Construction of a cDNA Library from the Testis and Sequence Analysis of the Ubiquitin Gene from Rana nigromaculata

黑斑蛙精巢组织cDNA文库的构建及泛素基因序列的分析(英文)

A full-length cDNA library from the testis of dark-spotted frogs （ Rana nigromaculata ） was constructed with the SMART （switching mechanism at 5＇ end of RNA transcript） technique. Total RNA was extracted from the testis and reverse transcripted into full-length cDNA using PowerScript reverse transcriptase. The first-strand cDNA was amplified using long-distance PCR （LD-PCR）. After Sfi Ⅰ digestion and fractionation, cDNA （ 〉 500 bp） was ligated to λ TriplEx2 vector and packaged with GigapackⅢ Gold Packaging Extract. The titers of optimal primary libraries were 2.0×10^6 pfu/mL and 2.4 × 10^6 pfu/mL and the tlters of the amplified libraries were 0.48 × 10^9 pfu/mL and 3.0 × 10^9 pfu/ mL, respectively. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. The libraries were converted into pTriplEx2 plasmids in E. coli BM 25.8 strain. The insert sizes were measured by PCR which showed most fragments were over 500 bp and the average length was 1.0 kb approximately. A positive clone of 1 171 bp was sequenced and named RnUb based on sequence similarity with the known ubiquitin genes in Gen- Bank. This sequence was a full-length cDNA with complete coding sequences, which indicated that the library built a base for screening the full-length cDNA. These data showed that this library attained to the requirements of a standard cDNA library. This library provided a useful resource for the functional genomic research of Rana nigromaculata.

采用SMART(switching mechanismat 5 end of RNA transcript)技术构建了黑斑蛙(Rana nigromaculata)精巢组织全长cDNA文库。一步法提取成体蛙精巢组织总RNA,用PowerscriptTM反转录酶逆转录合成第一链cD-NA;再用LD-PCR合成双链cDNA;经过SfiⅠ酶切和Chroma spin-400柱分离后,500bp以上的片段与λTriplEx2载体连接,再用GigapackⅢGold Packaging Extract包装蛋白包装,即获得原始文库。原始文库进行扩增后得到扩增文库。经检测原始文库的滴度分别为2.0×106pfu/mL和2.4×106pfu/mL,扩增后的文库滴度分别为0.48×109pfu/mL和3.0×109pfu/mL,重组率均在90%以上。通过E.coliBM25.8菌株将文库转化为pTriplEx2质粒,挑选一阳性克隆进行PCR检测,其插入片段平均长度约为1.0kb。挑取一阳性克隆分别从5′端和3′端进行测序,得到一长约1171bp的序列。经序列分析知,该序列含有完整的编码框,可编码305个氨基酸,是一全长cDNA序列。提示所建文库是可以用于全长cDNA的筛选。结果表明,所构建的黑斑蛙精巢组织cDNA文库的各项指标均满足建库的基本要求。该文库将为蛙类及两栖类的已知或未知的功能基因及新基因的获得及其研究提供可靠资源;另外,该文库还将为研究蛙类动物的性别决定和分化相关基因及其表达提供直接的分子资料。