太子参类胡萝卜素双加氧裂解酶家族4成员的克隆及表达特性分析

Cloning and expression analysis of four carotenoid cleavage dioxygenases genes from Pseudostellaria heterophylla

为探讨太子参环肽成分与品质形成的分子机制,利用生物信息学方法从太子参转录组数据库中筛选出太子参类胡萝卜素双加氧裂解酶(carotenoid cleavage dioxygenases,CCDs)新基因,并设计引物对其进行全长扩增及克隆、生物信息学分析和基因表达分析。克隆得到的4条CCDs序列全长分别为1 617,1 461,1 746,1 875 bp,并根据其功能依次命名为Ph CCD1,Ph NCED2,Ph NCED3,Ph CCD4。序列分析结果表明,4个基因均含有REP65结构域,且均含有与亚铁离子结合的位点。系统进化分析表明,Ph NCED2,Ph NCED3聚为NCEDs一支,Ph CCD1,Ph CCD4聚为CCDs一支,且与拟南芥Arabidopsis thaliana的At CCDs家族相比,Ph CCD1与At CDD1同源性最高,Ph CCD4与At CCD4同源性最高,Ph NCED2,Ph NCED3与At NCED3同源性最高。实时荧光定量分析显示Ph CCD1和Ph CCD4 2个基因的表达主要在地上部分,其中Ph CCD1在叶中的表达量最高,Ph CCD4在茎叶具有高表达,可能参与太子参类胡萝卜素的生物合成;Ph NCED2和Ph NCED3 2个基因的表达主要在地下部分,其中Ph NCED2在须根中表达量最高,Ph NCED3在块根木部和皮部表达量较高,可能是脱落酸生物合成的关键酶基因。研究首次得到太子参CCDs基因,为进一步阐明太子参植株对于外界胁迫的应答机制,进而探讨太子参品质形成的生物途径奠定基础。

To investigate the molecular mechanism of quality formation of Pseudostellaria heterophylla,the carotenoid cleavage dioxygenases（ CCDs） genes were cloned from the transcriptome database of P. heterophylla,and analyzed them with bioinformatics analysis and expression analysis. The sequence length of four new gene were 1 617,1 461,1 746,1 875 bp,and subsequently,named as Ph CCD1,Ph NCED2,Ph NCED3 and Ph CCD4 according to its genetic relationship with Arabidopsis thaliana. The sequence analysis showed that four new gene were all containing REP65 domains and binding sites of ferrous ion,such as histidine,glutamates and aspartates. Analysis phylogeny showed that Ph NCED2 and Ph NCED3 were the cluster of NCEDs,Ph CCD1 and Ph CCD4 were the cluster of CCDs. In addition,Ph CCD1 and At CDD1 of Arabidopsis thaliana,Ph CCD4 and At CCD4 of A. thaliana,Ph NCED2,Ph NCED3 and AtNCED3 of A. thaliana have high similarities. Analysis of real-time fluorescence quantitative showed that Ph NCED2 and Ph NCED3 were expressed mainly in underground part,the expression quantity of Ph NCED2 reached the highest in fibrous root,Ph NCED3 keeps higher in phloem and xylem,it may be the key enzymes of ABA biosynthesis genes. Moreover,Ph CCD1 and Ph CCD4 were expressed mainly in aerial part,the expression quantity of Ph CCD1 reached the highest in leaf,Ph CCD4 keeps higher in stem and leaf. It may be involved in the biosynthesis of carotenoids for P. heterophylla. The study obtained CDDs gene of P. heterophylla for the first time,this would lay the foundation of developing the response mechanism of P. heterophylla about external stress further,and then exploring the biological approach of quality formation in P. heterophylla.