Abstract:Plants provide an immense reservoir of natural secondary metabolites. Secondary metabolites and those involved enzymes accumulate in various compartments in specific plant tissues. The biosynthesis of diverse groups of secondary metabolites is often complicated, tightly controlled via network interconnections, metabolite levels, metabolite channeling and multi-enzyme complexes, and so on. Secondary metabolite profiles could be genetically altered by two strategies, i.e. single gene modification and multiple gene modification; which thus has opened a feasible and prospective platform for secondary chemicals production in plant.

Abstract:sall4, a member of sall4 gene family, plays important roles in embryo development; organogenesis as well as pluripotency maintenance and re-establishment. There are two isoforms of Sall4, Sall4A and Sall4B. The sequence of porcine sall4 gene is still not reported. Because of its distinct role in maintaining the pluripotent state of stem cells, we cloned and sequenced porcine sall4 gene and assessed its expression in pig tissues and embryos. One 2 372 bp nucleotide sequence representing the full-length cDNA of pig sall4 was obtained by 5￠and 3￠RACE. Analyses of putative protein sequence showed a 70% to 80% identity with isoform Sall4B of human and mouse. Comparing with Sall4A, the identity reduced to 30% to 55% because of the loss of a zinc-finger domain-rich fragment. Assessment of sall4b expression in porcine tissues by Real-time PCR showed that it expressed most strongly in ovary and stronger in spleen, lung, heart and testis. For preimplantation embryos, the expression level was lower in 4-cell embryos compared with other stages. Immuno-fluorescence analysis of Sall4 on porcine preimplantation embryos indicated that it expressed in all the preimplantation embryos and located in nucleus, in blastocyst it preferentially limited in ICM cells. Expression pattern in early embryos suggest that pig sall4b is associated with pluripotency and might be a new and useful reprogramming factor for establishing pig induced pluripotent stem cell lines.

Abstract:Nisin is an antimicrobial peptide widely used in food industry. In this study, Nisin A production in Lactococcus lactis ATCC 11454 was improved by overexpression of Nisin A structural gene nisA through introducing a shuttle expression vector pMG36ek-nisA and an integrated vector pDG780-nisA into the host strain. The differences of growth profiles and Nisin A production level between the two obtained genetic engineering strains FMM1/FMM2 and the parent strain were investigated. Our results show that while the growth profile (the growth rate, biomass and ph) of FMM1 was similar to the parent strain, its Nisin A production increased 31%. In contrast, the biomass of FMM2 was notably lower than the parent strain, while its yield of Nisin A enhanced slightly. The transcription level of genes involved in Nisin A biosynthesis in both engineering strains was further detected by RT-PCR. We found that all the 11 Nisin A biosynthetic genes in FMM1 and FMM2 had a higher transcription level than those in the parent strain, and these genes exhibited more significant increasing degree of transcription level in FMM1 which hosted the autonomous replicating nisA gene. These data suggest that expression of nisA may act as a rate-limit factor in Nisin A biosynthesis. In conclusion, this work provides a new method to improve Nisin A production by increasing the transcription level of nisA, paving the way to further large-scale industrial production of Nisin A.

Abstract:cry1Ah1, one of holo-type cry genes, cloned in this laboratory from Bacillus thuringiensis strain has been patented in China, and it encoded a protein with strong insecticidal activity against certain lepidopteran insect pests, such as Chilo suppressalis. cry1Ah1 gene is exhibiting good application prospects. In order to improve the expression level of cry1Ah1 gene in rice, and investigate the effect of codon usage preference of gene expression, we designed five different optimized schemes for cry1Ah1 insecticidal critical fragment in accordance with bias of rice codon, to improve G+C content, removed the shear signal and unstable factors. Optimized cry1Ah1 genes were transformed into Escherichia coli Rosetta (DE3) respectively, and 65 kDa polypeptides was expressed normally in inclusion body separately. All of these expressed polypeptides showed insecticidal activity against 2nd-instar larvae of Plutella xylostella and neonate of Chilo suppressalis. After transformation with modified cry1Ah1 genes into Var nippobare, the transgenic rice seedlings were detected by PCR, the positive rate containing target gene was more than 87%. Afterwards, the results of real-time RT-PCR and ELISA assay indicated that the highest expression level of five modified cry1Ah1 genes was that using the highest frequent codons. Average expression amount of Cry1Ah1 polypeptides was 0.104% of total soluble proteins from the positive transgenic rice.

