Over-expression of HMG-CoA reductase and amorpha-4,11-diene synthase genes in Artemisia annua L. and its influence on artemisinin content
Over-expression of HMG-CoA reductase and amorpha-4,11-diene synthase genes in Artemisia annua L. and its influence on artemisinin content
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DOI：
10.1007/s00299-011-1099-6
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发表时间：
2011-10-01
影响因子：
6.2
通讯作者：
Abdin, M. Z.
中科院分区：
文献类型：
--
作者：
Alam, Pravej;Abdin, M. Z.
青蒿素是一种内过氧化物倍半萜内酯，是从黄花蒿属植物中分离得到的新型抗疟天然产物。然而，青蒿素在黄花蒿属植物中的低浓度（0.01-1.1%）是世卫组织推荐的用于治疗耐多药恶性疟原虫引起的疟疾的以青蒿素为基础的联合疗法（ACTs）商业化的主要制约因素。在此背景下，我们开展了体内产量提高计划。本研究利用花楸（Catharanthus roseus， L） Don中HMG-Co - A还原酶基因（hmgr）和黄花蒿（a.a annua L.）中amorphaa -4,11-diene synthase （ads）基因在黄花蒿中过表达，研究其对青蒿素产量的影响。利用hptII基因探针对从推定的转基因再生体中获得的转基因系进行整合和拷贝数评估，因为它是表达盒的一部分。转基因株系在Southern blots上显示hptII基因阳性带，证实了转基因的整合。一些转基因品系只有一个基因拷贝，而另一些则有多个基因拷贝。利用RT-PCR方法也证实了hmgr和ads在转录水平上的表达。高效液相色谱分析表明，这些转基因植物的青蒿素含量显著增加。其中一个转基因品系TR4的青蒿素含量是非转基因品系W的7.65倍（1.73 mg/gDW）。生化分析发现，青蒿素水平升高与HMG-Co A还原酶和非晶4,11-二烯合成酶活性相关。
Artemisinin, an endoperoxide sesquiterpene lactone, is a novel antimalarial natural product isolated from Artemisia annua L. plants. The low concentrations (0.01-1.1%) of this compound in A. annua L. plants is, however, a major constraint for commercialization of artemisinin-based combination therapies (ACTs) recommended by WHO for treating malaria caused by multidrug-resistant P. falciparum sp. In this context, in vivo yield improvement programs were undertaken by us. In the present study, HMG-Co A reductase gene (hmgr) from Catharanthus roseus (L) G. Don and amorpha-4,11-diene synthase (ads) gene from A. annua L. were over-expressed in A. annua L. plants to study their effects on artemisinin yields. The transgenic lines developed from putative transgenic regenerants were evaluated for integration and copy number of the transgenes using hptII gene probe, as it was a part of the expression cassette. The transgenic lines showed positive bands of hptII gene on Southern blots confirming the integration of transgenes. Some of the transgenic lines had single copy of the transgenes, while others had multiple copies. The expressions of hmgr and ads at the transcriptional level were also confirmed in each transgenic line employing RT-PCR assays. The HPLC analyses showed that the artemisinin contents were significantly increased in these transgenics. One of the transgenic lines, TR4, was found to contain 7.65-fold higher (1.73 mg/gDW) artemisinin than the non-transgenic plant (W). The increased artemisinin levels were found to be correlated with HMG-Co A reductase and amorpha4,11-diene synthase enzymatic activities in the biochemical analyses.