The Diversity Of Artemisinin Content In Population Of Artificial Cultivated Artemisia Annua L. And Genetic Polymorphism Analysis

Artemisia annua L. is a traditional herbal medicine in China. Artemisinin, which was isolated from Artemisia annua first by Chinese scientists in 1970s, is in repute as a new antimalarial drug because it is a powerful weapon to meet the challenge of drug-resistant haemoplasmodium. Artemisinin is also a promising potent drug in treatment of caner, human parasites such as schistosomiasis, pneumocystosis and AIDS complication.Artemisia annua is the only source of natural artemisinin at present. But, the great diversity of artemisinin content in population of artificial cultivated Artemisia annua L. resulted in low production efficiency of artemisinin and depressed the efficient utilization of farmland. Improvement on detecting method of artemisinin content and the researches on heredity and molecular marker of the trait are very important for new variety breeding of Artemisia annua with high content of artemisinin. By extending new variety of Artemisia annua with high content of artemisinin, output of artemisinin would be increased signally and the production cost would be decreased noticeablely.In this research, the measurement of artemisinin content is optimized firstly to make the process more quickly, simply and accurately for single-plant samples. Measurement of the artemisinin content of single-plant samples from population of cultivated Artemisia annua planted in Qingchuan region of Sichuan Province was performed by the optimized method. The relationship between diversity of artemisinin content in population of cultivated Artemisia annua and genetic polymorphism was analyzed. The preliminary study on effect of mutagenesis on artemisinin content and genetic polymorphism was carried out. The main results are as follows:1. The measurement of artemisinin content was optimized: 1g sample of dried leave powder of Artemisia annua was used for extraction with 10mL of heated benzinum (60～90℃) . The artemisinin was extracted at 50℃by ultrasonic wave for 5 minutes, repeated 3 times and collect infusion. The infusion was concentrated to extractum at 50℃by reducing the pressure. The extractum was dissolved and then diluted to 50ml with 95% ethanol. Then a portion of 10ml solution was mixed in 40 ml of 0.2% NaOH and incubated in 50℃water bath for 30 minutes. After cooling to room temperature, ultraviolet-absorption peak value was detected at 292nm in ultraviolet- visible spectrometer.2. Measurement of the artemisinin content of single-plant samples from cultivated Artemisia annua were measured by the optimized method. The average content of artemisinin was about 9.34‰, the maximum and minimum were 13.04‰and 6.12‰respectively. The results indicated that there were great diversity of artemisinin content in population of cultivated Artemisia annua, which resulted undoubtedly from genetic variation, and showed considerable potentialities of increasing production efficiency of artemisinin by breeding technology.3. Grinding the leave powder thoroughly in mortar was found to be also crucial to the accuracy of measurement of the artemisinin content, even the ultrasonic wave was used in extraction.4. Genomic DNA of Artemisia annua was extracted from dried leave of single-plant samples by the modified CTAB method. Quantity and quality of DNA samples were good enough for experiments of molecular marker or genetic polymorphism analysis of Artemisia annua. It is convenient for sampling of Artemisia annua plant and discriminating traditional Chinese medicinal materials in molecular level.5. The efficacy of stress-response-elements Restricted AFLP (SR-AFLP),a novel method designed for genetic polymorphism analysis and detection of novel genes associated with content of effective constituent in medical plants in the research, was proved. And this method was laid a foundation to the further study on the metabolic path of artemisinin.6. The relationship between the artemisinin content in Artemisia annua and two sites with stress response elements was revealed commendably, and two candidate SR-AFLP markers were found for artemisinin content.