To characterize the structural protein VP gene of muscovy duck (Cairina moschata) origin Goose parvovirus (GPV) PT strain, a pair of primers were designed by DNAStar software based on the published sequences of goose original GPV and Muscovy duck parvovirus (MDPV) in GenBank. High fidelity PCR was performed, and the obtained PCR products were cloned into the pMD18-T vector, sequenced and analyzed. Sequencing analysis demonstrated that the VP gene of GPV PT strain included 2 199 bp encoding 732 amino acids (GenBank number: JF926695). The VP gene of GPV PT strain showed a combined feature of GPV and MDPV. It shared a nucleotide identity of 98.8% and an amino acid identity of 98.8% with muscovy duck origin GPV DY strain, which were much higher than the identities with that of goose origin GPV and MDPV. The VP1 gene unique part was similar to that of MDPV and the VP2 gene had the characteristic of goose origin GPV, which have not been reported previously. The success of this study has provided clues for the origin of muscovy duck origin GPV, and even make it possible to study the evolution and phylogenesis of Waterflow parvovirus.

sbHLH1 gene encoded one of transcription factors in bHLH (basic helix-loop-helix) family, which is involved in cold tolerance. The expression of Bar gene can relieve the toxicity of glufosinate, a highly effective and low toxicity herbicide.The OsbHLH1 and Bar genes were transformed into rice cultivar Huai C17 (Oryza sativa L. subsp. japonica Kato) by Agrobacterium tumefaciens mediation. The herbicide resistance, cold tolerance and agronomic traits of transformants with Bar and OsbHLH1 genes were identified in this study. The existance of Bar gene and its expression in transgenic lines were identified by stress of glufosinate. The integration of OsbHLH1 gene and its expression were also detected and confirmed by PCR, Southern blot, RT-PCR and Real-time PCR. The T3 generation of the transgenic line No. 6 had been treated with 2℃ for 6 days at the germination stage. And the dead seedling of this transgenic line was 17.8% while that of control was 61.1%. Under stress of 8~10℃ for 7 days at the seedling stage, the root length of transgenic lines No. 5, 6, 9 and 11 of T3 generation were longer than that of control (P<0.05), and the root number of line No. 3, 5 and 11 were more than that of control (P<0.05) and the fresh weight of No. 3 line was higher than that of control (P<0.05). The increase of cold tolerance at germination and seedling stage of transgenic lines showed that the over-expression of OsbHLH1 gene could improve the cold tolerance of rice. Based on the investigation of the main agronomic traits, there were differences (P<0.05) in plant height, panicle length, 1000-grain weight, seed setting and theoretical yield between transgenic lines of T2 generation and control, which indicated that the over-expression of exogenous gene had obvious influence on rice agronomic traits. The new germplasm created in this study will be available for breeding of new japonica hybrid rice with cold and herbicide tolerance

Mediator is a large, modular protein complex. Interactions among mediator subunits are crucial for mediator complex to play the important roles in regulating transcription in vivo. We cloned tobacco(Nicotiana tabacum)NtMed6、NtMed18 and NtMed21 by homology searching method. Sequencing results showed that the full-length coding sequences (CDS) of NtMed6, NtMed18 and NtMed21 span 753, 651 and 366 bp respectively, which share high similarities (69.46%, 76.91% and 77.01% respectively) with their corresponding homologues in Arabidopsis thaliana. By screening different infiltration buffers, we optimized the method for detecting the subcellular locations of these fused genes by transient transformation assays performed with Agrobacterium-infiltrated tobacco (Nicotiana benthamiana) leaves. At the same time, we constructed the expression vectors of these three genes fused with enhanced yellow fluorescent protein (EYFP) genes driven by 35S promoter, and analyzed the function of linker between NtMed8 protein and EYFP in subcellular locations. The results revealed that YFP expression showed higher fluorescence intensity and cell density which scattered the fluorescent protein infected with the infiltration buffer of YEB+100 mmol/L AS and AA+100 mmol/L AS, and the linker with five amino acids improved the truth of subcellular locations. The four subunits of mediator NtMed8, NtMed18, NtMed6 and NtMed21 were all located in both nucleus and plasma membrane, but mainly in nucleus. To further confirm whether NtMed8 belongs to tobacco mediator subunits as well as the interactions between NtMed8 and other tobacco mediator subunits, bimolecular fluorescence complementation (BiFC) vectors containing coding sequence of EYFP N-terminus from amino acids 1-173 (pNYE) and coding sequence of YFP N-terminus from amino acids 151-238 (pCYE) driven by 35 S promote were constructed to subclone NtMed8, NtMed6, NtMed18 and NtMed21 full length ORFs into the BiFC vectors respectively. Then the subcellular locations with different combinations of the vectors by transient cotransformation assays performed with Agrobacterium-infiltrated tobacco (Nicotiana benthamiana) leaves were also detected. And the results showed that interactions exist between NtMed8 and NtMed18, NtMed8 and NtMed6 as well as NtMed18 and NtMed6. On the basis of these results, we conclude that BiFC is a good method which can be used in analysis of interactions between subunits of mediator

