To reveal the molecular mechanism of the high phosphorus efficiency in maize using existing genetic information and gene resources, a genetic linkage map was constructed for F2 population derived from maize (Zea mays L.) cross of 082(high p)×Ye107(low p) by means of SSR and AFLP. The QTLs for 3 root exudates related to phosphorus efficiency were analyzed according to the phenotypes of 241 F2∶3 families at two stages, two phosphorus treatments and two sites by composite interval mapping. Results showed that a total of 4 QTLs were identified for acid phosphatase and among which AP1-KXNP and AP9-KXNP were identified repeatedly in 4 different conditions. AP1-KXNP was located in the interval bnlg1268a-umc1290a on chromosome 1(bins 1.09) and AP9-KXNP in the interval P1M3/d- P1M3/g on chromosome 9(bins 9.04). Individual QTL explained 9%~16% of the variation of acid phosphatase and the total contribution of the two QTLs was 25%; Five QTLs were identified for organic acid in 4 different conditions , among which only one QTL was identified repeatedly in two different conditions and it was located in the interval P1M3/f-P1M7/g on chromosome 9(bins 9.03); A total of 4 QTLs were identified for H+, none of which was identified in 4 conditions repeatedly. It showed that 2 QTLs AP1-KXNP and AP9-KXNP for acid phosphatase were relatively stable and the 2 QTLs can be used for molecular marker-assisted selection for high phosphorus efficiency in maize.

Soybean cyst nematode (SCN) is a pest which can cause worldwide soybean yield losses seriously. The resistance of soybean to SCN is controlled by multiple genes. It is very important to detect the disease-resistant genes and breed the SCN disease-resistant cultivar. In order to understand the relationship between the gene and resistance to SCN, we analyzed the expression characteristics of candidate gene GmHslpro-1 on Soybean (Glycine max) resistant line Zhongpin03-5373 and susceptible cultivar Zhonghuang 13 using the Real-time PCR technique. We found that the relative expression of the gene raised after 1 d vaccinating SCN 4 Race (SCN 4) and it was 13.4 times higher than that of the control sample, it kept upward until inoculated 6 d. Analysis of variance showed that the relative expression of the gene was significant difference between inoculated and un-inoculated at significant difference (0.05 or 0.01 level). It indicates that GmHslpro-1 may be induced to express in the conditions of SCN 4 infection, and the gene has a certain resistance to SCN 4.

In the present study, we investigated the effects of exogenous pea ferritin gene (Pea-Fer) on the important biological properties of transgenic rice. Fer34-XS11 was obtained from a near isogenic line (NIL BC6F3) of donor parent (exogenous ferritin transgenic pure lines Fer34) and recurrent parent (Oryza sativa ssp.japanica) (Xiushui 11) by successive backcrossing and GUS analysis. Several important biological properties were investigated completely using Fer34-XS11 and Xiushui 11. The major results are as follows, (1)The iron content of Fer34-XS11 was increased significantly, but no obvious difference was observed in the other mineral elements, such as Mg, Ca, Mn and Zn; (2)The introduction of exogenous gene (Pea-Fer) had no significant effect on germination rate and seedling leafing rate; (3)No significant changes were found in the contents and composition of chlorophyll in leaves, but the net photosynthetic rate was dramatically enhanced in the transgenic rice; (4) no harmful effect were found on major agronomic traits as well as economic traits. All the results showed that the exogenous gene (Pea-Fer) was highly expressed in transgenic rice with increasing the iron content of rice grains. And transferring the exogenous gene(Pea-Fer) did not result in significant harmful effect on the important biological properties. The results provide some theory basis for breeding novel high-iron rice varieties by utilizing this foreign gene.

To reveal the disease-resistant mechanism of Chinese wild grape transcription factor in the process of grape resistant to powdery mildew, the highly resistant line Baihe-35-1 of Chinese wild Vitis pseudoreticulata W.T. Wang was used as the materials, and a 1 053 bp full-length cDNA encoding V. pseudoreticulata RING-finger protein1 gene (VpRFP1) was obtained using the transcript product induced by grape powdery mildew (Unicunula necator (Schw.) Burr.) at seven different stages as templates. The resultant PCR products were cloned to pMD19-T vector, sequenced and subcloned into pGEX-4T-1 vector. The recombinant expression clone were transformed into Escherichia.coli BL21 and induced expression by IPTG. A 64 kD fusion protein GST-VpRFP1 was obtained with the form of inclusion body. The fusion protein was purified by electrodialysis, and immunized the rabbit to obtain the polyclonal antibodies. The immunity-antigen reaction was tested by Western blot, showing a specific antigen-antibody recognition feature. The results suggested that the fusion protein GST-VpRFP1 was expressed at high levels 4 h after 0.1 mmol/L of IPTG induction (27℃). And the polyclonal antibodies obtained by immunized rabbit with the purified fusion protein can be used for the functional analysis of VpRFP1 gene.

