With the development of transgenic technology has matured and increased demand for Genetically modified (GM) crops, commercial growing of it has been rapidly developed in recent 14 years. Based on a large-scale research of the global GM crops, the paper analyses the situation and tendency of its development. It is a great significance for guiding the study and commercialization of GM crops.

Bovine embryo in vitro production is an efficient approach to provide embryos for embryo transfer industry and it includes three procedures, in vitro maturation, in vitro fertilization(IVF) and in vitro culture. The qualities of embryos derived from IVF play an important role in successful rate of embryo transfer and live calves. Many researches reported that cumulus cells secret many important factors, such as steroids hormone and cell growth factors, contributed greatly to bovine oocytes in vitro maturation, fertilization and culture of fertilized oocytes. The current paper reviews recent advances about the effect of cumulus cells on bovine embryos produced by IVF, in which the effect of cumulus cells on oocyte in vitro maturation, the secreting function, quality, and cocultural system of cumulus cells on efficiency of IVF are highlighted and discussed.

To develop a RT-PCR assay for the detection of novel duck reovirus(NDRV) and apply the developed assay to isolated strains and artificial infected samples, according to NDRV-NP03 S3 gene in the GenBank(GenBank:GQ888710), a pair of primers for amplying NDRV specifical fragment were designed and synthesized.RT-PCR technique detecting the RNA of NDRV was constructed.RT-PCR results showed that a 586 bp specifical fragment could be isolated only from the NDRV-NP03 strain RNA, and the sensitivity of RT-PCR reached to 2 pg NDRV-RNA.And the negative results were achieved from the other viruses, Muscovy duck reovirus(MDRV), Avian reovirus(ARV), Infectious bursal disease virus(IBDV), Muscovy duck parvovirus(MDPV), Goose parvovirus(GPV), Duck paramyxovirus(DPMV) and Duck hepatitis virus(DHV). The positive rate of RT-PCR method for detecting viral pathogens in 8 field NDRV isolates and 3 samples of the liver and spleen tissues of ducklings which were artificially infected by the strain of NDRV-NP03 was 100%. The results indicate that this RT-PCR method is sensitive and specific for detecting NDRV, and can be used in NDRV clinical diagnosis and epidemiology investigation.

This study aimed to isolate and clone root-specific promoters in rice and provide resources for breeding. Integrating of our lab's database, a root specific gene(TIGR Locus: Loc_Os05g19570) was obtained after RT-PCR. A promoter named P12 with 1 950 bp in length of the gene was cloned by PCR from the genomic DNA of MH63. The cloned promoter was fused with gus gene to construct expression vector, and then introduced into rice (Oryza sativa ssp. japonica) Zhonghua11 by Agrobacterium-mediated transformation. The result of GUS histochemical staining and quantitative analysis showed that the promoter had root specificity and time difference. Also, 5' end different length deletions of P12 were fused with gus gene and were transformed into rice, respectively. Quantitative analysis of GUS activity showed the expression level of gus gene in root was badly reduced after deletion the region of －1 059~－819 bp, it displayed some cis-elements maybe presence in this region. A root-specific promoter was cloned in this study.

Identifying the copy number of foreign gene in transgenic primary plants is the basic step in genetic engineering application. In this paper, Real-time PCR and the related PfaffI method, usually applied to studying transgenic expression，was used to analyze comparatively the differences of exogenous gene copy number of transgenic cotton(Gossypium hirsutum) T0 plants transformed with one same vector by three different methods, Agrobacterium-mediated, gene-gun bombardment and pollen tube injection, respectively. Results showed that in Agrobacterium-mediated T0 generation, the transgenic plants integrated one or two copy number accounted for only 26.2% of the total plants, respectively; In contrast, that of transgenic plants transformed by gene-gun bombardment and pollen tube injection were 60% and 42.8%, respectively. Transgenic lines with three to five copies accounted for 47.5%, 40% and 57.1%, respectively. In addition, in Agrobacterium-mediated T0 generation, transgenic lines with more than 5 copies accounted for 27.3%, while there was no transgenic plant with more than 5 copies in genetically modified T0 generation transformed by gene gun bombardment or pollen tube methods. These results provide some rationale for selection of transformation methods in crops transgene breeding.

