NMT调控茶叶咖啡碱代谢的功能蛋白质组学研究

Caffeine (1,3,7-trimethylxanthine) is an important secondary metabolite in tea (Camellia sinensis),and the available data support the operation of a xanthosine- - >7-methylxanthosine- - >7-methylxanthine- - >theobromine- - >caffeine biosynthetic pathway as the major route to caffeine. N-methyltransferase, the SAM-dependent methyltransferase involved in the methylation of purine ring in the last three steps of caffeine biosynthesis, play an important roles, and a total 47 genes related to the production of caffeine isolated from tea plants in our previous work, they share a high sequence homology in nucleotide sequence and the predicted amino acid sequence, but exhibit remarkable substrate selectivity, and the subtle mechanism why so many genes and enzymes involved in the caffeine biosynthesis of the last three steps remain unclear. In this study two kinds of tea plants including Camellia ptilophylla Chang which is a natural tea with low-caffeine and high theobromine content and Camellia sinensis var assamica cv Yinghong 9 which owned high-caffeine and low theobromine content were used, and the main work as follows: (1) to find out the relationship between the genes and the chromosomes and whether exist the alternative splice transcribe in genome by the technique of FISH and southern blotting;(2) to carefully understand the parameter in the transcribe level of gene and the translation level of gene with corresponding enzymes by the technique of quantitative PCR and western blotting;(3) to construction the relationship between the functional enzyme and the gene nucleotide sequence by the technique of bioinformatics and directed mutagenesis of gene;(4)to explanation the subtle difference in the property of enzyme catalysis and substrate discrimination by the technique of enzyme catalytic specific property analysis and circular dichroism spectra analysis. This work to research the caffeine biological metabolism in tea plants from the perspective of functional proteomics would make know the relationship among gene and enzyme protein and product, and would helpful to understand how to coordinate in regulation the biosynthesis of metabolite in tea plants among members in gene family.

咖啡碱是茶树体内重要的次生代谢产物，其生物合成主要以黄嘌呤核苷为底物，经三步转甲基化反应催化合成。NMT家族成员是催化反应的关键酶蛋白，其受NMT基因调控。本课题组已从南昆山毛叶茶、英红9号茶树品种中克隆出多达47个NMT基因，功能鉴定证明其具有不同的催化特性，但核苷酸和氨基酸序列高度同源，内在机理何在？本研究拟以低咖啡碱/高可可碱南昆山毛叶茶、高咖啡碱/低可可碱英红9号为研究材料：通过FISH杂交辅以southern杂交构建家族基因与染色体基因组的关系，表达是否存在可变剪切；通过定量PCR、western blotting技术回溯各酶蛋白与基因转录、翻译的关系；通过生物信息学结合基因定点突变，剖析酶蛋白与基因调控的关系；通过酶催化特性及圆二色谱分析，揭示各酶蛋白的差异及对底物识别特性。本研究工作的开展，对于从功能蛋白质组学角度，揭示NMT家族酶蛋白调控茶叶咖啡碱代谢机理具有重要意义。

咖啡碱是茶树体内的重要功能物质并受NMT基因的调控合成，项目组前期从茶叶中克隆了系列序列高度同源但功能差异的NMT基因。为深入探明多NMT基因调控茶叶咖啡碱合成机理，本项目设计探讨了茶叶NMT基因从基因组转录到cDNA是否存在可变剪切、基因序列尤其特定碱基对其功能的影响、基因酶蛋白与基因转录及翻译的关系、酶活性与其空间构象的关系、多基因是如何发挥功能的等研究内容。结果表明，不同特性的茶叶体内其调控咖啡碱合成的NMT基因多样性属普遍现象；不同NMT基因内的特定碱基对其酶蛋白催化功能具有特定意义，单个碱基突变引发氨基酸变化往往导致表达蛋白的催化活性变化；基因组southern杂交仅检测到单一NMT杂交带推测NMT从基因组DNA到mRNA过程存在可变剪切转录现象；不同NMT基因的转录水平从茶树新稍的芽头到其下的一叶、二叶均曾下降现象，同时发现在同一叶位咖啡碱合成酶基因的表达水平高于可可碱合成酶基因，但在基因翻译水平咖啡碱合成酶蛋白在新稍中的含量却反高于可可碱合成酶蛋白；不同NMT基因具有不同的底物识别特性和催化能力差异决定于其酶蛋白所形成的特定空间结构，并且发现家族基因蛋白间存在互作现象，并首次把茶叶体内合成的NMT酶蛋白定位在线粒体。本研究的开展及取得的结果，从功能蛋白质组学角度深入阐明了茶叶体内多NMT基因调控咖啡碱合成的机理，并为通过生物技术途径改良茶叶生物碱的合成提供了数据和技术的支持。