| Extreme environment living creatures, after a long period of adaptation and evolution, must have unique resistant substance and metabolic pathways.Chimonanthus praecox(Wintersweet) can blossom in the condition of low temperature below freezing, suggest that it must contain the survival material suitable for low temperature. The excessive expression of LEA protein in Chimonanthus praecox may be related to low temperature resistance. Using Chimonanthus praecox c DNA Library(structured by our laboratory), we cloned a LEA gene,Cp LEA5(KT727031)successfully.Then to determine the gene expression in prokaryotes and eukaryotes transformation system and detect the antifreezing activity of the expressed protein, compared with the control group, there were some antifreeze activity. This experiment studied biological activity and cell membrane permeability through processing the low temperature on the positive clone strains and wild type strains. Through the low temperature stress, the survival rate of positive clones were 20% higher than CK strains. Through the determination of electrical conductivity, the damage degree of positive clone cell membrane was only 55% of them of empty load strains. The integrity of the cell membrane was less destroyed than empty load strain, which indicated that the Cp LEA5 gene can enhance the expression of host strains resistant to low temperature. We also built a plant expression vector p TEV7: Cp LEA5, and perform genetic transformation of Arabidopsis thaliana by taking adventage of Agrobacterium mediated transformation; screening positive plants of Arabidopsis thaliana and getting expected Cp LEA5 transgenic Arabidopsis for freezing biological function identification.In this study, we explored a high anti-salt(alkali) ribosomal protein of Aspergillus glaucus(Ag RPS3 a E) in yeast expression system. According to the bioinformatics analysis results we learned Ag RPS3 a E encoded small subunit ribosomal protein with a size of 29.2 k Da, which belong to the eukaryotic ribosomal protein RPS3 Ae family. In order to determine the anti-salt and the protection mechanism of ribosomal protein Ag RPS3 a E, We made the expression of ribosomal protein Ag RPS3 a E in three different transformation system, the filamentous fungus Magnaporthe oryzae, the other two kinds of transgenic tobacco and Arabidopsis system. Based on all the excessive expression of Ag RPS3 a E ribosomal protein gene in the tested transformants, the result was that, compared with the control group, all the transformants were obtained more remarkable resistance. So we know the ribosomal protein Ag RPS3 a E function not only expressed in fungi, and can also play its role in stress resistance in plants. Based on the fact that ribosomal protein is household element not only in eukaryotic tissues but also prokaryotes, this study suggests that the Ag RPS3 a E ribosomal protein gene is a very ideal high salt resistance gene.For cultivating new crop varieties, the development and use of resistant genes of extreme environment living organisms have broad application prospects.After completing the content above, I also studied the sulforaphan of broccoli,in the university of Michgan U.S. Gulcoraphanin is a main secondary metabolite of broccoli, it and its hydrolyzate sulforaphan, related to defense mechine of plant. Sulforaphane can protect the damaged plant cells in stress conditions, also have been shown to be not only effective in preventing chemically induced cancers. In this study different methods were used todevelop broccoli sprout preparations. To compare their ability to deliver sulforaphane in vivo and to evaluate the pharmacokinetics and tissue distribution of sulforaphane and sulforaphane-GSH conjugate in mice after oral administration of the broccoli sprout preparations.Sulforaphane is a naturally occurring isothiocyanate in broccoli sprouts with cancer chemopreventive activity. The purpose of this study is to use different methods todevelop three broccoli sprout preparations to compare their ability to deliver sulforaphane in vivo and to evaluate the pharmacokinetics and tissue distribution of sulforaphane and sulforaphane-GSH conjugate in mice after oral administration of the broccoli sprout preparations. The sulforaphane-rich sprout preparation generated by our two-step procedure(SM preparation) contained the highest amount of sulforaphane, which was 11 times and 5 times higher than the freeze-dried sprouts with and without plant enzyme activities(FR and ST preparations), respectively. Oral administration of SM preparation produced the highest plasma response among all the three preparations, with the peak plasma concentration of sulforaphane 6 times and 2.3 times higher, and the AUC 7.9 and 2.2 times higher, compared to the other two preparations. Oral administration of ST preparation resulted in high level of glucoraphanin and relatively high concentration of sulforaphane in the plasma, suggesting oral absorption of glucoraphanin and incomplete conversion of glucoraphanin in the intestinal tract. The urinary excretion of intact sulforaphane was 49.99%, 78.96%, and 48.56% of a single oral dose over 24 h after administration of SM, ST, and FR preparations, respectively. This study provides a broccoli sprout preparation that can serve as a good source of sulforaphane, and these data can be utilized to guide the dose regimen of sulforaphane for us. |
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