Potato cultivar Atlantic is widely grown for potato chips in the world. However, this economically important potato cultivar exhibits very poor yields and traits under severe environmental stress. To develop an efficient plant transformation system that could be used to produce large scale transgenic potato plants with enhanced tolerance to environmental stress and therefore would be beneficial for potato processing industry, Agrobacterium-mediated transformation of internodal stem explants using both superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible SWPA2 promoter was performed. Comparing to leaf explants, stem internodal explants were less liable to damage during manipulation, more amenable to in vitro conditions. The addition of silver thiosulfate to the selection medium considerably promoted the shoot induction from explant-derived callus. Seven to nine shoots per stem explant were obtained. By combining the best treatments, this system yielded shoot induction frequency of 94.2% and transformation frequency of 80% of internodal stem explants. Stable integration of the transgenes was confirmed by PCR and Southern blot analyses. In conclusion, short duration (7～8 weeks), high efficiency and easy process make this system well suited for wider commercial applications of transgenic Atlantic potato plants.

To improve 3-ketosteroid-△1-dehydrogenase (KSDH) activity and the transformation level for androst-4-ene-3,17-dione,3-ketosteroid-△1 -dehydrogenase gene (ksdD) from Arthrobacter simplex was cloned into plasmid pWB980 and expressed in B. subtilis WB600 under the control of promoter P43. The molecular weight of expressed enzyme was about 55kDa by SDS-PAGE analysis. The activitities assayed by spectrophotometrical method of intracellular and extracellular soluble enzyme were 110 ± 0.5mU and 15 ± 0.6mU per milligram of protein respectively. The transformation rate of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was 45.3%. Compared with Arthrobacter simplex, the enzyme activity of KSDH expressed in B. subtilis was improved about 30 fold, and the transformation level of androst-4-ene-3,17-dione by the B.subtilis recombinant cells was improved about 10 fold. The recombinant B. subtilis cells used in biotransformation of steroids provided a new way for steroid medicines production.

Enhanced green fluorescent protein( EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin.Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo - peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his -EGFP B in frame with EGFP, myc and 6 × His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6 × his did not interfere each other and octo- peptide linker owned certain flexibility. The results suggest that pcDNA6/myc-his-EGFP B may be useful as a genetic tool for mammalian cells and a vector for gene therapy.

Objective: To research the activity of antibacterial proteins isolated from Musca domestica during larvae-pupae metamorphosis. Methods: The 5-day-old larvae of Musca domestica were injured with a needle. 24h later, the larvae which had changed into pupae were selected. The antibacterial protein was isolated by a four-step protocol including grinding, heating, CM-sepharose F. F. cationexchange chromatography and another CM-sepharose F. F. chromatography. Antibacterial activities of samples were tested by an ultrasensitive radial diffusion assay using Staphylococcus aureus as the indicator organism. The molecular weight of the isolated protein was determined by SDS-PAGE.Results: The molecular weight of this isolated protein was 43kDa, and its antibacterial activity was strong. Conclusions: A kind of protein with strong antibacterial activity can be produced in the Musca domestica during larvae-pupae metamorphosis.

Plant expression cassette for TaDREB from wheat was constructed into plasmid pBIR1.Aloe stems were used as explants for the transformation mediated by Agrobaterium.Infected tissues were selected using G418 to generate transformants.In total,58 resistant plantlets to the antibiotics were obtained from the infected explants.The designed primers according to the selective gene npt II and the target gene TaDREB were used to analyze all of the G418 resistant plantlets.PCR results demonstrated that TaDREB were successful transferred into aloe genomic with the transformation efficiency of 0.5%.The transgenic aloe plants were treated under 4℃ for two weeks and then at-20℃ for 30min.The treatment showed that the leaves of negative plants appeared severe evidence of freeze injury with brown,withered and translucent,while the positive plants appeared good growing condition.The activities of enzymes such as peroxidase(POD)and superoxide dismutase(SOD)of transgenic plants which were stressed for 14 days under low temperature were analyzed.The results indicated that the trend of SOD and POD activities in transgenic plants was down-up-up-up,and that in non-transgenic plants was down-up-down-down.The average value of relative electrical conductivity in the positive plants was 0.456 which was lower than 0.685 in the negative plants.It is supposed that transformation of the kind of gene could improve the resistant ability of aloe to low temperature.

