Fuyuan Jing

Development of transgenic Artemisia annua (Chinese wormwood) plants with an enhanced content of artemisinin, an effective anti-malarial drug, by hairpin-RNA-mediated gene silencing.

Artemisinin is an effective anti‐malarial drug isolated from Artemisia annua L. (Chinese wormwood), but the content of artemisinin in A. annua is low. In the present study we explored the possibility of using genetic engineering to increase the artemisinin content of A. annua by suppressing the expression of SQS (squalene synthase), a key enzyme of sterol pathway (a pathway competitive with that of artemisinin biosynthesis) by means of a hairpin‐RNA‐mediated RNAi (RNA interference) technique. A total of 23 independent transgenic A. annua plants were obtained through Agrobacterium tumefaciens‐mediated transformation, which was confirmed by PCR and Southern‐blot analyses. HPLC‐evaporative light‐scattering detection analysis showed that the artemisinin content of some transgenic plants was significantly increased, with the highest values reaching 31.4 mg/g dry weight, which is about 3.14‐fold the content observed in untransformed control plants. Real‐time reverse transcription–PCR analysis demonstrated that the expression of SQS was suppressed significantly, and GC–MS analysis showed that sterol was efficiently decreased in the transgenic plants. The present study demonstrated that genetic‐engineering strategy of RNAi is an effective means of increasing artemisinin content in plants.

Abscisic acid (ABA) treatment increases artemisinin content in Artemisia annua by enhancing the expression of genes in artemisinin biosynthetic pathway

Artemisinin, a sesquiterpene lactone endoperoxide derived from Artemisia annua L., is the most effective antimalarial drug. In an effort to increase the artemisinin production, abscisic acid (ABA) with different concentrations (1, 10 and 100 µM) was tested by treating A. annua plants. As a result, the artemisinin content in ABA-treated plants was significantly increased. Especially, artemisinin content in plants treated by 10 µM ABA was 65% higher than that in the control plants, up to an average of 1.84% dry weight. Gene expression analysis showed that in both the ABA-treated plants and cell suspension cultures, HMGR, FPS, CYP71AV1 and CPR, the important genes in the artemisinin biosynthetic pathway, were significantly induced. While only a slight increase of ADS expression was observed in ABA-treated plants, no expression of ADS was detected in cell suspension cultures. This study suggests that there is probably a crosstalk between the ABA signaling pathway and artemisinin biosynthetic pathway and that CYP71AV1, which was induced most significantly, may play a key regulatory role in the artemisinin biosynthetic pathway.

Cloning and characterization of trichome-specific promoter of cpr71av1 gene involved in artemisinin biosynthesis in Artemisia annua L.

Artemisinin, a sesquiterpene lactone endoperoxide derived from Artemisia annua L.(Asteraceae), is the most effective antimalarial drug. We used two methods: genome walking and thermal asymmetric interlaced polymerase chain reaction, to isolate the unknown 5′-flanking sequence of the cyp71av1 gene. The subsequent sequence analysis using bio informatics software revealed that there are several cis-acting elements inside the cyp71av1 promoter. The 5′-rapid amplification of the cDNA ends method was used to determine the transcription start site of the cyp71av1 gene. We then mapped it at the 18 base upstream of the ATG initiation codon. For simple functional characterization, we built fusion vectors between the 5′-deletion promoter and the gus reporter gene. The expression levels of the transferred vectors into A. annua L. were analyzed by the transient expression way. The β-glucuronidase assay results indicated that deletion of the region to −1551 bp did not lead to much damage in the GUS activity, whereas further deletion, to −1155 bp, resulted in a 5.5-fold reduction of GUS activity. In stabilized transgenic A. annua L. seedlings we observed that GUS expression was restricted to trichomes, which means that the promoter of the cyp71av1 gene is trichome-specific. Compared with the constitutive CaMV 35S promoter, which can express genes throughout the plant, influence on the trichome system through the trichome-specific expression promoter merely imperils plant growth. In addition, the promoter of the cyp71av1 gene contains several binding sites for transcription factors, which implies that the cyp71av1 promoter responds to more than one form of stimulation.