To search for new resource of chondroitin sulfate (CS) for therapeutics, glycosaminoglycan (GAG) was isolated from nasal proteoglycan(PG) of mackerel (Pneumatophorus japonicus Houttuyn). The disaccharide composition of CS in the GAG preparation was determined by anion-exchange HPLC after digestion with chondroitinase. The molecular size of GAG was determined by gel filtration, and the interaction of GAG with growth factors was analyzed to determine the kinetic parameter ka(association rate constant)、 kd (dissociation rate constant) and KD(equilibrium dissociation constant) through surface plasmon resonance (SPR) analysis. The content of GAG was 1.09 μmol/mg PG(as disaccharide unites) or 651 μg/mg PG. CS is the major ingredient of the GAG preparation (1.03 μmol/mg PG). Unsaturated disaccharide units produced from CS were ΔDi-6S(38.8%), ΔDi-4S(46.3%), ΔDi-0S(8.4%) and ΔDi-diSD(6.5%). Molecular size of GAG (CS) was approximately 78 kD. The kinetic parameter ka((mol/L)-1·s-1), kd(s-1) and kD(nmol/L) of GAG (CS) with the growth factors were (2.77±0.17)×105, (7.74±1.56)×10-5 and (0.28±0.06) for midkine (MK); (1.05±0.22)×104, (4.16±0.80)×10-3 and (417±131.3) for pleiotrophin (PTN); and (7.04±0.94)×105, (7.84±2.82)×10-3 and (11.1±3.80) for hepatocyte growth factor (HGF), respectively. High affinity with MK and HGF and higher affinity with PTN was observed, suggesting that the CS chains in mackerel nasal cartilage may regulate growth factor signaling and have a potential for therapeutic applications to some diseases

The large capacity of differential expression genes during oocyte maturation and the transition from maternal to zigotic control makes it possible to screen genes playing crucial roles in embryonic development by mRNA differential display reverse transcription PCR(DDRT-PCR). In order to explore the gene expression mechanism of in vitro maturation (IVM) of oocytes and in vitro development of pre-implantation embryos derived from in vitro fertilization (IVF) in different developmental stages in cattle, mRNA DDRT-PCR was employed to screen differential expression genes in germinal vesicle (GV) stage oocytes, 8-cell stage embryos and blastocysts. Real-time quantitative PCR (Q-PCR) was used to analyze the expression abundance of the differential expression genes in the three stages and in vitro matured MⅡ oocytes. Five differential displayed straps were discovered by DDRT-PCR. The results of sequencing and database analysis showed that the 5 fragments were corresponding to pyrophosphatase (inorganic) 1(PPA1), erbb2 interacting protein (ERBB2IP), restin-like (CEP350), ribosomal protein L27a (RPL27A) and IK cytokine, down-regulator of HLAⅡ (IK), respectively. Q-PCR and SPSS statistical analysis indicated that the mRNA level of PPA1, ERBB2IP and CEP350 decreased along with the procedure of embryonic development, whereas that of RPL27A turned to increase till blastocyst stage. The mRNA level of IK fluctuated before blastocyst stage and then decreased. Overall, the results of this research provide more information on effects of oocyte maturation on the following embryonic development and the mechanism of zygotic gene activation.

