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)

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。

Flavanone 3-hydroxylase（ F3 H） is a crucial enzyme involved in the flavonoid biosynthesis. To study the effect of F3 H on artemisinin in Artemisia annua L.,AaF3 H was cloned from Artemisia annua L. in this study. The completed open read frame of AaF3 H 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 AaF3 H transgenic plants were generated by transforming AaF3 H overexpression vector into A. annua,and the artemisinin content of AaF3 H transgenic A. annua was determined by HPLC. The results showed that overexpression of AaF3 H 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（ AaCYP71 AV1） and artemisinic aldehyde Δ11（ 13） reductase gene（ AaDBR2） was also increased in AaF3 H-overexpression transgenic A. annua plants by real-time quantitative PCR analysis. These results demonstrated that overexpression of AaF3 H gene was an effective method to improve the content of artemisinin in A. annua.