金花茶CnLCYE基因cDNA克隆及表达载体构建

Cloning of Camellia nitidissima Lycopene ε-cyclase cDNA and Construction of Sense Expression Vectors

利用RT-PCR和RACE技术,从珍稀观赏植物金花茶(Camellia nitidissima)花瓣中克隆得到了一个番茄红素ε-环化酶(lycopeneε-cyclase,LCYE)基因c DNA全长,命名为CnLCYE。碱基序列分析显示,CnLCYE基因全长2 149 bp,包含291 bp的5'非翻译区(untranslated regions,UTR)、265 bp的3'UTR和1 593 bp编码530个氨基酸的开放阅读框。该基因编码的蛋白质含有2个跨膜结构域,一个NADB_Rossmann superfamily结构域以及番茄红素ε-环化酶结构域(PLN02697)。CnLCYE蛋白二级结构以无规则卷曲为主,其次为α-螺旋,β-折叠所占比例最少。氨基酸序列比对分析结果显示,CnLCYE与茄科、蔷薇科等植物LCYE蛋白同源性都在70%以上,与普洱茶(Camellia sinensis var.assamica)LCYE同源性最高。根据该CnLCYE全长序列设计带有KpnⅠ和Bam HⅠ位点的全长扩增引物扩增基因全长,用KpnⅠ和Bam HⅠ双酶切后与表达载体p CAMBIA1300连接,成功构建了CnLCYE基因的正义表达载体,为深入研究CnLCYE基因的功能及其对花色的调控作用提供了帮助。

By the methods of reverse transcription PCR（RT-PCR） and rapid amplification of c DNA ends（RACE）,a full-length cDNA sequence of lycopene ε-cyclase（LCYE） gene was obtained and named CnLCYE from the petals of Camellia nitidissima, a rare and famous ornamental plant of China. According to the base sequence analyses, the cDNA sequence of CnLCYE was 2 149 bp and contained 291 bp of 5＇-untranslated region（5＇-UTR）,265 bp of 3＇-UTR, and an opening reading frame（ORF） of 1 593 bp encoding 530 predicted amino acids. CnLCYE protein contained 2 transmembrane domains, a NADB_Rossmann superfamily domain and a lycopene ε-cyclase conserved domain（PLN02697）. The analysis data of the second structrure of CnLCYE indicated that random coil was the largest structural element followed by alpha helix and β-sheet. Sequence alignment of amino acids revealed that CnLCYE shared more than 70% homology with LCYE from plants of Solanaceae, Rosaceae and the highest homology with Camellia sinensis var. assamica. The combined sense expression vector of CnLCYE was constructed successfully by connecting the product amplified by the primers which contained the enzyme sites of KpnⅠand Bam HⅠand then digested by enzymes of KpnⅠand Bam HⅠto express vector p CAMBIA1300. This research provided great help for studying the CnLCYE gene function and its roles on flower color.