秋石斛LCYB、LCYE基因克隆和表达分析及其与花色的关系

Cloning and expression analysis of LCYB and LCYE genes in Phalaenopsis-type Dendrobium hybrids and their relationship with flower color

关键词:  番茄红素β-环化酶, 番茄红素ε-环化酶, 基因克隆, 表达量分析, 花色, 类胡萝卜素

Key words:  lycopene β-cyclase, lycopene ε-cyclase, gene cloning, expression level analysis, flower color, carotenoids

摘要:

类胡萝卜素是秋石斛重要的花色素之一,为探究秋石斛花朵中类胡萝卜素生物合成过程,该文以黄花秋石斛品种‘金童'花芽为材料,克隆了类胡萝卜素合成途径下游分支上的2个关键合成酶基因DhLCYB和DhLCYE,并对其进行序列分析,利用RT-qPCR测定了其在不同花色秋石斛品种花发育不同阶段的表达特征,并利用靶向代谢组学技术研究类胡萝卜素组成及含量且分析色素组成和含量与花色及基因表达之间的相关性。结果表明:(1)克隆到了基因DhLCYB和DhLCYE的转录本,它们编码的氨基酸序列与同属的铁皮石斛、鼓槌石斛同源蛋白一致性最高。作为番茄红素的2个环化酶,DhLCYB和DhLCYE在蛋白质三维结构上十分相似。(2)DhLCYB和DhLCYE在不同秋石斛品种花发育不同阶段表达特征各异。DhLCYE在黄色花中表达量比紫红色花高且与叶黄素和α-胡萝卜素含量显著正相关; DhLCYB在不同花色品种中表达没有明显的规律,但其在S1时期的表达量与花药黄质、紫黄质等LCYB催化分支途径的β, β-类胡萝卜素类代谢物显著正相关。(3)从秋石斛花中共鉴定出48种类胡萝卜素,其中胡萝卜素类4种,叶黄素类44种; 总类胡萝卜素含量在黄色品种中高于紫红色品种。(4)15种类胡萝卜素及其衍生物、总类胡萝卜素含量与花色的色相a^*值显著负相关,与b^*值显著正相关。该研究为秋石斛花色改良提供了基因资源和育种思路。

Abstract:

Carotenoids is one of the important floral pigments in Phalaenopsis-type Dendrobium hybrids(Den-Phals). In order to explore the biosynthesis of carotenoids in Den-Phals flowers two key synthase genes DhLCYB and DhLCYE on the downstream branch of carotenoid synthesis pathway were cloned from the flower buds of Den-Phals cultivar Dendrobium ‘Thongchai Gold', and their sequences were analyzed, and their expression characteristics at different stages of flower development in Den-Phals with different flower colors were measured using RT-qPCR, and their correlation with the composition and content of carotenoids were analyzed, which were investigated through targeted metabolomics assay. The results were as follows:(1)Transcripts of DhLCYB and DhLCYE genes were cloned. Their amino acid sequences showed the highest homology to those of species of the same genus, D. candidum and D. chrysotoxum, showed the highest homology. As two lycopene cyclase enzymes, DhLCYB and DhLCYE showed highly similar in protein 3D structure.(2)DhLCYB and DhLCYE had different expression characteristics at different stages of flowering in Den-Phals. DhLCYE had a higher expression level in yellow flowers than that in purple-red flowers, and was significantly positively correlated with the content of lutein and α-carotene; there was no regular pattern in the expression of DhLCYB in various cultivars with different colors, but its expression level during the S1 stage was significantly positively correlated with the β, β- carotenoid metabolites of LCYB catalytic branching pathways such as antheraxanthin and violaxanthin.(3)A total of 48 kinds of carotenoids were identified from the flowers of Den-Phals, including 4 carotenes and 44 xanthophylls; the total carotenoid content was higher in yellow cultivars than that in purple-red ones.(4)The content of 15 kinds of carotenoids and their derivatives, as well as total carotenoids, was significantly negatively correlated with the chroma a^* value and significantly positively correlated with b^* value. This study provides genetic resources and breeding ideas for flower color improvement of Den-Phals.