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茶叶科学 ›› 2006, Vol. 26 ›› Issue (4): 243-248.doi: 10.13305/j.cnki.jts.2006.04.002

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茶树咖啡因合成酶基因RNA干涉表达载体构建

张广辉1, 2, 梁月荣1, *, 陆建良1, 董俊杰1   

  1. 1. 浙江大学茶学系,浙江 杭州 310029;
    2. 江苏省徐州生物工程学校,江苏 徐州 221006
  • 收稿日期:2006-04-11 修回日期:2006-09-13 出版日期:2006-12-25 发布日期:2019-09-11
  • 通讯作者: * 梁月荣, E-mail:yrliang@zju.edu.cn
  • 作者简介:张广辉(1973- ),河南孟津人,在读博士研究生,从事茶树遗传余种与分子生物学研究。
  • 基金资助:
    国家自然科学基金(30571192)、浙江省科技项目(2004C32079)

Construction of Tea Caffeine Synthase Gene RNAi Vector

ZHANG Guang-hui1, 2, LIANG Yue-rong1, *, LU Jian-liang1, DONG Jun-jie1   

  1. 1. Department of Tea Science, Zhejiang University, Hangzhou 310029, China;
    2. Xuzhou Higher Vocational School of Bioengineering, Xuzhou 221006, China
  • Received:2006-04-11 Revised:2006-09-13 Online:2006-12-25 Published:2019-09-11

摘要: 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。

关键词: 茶树, 咖啡因合成酶基因, RNA干涉, PCR

Abstract: 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.

Key words: tea plant (Camellia sinensis), caffeine synthase, RNA interference, PCR

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