转录因子和miRNAs协同调控白化茶树新品系‘千江月’茶氨酸合成的分子机制

Theanine is the most abundant free amino acid in tea plant (Camellia sinensis). The rare albino tea cultivars (strains) usually have high level of theanine, and their young shoots are valuable materials for processing quality green tea. Their theanine content varied greatly during the different albino stages, leading to limiting the use of their superiority, while transcriptional and post-transcriptional regulatory mechanisms of the theanine biosynthesis remain largely unclear. A new albino clonal tea strain ‘Qianjiangyue’ previously identified by our research group. Its free amino acid content was up to 8.6% at the albino stage, but was significantly different at different leaf color stages. At four different stages (purple, yellow jade, light green and green stage), samples of ‘two and a bud’ (one young shoot with two leaves and a bud) from ‘Qianjiangyue’ were collected. The ‘GT-01’ as the control, a clonal green tea strain, has low and stable amino acid content, as well as close genetic relationship with ‘Qianjiangyue’. To investigate the differential profiles of metabolites involved in the theanine biosynthesis during four different color stages, the ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) analysis was conducted. Through RNA sequencing, we will try to understand the expression characteristics of the structural genes encoding enzymes for the theanine biosynthesis. Through combining with small RNA and degradome sequencing, the transcription factors (TFs) and microRNAs (miRNAs) in the regulation of theanine biosynthesis could be screened and identified. The coordinated regulation mechanisms of TFs and miRNAs involved in the theanine biosynthesis could also be elucidated using the correlation analysis and co-expression analysis. Additionally, the potential TFs and miRNAs coordinated regulatory network for theanine biosynthesis in ‘Qianjiangyue’ could be presented. The results will further deepen the regulation mechanisms of the theanine biosynthesis in tea plant. The proposed project will also serve as a valuable resource for the improvement of quality of tea and the oriented breeding in tea plant.

茶氨酸是茶树体内含量最高的游离氨基酸，适制名优绿茶的珍稀白化茶树品种（系）不同叶色期茶氨酸含量差异大，限制了其优势发挥，而茶氨酸生物合成的转录和转录后调控机制尚未明晰。项目组鉴选的特异白化茶树新品系‘千江月’，白化期游离氨基酸含量高达8.6%，但不同叶色期差异显著。本项目拟以‘千江月’紫红期、玉黄期、浅绿期和绿期的一芽二叶为材料，采用叶色常绿、氨基酸含量较低且比较稳定、亲缘关系近的‘GT-01’品系为对照，通过UPLC-Q-TOF-MS分析，明确茶氨酸合成途径中代谢物的动态变化特征；通过转录组测序，探明茶氨酸合成途径结构基因的表达特性；结合小RNA测序和降解组测序，筛选、验证调控茶氨酸合成的转录因子和miRNAs；通过关联分析和共表达分析，探析转录因子和miRNAs协同调控茶氨酸合成的分子机制，为进一步深入研究茶氨酸合成调控机制、茶树品质改良、茶树定向育种提供理论基础。

多数优异白化茶树品种适制名优绿茶，但其新梢色泽与氨基酸等主要滋味物质在不同生长时间点差异大，导致其绿茶品质不稳定，限制了其优势发挥。针对茶树新梢色素和主要滋味物质累积和代谢结果多样、规律不清的问题，本项目采用常绿和白化不同基因型茶树，研究了不同生长时间点白化茶树新梢（一芽二叶）色素和主要滋味物质的变化特征及其相关性，这些物质生物合成代谢的动态变化特征、合成代谢途径酶基因的表达特性、miRNA和转录因子的协同调控机制。结果表明，白化茶树新梢颜色越黄（白），类胡萝卜素、游离氨基酸含量越高，叶绿素a、叶绿素b、茶多酚含量越低，叶绿素a/b值较高，花青素含量适中，茶树新梢色素含量与其主要生化成分含量关系密切。白化茶树新梢色素、儿茶素、茶氨酸等差异代谢及其差异表达酶基因主要存在于白化期，而咖啡碱代谢较稳定。确定了新梢色素、儿茶素、咖啡碱、茶氨酸生物合成途径酶基因的表达特征；构建了转录因子-酶基因调控网络模型，初步阐明了这些物质的合成代谢机理；揭示了转录因子-酶基因-色素和主要滋味物质丰度之间的相关性；发现一些酶基因的表达、一些转录因子-酶基因的互作关系具有基因型特异性。构建了两种基因型茶树不同生长时间点新梢色素、儿茶素、咖啡碱和茶氨酸生物合成代谢途径的miRNA-靶基因（转录因子或酶基因）共表达调控网络模型；解析了miRNA和转录因子协同调控这些代谢物的生物合成机制；进一步揭示了这些调控机制在白化和常绿基因型茶树之间的差异特征。这些发现将提高我们对茶树新梢色素和主要滋味物质生物合成代谢调控机理的理解，为茶树遗传改良、定向育种与开发利用提供基础。