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咖啡碱是茶叶中的重要功能物质,N-甲基转移家族酶是催化黄嘌呤核苷3步转甲基化反应合成咖啡碱的关键调控酶。本研究以英红9号茶树为材料构建c DNA文库,采用同源探针筛选c DNA文库获得2个NMT基因,通过体外原核表达检测其催化功能,并用基因瞬时表达技术和酵母双杂交技术对其编码蛋白进行亚细胞定位和互作分析,获得结果:1克隆得到2个NMT基因并分别命名为YHNMT4、YHNMT14,其ORF全长分别为1 095 bp和1 066 bp,各编码365个和355个氨基酸,原核表达蛋白均具有催化7-甲基黄嘌呤合成可可碱的功能,虽前者催化合成可可碱的能力强于后者,但两者均无催化可可碱合成咖啡碱的功能;2两克隆可可碱合成酶基因在水稻原生质体瞬时表达系统中的检测表明其编码蛋白均定位于细胞质内的线粒体;3酵母双杂交表明YHNMT4与YHNMT14表达蛋白具有互作关系,YHNMT4自身表达蛋白存在互作关系,YHNMT14自身表达蛋白无互作关系。这些结果为深入研究NMT基因功能和咖啡碱合成分子调控机制提供了基础。
Caffeine is an important functional substance in tea, and N-methyltransferase family of enzymes is a key regulatory enzyme that catalyzes the synthesis of caffeine by 3-step transmethylation of xanthine. In this study, c DNA library was constructed with the English Red 9 tea tree. Two NMT genes were screened by c-DNA library using homologous probes. The catalytic function was tested by prokaryotic expression in vitro. The gene transient expression and yeast two-hybrid The encoded proteins were subcellularly located and their interaction analysis was performed. The results showed that: 1, 2 NMT genes were cloned and named as YHNMT4 and YHNMT14 respectively. The full-length ORFs of ORFs were 1 095 bp and 1 066 bp respectively, encoding 365 and 355 Amino acids and prokaryotic proteins all have the function of catalyzing the synthesis of theobromine with 7-methylxanthine. Although the former is more capable of catalyzing the synthesis of theobromine than the latter, neither of them catalyzes the function of theobromine in the synthesis of caffeine. Detection of the theobromine synthase gene in the transient expression system of rice protoplasts showed that the encoded proteins were located in the cytoplasm of the mitochondria; 3 Yeast two-hybrid showed that YHNMT4 and YHNMT14 expression protein interaction, YHNMT4 self-expressed protein interaction , YHNMT14 itself expressed protein no interaction. These results provide a basis for further study of NMT gene function and caffeine synthesis molecular regulation mechanism.