To explore the function of bovine natural resistance-associated macrophage protein-1(Nramp1) and seek for the objective genes for resistance breeding against intracellular pathogen via transgenic technology, Nramp1 gene N-terminal fragment was amplified from total RNA of Qinchuan cattle's(Bos taurus) peripheral blood by RT-PCR, inserted into pMD18-T simple vector, and secondly subcloned into pGEX-4T-1 vector(named pGEX-N2). And the recombinant plasmid pGEX-N2 was transformed into Escherichia coli BL21(DE3) strain. SDS-PAGE analysis showed that GST-Nramp1-N fusion protein was satisfactorily expressed by optimizing the concentration and induction time of IPTG. Eventually, GST-Nramp1-N fusion protein was purified by glutathione Sepharose 4B. The results demonstrated that Qinchuan Cattle's Nramp1 gene N-terminal fragment was 186 bp; Compared to gene order of Nramp1 published at GenBank(U12862.1), it had a single nucleotide mutation. This nucleotide mutation made the 49th amino acid which encoded by the Qinchuan cattle's Nramp1 gene N-terminal fragment changed to the alanine. Recombinant plasmid pGEX-N2 was expressed efficiently in the Escherichia coli BL21(DE3) under the optimized induction condition (0.1 mmol/mL IPTG for 10 h at 35℃) and its expressive product (33 kD) was soluble; In brief, the findings that depurated GST-Nramp1-N fusion protein obtained in the present study provides a good foundation for further research to determine the biological activity of bovine Nramp1 and lay a pathway for resistance breeding by animal transgenic technology.

In order to explore the effect of uncoupling protein gene(UCP) on birth weight and content of reactive oxygen species(ROS) in chickens (Gallus gallus), the birth weight, UCP C353T mutation genotyping and muscle ROS contents from three chicken breeds were analyzed. The results indicated Silky chicken had higher ROS contents than that of White Leghorn and Shouguang chickens, and ROS contents for genotype TT of UCP appeared notably higher than that of CT and CC genotypes. ROS contents presented a positive correlation with chicken birth weight(r=0.5011). The birth weight for genotype TT of UCP demonstrated notably higher than that of CT and CC genotype. The findings showed UCP genotypes can impact chicken birth weight for selections.

In order to analyze the relationship between the Pepper Nicotinamide Adenine Dinucleotide Phosphate Gene gene(canNADPH) and the resistance to Phytophthora capsici, make clear the role of canNADPH gene resistance to P. capsici, the pBI121-NADPH expression vector was constructed by the vector pBI121. Pepper (Capsicum annuum) susceptible cv. B12 was transformed with pBI121-NADPH by Agrobacterium tumefaciens-mediated transformation. Kanamycin-resistant regenerated lines were obtained, PCR and RT-PCR molecule detection confirmed that the five transgenic lines carried the target gene. The resistance of transgenic plants to P. capsici was identified by in vitro leaf technique. Results showed that the incidence of transgenic plants were reduced about 22.2% than those of the non-transgenic peppers, and the disease index of transgenic plants were also reduced 46.5%. The result shows that the canNADPH gene plays an important role at the resistance to P. capsici.

Antithrombin(AT) is the major inhibitor of thrombin and other serine proteinases comprising the coagulation cascade enzymes. Furthermore, it has anti-inflammatory properties. The nucleotide sequence of full-length cDNA clone of ayu(Plecoglossus altivelis) AT gene (EMBL accession: No. FN429980) was obtained from the constructed liver cDNA of ayu. The whole cDNA length of ayu AT gene was 1 487 bp, consisting of a 1 362 bp open reading frame(ORF). The predicted mature ayu AT consisted of 453 amino acids preceded by a signal peptide of 23 residues. The structure of mature AT in ayu was similar with those in the mammalian AT, including serine protease active site near the carboxyl-terminal, three disulfide bonds formed by six conserved Cys residues and the four N-glycosylation sites. Sequence comparison showed that ayu AT had the highest amino acid indentity to Atlantic salmon(Salmo salar), which was 81%. Phylogenetic analysis showed that the evolutionary relationship of AT among different species was identical with the classification relationship of different species accepted currently and ayu AT grouped constantly with those previously reported from Atlantic salmon, tiger pufferfish(Takifugu rubripes), and zebrafish(Danio rerio). In healthy ayu, AT mRNA was mainly expressed in the liver, and weakly in the spleen, the kidney and the brain. Following Listonella anguillarum infection, liver AT mRNA expression change was determined by quantitative Real-time PCR(qRT-PCR) method. AT transcripts were significantly down-regulated in ayu liver at 4 and 8 h post injection(hpi) (P<0.05). However, AT transcripts were significantly up-regulated at 12~36 h when bacteria developing(P<0.05), suggesting that AT may be involved in the immune response of Ayu.

