The Germplasm Innovation Of Artemisia Annua L. And Separation Of Dihydroartemisinic Acid From Artemisinin Production Waste

Malaria is one of the most important parasitic diseases in the world, affecting at least 300 millions people a year, and resulting in more than 1 million deaths. Artemisinin, an endoperoxide-containing sesquiterpene lactone isolated from the aerial parts of Artemisia annua L. （sweet wormwood）, have proven efficacy in killing Plasmodium falciparum parasites in severe as well as uncomplicated malaria. The demand of artemisinin has increased sharply since the World Health Organization recommended its use as part of the artemisinin combination therapies. Besides anti-malaria, artemisinin proved effective against hepatitis B, schistosomiasis, several blood parasitic protozoans, and against a variety of cancer cell lines including breast cancer, human leukemia, colon, small-cell lung carcinomas and drug-resistant cancers. As artemisinin cannot be synthesized chemically in an economically feasible way, A. annua is the only practical source of this valuable drug. Unfortunately, A. annua contains only very small amounts of artemisinin resulting in high cost for artemisinin production. How to supply artemisinin in a costly efficient way has received more and more attention. The aim of the project described in this thesis is to provide advanced technique system and basic theory for production of artemisinin in a low cost way through germplasm innovation and high efficient utilization of artemisinin production waste. And the results were obtained as below:1.By comparison of different explants of A. annua, it was found that internode stem was resistant to> 500 mg/L Carb and it was the best start material for A. annua transformation mediated by Agrobacterium tumefaciens. The factors influencing A.tumefaciens-mediated transformation of A.annua, including selection pressure, preculture period, infection time and composition of infection bacterium suspension, were explored to optimize the transformation system. Then a system of high efficiency of genetic transformation and regeneration of A. annua was established.2. A seriers of excellent germplasts were generated by combining three major mutagenesis systems, including somatic variation, EMS and UV treatment. The stable increased adventitious rooting （iar） mutant displaid increased adventitious rooting. Except for the increased number of roots, the overall plant morphology, including leaf and stem structure, was unchanged in the iar mutant. No difference was found between wild-type and iar in the structure or density of trichomes on the leaves. SEM analysis indicated that there was no marked ultrastructural changes on stems between wild-type and the iar mutant. Beside iar mutant, a branch angle decreased mutant, an anti-powdery mildew mutant, and a leaf increased mutant were obtainded. These results implied that combination mutagenesis represents a useful technology for enhancement of A. annua germplasm.3. Metabolic and morphological analyses of the iar mutant coupled with in vitro assays were used to elucidate the relationship between plant secondary metabolites and AR formation. Results showed there were no significant differences between wild-type and iar in the amounts of photosynthetic pigments, including chlorophyll and carotenoids. The carbohydrate levels of the iar mutant and wild-type were analyzed by GC （gas chromatography） and, again, no significant differences were found. Moreover, the total phenol content in iar and wild-type were examined according to Folin-Ciocalteu method, and no significant differences were found. The only detected differences between the iar mutant and wild-type were rooting capacity and borneol/camphor content.No camphor was detected in the iar mutant, whileas the borneol content in the iar mutant was about 15-fold higher than that in wild-type. Consistent with this, treatment with borneol in vitro promoted adventitious rooting in wild type. The enhanced rooting did not continue upon removal of borneol. The iar mutant displayed no significant differences in AR formation upon treatment with camphor. Together, our results suggest that borneol promotes adventitious rooting whereas camphor has no effect on AR formation.4. To understand the molecular mechanism of AR formation, completely unfolded leaves of A. annua were analyzed by the iTRAQ proteomic technique coupled with LC-MS-MS. A total of 1292 proteins were detected in young and mature leaves. BLASTp analysis （Figure 3） using the GenBank non-redundant （nr） protein database revealed that 1025 （79.3%） proteins showed significant similarity to proteins with known functions,112 （8.7%） showed significant similarity to unknown proteins, and the other 155 （12%） showed no significant similarity to any proteins in public databases. The resulting identified proteins in leaves were categorized into 22 putative functional groups based on the functional categories, covering RNA synthesis, signal transduction, defense and stress, protein synthesis and fate, and metabolism, secondary metabolism, energy, cell growth/division, transcription, protein synthesis, protein destination/storage, transport, cell structure, signal transduction, disease/defense and unknown protein.5. In order to discover the molecular mechanism under which adventitious root ablity is increased in the iar mutant of A. annua, the full-length cDNA of a putative borneol dehydrogenase gene （AaBDH） from A. annua was cloned by rapid amplification of cDNA ends. The completed open read frame of AaBDH was 1415 bp and it encoded a 885-amino acid protein with a predicted molecular mass of 31.04 kDa and a pI of 6.16. AaBDH showed 70%,69%,68%,70% of amino acid identity to Solanum lycopersicum S1ZBS （XP₀04249781.1）, Ppulus trichocarpa PtADH （EEE92928.1） Morus notabili MnMLAS （EXCO 1949.1）, and to Ricinus communis RcSAD （EEF32231.1）, respectively. The recomibant protein was obtained by heterogenous expression of AaBDH in a strain of E. coli BL 21 and purified by affinity chromatography. The function of AaBDH was characterized by enzyme assay in vitro, and the results showed that AaBDH bear the ability to convert borneol to camphor with the present of NAD+ or NADP+.6. A quantification method of dihydroartemisinic acid using gas chromatography with flame ionization detector was established in the thesis. This method is simple, sensitive, accurate and reproducible with over 98% recoveries. The limit of detection was less than 3 μg/mL and the limit of quantification was less than 9 μg/mL. The quantification of for dihydroartemisinic acid could be finished within 20 min, allowing samples as low as 100 mg dry weight to be analyzed. The method could be applied to dihydroartemisinic acid level analysis of commercial plant extracts, dihydroartemisinic acid products, and A. annua. The determination method provides a powerful analytical tool for development of dihydroartemisinic acid products, molecular biologic study on dihydroartemisinic acid, high-through-put screening high yield clone in an early stage or real-time monitoring of crop quality.7. A method to extract and purify dihydroartemisinic acid from artemisinin production waste was developed. The single factors, including NaOH concentration, power, solid-liquid ratio, extraction time, and pH value were investage using single factor experiment. Afterward, response surface analysis was performed to optimize the extraction condition. The optimum extraction conditions were as bleow:untrasonic extraction with 7-fold 0.3% NaOH under a power of 75.2 W for 48.7 min, and participation with pH value 1.0. Using the adsorption and de-sorption amount of dihydroartemisinic acid as determination index, the type of anion exchange resin, the type of eluent, the concentration of eluent, the volume of sample solution, the flow rate of eluent and the volume of eluent were investigated through gas chromatography.717 anion exchange resin showed the best capacity to purify dihydroartemisinic acid from artemisinin production waste. The optimum separation conditions were as below:the best eluent was equal volum of 10% ammonium chloride and 80% ethanol; the loading amount of sample solution was 3 mg per miaroliter of resin; the optimum elution volume was 3BV. The present work should provide an effectively solution way to decrease the pollution of artemisinin production waste, and to transfer waste into treasure, which would bring potential economic value.