【作者】 冯丽玲；

【导师】 曾庆平；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2007， 硕士

【摘要】 青蒿素是由我国科研人员自主研发并为国际所公认的一种抗疟有效单体。野生青蒿中青蒿素含量过低，难以满足疟疾治疗中对青蒿素的迫切需求。由于常规育种在青蒿素高产方面的作用有限，因此在青蒿素合成代谢途径已被阐明的情况下，通过基因工程对野生青蒿的青蒿素生产能力进行人工操纵不失为大幅度提高青蒿素含量的最佳选择。已有证据表明，当植株接触真菌刺激物时，其鲨烯合酶基因(SS)表达下调，从而导致类固醇积累减少，同时倍半萜合成增加，暗示类固醇合成阻遏可能导致倍半萜合成途径活化。为了培育青蒿素高产的转基因青蒿植株，我们用Ti质粒衍生的双元载体上携带反义SS基因的工程农杆菌对青蒿外植体进行遗传转化。本文描述了转反义SS基因后内源SS基因的抑制及其后果：在所获得的3棵转基因植株中，通过Southern印迹杂交已对反义SS基因整合的拷贝数进行计数(1个至多个拷贝)。半定量RT-PCR分析结果显示，反义SS基因的功能表达已导致内源SS基因表达下调，使SS mRNA水平下降，类固醇含量也从0.8mg／g鲜重降低到0.4-0.5mg／g鲜重，降幅达50％。转基因青蒿植株青蒿素含量(12.3mg／g干重)远比非转基因青蒿植株青蒿素含量(4.5mg／g干重)为高。为了进一步将碳流引向青蒿素，我们对转基因植株进行了短暂的冷胁迫处理(4℃，30min)，结果使青蒿素含量进一步提高，其中最高者达到16.6mg／g干重。因此，采取类固醇合成阻遏与冷诱导青蒿素合成基因高效表达的联合策略可以显著促进青蒿素的高产，可望最终培育出转基因高产青蒿素新品系。更多还原

【Abstract】 Artemisinin, an effective anti-malarial component, is independently developed byChinese scientists and accepted by the community. The trace amount of artemisinin inwild-typed A. annua is a bottleneck to meet the urgent need at the maximum extentfor artemisinin-based malaria therapy. Due to the limitation of conventional breedingon the over-production of artemisinin and the elucidation of metabolic pathwayresponsible for artemisinin biosynthesis, the artificial manipulation of artemisininproductivity in planta by virtue of genetic transformation can be a reasonable optionfor greatly enhancing the yield of artemisinin. Evidence has been documented thatwhen exposed to fungal elicitors, squalene synthase gene (SS) in plants is specificallyexpressed in a down-regulated manner, which leads to a decreased accumulation ofsterols but an increased biosynthesis of sesquiterpenes, suggesting that the suppressionof steroid biosynthesis has resulted in the activation of sesquiterpenoid biosynthesis.For enhancing artemisinin production by the down-regulation of key enzyme encodinggenes for squalene biosynthesis, we describe here the suppression and consequence ofendogenous SS gene in A. annua by an antisense strategy, i.e. A, annua is transformedwith an anti-sense SS gene, which is carried within an engineered Agrobacteriumstrain harboring a Ti-derived binary vector. In three transgenic plants available fromthe underlying experiment, the integrated copy numbers of anti-sense SS gene, fromone copy to more copies, have been counted through Southern blotting. Upon thefunctional expression of anti-sense SS gene in transgenic plants, a dramaticallydropped level of SS mRNA has been determined by semi-quantitative RT-PCR assay.Correspondingly, the down-regulation of SS gene is confirmed by an extremelydecreased content of steroids from 0.8mg/g fresh weight to 0.4-0.5mg/g fresh weight, intransgenic plants. Furthermore, for re-direction of carbon source towards artemisinin,we have treated transgenic plants transiently by chilling stress (4℃, 30min), whichleads to an over-production of artemisinin up to 16.6mg/g leaf dry weight as comparedwith untreated transgenic plants (12.3mg/g leaf dry weight) or non-transgenic plants(4.5mg/g leaf dry weight), demonstrating a marked enhancement of artemisininproduction promoted by a coupled strategy of down-regulation of SS gene withchilling-induced over-expression of artemisinin biosynthesis responsible genes. Asconsequence, this strategy should facilitate the molecular breeding of novel transgenicA.annuca varieties with higher artemisinin yield.更多还原