黄鳝性腺抑制差减文库的构建和分析

Construction and analysis of gonad suppression subtractive hybridi-zation library of rice field eel(Monopterus albus)

应用抑制性差减杂交技术构建了黄鳝（Monopterus albus）IV期卵巢和间期性腺的差减cDNA文库。正向差减杂交以间期性腺为试验方,IV期卵巢为驱动方;反向差减杂交以IV期卵巢为试验方,间期性腺为驱动方。将获得的差减cDNA片段连接质粒载体,然后转化大肠杆菌DH5α,最后获得的正、反向差减文库分别含461、678个重组子。经PCR扩增鉴定插入片段范围为200-2 000 bp。随机选取正、反文库共100个阳性克隆测序分析,得到90个有效基因片段,与GenBank进行BLASTx和BLASTn同源比对。进一步从文库中选取2个基因用于半定量RT-PCR,对文库质量进行验证。结果表明,所建文库能够达到富集这两期性腺差异表达基因的目的。黄鳝性腺差减文库的构建,为进一步分离、鉴定黄鳝性腺发育和性逆转的相关基因奠定了基础。

By using suppression subtractive hybridization（SSH）,subtracted cDNA libraries were constructed from rice field eel（Monopterus albus） gonad at IV ovary and ovotestis stage.Two-directional（forward and backward） SSH was performed under cDNAs of rice field eel gonad at IV ovary and ovotestis stage.The cDNA fragments were inserted into plasmid vectors,and then transformed into Escherichia coli DH5α.Finally,forward and backward subtracted cDNA libraries（461 and 674 clones,respectively） were obtained.By PCR detection,length of the subtractive cDNA fragments cloned into the vector ranged from 200 bp to 2 000 bp.One hundred positive clones were selected and sequenced ran-domly from the forward and the backward libraries,and ninety gene fragment sequences were obtained.EST analysis of these sequences was performed with Blastx and Blaxtn by comparing sequences with GenBank database.Two se-quenced genes were further analyzed by semi-quantitative RT-PCR method.The results showed that the library can enrich differentially expressed genes in the two phases gonad.