cDNA Library Construction Using Leaves of Macleaya Cordata and Expressed Sequence Tags Analysis
|School||Hunan Agricultural University|
|Course||Medicinal plant resources engineering|
|Keywords||Macleaya cordata cDNA library EST analysis|
Macleaya cordata is a genus of the Papaveraceae family, which are perennial herb plants. It is riched in alkaloids, mainly for sanguinarine(SA), chelerythrine(CHE), protopine(PRO), allocryptopine(ALL). Pharmaceutical researches showed that alkaloids were its major bio-active components. It has been found that SA and CHE had antiseptic and antitumor activities and an antimicrobial drug, Although the alkaloids from Macleaya spp. exhibited a great variety of pharmacological activities and have been applied widely, the molecular and genetics study of it was rare. There are only less than20sequences from M. cordata in NCBI GenBank till the paper has been published. Considered these, it is essential to to study its molecular genetics and alkaloid biosynthesis. This research constructed the cDNA library by using leaves of Macleaya cordata, and makes a preliminary analysis of the EST. the following are some result:(1) Comparing the experimental results, the traditional TRIZOL method has improved. Using the improved method, we can obtain good quality of total RNA extracted by leaves.(2) The cDNA library has been constructed. Synthesize double-stranded cDNA was synthesized, then collect segments over500bp, followed by connecting to carrier PBluescript II SK(+) and transfecting DH5a E.coli. Finally the cDNA library is constructed. The library qualification evaluation showed:the titer of Primary cDNA library was4.3x10, the titer or amplined library was5.05×108, the fragment size of inserted was0.5-2kb.The cDNA library was constructed successfully, and could be satisfied for further studying on expressed sequence tags (ESTs) analysis.(3) The preliminary analysis of the EST was carried on. Based on cDNA library, large-scale sequencing puts out600ESTs. We get486valid sequences of more than50bp after wiping off carrier and low-quality sequences are got. These ESTs stitch to395unigenes. There is186unigenes match GO annotation after BLAST2GO, which was classified to biological processes, cell component and molecular function, and186unigenes match KEGG annotation after KAAS, which was classified to36metabolic pathways. The results weupdate to the Genenbank database and now can down;oad from the website. The ID is:JK752513-JK752907.