Establishment of a sensitive and accurate quantitative labeling system for transgenic components of Genetically Modified Organisms (GMOs) is a crucial step to employ the biosafety management. Real-time quantitative PCR methods have been applied to quantitatively detect the transgenic components as a commonly used technique. By using this method, three inserted copies were detected in Kefeng-6, an insecticidal transgenic rice(Oryza sativa) harboring bivalent gene (cry1Ac+CpTi). Via using absolute quantitative and relative quantitative method to detect the inserted cry1Ac genes and its event-specific sequence in Kefeng 6, we found out that the detection both inserted genes and its event-specific sequence could meet the needs for the quantitative detection. As for 100 ng of genomic DNA, In relative quantitative PCR, the detection threshold of relative amount of cry1Ac gene and its specific flanking sequence was 0.1% and 0.5%, respectively; In absolute quantitative PCR, the detection threshold of the event-specific for Kefeng 6 was approximately five copies. We validated the accuracy of above methods by detecting four mixed samples with known GM contents. To quantitate the GM ratio of a product, the relative quantitation results were not affected either by multi-copy gene or single-copy gene. This study could provide a reliable reference to set up the threshold in quantitative detection of transgenic product.

Potato late blight, ranks as world agriculture's most destructive disease, which are caused by Phytophthora infestans (Mont.) de Bary, and potato production has been seriously limited by this disease. Research on molecular mechanism of resistance against P. infestans is a very important issue in potato breeding program. To date, many ESTs involved in potato late blight resistance have been isolated by the molecular biology methods such as suppression subtractive hybridization, DNA microarray and cDNA-AFLP. To screen and analysis of key candidate genes of potato against P. infestans, functional studies of these ESTs are the most urgent thing. In the present study, two potato ESTs EL732276 and EL732318 which involved in late blight resistance were silenced in Nicotiana benthamiana using virus-induced gene silencing(VIGS). The gene silenced N.benthamiana leaves were inoculated with P. infestans and length grow rate (LGR) of lesions were measured. The RT-PCR results showed that the fragments of candidate genes EL732276 and EL732318 have been successfully cloned into Tobacco rattle virus (TRV) vector. The N.benthamiana leaves infected with TRV carrying the phytoene desaturase gene(PDS) were bleached, indicating that the VIGS system was successful. One month late, the N.benthamiana leaves infected with TRV carrying the genes EL732276 and EL732318 fragment individually were inoculated with P. infestans. The results showed that, compared to the control, LGR values were significantly increased in the EL7322276 and EL732318 silenced N.benthamiana plants, suggesting that P. infestans resistance was dramatically decreased after these two genes silenced in N.benthamiana. This founding was consistent with the results that water soaked lesions and white mycelium obviously observed on EL732276 or EL732318 silenced N.benthamiana leaves after P. infestans inoculation, whereas the lesions developed very slowly on the control leaves which infected with TRV empty vector. The results suggest that functions of potato genes involved in late blight resistance can be rapidly confirmed in N.benthamiana by VIGS technique

causes abnormality of plant growth. To reduce the burden of plant metabolism and to prevent energy waste, we have used a Leafy promoter from genome of Arabidopsis thaliana instead of CaMV35S promoter to govern the expression of a modified 5-enolpyruvylshikimate-3-phosphate synthase gene (CP4EPSPS) with addition of the transit peptide-coding sequence of rubisco small ribosome subunit. Meanwhile, the Leafy promoter was used to regulate gus, a reporter gene. Two plant expression vectors, p3300-Leafy-gus and p3300-Leafy-CP4EPSPS were constructed and used to transform tobacco W38 through Agrobacterium-mediated procedure. Under these conditions, stable expression of the CP4EPSPS and gus genes was only observed in stem apex and extremely young apical leaves of transgenic tobaccos. The gus activity was not detectable in mature leaves, stems or roots. It is demonstrated that the budlet of transgenic tobaccos shows tolerance to herbicide glyphosate