In order to obtain active Arabidopsis thaliana alpha-dioxygenase 2 (AtDOX2), the encoding sequence of AtDOX2 was cloned into pPIC9k to obtain pPIC9k-AtDOX2. The recombinant vector was linearized and electrophorated into Pichia pastoris strain GS115. After G418 selection, PCR analysis and optimization of methanol inducing time，the high level expression strain of GS115/pPIC9k-AtDOX2 was obtained. SDS-PAGE analysis revealed that the expression level of recombinant protein reached to top at 96 h after inducing by 0.5% methanol and the target protein accounted for 15% of the extracellular total protein. The 70 kD recombinant AtDOX2 was purified by the Ni-NTA chromatography column, and the purity of recombinant protein was more than 80%. The peroxidase activity of the purified protein was tested by the 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) assay, the result showed that the recombinant protein had peroxidase activity, and the peroxidase activity of AtDOX2 could be activated by Ca2+ and Mg2+ and inhibited by EDTA, imidazole and Mn2+. The α-dioxygenase activity was analyzed by 2,4-dinitrophenyl hydraz(2, 4-DNP) assay, the result showed that the recombinant protein had dioxygenase activity, and the α-dioxygenase activity of AtDOX2 could be activated by Ca2+ too. Results indicate that the ecombinant AtDOX2 was expressed and purified by yeast system, and it acts as a dual functional enzyme of peroxidase and dioxygenase.

It is meaningful for resistance management to study the resistance mechanism of Asian corn borer (Ostrinia furnacalis) to Cry1Ab. cDNA of aminopeptidase N (APN) from Cry1Ab toxin susceptible and resistant strains of Asian corn borer was cloned and sequenced. Ofapn3, a member of aminopeptidase N families, was identified. It contained 3 591 nucleotides with a 3 045 bp open reading frame that encoded 1 014 amino acids. Putative protein sequences included 8 and 4 potential N- and O-linked glycosylation sites and a zinc metal binding site motif of HEXXHX18E, which was typical of the active sites of the zinc-dependent metalloproteases, and a highly conserved GAMEN motif, which was to form part of the active site. The sequences had a cleavable N-terminal signal peptide with 18 amino acids and a glycosylphosphatidylinositol (GPI) anchor signal peptide with 22 amino acids in C-terminal, which were also typical characters of lepidopteran APN proteins. Sequence analysis indicated that the deduced protein sequences were most similar to Onapn3 (GenBank accession No.ABL01483) from Ostrinia nubilalis with 96.6% sequence identity. The predicted protein molecular weights and isoelectric points were 115.1 kD and 4.44, respectively. Forty nucleotide differences were observed in the open reading frames which translated into 10 amino acid differences in the putative protein sequences. The sequences reported in this paper have been deposited in the GenBank database (accession No. EF538427and EU137839 for Ofapn3 from Cry1Ab-resistant strain ACB-AbR and susceptible strain ACB-BtS).

The plant Na+/H+ antiporter(NHX) adjusts the pH and Na+ balance，reducing the salt-stress damage to the plant. A full length cDNA from Moso bamboo(Phyllostachys pubescens)，designated PpNHX1(GenBank accession No.GU295174), was isolated by RT-PCR and RACE according to the homologous region of plant Na+/H+ antiporter genes. The sequence with 2 290 bp in length, contained an open reading frame of 1 635 bp which encoded a predicted polypeptide of 545 amino acids. The amino acid sequence of PpNHX1 was about 88% and 89% identical to the NHX1s from Oryza sativa and Phragmites communis, respectively. PpNHX1 protein shared high homologies with other reported Na+/H+ antiporter. Phylogenetic analysis showed that PpNHX1 shared the same group with vacuolar-type Na+/H+ antiporter, and distinguished from plasma membrane-type Na+/H+ antiporter. RT-PCR results revealed that PpNHX1 expression under 200 mm/L NaCl stress was continuously enhanced in the roots, stems and leaf of Moso bamboo under different NaCl stress time, and after 1 h of treatment the expression of PpNHX1 in the root was higher than that of leaf and stems, but after 4 h the expression of PpNHX1 in leaf was the highest. Therefore, we can infer that the expression of PpNHX1 has a close relation with the ability of salt tolerance of Moso bamboo.

