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采用CloneMinerTMcDNA文库构建试剂盒构建了患败血症的鲢(Hypophthalmichthys molitrix)肝和肾的cDNA文库。经检验,文库的滴度为1.34×107 cfu/mL,总库容为2.68×107 cfu,阳性克隆率为97.5%,平均插入片段大于1.2 kb。对80个随机挑选的克隆进行两端测序,结果显示有74个测序成功。经在GenBank上BLAST比对,其中57个为已知功能基因,属于45个不同基因,另外17个没有明显的同源序列。在已知功能的57个克隆中,含有全长编码区序列的克隆共有49个,全长cDNA比率为61.25%。采用PCR法对文库进行筛选,获得一个含MHC class I完整编码区的序列,该序列具有典型的脊椎动物MHC class Ⅰ的序列特征。本研究完成鲢肝和肾较高质量的cDNA文库的构建,旨在为今后开展鲢功能基因研究奠定基础。
The CloneMinerTM cDNA library construction kit was used to construct a cDNA library of liver and kidney of septicemia silver carp (Hypophthalmichthys molitrix). The test showed that the library titer was 1.34 × 107 cfu / mL, the total storage capacity was 2.68 × 107 cfu, the positive cloning rate was 97.5%, and the average inserted fragment was greater than 1.2 kb. Totally 80 randomly selected clones were sequenced at both ends and 74 were sequenced successfully. After BLAST alignment in GenBank, 57 of them were known functional genes, belonging to 45 different genes, and the other 17 had no obvious homologous sequences. Among the 57 clones with known function, there were 49 clones with the full-length coding region, and the full-length cDNA was 61.25%. The library was screened by PCR, and a sequence containing the complete coding region of MHC class I was obtained. This sequence has typical sequence characteristics of vertebrate MHC class Ⅰ. This study completed the construction of higher quality cDNA libraries of silver carp liver and kidney, which laid the foundation for future research on the gene of silver carp.