春兰与大花蕙兰杂交种红花呈色机理初探

Preliminary Study on the Coloring Mechanism of Red Flowers in a Hybrid Between Cymbidium goeringii and Cymbidium hybridum

为探究兰属杂交种红花的呈色机理,选择绿花春兰和黄花大花蕙兰的红花杂交种黄金梅为试验材料,通过小花蕾期和始花期花瓣的转录组测序分析,筛选影响花色的关键结构基因及调节基因,实时荧光定量PCR(qPCR)验证基因的差异表达,分光光度计法检测花瓣色素(类黄酮、叶绿素A、叶绿素B、花青苷)的含量。结果表明,通过测序共获得113 780条单基因(Unigene),其中200~300 bp的Unigene占比42.68%;44 088个Unigene获得注释信息;与小花蕾期花瓣比较,始花期花瓣上调基因有1 855个,下调基因有2 494个;差异表达基因(DEGs)被GO数据库注释划分为47个功能组;1 219个DEGs被KEGG数据库注释,涉及166条代谢途径;根据KEGG代谢通路及基因注释结果,筛选出与花色相关的54个关键结构基因和21个转录因子;花青苷合成基因DFR、ANS、3,5GT及转录因子Zm1、Hv1、MYB305表达量在始花期上调,在NCBI库BLAST同源基因发现黄金梅DFR、ANS、3GT基因与大花蕙兰的基因同源性最高,5,3GT、3,5GT、Zm1基因与蝴蝶兰和石斛兰的基因同源性最高,转录因子Hv1、MYB305与建兰的基因同源性最高。qPCR参试基因表达量变化与转录组测序结果趋势一致。各阶段花瓣总黄酮的含量显著高于类胡萝卜素和叶绿素含量;始花期花瓣中花青苷含量升高。本研究结果为兰属杂交种花色基因的功能分析提供了参考。

In order to explore the coloring mechanism of red flower in Cymbidium hybrids, the hybrid Huangjinmei with red flowers between C.georigingii with green flowers and C.hybridum with yellow flowers was selected as the experimental material. The key structural genes and the regulatory genes affecting the color were selected based on the transcription sequencing data of the petals at little flower bud and initial bloom stages. The differentially expressed unigenes(DEGs) was verified by qPCR, and the contents of the pigments(carotenoid, chlorophyll a,chlorophyll b,anthocyanin,total flavonoids) in the petals were detected by the spectrophotometer. The results showed that a total of 113 780 Unigenes was obtained by transcriptome sequencing, the ratio of Unigenes with length of 200-300 bp was 42.68%, among which 44 088 Unigenes were annotated. Compared to the little flower bud stage, there were 1 855 DEGs were up-regulated and 2 494 were down-regulated at initial bloom stage. DEGs were assigned into 47 gene ontology(GO) terms, and 1 219 DEGs were assigned into 166 kyoto encyclopedia of genes and genomes(KEGG) pathways. According to the KEGG pathway and the gene annotations, 54 key structural genes and 21 transcriptional regulatory genes associated with the flower color were screened. The expression levels of DFR, ANS, 3,5GT genes and Zm1, Hv1, MYB305 transcription factors of anthocyanin biosynthesis was up-regulated at initial bloom stage. Blasting to the homology genes in the NCBI, DFR、ANS、3GT genes were found having the highest homology with the genes of Cymbidium hybrid cultivar, 5,3GT、3,5GT、Zm1 genes having the highest homology with the genes of Phalaenopsis equestris and Dendrobium catenatum, and the transcription factors Hv1、MYB305 having the highest homology with the genes of C.ensifolium. The qPCR results showed that the expressions of genes were consistent with the resulting transcription sequencing. The contents of total flavonoids in the petals were higher than the contents of carotenoid and chlorophyll at different stages, and the contents of anthocyanin went up at initial bloom stage. The results of this study provide a reference for the study of the function analysis of flower color genes in Cymbidium hybrids.