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本研究以日本通草蛉Chrysoperla nipponensis(Okamoto)为材料,采用Oligo(dT)引物定向克隆构建cDNA文库并进行EST序列测定,旨在以基因库的形式进行种质资源的保存,为其遗传改良奠定基础,并为探讨其分类地位提供分子依据。对该文库质量分析表明:库容量为1.0×106,重组率为80.0%,平均插入片段为512bp。测序后最终成功得到323条表达序列标签(expressed sequencetags,ESTs)序列,经Phrap程序聚类拼接后得到236条单基因簇(unigene),包括86个重叠群(congtigs)和150个单拷贝(singlets)。使用NCBI中的BlastN和BlastX程序对236条ESTs进行本地化搜索,BlastN的结果表明:180条ESTs(76.3%)没有注解,56条ESTs(23.7%)与GenBank上公布的序列有较高的同源性,其中一条序列被确定为该种的16SrRNA基因,利用MEGA软件构建了基于该16SrRNA序列草蛉科的系统发育树,结果显示通草蛉属Chrysoperla与叉草蛉属Dichochrysa、玛草蛉属Mallada、草蛉属Chrysopa的亲缘关系比较近,这与传统分类相吻合。BlastX的比对结果为197条ESTs(83.5%)有功能注解,39条ESTs(16.5%)无注解或score值小于100。使用GO(geneontology)数据库对236条ESTs序列进行功能注释,结果表明:142条ESTs(59.7%)有注解,并表达出40多种基因产物。
In this study, a cDNA library was constructed by using Oligo (dT) primer directed cloning from Chrysoperla nipponensis (Okamoto), and the EST sequence was determined in order to preserve the germplasm resources in the form of genebank and lay the foundation for its genetic improvement Basis, and to provide a molecular basis for exploring its taxonomic status. The quality analysis of the library showed that the library size was 1.0 × 106, the recombination rate was 80.0%, and the average inserted fragment was 512bp. A total of 323 sequences of expressed sequence tags (ESTs) were successfully obtained after sequencing, and 236 unigene were obtained by phylogenetic tree clustering, including 86 congtigs and 150 single copies ). The 236 ESTs were searched locally using BlastN and BlastX programs in NCBI. BlastN results showed that there were no annotations for the 180 ESTs (76.3%), and 56 ESTs (23.7%) were highly homologous to the published sequences in GenBank One of which was identified as 16S rRNA gene of this species. The phylogenetic tree based on this 16S rRNA sequence was constructed by using MEGA software. The results showed that both Chrysoperla and Dichochrysa, Mallada , Chrysopa grass is relatively close genetic relationship, which is consistent with the traditional classification. The BlastX alignment gave functional annotations of 197 ESTs (83.5%), 39 ESTs (16.5%) with no comments or a score of less than 100. The functional annotation of 236 ESTs using GO (geneontology) database showed that 142 ESTs (59.7%) were annotated and more than 40 gene products were expressed.