Ditylenchus destructor is a quarantine plant-parasitic nematode that distributed worldly. The genetic variation of D. destructor may be modified with the change of ecological environment. The effect of ecological factor on the genetic structure may be induced by the genetic variation of different population of Ditylenchus destructor. The genetic diversity and genetic differentiation of D. destructor were analyzed with inter-simple sequence repeat(ISSR) molecular marker in this study. The effect of ecological factor on genetic diversity of D. destructor was analyzed with SPSS software. Results showed that genetic diversity was relative high in D. destructor. At the population level, the higest genetic diversity existed in the Taihe population of Anhui Province, the lowest genetic diversity existed in the Changli population of Hebei Province. At the group level, the genetic diversity of five groups was arranged as Anhui＞Shandong＞Jiangsu＞Hebei＞Beijing. Relativey low genetic differentiation and high gene flow were existed in five groups with which coefficient of genetic differentiation(Gst) was 0.0725 and gene flow(Nm) was 6.3933. Correlation analysis showed a positive relationship between Shannon genetic diversity and altitude, longitude, latitude and annual precipitation, while a negative relativeship existed between Shannon genetic diversity and average temperature, maximum temperature and minimum temperature. A positive relationship also existed between genetic distance and geographical distance. The results imply that D. destructor adapts to low temperature and it will be suitable to propagate in northern China. The management of seedling and seedtuber of sweet potato(Solanum tuberosum) shall be reinforced to prevent it's distribution and spreading in northern China.

Metabolic detoxification of carboxylesterases (CarEs) is a major mechanism for the development of resistance in insects to organophosphates (OPs), synthetic pyrethroids (SPs) and carbamates (CBs). In order to elucidate the role of CarE genes from Helicoverpa armigera in metabolic resistance, two CarE genes, 001f and 001g，were successfully expressed in the Sf9 insect cell lines using the Baculovirus Expression Vector System (BEVS). Both of the expressed CarEs were subjected to native PAGE and stained with artificial substrate α-naphthyl acetate(α-NA), and showed high activity against α-NA according to the darker stained bands. The resulting stained bands (closely together) indicated that the relative mobility (Rm) of CarEs fell in two zones (0.27~0.36 and 0.37~0.48). The kinetic assays with α-NA and para-nitrophyl acetate (p-NA) using a spectrophotometer showed the two CarEs had high affinity to the former substrate (Km=(3.9±0.6) and (3.3±0.4) μmol/L, respectively). They also produced a considerably high rate constant (kcat/Km=(26.5±1.9) and (95±9.3) μmol-1·s-1·L, respectively) agianst α-NA, but a relatively lower rate constant against p-NA (kcat/Km=(4.47±0.33) and (12.8±0.10) μmol-1·s-1·L, respectively) compared with that of α-NA. It's showed that the two CarEs had high hydrolytic activity against the artificial substrates. Thus, 001F and 001G esterases are probabaly associated with metabolic resistance of insecticides, and may have relatively high detoxification activity against some traditional chemical insecticdes such as OPs, SPs and CBs.

The translationally controlled tumour protein (TCTP) is a highly conserved protein and appears to be widely expressed in all eukaryotic organisms. TCTP is a multifunctional protein, including many cellular processes, e.g. cytoskeleton regulation, cell growth and proliferation, apoptosis, tumor reversion, cell differentiation, inflammatory response and protection of cells against various stress conditions. TCTP is also a transcription factor for pluripotency genes Oct4 and Nanog. This review focuses on TCTP expression regulation and its biological functions.

