黄秋葵番茄红素β-环化酶LCYB基因的克隆与分析

Cloning and Expression Analysis of a Lycopene Beta-cyclase Gene in Hibiscus esculentus L.

应用RT-PCR和RACE技术克隆得到cDNA全长1797 bp的黄秋葵(Hibiscus esculentus L.)番茄红素β-环化酶基因LCYB,开放阅读框(ORF)包含1509个碱基;预测编码503个氨基酸,理论分子量(Mw)为56.288 kD,等电点(p I)为4.577;编码的蛋白与陆地棉(Gossypium hirsutum L.)、黄麻(Corchorus capsularis L.)和可可(Theobroma cacao L.)同源蛋白的相似性均在88%以上,显示其高度的保守性,将基因命名为HeLCYB,GenBank登录号为:KX257998。Motif Scan分析显示,蛋白氨基酸序列88~481位为HeLCYB保守结构域,并在88~113位含有1个FAD结合域。通过荧光定量PCR分析表明,HeLCYB基因在黄秋葵根、茎、叶、花和果荚中均有表达;叶片生长中以成熟叶中表达最高,果实发育中以花后7d高表达。建立与优化了黄秋葵类胡萝卜素超高效液相检测体系,结果显示黄秋葵中主要含有β-胡萝卜素和叶黄素。β-胡萝卜素在成熟叶中含量最高,果实以花后7 d含量最高,与HeLCYB基因的表达呈正相关。本研究揭示了HeLCYB基因表达和类胡萝卜素积累的特性,为开展黄秋葵类胡萝卜素分子调控机制研究奠定了基础。

In this study, a LCYB gene was cloned from Hibiscus esculentus L. by RT-PCR and RACE technique. The full-length cDNA sequence of LCYB was 1797 bp ,which contained a 1509 bp open reading frame （ORF） that encoded 503 amino acids, with a predicted molecular weight （mol. wt） of 56. 288 kD and a hypothetical isoelectric point （pI） of 4. 577. It shared over 88% identity with the homologous proteins from Gossypium hirsutum,Corchorus capsulars, and Theobroma cacao, showing that it was highly conservative. This gene was named HeLCYB and the GenBank accession number was KX257998. Motif Scan analysis showed that HeLCYB protein had a conservative structure domain and a FAD binding domain in the position of 88-481 and 88-113 sites, respectively. Real-time PCR analysis revealed that HeLCYB expressed in different tissues of Hibiscus esculentus L. , including roots, stems, leaves, flowers and fruits, and the level was the highest in mature leaves and 7 days after flowering during the process of leaf and fruit development, respectively. What＇s more, an ultra-high performance liquid chromatography （UPLC） method for the determination of Hibiscus esculentus L. carotenoid contents was explored. Hibiscus esculentus L. mainly contained lutein and β-carotene, and β-carotene contents was also the highest in mature leaves and 7 days after flowering during the process of leaf and fruit development respectively, which probably related to the changes in HeLCYB genes expression. These results revealed the expression of HeLCYB gene and the accumulation characteris- tics of carotenoid contents, and provided a base for the study of carotenoid molecular regulation mechanism in Hibis- cus esculentus L..