咖啡因合成酶（TCS）是茶树咖啡因生物合成途径中的一个关键酶,催化7-甲基黄嘌呤转变为可可碱和可可碱转变为咖啡因。抑制TCS基因表达是培育低咖啡因茶树的最有效途径。用RT-PCR方法扩增茶树TCS基因的两个cDNA片段,并克隆入T-载体中。干涉载体和TCS基因片段的T克隆经限制性内切酶两次双酶切与连接,将两个TCS基因片段分别反向重复插入到干涉载体pFGC5941,构建了TCS基因的RNA干涉载体,分别命名为pFGC5941-TCS02和pFGC5941-TCS03。经PCR和DNA测序获得验证。TCS基因RNA干涉载体的构建为培育低咖啡因茶树奠定了基础。
Tea Caffeine synthase (TCS) is one of the key enzymes involved in caffieine biosynthsis in tea plant (Camellia sinensis),which catalyses conversions of 7-methylxanthine to theobromine and theobromine to caffeine. Inhibition of TCS gene expression can leads to breeding low caffeine tea cultivars. Two cDNA fragments of TCS gene were amplified by RT-PCR, and ligated into T-vector. The two TCS gene fragments were inserted into RNAi vector pFGC5941 in reverse direction after double digestion with two pairs of restriction endonucleases. The insertion of two fragments, namely pFGC5941-TCS02 and pFGC5941-TCS03, into the RNAi vector were confirmed by PCR and DNA sequencing.
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