为明确紫娟茶树（Camellia sinensis cv. Zijuan）叶片发育中花青素积累特性及合成途径上相关基因的表达特点,利用液质联用法（HPLC-MS）、转录组测序（RNA-Seq）和数字基因表达谱技术（DGE）,分析了紫娟茶树芽、第二叶、开面叶和成熟叶4个发育期花青素的种类、含量及合成相关基因的表达水平。结果表明,花青素积累量随叶片发育先增加后减少,第二叶含量最大（9.87βmg·g-1）、成熟叶含量最小（0.11βmg·g-1）,与叶色表现相吻合。结构基因PAL在芽、第二叶和开面叶高表达,在成熟叶下调表达;C4H、4CL、CHS、CHI、F3H、F3'H、F3'5'H和ANS表达模式相似,表达量均随叶片发育而降低,在芽期高表达,在成熟叶全部下调表达;FLS在第二叶上调表达,在成熟叶下调表达,与花青素积累情况一致;DFR在各发育期均有上调和下调表达。GT和ACT表达模式相似,在第二叶、开面叶和成熟叶上调表达;ANR和LAR表达模式相似,在芽、第二叶和开面叶高表达,在成熟叶下调表达。bHLH、MYB和WDR在各发育期均有上调或下调表达。说明紫娟茶树叶片不同发育阶段结构基因和调控基因的表达水平不同,导致花青素积累存在差异,具有一定的时间表达特异性。
In order to explore the regulatory factors and structural genes of anthocyanin biosynthesis in leaves of different developmental stages in C. sinensis cv. Zijuan, anthocyanin contents and compositions were analyzed by high performance liquid chromatography-mass, and the expression of synthesis-related genes were tested by RNA-sequencing and digital gene expression (DGE) profiling technology. HPLC-MS analysis showed that the anthocyanin content was consistent with color changes, increased firstly and then decreased with the leaf positions of Zijuan, with the highest anthocyanin content in the second leaf (9.87βmg·g-1) and the lowest content in mature leaf (0.11βmg·g-1). DGE determination results showed that the expression levels of PAL gene were relatively high in bud, second leaf and open surface leaf, but low in mature leaf. C4H, 4CL, CHS, CHI, F3H, F3'H, F3'5'H and ANS showed a pattern of declining expression as PAL in different leaf positions of Zijuan. FLS was firstly up-regulated in the second leaf and then declined in the mature leaf, which was consistent to the anthocyanin changes. DFR showed no clear expression pattern. GT and ACT exhibited similar expression patterns, which were up-regulated in the second, open surface and mature leaves. ANR and LAR showed similar expression patterns, which were high in bud, second and open surface leaves, but low in mature leaf. The gene expression of bHLH, MYB and WDR showed different expression patterns in different leaf positons of Zijuan. These results suggested that the temporal expression specificities of structural and regulatory genes may significantly affect the accumulation of anthocyanin in Zijuan.
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