This study was performed to explain effect of Sertoli cells on spermatogenesis. Testes of porcine from 7 d-old, 21 d-old, 35 d-old were used, and the methods including cell count, immunohistochemistry, flow cytometry and western blot were used. The results were as follows: (1)from 7 d to 21 d , the boundary between Sertoli cell and other cells in seminiferous tubules was more significant. From 7 d to 35 d, the number of Sertoli cells and Leydig cells and of per gram of testis increased(P<0.05), and the number of both cells were the most at 21 d(P<0.05). After that period, the number of both cells of per gram of testis began to decrease(P<0.05). However, the number of germ cells of per gram of testis continued to increase(P<0.05), but round spermatids could not be found in the seminiferous tubule. (2) form 7 d to 35 d, both of Sertoli cells and Leydig cells could express GATA-4, and the integrated optical density(IOD) of GATA-4 of Sertoli cells was the most at 21 d(P<0.05), but that of Leydig cells between at 21 d and 35 d had no difference(P>0.05). (3) form 7 d to 35 d, total testical cells had proliferation capacities, while PI and SPF in testes were the most at 21 d(P<0.05). During the periods, three types of cells(Sertoli cells, Leydig cells and germ cells) could express PCNA, and the IODs of PCNA of three cells were the most at 21 d(P<0.05). the expression of PCNA(Proliferating Cell Nuclear Antigen) in the testes was the most at 21 d(P<0.05). (4) form 7 d to 35 d , Connexin 43(Cx43) was mainly expressed in cytoplasm of Sertoli cells, and the IODs of Cx43 of Sertoli cells and Leydig cells were the most at 21 d(P<0.05). The results infer that it is an important proliferation peroid form 7 d to 35 d, but these immature sertoli cells could not support spermatogenesis.

To evaluate the influence of miR-196a-1 on 3T3-L1 adipocyte differentiation, the precursor of miR-196a-1 was amplified from the 3T3-L1 cells genomic DNA, and then miR-196a-1 expression vector was constructed, and the miR-196a stable expressing cells were obtained. The transfection of pcDNA3.0-miR-196а-1 into 3T3-L1 cells was followed by the positive clones selection of G418 and the miR-196a stable expressing cells were obtained. The miR-196a stable expressing cells were treated with an adipogenic stimulus, and the expression of adipogenic marker genes and the lipid accumulation were detected. The results showed that miR-196a-1 was up-regulated during adipocyte differentiation. The target sequence which was inserted into pcDNA3.0 was about 340 bp, and the sequencing result was consistent with miR-196a-1. The positive clones could express miR-196a-1 stably, and the expression level of miR-196a-1 increased nearly 10-fold compared to control cells. In the miR-196a-1 stable expressing cells, the mRNA levels of adipogenic marker genes: peroxisome proliferator-activated receptor γ(PPARγ), CCAAT enhancer binding protein α(C/EBPα) and lipoprotein lipase(LPL) and the lipid accumulation increased markedly after adipogenic induction. These results suggest that miR-196а-1 was involved in adipogenesis. The miR-196a-1 stable expressing cell line can be used for further studies of the function and mechanism of miR-196a-1 in the adipogenesis.

To explore the species distribution and probiotics function of enzyme-producing bacteria in gut of the silkworm, protease-producing bacteria strains were isolated from the inclusion in 4th larval guts of the silkworm Bombyx mori by NA mediums and casein-NA mediums and 16 Sr DNA sequences were used to identify the species. Meanwhile, the enzyme producing activities of the three strains and optimum fermentation conditions of Haoyue NA1 strain were investigated. The results showed that three strains Haoyue NA1, 951 NA3 and 951 NA6 belonged to Acinetobacter were isolated and confirmed. The maximum protease activity with 29.5 U/mL was found in Haoyue NA1 and the optimum fermentation conditions were corn powder 10 000 mg/L, soybean powder 10 000 mg/L, MgSO4 400 mg/L, NaC1 15 000 mg/L and K2HPO4 1 000 mg/L with a liquid volume of 80 mL in 150 mL bottol and cultured at 35 ℃, pH 9.0 under 180 r/min for 48 h. In this condition, the maximum protease activity produced by Haoyue NA1 reached 50 U/mL. This research can play an important role on the research of distribution of microrganisms in gut of the silkworm and on the control of the gut probiotics function.

