万寿菊胡萝卜素羟化酶基因 TeCHYE 克隆与分析

Cloning and analysis of carotene hydroxylase gene TeCHYE in Tagetes erecta L.

α-胡萝卜素ε环羟化酶(α-caroteneε-ring hydroxylase,CHYE)是植物类胡萝卜素合成途径下游关键酶,催化叶黄素的形成。万寿菊(Tagetes erecta L.)是菊科园艺作物,富含类胡萝卜素,尤其是叶黄素,可用于天然色素的提取。文章根据万寿菊的转录组数据,通过花组织RNA提取、反转录合成cDNA,以克隆获得编码万寿菊α-胡萝卜素ε环羟化酶的基因TeCHYE,连接于克隆载体pEASY-Blunt。序列分析结果显示,该基因为读码框全长1656 bp、编码相对分子质量约62 kDa的亲水性蛋白。进化分析表明,CHYE基因在植物中保守存在,万寿菊TeCHYE与同科植物向日葵HaCHYE同源性最高。进一步对万寿菊叶片、不同发育时期花组织中叶黄素的质量比及其TeCHYE基因表达水平进行分析,结果显示,TeCHYE在花的发育过程中表达水平显著升高,表明其参与了叶黄素在万寿菊花中的合成积累。

α-caroteneε-ring hydroxylase(CHYE)is a key downstream enzyme of the carotenoid synthesis pathway in plants,catalyzing the formation of lutein.Marigold(Tagetes erecta L.)is an ornamental crop of Compositae,which is rich in carotenoids,especially lutein,to be used for natural pigments extraction.In this paper,cDNA from marigold flower was synthesized by RNA extraction and reverse transcription to clone the Tagetes erecta L.α-caroteneε-ring hydroxylase(TeCHYE)gene based on the transcriptome data.Then,TeCHYE was ligated to the cloning vector pEASY-Blunt.The sequence analysis indicated the 1656 bp open reading frame of TeCHYE,encoding a hydrophilic protein with relative molecular weight of 62 kDa.The phylogenetic analysis suggested that CHYE gene conserves in plant species,and TeCHYE showed the highest identity with homologous HaCHYE from sunflower belonging to the same family.The content of lutein,as well as the expression level of TeCHYE,in marigold leaves and flower tissues at different development stages were furtherly analyzed.The significantly increased level of TeCHYE in developmental flowers suggested its involvement in the synthesis and accumulation of lutein in marigold.