To identify QTL related with fiber qualities in upland cotton (Gossypium hirsutum L.), a composite cross population was derived from the cross between (Yumian 1 × CRI 35) F1 and (Yumian 1 × Belshinuo) F1. A total of 155 SSR polymorphic primer pairs were obtained by selecting 6 565 primer pairs among upland cotton cultivars, CRI 35, Yumian 1 and Belshinuo. Genetic linkage analysis was conducted on 158 loci, which were obtained from genotyping a composite cross population, [(Yumian 1 × CRI 35) × (Yumian 1 × Belshinuo)] F1, and a genetic linkage map was constructed. The map contained 102 SSR loci and covered a whole length of 1 199.0 cM. Based on multiple QTL mapping method, eleven QTLs for fiber quality traits were identified, including four for fiber length, two for uniformity, and five for fiber strength. Eleven QTLs were mapped on four D-genome chromosomes. The present study shows that different upland cotton cultivars have different alleles of identical QTL for the same fiber quality trait. Composite cross population with three parents can increase the polymorphic ratio of DNA marker, and detect the different alleles of identical QTL in different backgrounds.

To study the effect of 40S ribosomal protein S15a (RPS15a) on the formation of multi-ovary in wheat, the EST highly similar to RPS15a gene from the multi-ovary SSH-cDNA library was used as a query probe to blastn the GenBank databases. Based on the assembled homologous cDNA sequence, both cDNA and DNA sequences encoding a RPS15a were isolated and characterized from multi-ovary line by RT-PCR. Furthermore, expression patterns of the gene in different material, different development stages of ears in multi-ovary line and different tissues of multi-ovary line were analyzed by semi-quantitative RT-PCR. Results showed that the cDNA sequence (GenBank accession No.HM055513) was 413 bp in length and the open reading frame encoded a peptide of 131 amino acids. The DNA sequence (GenBank accession No.HM063421) was 642 bp in length, which contained three extrons and two introns. According to expression analysis, the expression of the gene in 2~4 mm ears of multi-ovary line was higher than that in mono-ovary line. This gene was upregulated with the development of young ear, and the highest levels of expression at period of 8~9 mm. Besides, the gene was expressed in various tissues and relatively higher at the stem shoot meristem than that of other parts examined. Thus conclusively the RPS15a gene is differentially expressed in multi-ovary and mono-ovary lines and may involve in the information of multi-ovary.

Plant genome DNA methylation plays an important role in regulating gene expression and epigenetic change responded to various stresses. In this study, Maize(Zea mays) of high resistance inbred line R15 and high susceptible inbred line 478 were adopted by inoculating sheath blight pathogen (Rhizoctonia solani Kühn), and pathological changes in maize inbred line R15 were observed by electronic microscope and digital camera technology. The result showed that host was infected by mycelium through the leaf sheath stomata and the change of lesion size revealed the pathogen infection on the host has a certain gradient. The patterns of methylation were analyzed in high resistant inbred line R15 and high susceptible inbred line 478 by methylation-sensitive amplified polymorphism(MSAP), which are all inoculated for 0, 6, 12 and 24 h, respectively and amplified by 24 pairs of selective primers. The results showed hemi-methylation level increased but total methylation level first increased and then decreased in both R15 and 478. However, the level of increase in R15 was higher than that of 478. In conclusion, DNA methylation is closely related with maize resistance to Rhizoctonia solani Kühn and plays an important role in regulating gene expression responded to maize resistance.

Banded leaf and sheath blight (BLSB) becomes an emerging worldwide problem in maize production, which caused by the fungal pathogen Rhizoctonia solani Kühn. High-resistance lines R15 and susceptible lines Ye478 were selected as materials to elucidate the expression mechanisms of protein in maize leaves infected by R. solani Kühn, Total proteins were extracted from inoculated leaves during different times(0, 12, 24, 36 and 48 h) respectively and then analyzed by IEF/SDS-PAGE gel electrophoresis technique. Eight hundred protein spots were obtained, and of which, several proteins showed differences in expression levels and fifteen different protein spots were selected to analysis by ionization flight time mass spectrum facilitated by matrix auxiliary laser, and combining with the bioinformatics analysis and on-line database. It is showed that several differential proteins were induced by R. solani Kühn in different maize inbred lines. Of which, Shikimate kinase Ⅰ, Ferredoxin- thioredoxin reductase catalytic chain (FTR) increased their expressions which induced the resistance system startup, while photo-systemⅠreaction center subunit N, Ribulose-bisphosphate arboxylase/oxygenase large subunit ruduced their expressions which ihibited the material and energy metabolism, and then made the susceptible plants death.

