CAJ | 학술논문

采用SMART(switching mechanism at 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cDNA; 再用LD-PCR合成双链cDNA; 经过Sfi Ⅰ酶切和Chroma spin-400柱分离后, 500 bp以上的片段与λTriplEx2载体连接, 再用Gigapack(R) Ⅲ Gold 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2.0×106 pfu/mL和2.4×106 pfu/mL, 扩增后的文库滴度分别为0.48×109 pfu/mL和3.0×109 pfu/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1.0 kb. 挑取一阳性克隆分别从5'端和3'端进行测序, 得到一长约1 171 bp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.
채용SMART(switching mechanism at 5' end of RNA transcript)기술구건료흑반와(Rana nigromaculata)정소조직전장cDNA문고. 일보법제취성체와정소조직총RNA, 용PowerscriptTM반전록매역전록합성제일련cDNA; 재용LD-PCR합성쌍련cDNA; 경과Sfi Ⅰ매절화Chroma spin-400주분리후, 500 bp이상적편단여λTriplEx2재체련접, 재용Gigapack(R) Ⅲ Gold Packaging Extract포장단백포장, 즉획득원시문고. 원시문고진행확증후득도확증문고. 경검측원시문고적적도분별위2.0×106 pfu/mL화2.4×106 pfu/mL, 확증후적문고적도분별위0.48×109 pfu/mL화3.0×109 pfu/mL, 중조솔균재90%이상. 통과E. coliBM25.8균주장문고전화위pTriplEx2질립, 도선일양성극륭진행PCR검측, 기삽입편단평균장도약위1.0 kb. 도취일양성극륭분별종5'단화3'단진행측서, 득도일장약1 171 bp적서렬. 경서렬분석지, 해서렬함유완정적편마광, 가편마305개안기산, 시일전장cDNA서렬. 제시소건문고시가이용우전장cDNA적사선. 결과표명, 소구건적흑반와정소조직cDNA문고적각항지표균만족건고적기본요구. 해문고장위와류급량서류적이지혹미지적공능기인급신기인적획득급기연구제공가고자원; 령외, 해문고환장위연구와류동물적성별결정화분화상관기인급기표체제공직접적분자자료.
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 Gigapack(R) Ⅲ Gold Packaging Extract. The titers of optimal primary libraries were 2.0×106 pfu/mL and 2.4×106 pfu/mL and the titers of the amplified libraries were 0.48×109 pfu/mL and 3.0×109 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 GenBank. 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.