| Panax Ginseng is always considered as one of the most potent medicinal plant and used widely in traditional medicine. Ginseng hairy root was induced from the explant of ginseng by Agrobacterium rhizogenes(A4 strain in this study)and belonged non-natural plant. Compared with the cell development, hairy root grows quickly, the live material content is high and the hereditary is stability. Ginsenoside is a main effective composition in ginseng hairy root, with many important pharmacological functions, at present, the people wanted to get ginsenosides by culturing ginseng hairy root and achieved to factory production. In order to improve ginsenosides content of ginseng hairy root and satisfy the demand of medication product, we studied factors as nutrition components, culture conditions, precursors and elicitors. We want to change the metabolic pathway to improve the secondary metabolite by adding MeJA in medium of ginseng hairy root. With the development of bioengineering, it gradually becomes the hotspot to improve the product of supersession production by changing plant biosynthetic pathways.The elicitor MeJA could improve the growth speed of ginseng hairy root when its concentration was at 200μmol·L-1, the results showed the biomass increased 13.82 fold and total ginsenoside content was 2.28 percent,35.71 percent higher than contrast. Meanwhile, hairy root cultured in bioreactor showed that biomass increases 23.14 fold, total ginsenoside content was 2.04 percent, the content ginsenoside Rbi and Rd is obviously higher than contrast. The ginseng hairy root grows quickly without any hormone and the ginsenosides content is high, so the modified bioreactor is suit to cultivate it.It was assumed that the different content of saponin was the result of differential expression of the genes involved in saponin biosynthes. Therefore, it was very important to clone the genes and investigate their functions. The total RNA was prepared with Trizol reagent separately from ginseng hairy root induced by MeJA and contrast. The SSH library was constructed by suppression subtractive hybridization technology. There were 117 clones from library by blue-white spot screening, and among them 97 clones were sequenced, the recombinant rate of library was above 85%. The efficiency of suppressive subtractive hybridization was 212 fold. The detection of bacterial colony PCR showed the insert fragments size ranged from 0.2-1.0 kb, average size was about 0.5 kb. All these results indicated that SSH library was qualified for further studies. Successful construction of SSH library of ginseng hairy root is essential for cloning of genes known and is also an initial key for screening and cloning of new genes.97 high-quality expressed sequence tags were obtained by single-pass sequencing from 117 recombinant clones, and 84 unique sequences were formed after cluster analysis by Seqman program of DNAStar software. The sequences exhibited homology to previously known ones of GenBank in the level of protein or nucleotide by analysis of blastx and blastn. Function analysis by Interproscan and the Gene Ontology showed that function-known genes were 53 accounted for 63.1% of tatol genes and classified into 10 categories, most of them related to metabolism and secretory pathway. Function prediction revealed that 16 genes related to metabolism,5 genes related to response to stress,6 genes related to biosynthesis,7 genes related to tRNA modification, GTPase, ion binding et al,5 genes related to protein folding proteolysis,1 gene related to transcription regulation,4 genes related to signal transduction,4 genes related to secretory pathway,2 genes related to electron transport,3 genes related to cell organization. Function-unkown genes were 31 genes and accounted for 36.9%.EST sequences of PRB3, PRB6 related to ginsenoside synthesis were analyzed by Blastx in Genebank, and homology analysis with squalene synthase and squalene epoxidase enzyme reached 97% respectively. To find the open reading frame and the conserved domain of two predict proteins. Successful construction of SSH library induced by MeJA of Ginseng hairy root, is an initial key for cloning and expressing the genes, furthermore, is essential for finding the new genes and the new functions in ginsenoside analysis pathway.
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