To obtain the fusion protein of Lycopersicon esculentum Metacaspase (LeM) and study the characteristics and function of it, we cloned the open reading frame of LeM in Lycopersicon esculentum by RT-PCR, and successfully constructed the prokaryotic expression vector pET28a-LeM for LeM expression. The recombinant plasmid pET28a-LeM was transformed into Escherichia coli BL21(DE3)PlysS and Rosetta for expression, induced by IPTG, and the conditions of the expression was optimized. SDS-PAGE revealed that the best expression was induced by 1 mmol/L IPTG at 37℃ and the recombinant protein expressed mainly as inclusion body in E. coli BL21(DE3)PlysS while as fusion protein in E. coli Rosetta at the same condition. Enzyme activity assay showed that the activity of LeM in E.coli Rosetta was 33 RFU/min, which was much higher than 7 RFU/min of E. coli BL21(DE3)PlysS. We demonstrate that the strain has a great influence on the expression of LeM fusion protein.

Isoprenoid biosynthesis via mvalonate-independent pathway is very important to tobacco resistance and leaf quality. 1-deoxy-D-Xylulose-5-phosphate Reductoisomerases(dxr) is a key enzyme in biosynthesis of isopentenyl diphosphate, which is the precusor for monoterpenoid, diterpenoid and tetratepenoid compounds. To regulate the terpenoid metabolism pathway for tobacco improvement, some important genes such as dxr should be studied firstly. In this paper, dxr gene was cloned successfully from tobacco(Nicotiana tabacum) cultivar K326 leaf by RT-PCR. The cDNA code region was 1 422 bp long and encoding 437 amino acids. Sequence analysis by Clustal W declared that this fragment was highly homologous to dxr gene of other species. It shared 93.6% amino acid homologous to Lycopersicon esculentum, 87.9% to Catharanthus roseus, 86.3% to Antirrhinum majus, 84.6% to Mentha piperita, 84.2% to Zea mays, 82.9% to Arabidopsis thaliana, and 53.5% to Nostoc sp. PCC7120. The expression vector pET21b-dxr was constructed and expressed in Escherichia coli by IPTG. And a fragment about 50 kD was achieved as expect. Expression pattern of dxr gene in tobacco was investigated also by RT-PCR. This gene highly expressed in tobacco flower, leaf, stem and trichome, low expressed in seed and root. These results are helpful to regulate and control the terpenoid composition in tobacco by dxr gene manipulation in further research.

Many Chinese wheat landraces appear typically strong seed dormancy and pre-harvest sprouting (PHS) tolerance, which are presumed to be controlled by one or two main genes. In this study, we tried to investigate the genetic basis for strong seed dormancy of wheat(Triticum aestivum) landrace, Wanxianbaimaizi, using two recombinant inbred line (RIL) populations from the crosses of Wanxianbaimaizi/Jing 411 and Wanxianbaimaizi/ Zhongyou 9507, respectively. The populations were genotyped with 1 754 SSR markers and a gene-marker (Vp1-b2). By analyzing the data of grain dormancy covering two sites and four years, two major QTLs were detected using composite interval mapping, one was on chromosome 3AS flanked by Xbarc57 and Xbarc294 with linkage distances of 5.8 cM and 8.5 cM in the two populations, and the other on 3BL linked to Vp1 and Xwmc446 with the distance of 8.1 cM. The two QTLs were designated Qsd.ahau-3A and Qsd.ahau-3B, which explained 25.6%~ 48.3% and 23.5%~ 37.8% of phenotypic variation in the two mapping populations across four environments, respectively. These results demonstrated that the long period seed dormancy of Wanxianbaimaizi is due to the two loci with good stability in varied environments; Therefore, it is possible to obtain strong seed dormancy and resistance to PHS in white wheat cultivars by pyramiding the two major genes.

