柞蚕蛹全长cDNA文库的构建和随机EST测序

Construction of a Full-length cDNA Library From Antheraea pernyi Pupae and Random Sequencing of Expressed Sequence Tags

采用RNA转录5′末端转换（SMART）技术构建了柞蚕（Antheraea pernyi）蛹的全长cDNA文库。所构建cDNA文库的容量为5×10^5个独立克隆,插入片段长800-2500 bp,且90%的插入片段大于1 kb。随机挑取288个克隆进行表达序列标签（EST）测序,有效序列为250条,根据EST测序结果计算文库重组率达95%。经序列拼接得到175个unigenes,通过序列比对发现其中97个unigenes与GenBank中的已知基因高度同源,且有88条全长序列,基因的完整性比率达90%。在随机EST测序中获得了具有5′端和3′端非编码区的延伸因子-1α基因（Gen-Bank登录号：FJ788508）,该基因cDNA全长1743 bp,有一个1392 bp的开放阅读框,编码含463个氨基酸残基的蛋白,蛋白的理论分子质量为50.4 kD,等电点8.96。EST测序分析表明,柞蚕蛹cDNA文库符合构建基因文库的质量要求,该文库的构建将有助于柞蚕功能基因的克隆和研究。

By using switching mechanism at 5′ end of RNA transcript（SMART） approach,we constructed a full-length cDNA library from Antheraea pernyi pupae.The cDNA library contained 5×10^5 individual clones.The inserted fragments were 800 to 2 500 bp long and 90% of the fragments were over 1 000 bp.288 clones were selected randomly for expressed sequence tag（EST） sequencing,among which 250 yielded valid sequences.The calculation based on EST sequencing result gave out that recombination rate of the library reached 95%.Online assembly of the above EST sequences yielded 175 unigenes,among which 97 were homologous with known genes in GenBank.Out of them,88 were in their full-length form,meaning that the percentage of integral genes reached 90%.A full-length elongation factor-1α cDNA（GenBank accession No.FJ788508） of 1 743 bp with 5′ and 3′ untranslated regions was isolated by EST sequencing.This cDNA contained an ORF of 1 392 bp coding for a protein of 463 amino acids.The predicted molecular weight and isoelectric point of this protein was 50.4 kD and 8.96,respectively.EST sequencing analysis showed that the constructed cDNA library from Antheraea pernyi pupae reached the quality requirement of gene library.This library would greatly facilitate molecular cloning and functional studies of certain genes in Antheraea pernyi.