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咖啡碱是嘌呤碱中最重要的一种,对茶叶的滋味等品质起重要的作用,对于咖啡碱代谢途径的研究有重要意义。根据NCBI登录的TIDH基因(编码咖啡碱合成途径的一个关键酶——次黄嘌呤核苷酸脱氢酶)cDNA设计引物,克隆获得了一条基因组DNA的中间片段,长4172bp(TIDH3)。从中国茶树资源核心种质中选取咖啡碱含量有代表性的95份资源进行了两个年度、春秋两季的咖啡碱含量HPLC测定,大部分材料的咖啡碱含量在2.50%～4.50%,4次重复的平均值为3.50%,变异系数在15.52%～19.25%,表明茶叶咖啡碱含量在不同年份与季节都是相对稳定的。经PCR扩增、测序和序列多态性分析,TIDH3中的一个759bp片段的总核苷酸多样性指数πT和θW分别为0.006和0.012,同义突变多样性πsyn=0.009大于非同义突变多样性πnonsyn=0.006。Ka/Ks=0.600<1,认为TIDH3基因受到负向选择作用。TIDH3基因连锁不平衡分析表明,基因内的连锁不平衡衰减速度较快,在约300bp范围内r2值降低到了0.2。通过关联分析,获得两个与咖啡碱含量显著相关的SNP位点,遗传贡献率分别为10.43%和5.68%。
Caffeine is the most important one of the purine bases, which plays an important role in the quality of tea, and plays an important role in the research of caffeine metabolic pathway. According to the primers designed by NCBI TIDH gene (a key enzyme in caffeine synthesis pathway) -inosine dehydrogenase (cDNA) gene, an intermediate fragment of genomic DNA was cloned and was 4172 bp long (TIDH3). 95 representative caffeine sources were selected from the core germplasm resources of Chinese tea tree for determination of caffeine content in two years, spring and autumn. The content of caffeine in most of the materials was 2.50% -4.50%. Four replicates The average value was 3.50% and the coefficient of variation was 15.52% ~ 19.25%, indicating that the content of caffeine in tea was relatively stable in different years and seasons. The results of PCR amplification, sequencing and sequence analysis showed that the total nucleotide diversity index πT and θW of a 759bp fragment in TIDH3 were 0.006 and 0.012, respectively. The diversity of synonymous mutations πsyn = 0.009 was greater than that of non-synonymous mutations Πnonsyn = 0.006. Ka / Ks = 0.600 <1, suggesting that the TIDH3 gene is negatively-selected. The linkage disequilibrium analysis of the TIDH3 gene showed that the linkage disequilibrium within the gene decays more rapidly, reducing the r2 value to about 0.2 in the range of about 300 bp. Two SNPs that were significantly correlated with the content of caffeine were obtained through association analysis, and the genetic contribution rates were 10.43% and 5.68% respectively.