Investigating patterns of linkage disequilibrium(LD) is helpful for a more in-depth understanding of the genome-wide mechanisms for the genetic variation response to the natural and artificial selection in model and domestic animals. LD analysis for the candidate genes on fatness in chickens can provide more important information about the genetic control of fat deposition and make the molecular markers associated with fatness more powerful. In this study, polymorphisms of perpxisome proliferator-activated receptor-γ (PPARγ), apolipoprotein B(ApoB), uncoupling protein(UCP) and other 10 candidate genes on chicken fatness among individuals were detected by DNA sequencing, denaturing high performance liquid chromatography(DHPLC), single-strand conformation polymorphism(SSCP), restriction fragment length polymorphism(RFLP) and labelled primers(LP) methods. LD investigation among the polymorphisms of the candidate genes was performed in the 8th generation populations of the Northeast Agricultural University broiler lines divergently selected for abdominal fat content, two common measures of LD, |D'| and r2, were used in this study. The results showed that the value of |D'| and r2 often did not match very much within the candidate genes. The value of |D'| usually was larger than value of r2, sometimes even |D'|=1, while r2 was close to zero. Furthermore, in different genes, even within the same gene, LD patterns of different regions were usually different, and there was no a linear correlation between physical distance and the value of |D'| or r2. In addition, in some genes, such as ApoB, the values of |D'| and r2 between nonadjacent polymorphisms were much larger than those between the adjacent polymorphisms. Finally, there were different LD patterns of polymorphisms in some genes, such as PPAR-γ between lean and fat chicken lines. The results in this study also suggested, as a LD measure parameter, r2 is more useful than |D'|in population genetics analysis.

The experiment was conducted to study the response of bovine mammary epithelial cells induced by heat stress. Bovine mammary epithelial cells were exposed to 42 ℃ as the heat stress model and 38℃ as control, the transcription of targeted genes including heat shock proteins(HSPs) family, apoptosis, mlik protein and fatty acid biosynthesis, were detected by RT-qPCR. Results showed that the mRNA expression level of hsp27, hsp70 and hsp90, and heat shock factor-1(hsf-1) increased after 0.5 h at 42 ℃, and among them hsp70 increased sharply, then decreased with the extended time of treatment. The apoptosis genes Bcl-2 and Bax had different transcription, Bcl-2 upregulated during 24 h, while Bax upregulated first and downregulated later. Heat stress decreased the mRNA level of milk protein and fatty acids genes (α- and β-casein(CSN2), butyrophilin(BTN), and fatty acid synthase), increased transcription of fatty acid biosynthesis correlative genes (acetyl CoA carboxylase (ACACA), proxiomepro liferater activatedreceptor-gama (PPARG), and sterol response element binding protein (SREBF-1)), but did not influence the expression of roxiomepro liferater activatedreceptor-alfa(PPARA). It is suggested that heat shock induced the stress responses of bovine mammary peithelial cells, remarkably impacted the secretion of milk protein and fat, and arose tolerance of cell itself.

The aim of this study was to investigate the effect of different sizes and treatments of donor cells on the in vitro development of bovine embryos after genetic modification and selection, so as to establish an effective system for production of transgenic bovine embryos. Bovine fetal fibroblasts were transfeccted with the recombinant plasmid of mammary gland specific expression vector pEBH which containing human lysozyme (hLYZ) gene. Transgenic positive cells which were obtained through G418 selection were used as donor cells for production of transgenic cloned embryos. The result showed that genetic modification and screening of donor cells were harmful for the development of transgenic cloned embryos, the blastocyst rate decreased significantly; referred to internal diameter of injective needle, cells with the diameter of 15~20 μm were selected and used as nuclear transfer donor cells, the fusion rate and blastocyst rate of transgenic cloned embryos were significant higher than that of the other groups(P < 0.05); Compared with serum starvation and contact restrain groups, the cleavage rate and blastocyst rate of transgenic cloned embryos which were constructed with plant cells were significantly higher (P < 0.05); The expression of GFP was observed in each stage of in vitro development in all transgenic cloned embryos, but the expression levels seemed to be vary among individuals and even among different developmental stages of the same individual. We chose 10 embroys randomly for PCR detection and found that all of the embroys were positive. Above all, we obtained hLYZ transgenic cloned embryos by somatic cell nuclear transfer technique, the reconstructed embryos can develop to blastocysts successfully; and when plant cells with diameter of 15~20 μm were used as donor cell, the developmental competence of somatic cell cloned embryos is improved significantly.

