestion with SfiⅠ and size fractionation using CHROMA SPIN-400 TM Columns, SMART cDNA was ligated into the SfiⅠ-digested λ TriplEx2 TM Vector. The ligation mixture was packaged into lambda capsids using the Gigapack Ⅲ Gold packaging extract (Stratagene) to generate full-length testis and ovary cDNA libraries of Marsupenaeus japonicus. The titer of un-amplified cDNA libraries was 1.04×106 for the testis library and 1.2×106 for the ovary library. The titer of amplified cDNA libraries was 7.3×108(Pfu/ml)for the testis library and 9.1×108(Pfu/ml)for the ovary library respectively. The cDNA inserts sizes were between 400 bp-3 kb. These results indicate that the quality of these cDNA libraries is good enough for further cloning of testis and ovary specific expression genes.