In order to obtain high yield of the HrpNEcc protein with a lower total cost,fermentation and lactose induction conditions for recombinant E.coli BL21(DE3)/pET30a(+)hrpN Ecc were optimized in flasks and the recombinant E.coli was fermentated in 7L fermenter.The optimized incoulum concentration was 5% and the optimized nutrient medium was TB medium.The HrpNEcc protein yield reached 417.60mg/L by adding 3g/L exogenous inducer lactose in the growth prophase of log-phase for the recombinant E.coli.The HrpNEcc protein yield was higher 36.73% than that of the case of no any inducer,and was higher 16.85% than that of the case of adding IPTG.The wet weight of cell pellet of the recombinant E.coli reached 57.24g/L after fermentation in 7L fermenter,the HrpNEcc protein reached 3.29g/L,about 50.2% of total cellular protein.

ACEI,ACEII and Xyr1 are transcriptional factors that regulate cellulase gene expression in Trichoderma koningii.In vitro experiments have shown that ACEI and Xyr1 bind to the cbh1 promoter fragment(-304 to-18) and regulate the gene transcription.However,whether ACEII binds to this 287bp fragment is still unclear.To further elucidate the regulatory mechanism of ACEII for cellulase,DNA-binding domains of ACEII from T.Koningii were expressed in E.coli.It could not show binding to the cbh1 promoter fragment(-304 to-18) by electrophoresis mobility shift assays,suggesting that it is Xyr1 but not ACEII binds playing an essential role during induction of cbh1 gene transcription.

Objective:? -Hemolysin of Staphylococcus aureus,which was expressed in E.coli BL21(DE3) with recombinant pET32a+-?-HL plasmid,was purified with gel filtration chromatography(GFC),and then the engineered subunit vaccine was developed. The immunity effectiveness of this vaccine was evaluated on mouse models.Method:The purified fusion protein was analyzed in SDS-PAGE,and subjected to the evaluation of its median hemolytic dose potency(HD50) was finally analyzed with rabbit erythrocyte. Protein concentration was determined by the method of Bradford. Antibody titers were evaluated on ELISA,and then challenged to gain the immunity protect index.Results:There is an expected protein band with molecular mass of 53kDa in SDS-PAGE,and the concentration is 0.1278mg/ml. The hemolysis activity is 8012.5 HU/mg. There are specific antibodies acquired in blood-serum from mice after vaccined and the antibody titers rising until it has arrive the max,then following down.Conclusion:The purified fusion protein has good fineness and hemolysis activity,the antibody titers initiated by the protein vaccine go with regulation and the immunoprotection is satisfied.

The codeinone reductase gene (cor) and the berberine bridge enzyme gene (bbe) were cloned from young leaf of opium poppy(Papaver somniferum L) by RT-PCR and the coding sequences of these gene were analyzed. The result demonstrated that the cloned COR gene sequence was highly homologous to the other COR gene family members showing 98.96% identity to COR1.1. The cloned BBE gene sequence was 94.84% identified with the released BBE genes in GenBank previously. Based on the cDNA sequences of COR and BBE,two fragments about 400～500 bp from each gene with lower identity among them were cloned. The fusion gene BC(744 bp) is fused by the PCR technique. Then the promoter CaMV 35S driven,containing'forward BC fusion fragments-reverse pdk intron-reverse BC fusion fragments',plant siRNA expression vector were constructed based on the vectors pHANNIBAL and pART27. Inhibition efficiency of the expression vector to the morphine synthesis was studied by transforming papaver nudicarule.The work will lay the foundation for breeding a low morphine and high thebaine poppy.

The purpose is to obtain optimal cultural conditions for Ganoderma lucidum in the solid medium mainly containing the residual of Pteridium aquilinum,and to provide theoretical foundation for the further use of this material from Pteridium aquilinum.Response surface methodology was applied to optimize the main culture conditions including proportion of the residual compound in medium,water content in medium and culture temperature.The results showed that proportion of residual of Pteridium aquilinum in medium,water content in medium and cultural condition had noticeably significant effects on daily average growth rate of mycelium of Ganoderma lucidum(p