| Paclitaxel,a famous anti-tumor drug, extracted from the Taxus, is widely used in anti-cancer research. Paclitaxel can effectively inhibit breast cancer, colorectal cancer, liver cancer and some others kinds tumor cells growth, and induce these cells apoptosis, necrosis. Paclitaxel is the secondary metabolites of taxus chinensis cells. How to improve the taxus chinensis cell proliferation, and efficiency improve the taxus chinensis cell secondary metabolism, is playing a very important role in enhancing the synthesis of Paclitaxel. In this paper, we pick one-year old new taxus media leaves from the test field of China West Normal University, College of Life Science. In this paper, we conduct some experiments such as callus induction; callus subculture; suspension cell cultures; and add different concentrations of Nitric oxide; Salicylic acid; Jasmonic acid methyl; Hydrogen peroxide to deal with the taxus media Leaves and cells. To explore the mechanism about Nitric oxide; Salicylic acid; Jasmonic acid methyl; Hydrogen peroxide and Nitric oxide combine Salicylic acid; Jasmonic acid methyl; Hydrogen peroxide impacts on taxus media callus induction, cell proliferation, paclitaxel synthesis and the gene expression relatives paclitaxel synthesis. We use high performance liquid chromatography to detection the content of paclitaxel in taxus media callus and cells. Then we use real-time fluorescent quantitative PCR to test the gene expression relatives paclitaxel synthesis. From these experiments we can conclude the following conclusions:(1) MS+3% Sucrose+0.7% Agar+1.5 mg/m L 2,4-D+1 mg/m L 6-BA+0.3 mg/m L NAA was very suitable for taxus media callus induction. The callus induction rate was about 67%, and the callus color was very lighter and callus embryogenic Satisfactory.(2) B5 + 3% Sucrose + 0.7% Agar + 2.0 mg/m L 2, 4-D + 1 mg/m L 6-BA + 0.3 mg/m L NAA was very suitable for taxus media callus subculture. cell proliferation 2.1 times per 30 days and callus colour was milky white, fluffy, grew very well.(3) MS+ 3% Sucrose + 2 mg/m L 2, 4-D + 0.75 mg/m L 6-BA + 0.5 mg/m L N-nitro-l-Arginine + 10 mg/m L Vitamin C was very suitable for taxus media callus suspension culture.(4) Add 20μmol/m L, 40 μmol/m L Arginine the induction rate of taxus media callus were 26.67% and 23.33%, and add 20 μmol/m L, 40 μmol/m L nω-nitro-l-Arginine induction rate of taxus media callus were 13.33% and 6.67%, after adding 40 μmol/m L Arginine and 40 μmol/m L nω-nitro-l-Arginine, induction rate was reduced to zero, which suggesting that with the concentration increase of Arginine and nitric oxide, callus induction were suppressed. We guesstimated that adding these additions had great affect on the culture medium PH.(5) Add 20 μmol/m L Arginine and didn’t add Arginine, the callus size difference was very apparent, add 20 μmol/m L Arginine callus was bigger than another one. We added the nω-nitro-l-Arginine Same concentration further, the callus size was restrained, and the callus growth became more slowly, and callus colour and lustre became deeper, cell division, indicated that nitric oxide can promote the taxus media cells proliferation.(6) In the suspension cells line when the concentration of the Arginine increase, the promotion of cell proliferation became more more distinct. When adding nω-nitro-l-Arginine, the promotion of cell proliferation was significantly inhibited, and with the increase of the concentration of nω-nitro-l-Arginine, the inhibition of cell proliferation was becoming more and more intensity. Indicate nitric oxide can promote taxus media suspension cell proliferation.(7)With the increase of concentration of Salicylic acid, callus induction rate also gradually to increase, although the increase is not so obvious. Show that the Salicylic acid can promote taxus media callus induction. After adding the Arginine, 40 μmol/m L Salicylic acid +20μmol/m L nω-nitro-l-Arginine, taxus media callus induction rate was 36.67%,add nitric oxide inhibitors nω-nitro-l-Arginine, 40 μmol/m L Salicylic acid+20μmol/m L Arginine + 20 μmol/m L nω-nitro-l-Arginine callus induction rate reduced to 23.33%. Show nitric oxide and Salicylic acid is synergy in promote taxus media callus induction.(8) Jasmonic acid methyl to taxus media callus induction has certain inhibitory effect. And nitric oxide and Jasmonic acid methyl is negative synergy in taxus media callus induction, nitric oxide and Jasmonic acid methyl taxus callus induction is not an ideal combination, not suitable for use in the process of callus induction.(9) Hydrogen peroxide to taxus media callus induction has certain inhibitory effect. And nitric oxide and Hydrogen peroxide is negative synergy in media taxus callus induction, nitric oxide and Hydrogen peroxide taxus callus induction is not an ideal combination, not suitable for use in the process of callus induction.(10) By use Trizol reagent method and CTAB method to extracted media taxus total RNA, found that The CTAB method is a good method of media taxus total RNA extraction.(11) By use fluorescence quantitative detection test of related enzyme gene expression in paclitaxel synthesis and use high performance liquid chromatography to detection the content of in taxus media callus and cells. Find out nitric oxide can promote paclitaxel synthesis, and paclitaxel synthesis relatives enzyme gene expression.(12) Salicylic acid also can promote paclitaxel synthesis, and paclitaxel synthesis relatives enzyme gene expression. And Salicylic acid combine nitric oxide can promote paclitaxel synthesis, and paclitaxel synthesis relatives enzyme gene expression better.(13) Jasmonic acid methyl can promote media taxus cell synthesis of paclitaxel, and promote the synthesis of paclitaxel related enzyme gene expression increase. And Jasmonic acid methyl combine nitric oxide can promote paclitaxel synthesis, and paclitaxel synthesis relatives enzyme gene expression better.(14) Hydrogen peroxide can inhibit media taxus cell synthesis of paclitaxel, and inhibit the synthesis of paclitaxel related enzyme gene expression increase. And Hydrogen peroxide combine nitric oxide can inhibit paclitaxel synthesis, and paclitaxel synthesis relatives enzyme gene expression more intensity. |
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