基于转录组测序的紫芽茶树花青素合成相关基因分析

Transcriptome Analysis of Anthocyanin Synthesis Related Genes in Purple Bud Tea Plant

为筛选调控茶树芽叶紫色性状相关候选基因,以紫芽茶树品种紫娟、绿芽茶树品种云抗10号和福鼎大白茶为材料,利用液质联用法(HPLC-MS)、转录组测序(RNA-Seq)和实时荧光定量PCR技术(qRT-PCR),分析了紫芽和绿芽茶树品种芽叶花青素、儿茶素含量及合成相关基因的表达水平。结果表明,紫娟、云抗10号和福鼎大白茶芽叶花青素含量分别为5.05 mg/g、0.08 mg/g和0.15 mg/g,紫芽与绿芽茶树间花青素含量差异显著。3个茶树品种儿茶素含量分别为15.12 mg/g、19.52 mg/g和15.09 mg/g,差异不显著。转录组测序共获得255079条转录本序列(Transcripts),组装出166118条单基因簇(Unigenes)。所得单基因簇注释为GO分类中54446条,KOG分类中30666条,KEGG分类中20336条。DEG-Seq分析得到紫芽与绿芽茶树品种间共有434条差异表达基因,涉及到38个代谢通路。进一步筛选到类黄酮生物合成途径相关基因112条,有15条基因在紫芽与绿芽茶树品种间差异表达,其中PAL(Cluster-1850.70337)、CHS(Cluster-21971.0)、ANS(Cluster-1850.80848)和UFGT(Cluster-1850.77163)基因在紫芽茶树品种中上调表达,在绿芽茶树品种间表达差异不显著,表达趋势与花青素积累情况相似。FLS(Cluster-1850.81068)基因在绿芽茶树品种中均为上调表达,可能与儿茶素合成相关。qRT-PCR随机检测9个差异表达基因,表明不同茶树品种间差异表达基因的相对表达水平与转录组测序结果基本一致。因此,从基因表达模式推测,PAL、CHS、ANS和UFGT基因可能在紫芽茶树芽叶花青素生物合成途径中起关键作用。本研究探索了茶树芽叶紫色性状的转录组信息,筛选到一些差异表达基因,为进一步研究茶树芽叶呈色机制提供参考。

In order to identify the candidate genes that are involved in purple color of tea plants,three tea genotypes consisting of purple bud‘Zijuan＇,green bud‘Yunkang No. 10＇and‘Fuding Dabaicha＇were used. We determined the contents and compositions of anthocyanin and catechin by high performance liquid chromatography-mass（ HPLC-MS）,and also analyzed gene expression of synthesis-related genes by RNA-sequencing（ RNA-Seq） and quantitative real-time（ qRT-PCR）. HPLC-MS analysis results showed that the contents of anthocyanin in ‘Zijuan＇,‘Yunkang No. 10＇and‘Fuding Dabaicha＇were 5. 05 mg/g,0. 08 mg/g and 0. 15 mg/g,respectively. The anthocyanincontent was significantly different in purple bud vs. green bud tea cultivars（ P 0. 05）. Whereas the catechin contents of three tea cultivars were 15. 12 mg/g,19. 52 mg/g and 15. 09 mg/g,respectively,no significant difference was detected. By RNA-Seq,a total of 255079 reads was obtained and assembled into 166118 unigenes. Of that,54446 were assigned to GO terms,30666 were assigned to KOG categories,and 20336 were assigned to KEGG pathways. DEG-Seq analysis revealed 434 differentially expressed genes between purple bud vs. green bud tea cultivars,belonging to 38 metabolic pathways. 15 of 112 genes in anthocyanin biosynthesis pathway were differentially expressed between purple bud vs. green bud tea cultivars. Out of that,PAL（ cluster-1850. 70337）,CHS（ cluster-21971. 0）,ANS（ cluster-1850. 80848） and UFGT（ cluster-1850. 77163） were up-regulated in purple bud tea cultivar,while no significantly difference between green bud tea cultivars was detected. In addition,FLS（ Cluster-1850. 81068） genes were up-regulated in green bud tea cultivars,possibly associated with the catechin accumulation. Therefore,this study revealed a set of differentially expressed genes that associated with the anthocyanin synthesis of purple bud tea plan,and particularly,PAL,CHS,ANS and UFGT genes may play central role.