The present study established a porcine intramuscular preadipocyte in vitro model to provide a new experimental method for the study of intramuscular fat metabolism. The 5-day old crossbred pig was purchased and intramuscular tissue was digested by collagenase type Ⅱ, digesta were passed through 400 screen mesh filter to isolate digested cells. Cells were subjected to centrifugation at 1 500 r/min, then resuspend the cells in DMEM/F12 supplemented with 10% fetal bovine serum(complete medium), and the intramuscular preadipocytes were separated by differential velocity adherent technique: A media change after 2 h of seeding to remove the non-adherent cells. Subcultured cells after 48 h of constituted a confluent monolayer, complete medium supplemented with 0.5 mmol/L 3-isobutyl-1-methyl xanthine (IBMX), 1 μmol/L dexamethasone (DEX), 10 mg/L insulin (INS) induced cultured for 48 h, then changed the medium to complete medium supplemented with 10 mg/L insulin induced cultured for 48 h, at last, changed the complete medium cultured for up to 90% of the cells secreted lipid droplets. Results: The cells adhered to the wall showed a short spindle or a prism appearance, and had a high rate of lipid accumulation. Their dynamic morphological changes, growth curve and stained with oil red O, all verified their intramuscular preadipocyte identity. And the expression of adiponectin was detected by both general PCR and Real-time flurescent quantitive PCR. This study successfully cultured porcine intramuscular preadipocyte by differential velocity adherent technique, and under controlled conditions, the preadipocytes replayed their hyperplasia process in vitro.

N-methyl-D-aspartate (NMDA) is one of amino acid derivatives that exists in the body of animals and plays an important role in participating the effect of regulating secretion of hypothalamuic-putuitary-growth axis. The experiment was concluded to study the effect of NMDA on gene expression and secretion of somatostatin(SS) in the cultured rat(Rattus norvegicus) hypothalamus neuron cell and approached to mechanism of this process. Results showed that NMDA could significantly increase the gene expression and secretion of SS. Furthermore, NMDA could enhance the level of intracellular Ca2+ and reduce the cAMP obviously, which suggested that NMDA regulates SS secretion and gene expression by Ca2+ and cyclic adenosine monophosphate(cAMP) in hypothalamus neuron cells.

To detect the expression and distribution of integrin αvβ6 of Foot-and-mouth disease virus(FMDV) in the tissues and organs of healthy sheeps(Ovis aries), and explore the biological basis of tissue tropism in the FMDV infection, tissue profile of integrin αvβ6 in healthy sheeps was detected by indirect immunohistochemistry assay(IHA). Results showed that buffy idio-positive reactant was appeared in cell membrane and cytoplasm of epithelium of sheep tongue, nose, lip, palatum molle, pharynx, larynx, trachea, lung and pedal coronary band, respectively. These results suggest that integrin αvβ6 receptor is generally expressed in tissues and organs of sheep, and mainly expressed in cell membrane and cytoplasm. Importantly, integrin αvβ6 is expressed at high levels in organs of respiratory system and pedal coronary band epithelium in sheeps.

The development of expressed sequence tags(ESTs) from moso bamboo has provided a useful source for mining novel simple sequence repeat(SSR) markers,which would be used for the taxonomy of bamboo. Four hundred and eight simple sequence repeats(SSR)were obtained by screening 3 087 EST sequences of moso bamboo(Phyllostachys pubescens). Among the SSR-ESTs, trinucleotides(49.75%) were most abundant, followed by dinucleotides(43.14%), penta-nucleotide(4.41%), tetra-nucleotide(1.72%) and hexa- nucleotide (0.98%). Among the identified motif types, GAG/CTC and AG motifs had the highest frequencies, accounting for 18.72% and 71.02% respectively. Based on the SSR-ESTs sequences, 25 EST-SSR primer pairs were designed for detection of the amplification efficiency, polymorphism and transferability in 12 bamboo speices, 18 primer pairs of which could generate stable and clear bands, including 16 primer pairs with polymorphisms which accounted for 64% of designed primers. Bands ranged mainly from 100 bp to 500 bp. Phylogenetic analysis showed that 12 species of bamboo were divided into two groups: Clump bamboos and monpodial bamboos, conforming to the traditional taxonomy results. The results showed that the EST-SSR markers from Moso bamboo ESTs are convenient and practicable and are of great value in evaluation of bamboo genetic diversity.