Abstract:CYP2E1 enzyme encoded by cyp2e1 gene plays an important role in metabolism of heterogeneous organics in mammalian liver cells. The transgenic plant with cyp2e1 can metabolize various low molecular weight organic pollutants. However, it is unclear the mechanism of expression control of cyp2e1 in transgenic plant. In this study, plasmid pSLD50-6 with cyp2e1 and pKH200 with gus as control were transformed into Agrobacterium tumefaciens GV3101 separately. Then, the cyp2e1 or gus genes were transferred into tobacco (Nicotiana tabacum) and the transgenic plants were regenerated via Agrobacterium tumefaciens method. Real-time quantitative PCR (qRT-PCR) was used to analyze the cyp2e1 gene expression. The expression of cyp2e1 in transgenic tobacco with cyp2e1 decreased obviously treated by ethyl alcohol and reduced slightly by benzene and toluene, while it enhanced by acetone, formaldehyde and oxygen deficit in different levels. In addition, the gene expression of NADPH-P450 oxidoreductase and cytochrome b5 enzyme in the transgenic tobacco with cyp2e1 were increased significantly treated by benzene, which showed that NADPH-P450 oxidoreductase and cytochrome b5 enzyme in transgenic tobacco have relation with CYP2E1 detoxication process. It suggested that the NADPH-P450 oxidoreductase and cytochrome b5 enzyme in transgenic plant formed the requirement in mammalian and participated in the electron transport chain of CYP2E1 enzyme catalytic process.

Abstract:Heat shock protein 27 (HSP27) is a member of the small heat shock proteins (sHSP) and has multiple functions, it also plays an important role in the life cycle of some viruses. To investigate the regulatory effect of HSP27 during influenza virus infection, we cloned and expressed human HSP27 in both prokaryotic and eukaryotic cells, and demonstrated that HSP27 interacted with influenza A virus NS1 protein both in vivo and in vitro. Luciferase assay showed that HSP27 inhibited the expression of interferon-β (IFN-β) in infected cells, and independent of its phosphorylation. Moreover, HSP27 enhanced the inhibitory effect of NS1 on the expression of IFN-β. Further analysis indicated that HSP27 exerted the inhibitory effect probably through influencing MDA5 of the RIG-I like helicase (RLH) pathway. The results suggested that HSP27 play a role in the innate immunity of infected cells, contributed to our understanding of the regulatory effect of host factors during influenza virus infection.

Abstract:To study the attachment, proliferation and differenciation of neural stem cells (NSCs) on surface modified PHBHHx films and to establish the theory of PHBHHx application in NSCs-based brain tissue engineering. PHBHHx film was fabricated by a solution-casting method, and the morphology of the ?lm was observed under scanning electron microscopy(SEM). The films were treated by NaOH or lipase, then the surface hydrophilic property was characterized using water contact angle measurement. NSCs were isolated from the cerebral cortex of rat embryos on embryonic day 14.5, and cultured on surface treated PHBHHx films. The morphology of NSCs attached on the film was visualized under SEM, and the survival and differentiation of NSCs were observed through immunocytochemical staining. Compared with the untreated PHBHHx films, the water contact angle of NaOH or lipase treated PHBHHx films decreased dramatically, and the number of NSCs attached significantly increased. NSCs survived well on treated PHBHHx films and differentiated into neurons and glial cells. The amelioration of hydrophilic property of PHBHHx film improved its biocompatibility with NSCs. PHBHHx can serve as a novel CNS tissue engineering biomaterial applied for NSCs transplantation, brain repairing and regeneration.

Abstract:To investigate the protective effect of polyethylene glycol (PEG) modified recombinant cytoglobin (PEG-rCygb) on acute liver damage in mice. The acute liver injury model of KM mice was induced by CCl4 and then treated with PEG-rCygb, The liver and blood samples were collected for biochemical and histopathological analysis. The results showed that PEG-rCygb reduced the liver mass index and decreased significantly the levels of alanine amiotransferase (AST) and aspartate transaminase (ALT) in mouse serum. In liver tissues, the content of malondialdehyde (MDA) was decreased, whereas the content of glutathione (GSH) was increased in PEG-rCygb treated group. PEG-rCygb also elevated the activities of total super oxidedismutase (T-SOD) and catalase (CAT) in liver tissues. HE staining of liver tissue slices revealed that PEG-rCygb relieved fatty degeneration of liver, decreased inflammatory factors and reduced liver cell injury. Further in vitro experiments indicated that the protective effects of PEG-rCygb on hepatic stellate cell (HSC) against H2O2 were enhanced compared with that of rCygb. All results indicated that the PEG-rCygb promoted oxygen free radical scavenging ability and prevented acute liver injury in KM mice induced by CCl4.