Satellite cells are regarded as a population of muscle-specific committed progenitors which are responsible for the postnatal maintenance, growth, repair and regeneration of skeletal muscles. Although satellite cells have been first isolated for 50 years, the isolation protocols are still to be modified. In this study, the combination of two-step enzyme digestion and purification with different speed adherence protocols were used to isolate rat （Rattus norvegicus） satellite cells. Analyses of the cells with desmin immunocytochemical staining indicated that the cells were derived from skeletal muscle; RT-PCR of Myf5 and Myod1 showed that they expressed muscle-specific transcription factor; Myotubes began to form at 48 h in induced-culture medium; Lipid droplets began to appear in cells after 1 week，and the number of adipose cells increased significantly after 2 weeks. The results suggested that isolated satellite cells could be induced into adipogenesis, and had the abilities to develop into myobubes and differentiated into adipose cells.

In order to investigate the association single nucleotide polymorphisms in GH gene with growth and reproductive traits in Qingyuan partridge chicken, the parallel typing system based on PCR-ligase detection reaction(LDG) was established in the present study, and used to detect the polymorphisms of G662A in intron 1 and T3094C and C3199T loci in intron 4 of growth hormone (GH) gene as well as its genetic effects on growth and reproductive traits in Qingyuan partridge chicken (Gallus gallus). The results were as follows: three loci exhibited polymorphisms and were coincidence with direct sequencing. There was no significant effect on growth and reproductive traits at locus G662A; At locus T3094C, individuals with TT genotype had significant older age at first egg and significant higher body weight at the age of 18 weeks than that of CC and CT genotype birds. At locus C3199T, TT genotype birds had significant higher average egg weight at the age of 50 weeks than that of CC and CT genotype birds. The combine genotypes also had significant genetic effects on average egg weight at first laying, average egg weight at the age of 50 weeks, and growth traits. However, it seemed that the effect of GH gene on growth traits was more significant; individuals with AACTCC combine genotype can be used as the molecular genetic marker to select Qingyuan partridge chicken for high body weight.

In this study, we detected gene expressions of Wnts and their related molecules in ectoplacental cone (EPC) from 8-day p.c. pregnant mouse(Mus musculus) uterus based on RT-PCR. The localizations of β-catenin (the key molecule of canonical Wnt pathway) and Dkk1 (an inhibitor of Wnt pathway) were determined by immunofluorescence in EPC. We also primarily studied the function of canonical Wnt pathway during the trophoblast invasion by EPC outgrowth in vitro. Our results showed that 11 of 19 Wnt molecule members, 10 Frizzled receptors, 5 sFRP members ( inhibitors of Wnt pathway), Dkk and kremen1/2 were all expressed in mouse EPC. The β-catenin had strong expression in nucleus of trophoblast cells which were derived from the cultured EPC fraction. The outgrowth diameter of trophoblast increased after treatment of Dkk1, suggesting that the canonical Wnt pathway possibly inhibits the trophoblast invasion. Our study provided a theoretical basis for revealing the role of Wnt signaling pathway in the process of trophoblast invasion during murine placentation.

Leptin receptor(LEPR) plays a pivotal role in regulating leptin function as leptin binding protein. Variation in the LERP gene has been associated with obesity in human and fat deposition in cattle, swine and other animals. In this study, three pairs of primers were designed respectively referring to bovine(Bos tauras) LERP exon 4, exon 5 and exon 8 as well as their contiguous parts sequence in GenBank, so as to investigate the variations in corresponding part of LERP gene in Tianzhu white yak, Gannan yak and Qinghai yak(Bos grunniens) by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. The polymorphisms were observed at LERP exon 4 and exon 8 as well as their contiguous parts in yak. Two allelic variations containing five single nucleotide polymorphisms(SNPs) were observed including two missense mutations at LERP exon 4 and contiguous parts in three yak populations. Allele A was the most common allele and the polymorphism was low with PIC less than 0.25 at this region of LEPR gene in yak. Five SNPs including three missenses mutations were checked at LERP exon 8 and contiguous parts in yak, but alleles distributed disproportionably among three yak populations, such as alleles A→F observed in Tianzhu white yak, but alleles A→C in Gannan yak and Qinghai yak. Allele A was the most common allele, and that allelic and genotype frequency were significantly deviated from Hardy-Weinberg balanced (P<0.05). The polymorphism was higher or moderate with PIC more than 0.25 at LERP exon 8 in three yak populations. No polymorphism was found at LERP exon 5 and contiguous parts, but three SNPs were observed at intron 4 and intron 5 relative to bovine LERP gene sequence. Exon 8 for LEPR gene codes the amino acids consisting of C2 domain of LEPR where amino acids recognizes and binds ligand, for LEPR gene with much polymorphism, exon 8 would become a potential locus for genetic markers, in which it may be possible for traits regulation.

