The complete coding sequences of Eya gene family was amplified by standard PCR from human tissues or cells cDNA library. The product of PCR was cloned into the eukaryotic expression vector pcDNA3-FLAG, generating pcDNA3-FLAG-Eya1～4.Then human embryo kidney 293T cells were transfected with the recombinant plasmids and the expression of Eya genes were identified by Western blot. Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya cooperation with Six in 293T cells affected the Myogenin gene expression.The expression vectors of Eya genes were constructed and confirmed by restriction enzyme digestion and DNA sequence analysis. Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya in coordination with Six in 293T cells stimulated the Myogenin gene expression. This study further confirmed that Eya proteins are transcriptional activator of Six, can improve the activity of myogenin promoter.
To construct，express and purify recombinant human HPPCn protein and detect its biological acitivity in vitro. Methods：The prokaryotic expression vector pET-24a(+)-HPPCn was constucted by template pUC18-HPPCn, and then was transformed into E.coli BL21, inducing it with 0.4mmol/L IPTG for 4 hours. The expression and solubility of recombinant protein was detected by SDS-PAGE. The interest protein was purified by anion exchange chromatography and its immunogenicity was identitied by Western blot analysis. Its biological activity in vitro was detected by MTT and 3H-TdR assays. Results：The rhHPPCn was highly expressed after IPTG inducing, and 23% of HPPCn was resoluble in thalline . The purity of HPPCn protein reached over 94％ after twice anion exchange chromatography, and the Western blot analysis showed it could bind specifically to rabbit-HPPCn-polyclonal antibody. The MTT and 3H-TdR assays showed that the recombinant protein could remarkably promote proliferation of SMMC7721. Conclusion：rhHPPCn is successfully constructed and expressed in E.coli . The interest protein is successfully purified by twice anion exchange chromatography and is detected to promote the proliferation of SMMC7721.
Survivin is a protein that inhibits apoptosis and regulates cell division. In our study, The cDNA sequence of survivin was amplified by RT-PCR and sub-cloned into the prokaryotic expression vector pET-21b(+), followed by transformation into E.coli strain BL21(DE3) and induction with IPTG. The recombinant survivin protein fusing with 6xHis tag was expressed in E.coli in the form of inclusion body at the expression level over 60% of the total cell protein. Results of Western blotting showed that recombinant survivin reacted specifically with anti-human survivin antibody. After gel filtration, the recombinant protein reached the purity over 95%, which facilitate the study of diagnosing and inhibitor agents targeting survivin.
To study hepatic stem cells(HSCs) proliferation and characteristic in rotary cell culture system(RCCS) for ideal cell origin of liver tissue engineering and clinic liver diseases stem cells therapy. WB-F344, a kind of rat liver stem cell, was cultured in RCCS to investigate the effect of RCCS on hepatic stem cell characteristics, and the condition of two-dimensional (2D) monolayer cultivating as control. To analyze the changing of HSCs characteristics by morphological observation, flow cytometry（FACS）, RT-PCR and immunofluorescence. To the results, demonstrated that cells were grow well and packed together densely to form irregular aggregates in RCCS, and proliferation of the hepatic stem cells was superior with control, mRNA of AFP increased while ALB decreased, the protein of AFP expression increased while ALB decreased comparing to control. Indicating RCCS could afford appropriate environment to sustain the stem cells character of WB-F344 cells.
A strain ch-3, which produces cold-adapted lipase, was isolated from the frozen soil containing oil of Changji lipidic chemical plant, Xinjiang province. The morphology identification and 18S rDNA sequence analysis showed that it was Geotrichum candidum spp. and named as Geotrichum candidum ch-3. The optimal growth temperature of strain ch-3 is 20℃. Corn serum and bean oil in medium enhances the secretion of lipase. The enzyme purification was performed by double water phases extraction and gel filtration with Sephadex G-75. The optimal temperature and pH for lipase activity are 35℃ and 7.0 respectively. At 0℃, the lipase still has 66% relative enzyme activity. The lipase shows high thermolability, remaining 55% activity after incubation at 50℃ for 30min. The enzyme activity was stimulated by Fe2＋、Cu2＋、Mn2＋.
Bacillus subtilis ZC-7 was obtained by implantation with N+ ions beam to B. subtilis AS1.398, and compared with the AS1.398 neutral protease, the enzyme activity of ZC-7 neutral protease was about 1 times higher in previous research. A neutral protease was purified from the culture of B. Subtilis ZC-7 by the procedures including amoninium sulfate precipitation, ultrafiltration, DEAE-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography. By multi-step purification, the ZC-7 neutral protease was purified to 78.5 folds and its yield was 27.7%, at last, the specific activity of ZC-7 neutral protease was up to 4.1×105U/mg. Analysed by SDS-PAGE, the purified protease has shown a molecular mass of about 42kDa. The Km for casein hydrolysis was 3.67×10-3 ug/mL and the Vmax was 12.21ug/min. The optimum pH and temperature for hydrolysis of casein were 7.0 and 55℃, respectively. This protease was stable up to 40℃ within the pH range of 6.5 and 8.0. EDTA, isopropanol and alcohol nearly inhibited its activity while some ions such as Ca2+，Mg2+，Fe3+ can improve its activity. In addition, it could resist 1 mol/L H2O2.
