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园艺学报 ›› 2007, Vol. 34 ›› Issue (2): 371-376.

• 蔬菜 • 上一篇    下一篇

番茄dxs基因的克隆及在大肠杆菌中的颜色互补

潘夕春1;陈 敏1;刘 颜2;廖志华1*

  

  1. (1西南大学生命科学学院, 三峡库区生态环境教育部重点实验室, 重庆市甘薯研究中心, 重庆400715; 2重庆师范大学化学学院, 重庆400047)
  • 收稿日期:2006-09-30 修回日期:2007-03-05 出版日期:2007-04-25 发布日期:2007-04-25
  • 通讯作者: 廖志华

Clon ing of Deoxyoxylulose-5-phosphate Synthase Gene from Tomato and Its Color Complementa tion in E. coli

PAN Xi-chun1,CHEN Min1,LIU Yan2,and LIAO Zhi-hua1*   

  1. [1Key Laboratory of Eco-environm ents in Three Gorges Reservoir Region (M inistry of Education) , Chongqing Sweetpotato Research Center, School of Life Sciences, SouthwestUniversity, Chongqing 400715, China; 2College of Chem istry, Chongqing Normal University, Chongqing 400047, China]
  • Received:2006-09-30 Revised:2007-03-05 Online:2007-04-25 Published:2007-04-25
  • Contact: LIAO Zhi-hua

摘要: 用RT-PCR (反转录- 聚合酶链式反应) 的方法从番茄中克隆到5-磷酸脱氧木酮糖合成酶
(Deoxyoxylulose-5-phosphate synthase, DXS) 基因编码区(记为ledxs) , 并构建了ledxs原核表达载体pTrcLeDXS。将携带番茄红素生物合成途径上香叶基香叶基焦磷酸合成酶(geranylgeranyl pyrophosphate synthase, crtE) , 八氢番茄红素合成酶(phytoene synthase, crtB) , 八氢番茄红素脱饱和酶( phytoene desaturase, crt I) 3个关键酶基因的原核表达载体pAC-LYC转入大肠杆菌XL1-Blue。将pTrcLeDXS转入该重组工程菌, 获得番茄红素工程菌。将该菌用于构建颜色互补平板, 并进行发酵培养以检测番茄红素表达量。颜色互补平板上的菌斑呈现鲜艳的红色。经过21 h的培养番茄红素的产量达605.25μg·L-1。结果显示:ledxs能明显推动类胡萝卜素途径向番茄红素合成的方向流动。

关键词: 番茄, 5-磷酸脱氧木酮糖合成酶, 克隆, 番茄红素, 颜色互补, 发酵

Abstract: To investigate the function of tomato dxs gene and confirm it can facilitate the accumulation of the downsteam metabolites, the coding sequence of dxs gene ( ledxs) was cloned from tomato by reverse-transcrip tion polymerase chain reaction (RT-PCR) , and constructed into the prokaryotic exp ression vector pTrcLeDXS. The lycopene biosynthetic pathway in E. coli strain XL1-Blue was reconstructed by transforming with pAC-LYC carrying the three key enzymes, crtE, crtB and crt I, in the lycopene pathway. This engineered XL-2 Blue was transformed with pTrcLeDXS. Then the engineerd bacteria was used to construct the color complementation p late and detect the content of lycopene after been fermented. The engineered XL1-Blue harboring pAC-LYC and pTrcLeDXS became red because of the p roduction of lycopene. And after 21 h cultivation,the lycopene p roductivity reached 605.25μg·L-1. The results demonstrate that ledxs can facilitate the carotenoid pathway flow to lycopene biosynthesis, and p rove the function of ledxs by color comp lementation. The lycopene p roductivity of the engineered bacteria could meet the requirement of industrial p roduction and can be used for fermentation to produce lycopene.

Key words: Tomato, DXS, Cloning, Lycopene, Color comp lementation, Fermentation

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