In order to lay a foundation for Marker-assisted Selection (MAS) for Bian chicken's growth traits, single nucleotide polymorphisms (SNPs) of exon 1 of the myostatin(MSTN) gene were detected in Bian chicken(Gallus gallus) and three reference chicken (G. gallus) populations (the Jinghai, Youxi and Arbor Acre chickens) by PCR-SSCP. And the relationship with growth traits in the Bian chicken was analysed. Results showed that the Bian, Jinghai and Youxi chickens all displayed six genotypes (AA, AB, BB, BC, AC and CC), while only four genotypes (AB, BC, AC and CC) were detected in the Arbor Acre chicken. Sequencing revealed three mutations (G2 100A, G2 109A and C2 244G) of the MSTN gene. Chickens of BB genotype had significantly higher body weight than those of AA genotype (P<0.05) from 6 to 8 week of age and 12 week of age. Chickens of BB genotype had significantly higher body weight than those of AA and AC genotypes at 14 week of age (P<0.05). Chickens of BB genotype had significantly higher body weight than those of the other five genotypes from 16 to 18 week of age (P<0.05). These results preliminarily show that the MSTN gene is either a major gene that influences the body weight traits of the Bian chicken or a molecular marker in close linkage with them.

Excess fat deposition in livestock is of major concern to the meat industry. Various approaches have been attempted to solve this excessive fat deposition problem. In this paper, 150 female(♀) SD rats(Rattus norvegicus) weighting (110±10) g were allotted randomly into five groups (basic diet adding 0, 75, 500, 1 000 and 6 000 mg/kg of yolk powder containing antibody against adipocyte plasma membranes(APM), respectively. The trial lasted for 75 days after rats were slaughtered for carcass analysis and blood, and three different white adipose tissue depots (mesenteric fat, parametrial fat and perirenal fat) and hypothalamus were sampled. Concentrations of serum insulin and serum leptin were determined by radioimmunoassay (RIA). The total RNA was extracted from tissues to measure the abundance of Leptin, Bcl-2 and Bax mRNA in different adipose tissue depots and Ob-Rb mRNA in hypothalamus by RT-PCR with 18S rRNA internal standard. DNA and RNA content in different adipose tissue depots were determined. The results showed that yolk antibody treatment decreased celiac fat weight and index, but did no affect body weight gain, food intake of rats and gastrocnemius muscle weight. Basic diet adding 6000 mg/kg positive yolk powder decreased serum triglycerides concentration, increased serum free fatty acids (FFA) concentrations, reduced DNA content and concentration of parametrial fat and perirenal fat. Basic diet adding 75 mg/kg positive yolk powder reduced DNA content of parametrial fat and perirenal fat. Yolk antibody treatment decreased serum insulin, down-regulated Leptin mRNA expression in parametrial fat and perirenal fat, up-regulated Ob-Rb mRNA expression in hypothalamus, decreased Bcl-2 and Bax mRNA expression in parametrial fat, but did no affect Bcl-2 mRNA /Bax mRNA. The results also showed that the expression of Leptin mRNA in perirenal fat was higher than that in parametrial fat, latter was higher than that in mesenteric fat, and Bcl-2 and Bax gene expressions in parametrial and perirenal fat were higher than that in mesenteric fat. The results suggested that oral administration of yolk antibody against APM can decrease adipose tissue depots, and its possible mechanisms are: 1) IgY directly destory adipocyte and decline proliferation of preadipocyte; 2) IgY decrease fat deposition through metabolic changes of adipocytes.

Islet replacement therapy is limited by the shortage of donor islet cells. The aim of this article is to study the potential capacity of fetal porcine pancreatic stem cells(FPPSCs) as a xenograft donor cells. FPPSCs have a strong proliferation ability, and the expression of it's surface antigen was similar to the mesenchymal stem cells. It expressed not only the markers of pancreatic stem cells, but also the markers of ES cells. After 2 weeks induction using a serum-free protocol, the DTZ-positive islet-like cell clusters(ICC) were formed, and these ICCs expressed insulin and Glut-2. After induction, the expression of markers related to pancreatic stem cells decreased, while that related to insulin-producing cells increased, and the insulin and C-peptide content synthesized and secreted by FPPSCs increased remarkably(P<0.05). The blood glucose level of diabetic nude mice in differentiated cells transplantation group decreased, but quickly reached and maintained at a high level(>16.7 mmol/L). These results demonstrated that FPPSCs can be induced to differentiate into insulin-producing cells in vitro，and ameliorate the hyperglycemia of diabetic nude mice. It might provide unlimited resources for islet replacement therapy.