L-theanine ( γ-glutamylethylamide ) is the main free amino acid component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for optically pure L-theanine production with a new L-aminoacylases-production fungi Cunnighamella echinulata 9980 was developed. The optimum conditions for enzymatic catalysis were at pH 6.5 with 50 mM N-Acyl-DL-theanine and 40 mM CoCl2. After 12-h incubation at 50℃, 22.5 mM L-theanine was obtained, the conversion rate against N-Acyl-L-theanine being 90%. This is the first report that Cunnighamella echinulata and the aminoacylase were applied in preparation of L-theanine.
DC－12 is a Bacillus strain with strong fibrinolytic activity was screened from Douchi. In this thesis ,a novel breeding technology ---whole genome shuffling was applied to enhance the production of fibrinolytic enzyme. First of all ,a candidate mutant library was constructed by treating DC-12 with ultraviolet rays and HN02,respectively. Based on studying the optimum conditions for protoplast preparation and regeneration of DC-12,multi-parental whole genome shuffling with 4 strains was conceded by protoplasts electrical fusion ,then combines with the screening method of double-inactive protoplast , two strains with higher yield of fibrinolytic enzyme were obtained effectively, which can descend stably after five generations ,and their enzyme production reached 2710 IU/ml ,which was increased by 4-5 fold compared with the mutants , respectively.
Aim: To construct eukaryotic recombinant expression vector pCMV－Myc－fat1 and express fat1 gene in human kidney 293T cells. Methods: The cDNA of fat1 was amplified by RT-PCR from Caenorhabditis elegans. Constructed and identified the vector containing fat1 gene by endonucleases digestion and PCR. Expression plasmids were transfected into 297T cells; the expression of fat1 was detected by Western Blot; the distribution of this gene was analyzed by Indirect Immunofluorescense Assay (IFA). Results: Constructed eukaryotic recombinant expression vector pCMV－Myc－fat1;fat1 gene was effectively expressed in 293T cells and detected by Western Blot and laser scanning confocal microscope. Conclusions: We construct eukaryotic recombinant expression vector pCMV－Myc－fat1 successfully through DNA recombinant technique; The expression of fat1 was detected; the distribution of expressed fat1 was both on the cell membrane and in cytoplasm.
Abject To construct an α-hemolysin (α-HL)gene deficient S . aureus strains with the ability to produce staphylococcal enterotoxin B(SEB), and then to obtain the deletion mutation with the same gene background except α-HL gene with wild-type strain. Plasmid pMHL-α was constructed for homologous recombination of the α-HL system of S . aureus by inserting the neomycin resistance gene expressing unit and two α-HL -flanking regions into a shuttle plasmid pMAD. The recombination vector pMHL-α was constructed correctly by restriction analysis and PCR. Because S . aureus SM-01 has no ability to accept foreign DNA, the plasmid pMHL-α was firstly introduced by electroporation into restriction-deficient laboratory strain RN4220 and then transformed to the S . aureus SM-01 by protoplast transformation. Because the recombination vector is temperature sensitive for DNA replication, the wild-type strain SM-01 with recombination vector was incubated at the nonpermissive temperature while maintaining selective pressure to select the mutation of S. aureus α-HL delection strain. The deletion mutation of α-HL was proved by the antibiotic resistance, PCR analysis and the hemolysis assay. S. aureus α-HL deletion mutation was successfully constructed by homologous recombination with the main coding sequences of S . aureus α-HL were replaced by neomycin resistance gene. It could be of great value for providing some theories and approach to study genetic characters of S .aureus and construct genetically engineered bacteria for producing superantigen.
