桑泛素延伸蛋白基因片段的克隆及序列分析

Cloning and Characterization of Ubiquitin Extension Protein Gene Fragment of Mulberry

目的:利用3’RACE技术克隆植物泛素基因,是进一步研究其功能的基础。方法:本研究从桑树(丰驰桑)(Morus bomby-cis)幼叶中提取总RNA,反转录成cDNA,根据已报道的泛素基因序列设计1条正向引物,利用3’RACE(Rapid Amplification of cDNAEnd)技术进行扩增。结果:扩增出1条690 bp的泛素基因片段。该片段5’端为编码156个氨基酸残基的阅读框,3’末端有219bp的非翻译区。结论:同源分析表明,此cDNA序列为泛素延伸蛋白基因(Genebank登录号为DQ839403)。用Genedoc软件对该片段编码的氨基酸序列进行同源性分析的结果表明:桑树泛素延伸蛋白与马铃薯、烟草、陆地棉、黄瓜的泛素延伸蛋白以及苜蓿的核糖体S27A蛋白的同源性都在96%以上。

Objective：Cloning plant ubiquitin extension protein gene with 3′ RACE,that was the foundation of researching function. Methods：Total RNA was extracted from the young leaves of Morus bombycis and transcribed into cDNA. Using one universal primer and another designed from reported ubiquitin gene sequences, a 690 bp cDNA fragment were amplified with 3′ RACE （Rapid Amplification of cDNA End） method. Results： The fragment contains a 3′ UTR （Untranslated Region） 219 bp in length and a 5′ ORF （Open Reading Frame） which encodes 156 deduced amino acid residues. Conclusion：The deduced amino add sequence showed more than 96% similarity with the corresponding ubiquitin protein fragment of Solanum tuberosum, Nitotiana tabacum, Gossypium hirsutm, C. sativus and ribosomal S27A protein of M. multicaulis. The sequence information of the fragment indicates that this is the ubiquitin gene fragment.