Theacrine (1,3,7,9-tetramethyluric acid) is a natural product with remarkable pharmacological activities such as antidepressant, sedative and hypnotic activities, while caffeine (1,3,7-trimethylxanthine) has certain side effects to special populations. Hence, breeding tea plants with high theacrine and low caffeine will increase tea health benefits and promote consumption. In this study, we construct a F1 population by crossing “Zhongcha 302” (theacrine-free) and a tea germplasm “Ruyuan Kucha” (RY, theacrine-rich) to identify the causal gene for accumulating theacrine. The results showed that the content of theacrine was highly negative correlate with caffeine (R2 > 0.9). Bulked segregant RNA sequencing analysis, molecular markers and gene expression analysis indicating that the theacrine synthase (TcS) gene was the candidate gene. The TcS was located in the nucleus and cytoplasm, and the theacrine can be detected in stably genetic transformed tobacco by feeding the substrate 1,3,7-trimethyluric acid. Moreover, in vitro enzyme activity experiment revealed that the 241th amino acid residue was the key residue. Besides, we amplified the promoter region in several tea accessions with varied theacrine levels, and found a 234-bp deletion and a 271-bp insertion in RY. Both GUS histochemical analysis and dual-luciferase assay showed that TcS promoter activity in RY was relatively high. Lastly, we developed a molecular marker which is co-segregate with high-theacrine individuals in RY’s offspring. These results demonstrate that the novel TcS allele in RY results in the high-theacrine and low-caffeine traits and the developed functional marker will facilitate the breeding of characteristic tea plants.

中文翻译：

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一种具有高茶碱低咖啡因性状的新型 TcS 等位基因，在茶树育种中具有潜在用途

茶碱（1,3,7,9-四甲基尿酸）是一种天然产物，具有显着的抗抑郁、镇静、催眠等药理活性，而咖啡因（1,3,7-三甲基黄嘌呤）对特殊人群有一定的副作用。因此，培育具有高茶碱和低咖啡因的茶树将增加茶的健康益处并促进消费。在本研究中，我们通过杂交“中茶302”（无茶碱）和茶叶种质“乳源苦茶”（RY，富含茶碱）构建F1群体，以确定茶碱积累的因果基因。结果表明，茶碱的含量与咖啡因呈高度负相关（R2＞0.9）。大块分离RNA测序分析、分子标记和基因表达分析表明茶碱合酶(TcS)基因是候选基因。TcS位于细胞核和细胞质中，通过喂食底物1,3,7-三甲基尿酸可以在稳定遗传转化的烟草中检测到茶碱。此外，体外酶活性实验表明，第241个氨基酸残基是关键残基。此外，我们在不同茶碱水平的几个茶种质中扩增了启动子区域，并在 RY 中发现了 234 bp 的缺失和 271 bp 的插入。GUS 组织化学分析和双荧光素酶测定均表明 RY 中的 TcS 启动子活性相对较高。最后，我们开发了一种分子标记，它与 RY 后代中的高茶碱个体共同分离。