The Key problem in application of aqueous two-phase systems is polymer recycling. The phase systems should be recycled and reused due to reasons of production cost and reducing environmental pollution. The most feasible way is that using environmental sensitive and reversible dissolution copolymers to form aqueous two-phase systems. In this study, Immobilized penicillin acylase was used for bioconversion of penicillin G into 6-APA in aqueous two-phase systems consisted of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB. Partition coefficients of 6-APA was found to be: about 5.78, in the presence of 1% NaCl. Enzyme kinetic showed that reaction reached equilibrium at 7h or so. The 6-APA mole yields were 85.3%(pH 7.8, and 20 ℃) and this value was about 20% higher than control in reaction of single aqueous phase buffer. Partition coefficient of penicillin G(Na) was hardly changeable, while partition coefficient of product, 6-APA and phenylacetate acid was significantly changeable. Reason is due to Donnan effect of phase systems and hydrophobicity of products. The change of partition coefficients of products also affects bioconversion yield of products. In the aqueous two-phase systems, substrate, penicillin G, products 6-APA and phenylacetate acid are biased in top phase, while immobilized penicillin acylase is completely partitioned in bottom. Substrate, penicillin G enters into bottom phase, and it is catalyzed into 6-APA and phenylacetate acid, then the products enter into top phase. Finally, inhibition of substrate and products is removed to result in improvement of products yield. Moreover, immobilized enzyme has higher efficiency than immobilized cells and occupy smaller volume. Comparing with free enzyme, immobilized enzyme has higher stability, longer use life, completely partitioned in bottom phase and recycle. Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage. The light-sensitive copolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtrated 450 nm light, while pH-sensitive polymer PADB could be recovered by isoelectric point (pH 4.1) . The recovery of the two copolymers was 95-99%.
The effects of pretreatment methods of Jerusalem artichoke tubers on microbial lipids fermentation with an oleaginous yeast strain Rhodosporidium toruloides Y4 were investigated in shaking flask culture. The yeast strain accumulated substantial amount of lipids using either purple- or white-skinned Jerusalem artichoke tubers as sole carbon and energy source. When cells were cultured on the extracted juice or the acid hydrolysate, cellular lipid content reached 40% (w/w); While cultured on the pulp, the white-skinned tubers had higher lipid productivity, yielding 12.1 g lipids per100 g dried tubers. Major fatty acid constituents of microbial lipids were those contained 16- and 18-carbon atoms based on GC analysis, which is quite similar to traditional vegetable oil. Microbial lipids prepared from Jerusalem artichoke can be applied to biodiesel production.
Abstract The balance computation and metabolism analysis of carbon in xylose/glucose fermentation by Pichia stipitis was studied. A 400 h continuous chemostat culture was divided into 4 phases by adding different sugar concentration during each phase. By controlling temperature of （35±1）℃ and aeration of 100～150mL/min and stirring speed of 250～300 r/min, one steady state was established during each phase. The carbon recovery ratios of the four steady states were 118.0%, 105.6%, 113.5% and 94.7 %, respectively. By analyzing of metabolic effluence of carbon, it was found that approximately 50.0 % carbon from glucose and xylose was mainly turned into ethanol, while carbon was secondly emitted as CO2 and thirdly charged into yeast cells. Xylitol concentration was correlative with substrate xylose concentration. Aeration of 100～150mL/min was appropriate to continuous fermentation with mixture substrate of 30.0 g/L xylose and 30.0 g/L glucose .
The screening of the specific interaction between proteins displayed on phage displayed human liver cDNA library with methotrexate was carried out. Methods: With methotrexate immobilized on agarose gel, the affinity selection process of "binding-eluting-amplifying" was performed for five rounds to enrich the specific phage clones. PCR was performed to monitor the selection process, and the amplification products were sequenced and searched in GenBank by BLAST to find the obtained target proteins with high similarity. Results: The PCR products of the selected phage clones were proven with identical sequence with PI-3K related kinase SMG-1 isoform 1. Conclusions: The screening of specific proteins by using affinity selection of phage displayed cDNA library with small-molecule drug immobilized on agarose was an efficient and convenient method. It might be good to search for the candidate targets of the drug molecules, and might provide clues for the further studies of drug mechanism and toxocity.
Based on the conserved region nucleotide sequences of four viruses (Alfalfa Mosaic Virus, ALMV; Cucumber mosaic virus, CMV; Cucumber mosaic virus-satellite, CMV-sat; Potato virus Y, PVY) and one viroid (Potato spindle tuber viroid, PSTVd) which could be pathogenic on potato world and one inner control(18S rRNA), specific oligonucleotide probes and primers were designed and spotting for hybridization detection.There is examine the influence of probe concentration、hybridization time、hybridization temperature and spotting solutions on microarray signal.Finally there is validate the specificity of optimized plant virus detection array.The experiment indication: There is not significant influence on hybridization signals with the concentration of the oligo probes between 5-20 µM,PCR probe show the linear relationship in influence of hybridization signals. Microarray hybridization at 45℃ for 4 h, the intensity of hybridization signals was the most significant, and under this condition the influence tendency on oligonucleotide probes and PCR probes was nearly same. With the comparison of different spotting solutions, DMSO had the best spotting reproducibility. Through the whole condition optimized, the oligonucleotide probe microarray and PCR probe microarray were provided with better specificity in the hybridized detection.
