烟草叶片全长cDNA文库构建及EST序列分析

Construction and Sequences Analysis of Full-Length cDNA Library of Tobacco Leaves

【目的】构建烟草叶片全长cDNA文库并测序,发掘与烟叶发育、代谢和抗逆相关的基因,为进一步研究功能基因奠定基础。【方法】以烟草苗期叶片为试验材料,利用改进的Cap-trapper法构建烟草叶片全长cDNA文库。利用测序技术获得大量的EST序列,采用生物信息分析手段,对所测EST进行拼接和功能注释。【结果】构建了烟草叶片的全长cDNA文库,该文库滴度为1.2×106pfu.mL-1,平均插入片段在1.4 kb左右。利用该文库测序了5 280个克隆,获得5 233条高质量表达序列标签(EST),序列拼接出3 922个单一基因;同源比对分析表明,89.7%的基因与已知功能基因具有较高的同源性,这些基因涉及细胞生长、信号转导、翻译合成、抗逆反应和能量代谢等功能;并详细鉴定了部分与烟碱合成和抗病相关的基因。【结论】通过Cap-trapper法成功构建了高质量的烟草幼苗叶片全长cDNA文库;大规模EST测序分析挖掘到大量与烟草幼苗叶片发育、抗逆、抗病、烟碱代谢相关的侯选基因。

[ Objective ] The full-length eDNA library from tobacco leaves mRNA was constructed. EST data were generated and these gene families of development, metabolizability and resistance were detected and ananlyzed. [ Method] The Cap-trapper method was optimized to constructe a full-length enriched eDNA library of tobacco seedling leaves. The assembly and annotation of EST data were completed by bioinformaties program. [ Result] A high quality full-length eDNA library was constructed successfully from tobacco leaves. The titer was 1.2×10^6 pfu.mL^-1 and the average length of inserted eDNA fragments was 1.4 kb. A total of 5 280 clones were sequenced from the library, and 5233 expressed sequence tag （EST） sequences were obtained. The ESTs were assembled into 3 922 unigenes including 564 contigs and 3 358 singletons. The comparative analysis shows that approximately 89.7% of the unigenes have homologs in other plants. These genes were involved in cell development, signal transduction, transcription and synthesis, stress tolerance response, energy metabolism and so on. Nicotine synthesis and transform, and disease resistance genes were identified. [Conclusion] A high-quality full-length eDNA library of tobacco leaves was successfully constructed by Cap-trapper method. Meanwhile, development, nicotine synthesis and transform, stress resistance and disease resistance were identified by sequences analysis of EST.