This study is to supply a series of vectors for gene knockout, overexpression, and expressing fluorescent fusion proteins in the rice blast fungus. These vectors should be easily worked, time-saving, reliable, and conveniently for the research in the gene function of Magnaporthe oryzae. PCR, enzyme digestion, ligation and transformation were used to construct the plasmids．We cloned two promoters (SOD1 promoter and H3 promoter) which strongly expressed in the mycelia and conidia, built 3 vectors for knockout (pBS-SUR, pBS-BAS, pBS-NEO), 4 vectors for overexpression (pKD5, pKD6, pKD61 and pKD8), and 7 vectors for fluorescent fusion protein expression (pKD5-GFP, pKD5-RED, pKD6-GFP, pKD6-RED, pKD7-RED, pKD8-GFP and pKD8-RED)in M. oryzae and other filamentous fungi. These plasmids could be introduced into the fungi via protoplast transformation, or Agrobacterium tumefaciens-mediated transformation. The knockout mutants could be identified by PCR, Southern blot and Western blot, the fluorescence of the transformants was observed under the fluorescent microscope, and the expression level of genes in the transformants was assayed by Real-time PCR. We have used knockout constructs built by these knockout vectors (pBS-SUR, pBS-BAS, pBS-NEO) and pBS-HPH1 together to knockout 4 genes simultaneously in M. oryzae. After transforming pKD5-RED, pKD6-GFP or pKD6-RED into M. oryzae via Agrobacterium tumefaciens-mediated transformation, green or red fluorescent fusion proteins under the control of SOD1 or H3 promoter were expressed strongly in the hyphae; Real-time PCR results showed eGFP mRNA or DsRED2 mRNA level promoted by SOD1 promoter was 2.5 or 5.4 folds of β-tubulin, and DsRED2 mRNA level promoted by H3 promoter was 20.8 folds of β-tubulin in the mycelia. MoATG8-DsRED2 fusion protein produced by pKD6-RED could locate MoATG8 exactly in the nearby of vacuoles. Observation on DsRED2 fluorescent protein under microscope showed that DsRED2 under control of SOD1 promoter could express in several stages of M. oryzae, such as hypha, conidium and appressorium. These facts imply that above-mentioned 14 vectors can be used to knockout genes, overexpress genes, and express fluorescent fusion proteins in the rice blast fungus and other filamentous fungi, such as Fusarium.

Hlfoo(oocyte-specific linker histone H1) is a linker histone specifically expressed in oocytes and early embryos in mammalian, it plays a crucial role in oogenesis, fertilization and embryogenesis. The objectives of this study were to clone swine H1foo gene and construct a eukaryotic expression vector pVenus-H1foo. Firstly, we applied the 5'RACE and RT-PCR to obtain the complete CDS of swine H1foo gene, the sequence information was submitted to GenBanK (GenBank accession No.: HQ915640). The CDS of swine H1foo gene was linked into pMD19-T and confirmed by digestion of restriction enzyme, and then the H1foo CDS was cloned into pVenus to construct a eukaryotic expression vector pVenus-H1foo. After pVenus-H1foo transfected into Hela cells mediated by Lipofectamine 2000, it was efficiently expressed in hela cells and identified by observation of fluorescence microscopy and RT-PCR. In addition, after transcripted in vitro, the mRNA of H1foo-venus was microinjected into swine oocytes, the expression and location was identified under fluorescence microscopy. The result of sequence analysis showed that the CDS region of the swine H1foo gene included 1 041 nucleotides, encoding for 346 amino acids(Mol.wt.: 36.45kD), and the nucleotide similarity of H1foo between swine and bovine, human, mice was 75.7%, 67.9% and 54.3%,respectively. After transfection and microinjection, the H1foo was expressed and localized accurately both in Hela cell nucleus and swine oocyte nucleus. We had cloned swine H1foo gene and constructed the expression vector pVenus-H1fooin; The mRNA of H1foo-venus transcripted in vitro was expressed and localized accurately in swine oocytes, which greatly facilitates the further research of H1foo in oocytes maturation and nuclear transplantation.

To establish two-dimensional gel electrophoresis methods (2-DE) for porcine skeletal muscle proteome, the experimental conditions of 2-DE was optimized，and various extraction methods，different loading quantities and pH IPG strips，different adhesive concentration and gel staining were studied. The results showed that the process of grinding in liquid nitrogen and following by ultrasonication was more appropriate than that of grinding in liquid nitrogen, which could obtained better 2-DE map for low nucleic acid contamination and more protein spots. Compared with different pH IPG strips, 18 cm pH 4~7 IPG strips were better than pH 3~10 NL. For silver staining of 18 cm pH 4~7 IPG strip, the suitable loading quantity was 150 μg. Furthermore, the reproducibility of three gels in same sample was more 70%. Meanwhile, the common problems occurring in 2-DE experiments were analyzed. The results indicated that the 2-DE maps of porcine skeletal muscle were obtained with high resolution and reproducibility, and can be used for the further proteome analysis.