Artemisinic Preliminary Study Of The Molecular Marker System

Artemisinin is a new effective antimalarial drug extracted from the Chinese medicinal plant Artemisia annua L .The production of artemisinin is mainly depend on extracting from wild plant, but the yield is very low, and environmental factors can highly effect its content which has a great difference between different genotypes. Because of these heredity differences which play an important role in the process of chemosynthesis and accumulation of artemisinin, the contents of artemisinin are different in each plant. In order to solve the instability problem, we need to bring genetic breeding and quality evaluation into effect.As an method of genetic breeding and quality evaluation, molecular makers must be whole genome distributing, high polymorphic, and stability.A stable molecular system ISSR measures variability in the occurrence of two microsatellites close to one another, SRAP is a newly developed molecular marker system with the advantage of simplicity, moderate throughput, numerous co-dominant markers, high reproducibility and distributed evenly in genome, especially preferential targeting ORFs.Retrotransposon-based markers have a high degree of polymorphism. Retrotransposons are ubiquitous in plant genomes. Retrotransposon populations are highly plastic, showing great variation in copy numbers and genomic localization between even closely related species. These makers are mostly studied in crops rather than medicinal plans, in this paper, we use these makers to study Artemisia annua.ISSR and SRAP makers which based on PCR reaction can be effect by several factors. We analyzed the influence of some important parameters of PCR amplification, and established a stable and reproducible optimal ISSR and SRAP reaction system of Artemisia annua L.In order to establish retrotransposons-based markers, we need to isolate the sequence first. LTR retrotransposons has some protein coding domain which is generally synthesized as a polyprotein, and it contains the Gag domain, which encodes the protein forming the capsid of the virus-like particle; the integrase (IN), which inserts the cDNA copy into the genome; and the reverse transcriptase (RT) and RNase H (RH), which synthesize the cDNA from the RNA transcript. These conserved sequences make it possible to isolate retrotransposons.Consulting to the method of rapid isolation of LTR sequences established by Pearce, we get PCR primers designed from RNaseH conserved boxes. Template DNA was prepared by ligating adapters to partially restricted total genomic DNA, nest PCR was used to amplify the object gene, then the object gene was purified and ligate into a PMD18-T vector, recombination vector was transformed into E.coli by electroporation, positive clone examined by colony PCR reaction was selected and send to sequence. Sequence similarity analysis indicated that it is a kind of Ty3—gypsy retrotransposons. Genbank accession number is: EF422339.