Tissue Culture And Dihydroflavonol-4-reductase (dfr) Gene Cloning In Saussurea Involucrata

Saussurea involucrata Kar.et Kir.is a traditional medicial plant that is well know for its richness in flavonoids. Supply of this plant mainly from its wild populations,but due to the rate of survive is extremely low and destroied unlawful by people,and now this valuable plant species is under an enormous pressure for survival.In order to solve this problem ,increase the flavonoids in Saussurea involucrata.An alternative way to produce the bioactive compounds would be by tissue culture systems.In this paper we studies the effect of callus induction and differentiation on medium with plant hormones and different plant hormone and culture time on the callus growth and the production of total flavonoids of Saussurea involucrata.Metabolic engineering can be a useful approach to boost the accumulation of targeted metabolites.However,we also cloned DFR gene.The reasults can supply some references for the base on callus culture of DFR gene transform.1. Seeds Germination of Saussurea involucrataThe effects of different temperature,culture media and osmotic potentials on seeds germination of Saussurea involucrata are significant. The analysis of variance showed that there had the most significant difference (P<0.001) between the temperature and the germination rates. There were significant differences between culture media,osmotic potentials and seed germination rates (P<0.05). The optimal temperature of seeds germination is a variable temperature in 20/10℃;The suitable culture media of seeds germination is 1/2MS0 and germination hardly occurred in PEG -0.9 MPa.2. Effect of Plant Hormones on Callus Induction and Differentiation of Saussurea involucrataThe results showed that callus induction and differentiation of leaves with different plant hormones have significantly statistical differences. The results showed that the medium containing NAA was better than the others . For the comparison of whole explants, callus induction of leaves and roots, petioles and roots have significantly statistical differences. The induction effect using roots was better than that of leaves and petioles. When using NAA and 6-BA combination, callus induction of leaves and petioles has very significantly statistical differences and there are significantly statistical differences between leaves, petioles and roots. The induction effect using leaves was better than that of petioles and roots. Using Saussurea involucrata leaves, the MS medium containing 6-BA 1.00 mg/L and NAA 2.00 mg/L was the suitable medium for callus induction; The medium containing 6-BA1.00 mg/L and NAA0.20 mg/L was the suitable medium for differentiation; Then the regenerated plants were rooted on 1/2MS0 medium containing 6-BA 0.03 mg/L and NAA 0.30 mg/L3. Determination of Saussurea Involucrata Callus Total FlavonoidsWith the explants of leaves of Saussurea Involucrata, the effects of different plant hormone and culture time on the callus growth ,total flavonoids content and total flavonoids output of Saussurea involucrata were investigated in this test. The results show that MS+NAA2.00mg/L+6-BA1.00mg/L is the best induction medium.The value of the highest growth and the output of total flavonoids are 4.58g and 12.24mg respectively on this medium. The highest content of total flavonoids appeared on the medium with MS+NAA4.00mg/L+6-BA2.00mg/L and the value is 2.17mg/g.Callus growth can increa sed with the culture time prolong, but the content of flavonoids is not stable. The callus content and output of total flavonoids reached to the highest after being cultured 30 days and the respectively value is 2.7mg/g and 26.66mg.4.Isolation and Clone of dihydroflavonol-4-reducyase(DFR) genes of Saussurea involucrata and analysis their sequenceUsing Saussurea involucrata leaves,a clone representing a fragment of dihydroflavonol-4-reducyase(DFR) genes from Saussurea involucrata was isolated from a genomic DNA. Sequence analysis revealed that a band is 1,464 bp in size.The single stranded Molecular Weight were 44Kda, G+C content were 35.82%.The BLAST analysis indicated that the similarity between Saussurea involucrata DFR and Saussurea medusa DFR (EF672726) were 94.33% with the corresponding E-value of 0.0. Prediction of DFR protein have 6 alpha ,helix 3 beta turn ,no signal peptide and winded helix .