Ralstonia solanacearum is the agent of bacterial wilt. In order to provide the development of plant vaccine with avirulent mutants of R.solanacearum whose genetics are stable and insetion sites, this study constructed the avirulent mutants of R.solanacearum(strain Rs91) using EZ-Tn5 transposon mutagenesis. Through electroporation and screening, thriteen avirulent mutants were obtained. Results showed that the insertion sites of the 5 mutants were found to locate in phcA and phcS genes separately. The growth curves and the relative contents of Exopolysaccharide (EPSⅠ) of the 5 mutants were significantly lower than that of the original strain Rs91, but their optimum temperature and pH for the mutants to grow changed a few. The supernatants of the 5 mutants cultured in the TTC liquid medium were scanned by the UV-spectrophotometer, and the result showed that the absorbance of the mutants was higher than that of Rs91. The result of scanning was analyzed by UPGMA dendrogram. The cluster was displayed that the 5 avirulent mutants and Rs91 could be separated into 3 groups, e.g. the original strain Rs91 was in groupⅠ, the mutants of phcA- were in groupⅡ, and the mutant of phcS- was in groupⅢ. The 5 mutants were determined by the bioassay of potted tomato (Lycopersicon esculentum) and they did not fall ill after 15 days. The 5 mutants were determined to be avirulent R.solanacearum. This study provides foundation data for developing plant vaccine against bacterial wilt.

The hypersensitive response and pathogenicity(hrp) genes exist in Gram-negative plant pathogenic bacteria and are responsible for the pathogenicity of bacteria．They can induce hypersensitive response(HR) on non-host and resistant plants. hpaA, hrcT, hrcC and hrpG genes were cloned from the hrp gene cluster of Acidovorax avenae subsp. citrulli. Disruption mutants of strain xjl12 were successfully generated by a single cross-over event. Electron microscopy observation showed that the cells of hpaA and hrpG mutants lacked detectable flagella and obviously changed in cell morphology, while hrcT and hrcC mutants' flagella and cell morphology did not change. Surprisingly, hpaA, hrcT and hrcC mutants lost HR-induced capacity in tobacco(Nicotiana tabacam) and the pathogenicity in melon(Hami cantaloupe) leaves; while the hrpG mutant was significantly reduced. Furthermore, growth analysis results showed that colonization of hpaA, hrcT, hrcC and hrpG mutants were significantly decreased, respectively. Accordingly, the mutant phenotypes nearly recovered after complement. Overall, these results indicate that the hrp genes of bacterial fruit blotch of melon as a key component of type Ⅲ secretion system affect pathogenicity on host plants and disease resistance on non-host and resistant plants.

Loop-mediated isothermal amplification(LAMP) method is a novel gene detection technique with specificity, sensitive and rapidity, the products of which can be electrophoresed in 1.5% agarose gels, or visually inspected by adding nucleic acid dye(SYBR GreenⅠ). In this paper, a one step loop-mediated isothermal amplification(LAMP) assay was developed for detection of White spot syndrome virus(WSSV). A set of primers were designed from the VP26 gene sequence of WSSV. The assay was established to amplify WSSV genomic DNA by incubation at 65℃ for only 1 h, and required only a simple water bath or heating block to provide a constant temperature of 65℃. LAMP amplification products had a ladder-like appearance when electrophoresed on an agarose gel. With the addition of SYBR Green I, the presence of the target DNA could be detected by naked eyes. The detection limit of plasmid DNA (pMD19-T-VP26) by LAMP was 0.563 pg/μL which was found to be lower than that of the commonly used PCR method. The results indicate the suitability and simplicity of the test as a rapid, field diagnostic tool for WSSV.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was used to develop a specific, sensitive and convenient method for detection of H9 subtype of Avian influenza virus(H9-AIV). Six primers directing to eight recognition sites on the hemagglutinin(HA) gene of H9-AIV were used, and at least as 103 copies of the target gene fragment could be detected after reaction for 60 minutes at 63℃, with a sensitivity of 10-fold higher than that of RT-PCR. High species-specificity of the RT-LAMP method was confirmed by the assay of H1~H15 hemagglutinin(HA) subtypes Avian influenza viruses, Newcastle disease virus(NDV) and Infectious bronchitis virus(IBV). A mixture of calcein and Mn2+ was used as fluorescent reagent to make the result of RT-LAMP visible, and similar results could be obtained as the turbidity test. Furthermore, 109 clinical samples were assayed by RT-LAMP and RT-PCR, of which 61 and 46 samples were confirmed as positive by the RT-LAMP and RT-PCR, respectively. So we could draw a conclusion that the sensitivity of RT-LAMP was preferable to the RT-PCR assay.

