Anti-sense Squalene Synthase Gene Expression Mediated Over-production Of Artemisinin In Transgenic Artemisia Annua

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.