A total of 110 bio-products, extracted from both the supernatants by ethyl acetate and the precipitated thalli by methanol, derived from the fermented broths of 55 microorganisms including bacteria and molds isolated from deep seas in the tropical Pacific Ocean, were screened for antitumor activity by using 4 human cancer cell lines as the tested models and the techniques of methyl thiazolyl blue tetrazolium bromide（MTT）and sulforhodamine B(SRB) colorimetric assay; while the microbial strains were identified mainly by the molecular biology techniques.Some 90% of the tested 110 extracts appeared antitumor activity and 13 strains of the 55 isolates showed higher inhibitory capacities(effective concerntrations ≤16μg/ml）, which may imply a possiblility in their further medical application;whereas the percentage of positive extracts was higher from the thalli than that from the supernatants and of actived strains was higher in the bacteria than that in the molds according to this study.The 50 identitied strains belong to 29 species in 21 genera while the 13 higher actived strains distribute in 8 genera.These results probably released a pilot information in the research and development of deep sea microbial resources in China.
Compared with C3 plant, C4 plant had evident growth advantage, higher rates of water and nutrition using, and higher bio-yield than C3 plant, Sugarcane was one of the typical C4 plants. DNA was extracted from sugarcane leaves, primers were designed by the cDNA sequence of PPDK gene from GenBank. Then DNA was amplified by LA-PCR(Long Acute PCR) method, ligated into pMD18-T vectors, transformed E.coli. JM109, sequenced. Full-length PPDK gene sequence of sugarcane was obtained, the sequence was 13.5Kb in length. For convenient of the next experiment, two digest sites(XhoI and NotI) were introduced into primers, the full-length PPDK gene was splitted to two parts, each ligated into pMD18-T Simple vectors, transformed E.coli. JM109, whole PPDK gene clone of sugarcane was finished, preparation of transferring it into C3 plant was made. The strains were storaged in our lab.
Abstract RNA interference(RNAi) is an high efficient, specific gene silencing. Here we reviewed the discovery process of RNAi and its mechanism. In the further, RNAi will play important roles in characterizing new genes in the metabolic pathways, improving plant nutritional value, enhancing plant resistance and creating new varieties.
Castor (Ricinus communis L.), a high energy-stored and industrial raw-material plant species, is of great value in its application. Progresses in castor biotechnology were summarized in terms of tissue culture and genetic transformation in this paper. The results of studies on tissue culture indicated that application of TDZ (thiadiazuron) gave the maximum rate of shoot proliferation in various exogenous hormones when embryo axis was used as explant. On this ground, the in vitro regeneration system was established. The studies on castor genetic transformation showed that the Agrobaterium-mediated method was the most suitable one in different castor transformation methods. The hygromycin is suitable to selection of castor transfomants while castor embryo axis is insensitive to kanamycin. The possibilities of breeding of new castor varieties and improvement of castor production via biotechnology were also discussed.
The pathway for CoQ biosynthesis in microorganisms is a complex metabolic net. It is a conjunction of the following anabolic subnets: the biosynthetic pathways of shikimate, methionine (aspartate family), S-adenosylmethionine, isopentenyl diphosphate (IPP) and polyprenyl diphosphate (PPP) and the pathway for ring modification of ubiquinone. While bacteria derive the 4-hydroxybenzoate as the quinoid nucleus of the ubiquinone from the shikimate pathway and biosynthesize IPP from 2-C-Methyl-D-erythritol 4-phosphate pathway, eukaryotic microorganisms derive the 4-hydroxybenzoate or tyrosine as the quinoid nucleus of the ubiquinone from the shikimate pathway and biosynthesize IPP from the mevalonate pathway. CoQ10 has been used not only as a medicine but also as a food supplement because of its various physiological and biochemical activities (e.g. as an electron transporter in the respiratory chains of prokaryotes and eukaryotes and as an effective intracellular antioxidant, redox control of cell signaling and gene expression). Fermentation by microbes is the most effective way for CoQ10 production. The studies on the genetic improvement of the strains for CoQ10 production with the aid of molecular biological methods focused on four aspects: A. Amplification of 1-deoxy- D-xyluose 5- phosphate synthase and chorismate pyruvate-lyase level increases CoQ10 production; B. Cloning and expression of decaprenyl diphosphate synthase gene for production of CoQ10; C. Cloning and expression of 4-hydroxybenzoate polyprenyltransferase gene for production of CoQ10; D. Construction and introduction of multigene expression cassettes for production of CoQ10.
Unicellular green alga, Dunaliella salina (D.salina), is a biflagellar alga without cell wall, which is a kind of very important eukaryotic microalga. In the previous study, the research of D.salina focus on the morphology, the mechanism of salt tolerance andβ-carotene， however, with the rapid development of microalgal biotechnique, a lot of work about D.salina was reported by our research groups and other researchers in the recent years. In the area of molecular biology, the studies of D.salina mainly place emphasis on the cloning and analysis of important functional genes, regulatory sequences, and the expression of foreign genes using D.salina as host. This review reports the research advance in these aspects.
