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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。
The full length cDNA of lycopene ε-cyclase (LCYE) gene was cloned from the petals of the rare ornamental Camellia nitidissima by RT-PCR and RACE techniques and named as CnLCYE. The nucleotide sequence analysis showed that the CnLCYE gene was 2 149 bp in length and contained 291 bp of untranslated region (UTR), 265 bp of 3 ’UTR and 1 593 bp of open reading frame (ORF) encoding 530 amino acids. The gene encodes a protein containing two transmembrane domains, one NADB_Rossmann superfamily domain, and a lycopene ε-cyclase domain (PLN02697). The secondary structure of CnLCYE protein was random coil, followed by α-helix, with the least proportion of β-sheet. The results of amino acid sequence alignment showed that the homology of LCYE protein between CnLCYE and Solanaceae and Rosaceae was more than 70%, which was the highest homology with the LCYE of Camellia sinensis var. Assamica. According to the full-length sequence of CnLCYE, full-length amplification primers with KpnⅠ and Bam HⅠ loci were designed to amplify the full length of the gene. After digested with KpnⅠand Bam HⅠ, the full length cDNA of CnLCYE gene was ligated with p CAMBIA1300. Expression vector, in order to further study the function of CnLCYE gene and its role in regulating the color to provide help.