FecB mutation of bone morphogenetic protein receptor IB gene(BMPR-IB) is the major gene responsible for prolificacy in Booroola Merino and Small Tail Han sheep. In our research, 135 ewes with their two or more litters (totally 353) and 211 sires were selected. FecB mutation was detected by PCR-RFLP and the litter size of ewes with different genotypes were analyzed. The result showed that three genotypes (BB, B+ and ++) were detected in Duolang sheep(Ovis aries). Genotype frequency of BB, B+ and ++ was 0.005, 0.146 and 0.849, respectively. The Duolang ewes with genotype B+ had 0.51 (P<0.01) lambs more than those with genotype ++. No significant difference in ewes with genotype BB and B+ or BB and ++. Chi-square test showed that the Duolang sheep population was in Hardy-Weinberg equilibrium in FecB locus, which indicated that the population could not be affected by selection. It is the first time that BB genotype (one sire and two ewes) in FecB mutation was detected in Duolang sheep which reveals that the mechanism of high prolificacy in Duolang sheep can be the same as Booroola Merino and Small Tail Han sheep. In Duolang sheep industry, FecB mutation detection can be used to breed new strain of mutton and prolificacy.

Adiponectin(Adp) is a cytokine secreted mainly by adipose tissue. The function of Adp has attracted recent interest. Skeletal muscle satellite cells were separated from semitendinosus muscle of Wannanhua pig at 10 days of age. Muscle satellite cells were treated with 0, 1, 5 and 10 μg/mL recombinant adiponectin(rAdp) respectively for 12 h or 24 h. Adp, AdpR1, AdpR2, four isoforms of myosin heavy chain (MyHCI, MyHC2a, MyHC2b and MyHC2x), AMP-activated protein kinase(AMPK) and peroxisome proliferator-activated receptor alpha (PPARa) mRNA levels were determined by Real-time fluorescence quantitative RT-PCR. The results showed that: (1) mRNA expression of Adp and AdpR1 treated with 1 μg/mL rAdp was significantly increased, and AdpR2 mRNA expression was even significantly increased only at 24 h. But mRNA expression of Adp, AdpR1 and AdpR2 treated with 5 and 10 μg/mL rAdp were lower; (2) mRNA expression of MyHC1 and MyHC2x treated with 1 μg/mL rAdp were significantly increased, but MyHC2b mRNA expression was significantly decresed only at 24 h. mRNA expression of MyHC2b treated with 5 and 10 μg/mL rAdp was significantly decresed, but MyHC1 mRNA expression had no significant change. mRNA expression of MyHC2a treated with 10 μg/mL rAdp was significantly decresed. MyHC2x mRNA expression treated with 5 μg/mL rAdp was incresed significantly. (3) mRNA expression of AMPK treated with 1, 5 and 10 μg/mL rAdp was significantly increased only at 12 h. However, the low rAdp doseage group had higher AMPK mRNA level. PPARa mRNA expression was significantly increased treated with 1 μg/mL rAdp. But high rAdp dosage groups had no this effects. The results suggest that the function of adiponectin to skeletal muscle may be related to activating both AMPK and PPARa signal pathways by binding adiponectin receptor. Adiponectin can change composition of muscle fibers by increasing MyHC1, MyHC2a and MyHC2x, and decreasing MyHC2b gene expression.

