Construction of a cDNA Library from the Testis and Sequence Analysis of the Ubiquitin Gene from Rana nigromaculata (in English)
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Graphical Abstract
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Abstract
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 SfiⅠ digestion and fractionation, cDNA (>500 bp) was ligated to λ TriplEx2 vector and packaged with GigapackR 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.
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