Anchored RT-PCR-based Artemisia Annua EST Cloning, Sequencing And Bioinformatic Analysis

Since the successful completion of Human Genome Project (HGP) and RiceGenome Project (RGP), two huge milestones in exploring microcosmicbiology, the current molecular biological research has pushed forward toa new era of genomics. However, genome sequencing in an antimalarialtraditional Chinese herbal medicine, A. annua, has not yet initiated andheavily hampered the improvement of artemisinin over-production bygenetic engineering. To keep pace with the trend, we utilize anchoredreverse transcription-polymerase chain reaction (RT-PCR) as a technicalplatform for probing trancriptomics, by which a panoramic cDNA libraryhas been constructed by utilizing flowering A. annua plants as materials.Consequently, 93 sequenced cDNA clones in total have been available. Aftersoftware analysis and sequence comparison, 54 cDNA sequences have beenannotated, in which only one sequence representing the identical gene withthe sequence record in A. annua, 33 sequences representing the homologousgenes with sequence records in other plant species, and 20 sequencesrepresenting the novel genes without any sequence records are comprised.Now 20 annotated sequences have been accessed in a globally shared publicgene database, GenBank, and other 34 unannotated sequences have alsosubmitted in batches as the form of expressed sequence tags (EST).Furthermore, bioinformatic analysis dealing with virtual localiZation,clone classification and molecular evolution of encoding genes derivedfrom A. annua has been performed based upon the homology comparisonprinciple, by which 39 ancestral sequences orthologous to A. annua geneshave been browsed within the genomes of Oriza sativa and a comparativegenomics-based chromosome map has drafted for the A. annua genome. At thesame time, the full-length of cytochrome P450 reductase (cPR) cDNA and18S rRNA are employed for calculating their molecular evolution distanceand plotting the corresponding maximum pasimony phylogy tree, which willexpanse the research area of plant molecular taxonomy. Facilitated by theachievement of artemisinin research in this laboratory and otherinstitutions, an advanced artemisinin-specialized website, ArteLine, hasbeen preliminarily established for global access and free browsing in thefuture. In conclusion, the present study has enriched the availablegenetic resources of A. annua, and expedited genomics and post-genomicsin A. annua as well as other plants, which should be beneficial to the construction of physical map and finally the elucidation of whole genomicsequences in A. annua, thereby paving a wide path to reconstitution ofartemisinin biosynthetic pathway in vitro or in vivo by exploration ofartemisinin biosynthesis responsible metabolic pathway engineering.