过量表达黄烷酮3-羟化酶基因（AaF3H）提高青蒿中青蒿素的含量

doi:10.19586/j.20952341.2017.0073

生物技术进展 ›› 2018, Vol. 8 ›› Issue (1): 55-62.DOI: 10.19586/j.20952341.2017.0073

过量表达黄烷酮3-羟化酶基因（AaF3H）提高青蒿中青蒿素的含量

张婷婷1,马嘉伟1,王路尧1,唐克轩1,李杉2*,赵静雅1*

1.上海交通大学农业与生物学院, 交大-复旦-诺丁汉植物生物技术研发中心, 上海 200240; 2.华南理工大学生物科学与工程学院, 广州 510006

收稿日期:2017-07-03 出版日期:2018-01-25 发布日期:2017-08-08
通讯作者: 赵静雅，副教授，研究方向为植物代谢基因工程。E-mail：zhaojy@sjtu.edu.cn;李杉，副教授，研究方向为生物化学与分子生物学。E-mail：279814900@qq.com
作者简介:张婷婷，硕士研究生，研究方向为植物代谢基因工程。E-mail:1058563839@qq.com。
基金资助:
国家863计划项目(2011AA100605)资助。

Improving Artemisinin Content of Artemisia annua L. Through Overexpression of Flavanone 3-hydroxylase Gene (AaF3H)

ZHANG Tingting, MA Jiawei, WANG Luyao, TANG Kexuan, LI Shan, ZHAO Jingya

1.Joint Plant Biotechnology R&D Center of Jiao Tong University-Fudan University-Nottingham University, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; 2.School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China

Received:2017-07-03 Online:2018-01-25 Published:2017-08-08

摘要/Abstract

摘要： 黄烷酮3-羟化酶(flavanone 3-hydroxylase,F3H)是参与黄酮类化合物合成过程的一个关键酶。为了研究青蒿中F3H对青蒿素的影响，从青蒿中克隆到黄烷酮3-羟化酶基因(AaF3H)，全长为1 095 bp，编码364个氨基酸。Southern Blot证实AaF3H基因在青蒿基因组中只有1个拷贝。通过构建AaF3H过表达载体并稳定转化青蒿来获得转基因株系，再采用HPLC测定过量表达AaF3H的转基因青蒿植株中的青蒿素含量。结果表明，过表达AaF3H转基因青蒿植株中青蒿素的含量显著升高。通过实时荧光定量PCR分析，在转基因青蒿中，作为青蒿素合成的关键酶，紫穗槐-4,11-二烯合成酶基因（AaADS）、紫穗槐-4,11-二烯氧化酶基因（AaCYP71AV1）和青蒿醛Δ11（13）双键还原酶基因（AaDBR2）的表达量显著提高。研究结果表明过量表达AaF3H基因是提高青蒿中青蒿素含量的有效方法。

关键词: 青蒿, 黄烷酮3-羟化酶, 过量表达, 青蒿素

Abstract: Flavanone 3-hydroxylase (F3H) is a crucial enzyme involved in the flavonoid biosynthesis. To study the effect of F3H on artemisinin in Artemisia annua L., AaF3H was cloned from Artemisia annua L. in this study. The completed open read frame of AaF3H was 1 095 bp, and it encoded 364 amino acids. Southern blot showed that it was a single copy gene in A. annua genome. The AaF3H transgenic plants were generated by transforming AaF3H overexpression vector into A. annua, and the artemisinin content of AaF3H transgenic A. annua was determined by HPLC. The results showed that overexpression of AaF3H significantly increased the content of artemisinin. As the key enzymes in artemisinin synthesis, the expression of amorpha-4,11-diene synthase gene (AaADS), amorpha-4,11-diene 12-hydroxylase gene (AaCYP71AV1) and artemisinic aldehyde Δ11(13) reductase gene (AaDBR2) was also increased in AaF3H-overexpression transgenic A. annua plants by real-time quantitative PCR analysis. These results demonstrated that overexpression of AaF3H gene was an effective method to improve the content of artemisinin in A. annua.

Key words: Artemisia annua L., flavanone 3-hydroxylase, overexpression, artemisinin

张婷婷,马嘉伟,王路尧,唐克轩,李杉,赵静雅. 过量表达黄烷酮3-羟化酶基因（AaF3H）提高青蒿中青蒿素的含量[J]. 生物技术进展, 2018, 8(1): 55-62.

ZHANG Tingting1, MA Jiawei1, WANG Luyao1, TANG Kexuan1, LI Shan2*, ZHAO Jingya1*. Improving Artemisinin Content of Artemisia annua L. Through Overexpression of Flavanone 3-hydroxylase Gene (AaF3H)[J]. Curr. Biotech., 2018, 8(1): 55-62.

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过量表达黄烷酮3-羟化酶基因（AaF3H）提高青蒿中青蒿素的含量

Improving Artemisinin Content of Artemisia annua L. Through Overexpression of Flavanone 3-hydroxylase Gene (AaF3H)

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