茶树茶氨酸合成酶基因SNP与茶树高茶氨酸性状的关联分析研究

Theanine is a characteristic amino acid of tea plant and an important indicator of green tea quality identification. It has a wide range of health and pharmacological effects. Therefore, high theanine tea products are favored by consumers. The breeding of high theanine tea varieties is becoming more and more important. Theanine content can’t be observed directly from the phenotype. It takes 10 years to select tea cultivars by traditional methods. In addition, self-incompatibility and low efficiency of artificial pollination make it difficult to construct genetic research population. Tea plant genome is large and the density of genetic map is small, so few molecular markers can meet the requirements of assisted breeding. TS1 has the function of theanine synthesis, and its expression is positively correlated with theanine content..At present, several high theanine related QTLs are derived from F1 population. If the SNPs of TS1 is studied in the samples with more genetic diversity and combined with association analysis, more and more accurate markers may be obtained. The SNPs can be used in molecular-assisted breeding in a wide range of genetic backgrounds. In this project, theanine content of 205 tea germplasm resources was determined, promoter and CDS of TS1 were cloned, and SNPs were sequenced. Then SNP genotyping was performed by using MALDI-TOF-MS combined with MassARRAY, and the relationship between SNP and theanine content was studied by association analysis. It is expected that the SNP of TS1 gene can be obtained, and the SNP markers related to theanine can be developed.

茶氨酸是茶树特征性氨基酸，是绿茶品质鉴定的一个重要指标，具有广泛的保健药理作用，高茶氨酸茶产品受到消费者青睐，高茶氨酸茶树品种选育日益重要。茶氨酸不能从表型直接观察，传统方法选育茶树品种需10年，茶树自交不亲和，人工授粉效率低，构建遗传研究群体困难，且茶树基因组大构建的遗传图谱密度小，能达到分子标记辅助育种要求的甚少。茶氨酸合成酶TS1具有茶氨酸合成功能，其表达量与茶氨酸含量正相关。目前几个茶氨酸QTL均来自F1群体，若将TS1基因的SNP与关联分析结合，在遗传多样性更丰富的样本内开展研究，可能获得更多更精准的标记，用于广泛遗传背景下的分子辅助育种。本项目对205份茶树种质资源进行茶氨酸含量测定，克隆TS1启动子和CDS、测序搜寻SNP位点，飞行时间质谱结合MassARRAY技术进行SNP基因分型，关联分析法研究SNP与茶氨酸含量，预期可以获得TS1基因SNP，开发高茶氨酸关联SNP标记。

茶氨酸是茶树特征性氨基酸，是绿茶品质鉴定的一个重要指标。茶氨酸不能从表型直接观察，传统方法选育茶树品种需10年，茶树自交不亲和，人工授粉效率低，构建遗传研究群体困难。茶氨酸合成酶TS1具有茶氨酸合成功能，且当茶氨酸含量增加时，其表达量也增加。本研究克隆了茶树TS1基因，共1572bp，其中编码区序列共1071bp，编码356个氨基酸。克隆了TS1基因起始密码子上游2.4kp左右的序列，经与茶树基因组比对分析，确定了始密码子上游600bp左右的序列为TS1基因的启动子序列，其余序列还有待进一步验证。启动子序列含有的顺式作用元件主要有基础元件、光反应元件、逆境胁迫相关元件、生长发育调控元件、植物激素调控元件等5大类。建立了TS1基因的克隆方法，从212份资源中全部扩增出1.1kb的目的条带，有8份资源的测序峰图重叠较重，最终获得204份资源的测序结果用于SNP的发掘。经序列比对分析，发现TS1基因的编码区22个位点有碱基差异，其中21个位点都是位于密码子第3位，1个位点是位于密码子第1位，但该位点基因分型时因样本数量不足被过滤掉，过滤后共获得9个SNPs。对264份茶树资源进行了茶氨酸含量鉴定，黄金茶群体资源中茶氨酸具有较高的遗传多样性，4个湖南典型地方茶树种质资源（城步峒茶群体、江华苦茶群体、汝城白毛茶群体、安化群体）的差异代谢物主要为氨基酸类等物质，但茶氨酸在群体间不存在显著差异。将茶氨酸含量与9个SNPs进行关联分析，初步筛选出1个可能与茶氨酸相关的SNPs。此外发现1份白化茶树资源的一根枝条上的叶子全部返绿，收集了样品，正针对白化叶和返绿叶的代谢物差异和转录水平上的差异进行研究。本研究可为探索茶氨酸生物合成的分子机制以及茶树的遗传改良提供理论基础，为茶树品种选育早期鉴定提供参考。

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