Gene Cloning And Function Analysis Of Antimalarial Composition From Artemisia Annua L.

Annua L. (Artemisia) is an annual herb, whose main active ingredient artemisinin is the first choice for the treatment of malaria in the world. In recent years, it is found that the single artemisinin treatment is easy to cause drug resistance of Plasmodium, and the flavonoids in Artemisia can cooperate with the absorption of artemisinin, and reduce the production of drug resistance.3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) is the first key enzyme gene in artemisinin biosynthesis, overexpression of the gene can significantly improve the artemisinin content, but screening on the promoter activity analysis and core sequence of research has not been reported; at the same time, Flavanone 3-hydroxylase AaF3H and flavonols synthase AaFLS Artemisia annua flavonol biosynthesis key enzyme gene, its biological functions in vitro although has been validated, but also did not see its eukaryotic expression studies. Therefore, this study using gene cloning and GUS marker gene and Agrobacterium mediated conductance method tobacco transformation method, of HMGR promoter sub under the condition of high temperature, low temperature and dehydration of biological activity and core sequence is analyzed, and on the basis of full-length cDNA sequence of analysis AaF3H studied overexpression of genes AaF3H and AaFLS in tobacco, the thought of artemisinin and flavonoid compounds biosynthesis and their synergistic antimalarial follow-up study to provide reference. The main results are as follows:1. Use a. annua leaves as materials, to extract DNA, ?-glucoside acid enzyme (GUS) gene was used to as the report gene and constructed five different length expression vector, including the whole length of HMGR promoter, based on the cloned HMGR promoter sequences, and successfully obtained transgenic tobacco through the method of agrobacterium mediation, then GUS staining the tobacco. The GUS staining showed that the blue color could detect in roots, stems, leafs, flowers and fruit pods. And the leaf in the lower part could easily get the blue color than the leaf in the top part, this many due to the accumulation of HMGR metabolite product is more and the permeability is better in the mature leaves. The results also showed that the color shows shallow in heat and drought stresses, and no significant different in cold stress, the tobacco leaves with paragraph of P-HMGR-1, P-HMGR-2 and P-HMGR-3 showed blue color but did not find blue in leaves with paragraph of P-HMGR-4, P-HMGR-5 and control and the color of P-HMGR-1 and P-HMGR-2 are deeper than P-HMGR-3. HMGR promoter has a stable expression during the whole growth period of tobacco. HMGR promoter was not sensitive with cold environment, but sensitive with heat and draught resistance. The key area of HMGR promoter was in P-HMGR-3. And there are some regulatory elements but not the necessary part of HMGR promoter between the non overlap area of P-HMGR-3 and P-HMGR-2.2. We obtained AaF3H gene cDNA sequence through the SMART-RACE technology, the length is 2136 bp. The sequence analysis showed that the sequence contains a complete code area which is 1092 bp, encoding a protein with 364 amino acid composition. The protein molecular weight is 41.18 (KDa), the theoretical isoelectric point (pI) is 5.67, it is a stable hydrophilic protein, neither a secretion nor transmembrane protein, with a total of 13 phosphorylation sites, and belongs to the typical 2 og-Fe ?-foxy oxygenase super family. The secondary structure is given priority to random curl, followed by alpha-screw then chain extension. It's consistency with the chrysanthemum F3H protein as high as 97%.3. Use a. annua young bud leaves as materials, extract the total RNA, and reverse transcription. We cloned AaF3H gene and AaFLS gene, then integrated them into expression vector PCXSN, and import to the agrobacterium GV3101. to construct plant expression vectors PCXSN-AaF3H and PCXSN-AaFLS. Successfully transformed plant expression vectors PCXSN-AaF3H and PCXSN-AaFLS into the tobacco DNA respectively. Directly extract fresh tobacco leaves with methanol and formic acid, Liquid phase detection results show that AaF3H and AaFLS can significantly transform naringenin, and AaF3H gene has higher effect on naringenin, but over express AaFLS gene has a greater influence in chemical composition content in the naringenin biological pathways of tobacco.