Citrus canker is a severe bacterial disease of most commercial citrus species and cultivars around the world. The purpose of this study is to construct specificity and stability antibody for development rapid and reliable procedures for diagnosis and control of this pathogen. To construct the recombinant mouse anti-Xac disulfide-stabilized single chain variable fragment (sc-dsFv), we used mouse anti-Xac disulfide-stabilized as templates for genetic modification, amplified the genes of variable region of heavy chain(VH) and variable region of light chain(VL), and introduced a sequence encoding 5'-terminal 12 amino acid of heavy chain constant region CH1, as a linker, between VH and VL by overlap extension PCR. The recombinant gene was then cloned into expression plasmid pET24a(+),transformed and expressed into Escherichia coli BL21 (DE3). The products of expression were renatured and analyzed by SDS-PAGE and Western blot. The affinity of sc-dsFv to Xac lipopolysaccharides(LPS) was tested by BIAcore, the specificity and stability were detected by ELISA and Dot blot. The results showed that the linker sequence was introduced into VH and VL successfully, and the recombinant gene expressed at high level in E. coli BL21(DE3). The most of the express protein existed in the form of inclusion body. Purity of the protein was higher than 90 % after purified by Ni-NTA and renaturaton in vitro. Dot blot, ELISA and BIAcore determination demonstrated that sc-dsFv could bind antigen specificity and was more stable than that of sigle-chain variable fragment(scFv), that means the expression products were refolded correctly after renaturation. The expression yield of the antibody was higher than that of dsFv in the same conditions. Our study approved that a heavy chain constant region CH1 introduced between VH and VL genes of anti-Xac can improve the affinity, specificity and stability of expressive antibody. It will be useful for X. axonopodis pv. citri intuitive and rapid diagnosis.

The heterologous proteins are often expressed as inclusion bodies in Escherichia coli due to their inability to fold correctly. The chaperones can assist the heterologous protein folding and improve the protein aggregation in vivo. Using the E. coli strain DH5α genomic DNA as templates, six genes encoding chaperones including groEL, groES, dnaK, dnaJ, grpE and clpB were amplified respectively by PCR. The groEL, groES and grpE (Ⅰteam) were inserted into E. coli expression plasmid pCDFDuet-1, and the dnaK, dnaJ and clpB(Ⅱteam) were inserted into the expression plasmid pRSFDuet-1, respectively, to create the helper vector pR-GESP and pC-DJKL. Each resultant plasmid contained an artificial operon and every gene was controlled by a strong T7 promoter. The SDS-PAGE analysis showed that all proteins except DnaJ protein were expressed in the soluble extract from IPTG-induced E. coli cells containing the helper plasmid. The aggregation of maize(Zea mays)sirohydrochlorin ferrochelatase in E. coli cells was prevented by the coexpression of three chaperones GroEL, GroES and GrpE, but was not inhibited by the coexpression of the other chaperones DnaK, DnaJ and ClpB, as shown by SDS-PAGE. Overproduction of GroEL, GroES and GrpE chaperones partly suppressed the aggregation of the recombinant maize glutamate-1-semiadhyde aminotransferase, but did not function on changing the aggregation of recombinant maize uroporphyrinogen Ⅲ decarboxylase obviously, as revealed by SDS-PAGE. All results suggested that the different charperone team had the different effects on improving the souble expression in E. coli for three maize proteins chosen in this study.

Soil ecosystem is the important carrier of the agricultural ecosystem security and agriculturally sustainable development, and is the basis for human survival. The rapid development of genetic engineering and introduction of genetically modified crops (GMCs) have provided large economic benefits, but have also raised concerns over the potential impacts of these crops on environment. Among these environmental concerns, the unpredicted and complex impacts that GMCs might have on soil ecosystem, especially on its microbial community structure and function, have been recently increasingly studied, which has become one of the hot research directions in soil biosafety area. This review briefly summarizes the impacts of GMCs on microbial community in recent years, in which the advances on the effects of planted GMCs on rhizosphere microbial community diversity, both biological and technical approaches for the diversity research, and the countermeasures for assessing the risk of GMCs on rhizospheric microbes are highlighted and discussed.