Molecular Cloning And Functional Analysis Of AaF3H And AaFLS Genes In Flavonoids Biosynthetic Pathway From Artemisia Annua L.

Artemisia annua L. (A. annua) is a kind of annual herb. Nowadays, (A. annu a) and artemisinin are the one of the most international traditional Chinese medicine and resources since the discovery of the anti-malarial compound sesquiterpene artemis inin from A. annua by Chinese researchers. Artemisinin-based combination therapies h ave been recommended as the preferential treatment method of the drug-resistant mal aria by the WHO for its high efficacy and no serious side effects and gained great s uccess.But the plasmodium falciparum parasites resistant to artemisinin was detected i n lots of places all over the world especially the Southeast Asia and Africa since the year 2008, and internally generated global trend of spreading. But a lot of research fro m these years indicated that other functional compounds from A. annua flavonoids w ere found to synergize anti-malaria and anti-cancer compounds artemisinin, especially the flavonoids of the plant, Meanwhile, it can slows the evolution of malaria drug re sistance and overcomes resistance to artemisinin. However, the current researches wer e mainly focused on the biological activity of flavonoids and the synergize activity w ith artemisinin. Few papers were focused on the biosynthesis pathways of flavonoids from A. annua. Hence, In this research, two genes of Flavonoids biosynthesis from A. annua were cloned and characterized from A. annua. The results of this research were listed in the following paragraphs.1. The total RNA used for the reverse transcription(RT) reaction was isolated from buds and leaves of Artemisia annua L. the full length cDNA of flavanone 3-hydroxylase (F3H) were isolated from A. annua. for the first time by using PCR method. The completed open read frame of AaF3H was 1095 bp and it encoded a 364-amino acid protein with a predicted molecular mass of 41.18 kDa and a pI of 5.67. Subsequently, a BLASTP search of Genbank with the predicted amino acid protein sequence of AaF3H was performed and results showed that the deduced AaF3H shared 67%,76,77, and 78% of identity at the amino acid level with other plant F3Hs from Ginkgo biloba, Arabidopsis thaliana, Camellia sinensis and Citrus maxima, respectively. The recombinant protein of AaF3H was expressed in E.Coli BL21(DE3) with the expression vector pET-28a as His-tagged protein. At the final concentration of IPTG in 0.5mM and after cultivation at 28℃ for 8h, the effect of expression was very well.2. The recombinant protein of AaF3H was expressed in E.Coli E.Coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaF3H) catalyzed (-)naringenin to dihydrokaempferol with Km value of 164.35 μM. The optimum pH for AaF3H reaction was determined to be pH 8.5, and the optimum temperature was determined to be 35℃.3. The full length cDNA of flavonol synthase (FLS) were isolated from Artemisia annua L for the first time by using PCR method. The completed open read frame of AaF3H was 1008 bp and it encoded a 335-amino acid protein with a predicted molecular mass of 37.85 kDa and a pI of 5.35. Subsequently, a BLASTP search of Genbank with the predicted amino acid protein sequence of AaFLS was performed and results showed that the deduced AaFLS shared 86%,74%,74%, and 73% of identity at the amino acid level with other plant FLSs from Rudbeckia hirta, Gentiana triflora, Nicotiana tabacum and Solanum tuberosum, respectively. The recombinant protein of AaFLS was expressed in E.Coli BL21(DE3) with the expression vector pET-28a as His-tagged protein. At the final concentration of IPTG in 0.5mM and after cultivation at 28℃ for 8h, the effect of expression was very well.4. The recombinant protein of AaFLS was expressed in E.Coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaFLS) catalyzed (2R,3R)-dihydrokaempferol to kaempferol with Km value of 127.49 μM. The optimum pH for AaFLS reaction was determined to be pH 7.5, and the optimum temperature was determined to be 23℃.