Abstract:To establish an orthotopic transplant mouse model of hepatocellular carcinoma (HCC) labeled with secretary luciferase and to study its response to anti-tumor treatment with interferon-β gene therapy. We labeled the murine hepatoma Hepa1-6 cells with secretary Gaussia princeps luciferase (Gluc), and then injected Gluc labeled Hepa1-6 cells intrasplenically in C57BL/6 mice. We monitored blood Gluc to evaluate the tumor development and anti-tumor effects of hydrodynamic injection with interferon-β expressing plasmid. We successfully established the orthotopic mouse model of HCC by intrasplenic injection of Gluc labeled Hepa1-6 cells. The Gluc blood assay could reflect the amount of cancer cells in vivo, tumor progression, as well as anti-tumor effect of interferon-β gene therapy. In conclusion, Gluc labeled orthotopic transplant mouse model of HCC can ex vivo real-time monitor the tumor development and tumor response to treatments.

Abstract:We screened differential expression bone-related microRNAs (miRNAs) in serum of patients with osteogenesis imperfect (OI). First, we selected the reference gene (s) fit for quantitative detection of serum miRNAs by using geNorm and several other programmes. Then real-time fluorescent quntitative PCR was used to detect the expression level of bone-related miRNAs gained by means of miRanda, Targetscan and Pictar softwares caculation and reading literature. Then, the results were analyzed with the matched t test. All 6 candidate reference genes had a stable expression level in serum of healthy controls and patients with different characters, and the optimal number of reference genes is 4 (miR-16, let-7a, snRNAU6, miR-92a) after Pairwise Variations analysis (V4/5=0.133<0.15). For validating the universality of expression stability, we detected the relative expression value of miR-16, let-7a, snRNAU6 and miR-92a in another 8 healthy controls and 16 patients with OI and the result revealed that the expression of 4 genes remained stable (M<1.5). After measuring serum levels of more than 100 bone-related miRNAs in patients with real-time qPCR, 11 miRNAs showed differential expression, and bioinformatic analysis suggested these altered expressional mioRNAs had possibilities to participate in the process of OI. So the experiment indicated that there existed many differential expression bone-related miRNAs in serum of patients with OI, and these miRNAs had potentials to be promising biomarkers for serologic tests and diagnosis of OI.

Abstract:To study the function and potential application of nkx2.5, a critical gene for heart development, we constructed a recombinant adenovirus overexpressing nkx2.5 gene (Ad-Nkx2.5) with the AdEasy system. To evaluate the effect and mechanism of Ad-Nkx2.5 against oxidative injury, the H9c2 myocardial cells were infected with the recombinant adenoviruses Ad-Nkx2.5 or Ad-EGFP, and subsequently exposed to H2O2 to induce apoptosis. The anti-apoptotic potential of Ad-Nkx2.5 was validated by MTT assay for cell viability, Hoechst33342 staining for cellular morphology, and immunoblotting for caspase-3 activity. Ad-Nkx2.5 infection led to an increased survival rate of H9c2 cells and decreased the amount of caspase-3 in an active form. Additionally, overexpression of Nkx2.5 inhibited the release of cytochrome C from the mitochondria into the cytosol. Mechanismic studies showed that Nkx2.5 upregulated bcl-2 gene expression and significantly repressed H2O2-induced expression of bax detected by Real-time PCR. Additionally, H2O2 treatment did not affect the nuclear localization of Nkx2.5. These findings indicate that adenovirus-mediated nkx2.5 gene transfer exerted a protective effect on H9c2 cells against H2O2-induced apoptosis via mitochondrial pathway, and the Nkx2.5-mediated expression modulation of apoptosis-associated genes could be involved in this event.

Abstract:To express and identify fibronectin C-terminal heparin-binding domain (FNCH BD) polypeptides in Pichia pastoris expression system and study its function, the fragment of FNCHBD was amplified by PCR and inserted into pGEM-T vector. After sequenced, the fragment was inserted into pAo815SM vector, and then cloned into the expression vector pPIC9k. The recombinant plasmid was linerarized with restrict enzyme Sal I and transferred into the yeast host cell KM71 and GS115. The positive yeast clone was screened by G418 resistant, and the target protein was induced to express in the medium containing 0.5% methano1. The culture supernatant was collected and then was purified with membrane ultrafiltration and ion exchange chromatography. The purified product was analyzed with mass spectrogram, SDS-PAGE, Western blotting and heparin affinity chromatography. The results showed that the target protein was around 32 kDa and the purity of the product was above 95%. FNCHBD could be specifically recognized by fibronectin polyclonal antibody. These results suggest that FNCHBD could be expressed and purified successfully in Pichia pastoris, which provides a good strategy to further studies.