杂交兰及其花艺突变体中类黄酮成分差异的代谢-转录组联合分析

Integrated Metabolomics and Transcriptomics Analysis Reveals Differential Flavonoids Compounds of Cymbidium Hybrid and Its Mutant

为探索杂交兰花艺突变体变异的分子机理,该研究以杂交兰‘玉凤’及其花艺突变体‘双艺金龙’为材料,利用靶向代谢组学和转录组学鉴定两者中类黄酮化合物含量差异及其相关通路上的差异表达基因。结果表明:(1)代谢组分析发现,杂交兰‘玉凤’和‘双艺金龙’花瓣中共检测到271种类黄酮代谢物,其中黄酮醇类和黄酮类代谢物的相对含量约占总类黄酮的30%~50%;共检测到38个差异代谢物(15个上调,23个下调);‘玉凤’中差异最高的代谢物二氢山奈酚-7-O-葡萄糖苷(黄酮醇类)含量是‘双艺金龙’的124444倍,‘双艺金龙’中差异最高的代谢物3,5,7,3′,4′-五羟基-5′-异戊二烯基黄酮(黄酮醇类)含量是‘玉凤’的7244倍。(2)KEGG分析显示,差异代谢物显著富集在类黄酮、黄酮和黄酮醇生物合成途径。其中,在类黄酮生物合成途径上,根皮苷、黄腐醇、二氢山奈酚、二氢杨梅素和表没食子儿茶素含量升高,柚皮苷和二氢槲皮素含量降低;在黄酮和黄酮醇生物合成途径上,三叶豆苷和槲皮素含量均下降。(3)转录组分析共筛选到563个差异表达基因,与‘玉凤’相比,‘双艺金龙’中有220(39.1%)个基因上调表达,343(60.9%)个基因下调表达;KEGG分析发现,与类黄酮生物合成相关的差异基因(CCoAOMT、CHS、CHI、F3H和FLS)被显著富集。(4)qRT-PCR验证结果显示,‘双艺金龙’中类黄酮生物合成相关基因(CHS2、CHS3、CHI、F3H、FLS)以及转录因子基因HD-ZIP的表达量较‘玉凤’均显著下降,其表达量的变化趋势与代谢物含量的变化趋势一致。研究认为,类黄酮生物合成相关基因的低表达,使下游代谢物合成受阻,进而导致‘双艺金龙’花艺的产生;推测类黄酮生物合成途径中的基因可能是杂交兰花色变化的关键基因。

In order to confirm the molecular basis underlying flower color variegation between Cymbidium hybrid‘Yufeng’and its mutant‘Shuangyijinlong’,we used targeted metabolomics and transcriptomics to identify the differentially expressed metabolites(DEMs)and differentially expressed genes(DEGs)involved in flavonoid biosynthesis.The results showed that:(1)by using targeted metabolomics techniques,we identi ed 271 flavonoid metabolites,in which 30%-50%metabolites were flavonols and flavonoid metabolites.A total of 38 DEMs(15 up-regulated,23 down-regulated)were identified.Among these,the content of dihydrokaempferol-7-O-glucoside(flavonol)in‘Yufeng’was 124444 times of which was in‘Shuangyijinlong’,while the content of uralenol(flavonol)in‘Shuangyijinlong’was 7244 times of which was in‘Yufeng’.(2)KEGG analysis showed that flavonoid biosynthesis,flavone and flavonol biosynthesis were significantly enriched.In flavonoid biosynthesis pathway,the contents of phlorizin,xanthohumol,dihydrokaempferol,dihydromyricetin and epigallocatechin all increased,the contents of naringin and dihydroquercetin decreased.In addition,the contents of trifolin and quercetin decreased in flavone and flavonol biosynthesis pathway.(3)By the transcriptome,a total of 563 DEGs were identified,220(39.1%)DEGs of which were up-regulated and 343(60.9%)DEGs were down-regulated in the‘Yufeng’vs.‘Shuangyijinlong’comparison.KEGG analysis indicated that flavonoid biosynthesis-related DEGs(CCoAOMT,CHS,CHI,F3H and FLS)were found to be signiflcantly enriched.(4)QRT-PCR validation showed that the expression level of flavonoid biosynthesis-related DEGs(CHS2,CHS3,CHI,F3H and FLS)and transcription factor gene HD-ZIP were lower in‘Shuangyijinlong’than those in‘Yufeng’.In general,the expression of genes showed a same trend to the accumulation of metabolites,suggesting that the color change of‘Shuangyijinlong’might be attributed to the down-regulation of CHS,CHI,F3H and FLS that led to block the downstream metabolites synthesis.It is speculated that the genes involved in the flavonoids biosynthesis may be the key genes for the flower color variegation in Cymbidium hybrid.