黄皮西葫芦类胡萝卜素合成酶基因的鉴定及PSY1和LCYE2克隆分析

Identification of Enzyme Genes Related to Carotenoid Synthesis and Cloning Analysis of PSY1 and LCYE2 in Yellow Peeled Zucchini

类胡萝卜素是自然界中分布最为广泛的一大类色素,赋予植物花、果实等器官鲜艳的色彩,其合成过程中含有多种代谢酶基因。为了揭示黄皮西葫芦类胡萝卜素积累的分子机制,为进一步研究西葫芦类胡萝卜素合成酶基因的功能奠定基础,首先基于西葫芦基因组数据库对西葫芦类胡萝卜素合成代谢家族基因进行全基因组鉴定;进一步利用已公开发表的相关转录组数据筛选、qRT-PCR验证并克隆类胡萝卜素合成代谢关键酶基因。结果表明,在西葫芦基因组数据库中共发现48个类胡萝卜素代谢酶基因成员;根据Sweet REBA和Lady Godiva这2种不同颜色果皮材料不同发育时期果肉的转录组数据筛选出类胡萝卜素关键酶基因PSY1和LCYE2等在不同颜色果实发育时期存在显著性差异表达。qRT-PCR定量分析结果显示,除PSY1基因0 d和LCYE2基因2 d外,PSY1、LCYE2基因在黄皮西葫芦不同发育时期果皮中的相对表达量显著高于白皮西葫芦对应时期。对白皮西葫芦和黄皮西葫芦果皮PSY1和LCYE2等基因的全长CDS分别进行克隆、测序分析发现,白皮西葫芦和黄皮西葫芦2个材料的PSY1基因均比西葫芦参考基因组预测编码区序列多了一个51 bp的外显子,与已经公开的PSY基因序列(GenBank登录号:XM_023695146.1)一致;而LCYE2基因均与参考基因组无显著差异。系统进化树分析也表明,西葫芦PSY1与克隆报道的中国南瓜PSY(JN176311.1)同源,相似性高达98.80%。

Carotenoids are the most widely distributed class of pigments in nature,which endow plants flowers,fruits and other organs with bright colors.The synthesis process of carotenoids contains a variety of metabolic enzyme genes.In order to reveal the molecular mechanism of carotenoid accumulation in yellow peeled zucchini,lay a foundation for further studying the function of carotenoid synthase gene in zucchini,this study identified zucchini carotenoid anabolism family genes based on the zucchini genome database,and further screened and cloned the key enzyme genes for carotenoid synthesis and metabolism by publicly published transcriptome data and qRT-PCR verification.The results showed that 48 carotenoid metabolizing enzyme gene members were detected in zucchini genome;and two key genes PSY1 and LCYE2 were screened out and showed different expression during the different developmental stages based on the transcriptome data of Sweet REBA/Lady Godiva.qRT-PCR analysis also showed that,except for PSY1 gene 0 d and LCYE2 gene 2 d,the relative expression of PSY1 and LCYE2 in different developmental stages of yellow peeled zucchini were significantly higher than that of white peeled zucchini.The full-length CDS of PSY1 and LCYE2 genes were cloned and sequencing analyzed from white and yellow peeled zucchini,respectively,which suggested that PSY1 genes of both materials were 51 bp more than the predicted coding sequence in zucchini reference genome,and were all consistent with the published sequence of PSY gene(GenBank number:XM_023695146.1),while the sequences of LCYE2 gene was not significantly different from the reference genome.Phylogenetic tree analysis showed that zucchini PSY1 was homologous to the reported Cucurbita moschata PSY(JN176311.1)with the best similarity as high as 98.80%.