日本通草蛉cDNA文库构建及部分ESTs分析

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512 bp。测序后最终成功得到323条表达序列标签（expressed sequence tags,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16S rRNA基因,利用MEGA软件构建了基于该16S rRNA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(gene ontology)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。

cDNA library construction and analysis of some ESTs of Chrysoperla nipponensis(Okamoto) (Neuroptera: Chrysopidae)

Based on the construction technology of non-normalized cDNA library, the cDNA library of Chrysoperla nipponensis (Okamoto) was constructed, which aimed to preserve the resources in the form of cDNA library, to help to modify its genes and to provide the molecular basis for the classification of Chrysoperla nipponensis. The library capacity was 1.0×10⁶, the average size of insert cDNA was 512 bp, and the recombination frequency was 80.0％. In total 323 expressed sequence tags (ESTs) were generated successfully after sequencing, and 236 nonrepetitive sequences including 86 contigs and 150 singlets were obtained using cluster analysis with Phrap software. The 236 ESTs were analyzed with BlastN and BlastX of NCBI. The BlastN analysis showed that 180 (76.3%) from the 236 ESTs could not be compared, and the other 56 (23.7%) had a high homology with the available sequences from GenBank,of which one sequence was considered to be 16S rRNA gene. Based on the 16S rRNA sequences, phylogenetic tree was constructed by MEGA, and the result showed that the evolutionary relationship of Chrysoperla, Dichochrysa, Mallada and Chrysopa was relatively close, which coincides with the traditional classification. The BlastX analysis showed that among the 236 ESTs, 179 (83.5%) could be functionally annotated, and the other 39 (16.5%) had no functional information or a score less than 100. Finally, 236 ESTs were annotated by GO (gene ontology) database, and the results showed that 142 ESTs (59.7%) could be functionally annotated and expressed 40 products