In order to further reveal the characteristics of Largemouth bass ulcerative syndrome virus (LBUSV) isolated recently, and further study the group of LBUSV, DFV (Doctor fish virus) and LMBV (Largemouth bass virus), the full-length DNA methyltransferase (DNA MTase) gene was analyzed and expressed using prokaryotic system. A about 200 bp core fragment was amplified and sequenced, and the full-length of DNA MTase was identified by genome walking. The open reading frame (ORF) of DNA MTase gene was 663 bp encoding 220 amino acids (GenBank accession No.GU256634). Motif analysis indicated that LBUSV DNA MTase protein contained blocks Ⅰ, Ⅳ, Ⅵ and Ⅷ, and cofactor binding sites, substrate interacting sites and DNA binding sites were also found in LBUSV DNA MTase, and the proline-proline-cysteine tripeptide was proposed catalytic site. Additionally, the primers were designed according to DNA MTase ORF, and the PCR products were inserted in vector pBV220 and transformed to host Escherichia coli DH5α. After temperature inducement and SDS-PAGE analysis, the recombinant expression bacteria produced a special protein about 25 kD in molecular weight. The proportion of recombinant protein in total bacterial protein was about 30%. Characteristic analysis of DNA MTase gene showed that the predicted protein may play a role of DNA methyltransferase, and confirmed that the taxonomic status of LBUSV is genus Ranavirus of the family Iridoviridae.

The purpose of our study was to investigate the changes of plasma protein from the cows infected with Staphylococcus aureus. Dairy cows shown to be S. aureus-positive on bacteriological culture of milk from all 4 quarters, were classified as S. aureus subclinical mastitis. Two-dimensional gel electrophoresis was used to separate plasma protein from S. aureus subclinical mastitis and healthy dairy cows, and stained with Coomassie Blue G-250 solution. Differential expression proteins were detected by PDQuest 8.0 software and identified by ion trap mass spectrometer. The results showed that the ten protein spots were changed in cows subclinically infected with S. aureus mastitis, and six spots were identified as four proteins including haptoglobin, alpha 1 acid glycoprotein, transthyretin and serum amyloid protein A. Haptoglobin, alpha 1 acid glycoprotein, and serum amyloid protein A were up-regulated in plasma from S. aureus infected cows. ELISA detection of plasma haptoglobin showed that expression of haptoglobin was significantly higher in S. aureus infected cows than that in healthy controls (P<0.01). Dairy cows infected with S. aureus mastitis altered the protein level in plasma suggested that these differential proteins may be useful to clarify the host defense mechanisms and the response to invasion of mammary tissues by S. aureus mastitis.

In order to preparate polyclonal antibodies of ORF3 and ORF4 of Tobacco bushy top virus (TBTV) , ORF3 and ORF4 fragments were cloned from TBTV-BSLLi isolate by RT-PCR, and sub-cloned into pMD18-T. After correct sequencing, the ORF3 and ORF4 fragments were inserted into prokaryotic expression vector pET28a(+). Then the recombinant vector was transformed into Esherichia coli BL21(plysS) through heat shock, and 6×His-tagged ORF3 and ORF4 expression were induced by IPTG. The high purity protein of ORF3 and ORF4 were obtained through Ni affinity chromatography column. Their high titer specific antiserum were obtained by immunizing rabbit using the purity proteins．The DAS-ELISA results indicated that the antiserum could be used for specific detection of TBTV from field samples of tobacco(Nicotiana tabacum L.) and transmission vector. It provides the basic condition for serological detection of TBTV.

To investigate the mechanisms of feather degradation by Stenotrophomonas maltophilia YHYJ-1, the keratinase gene (KerF) which included complete sequence of 1 981 bp and a 1 734 bp open reading frame encoded 580 amino acid was cloned from S. maltophilia (GenBank Accession No. HM590650) and ligated into pET28a(+) expression vector. A recombinant plasmid pET28-KerF was constructed and transformed into Escherichia coli BL21(DE3), and finally the recombinant strain was obtained. Recombinant KerF with a molecular mass of 50 kD was purified to electrophoretical homogeneity after nickel affinity chromatography and had an optimal pH and temperature of 9.0 and 50℃, respectively. It was strongly inhibited by Mn2+, Co2+, Mg2+, Ni+, Ba2+, Zn2+, Ca2+ , Fe3+ and phenylmethanesulfonyl fluoride (PMSF), but stimulated by ethylene diamine tetraacetic acid (EDTA). These results indicated that keratinase KerF gene from S. maltophilia is expressed successfully and the KerF is considered to be a alkaline serine protease.

The objective of this study was to identify polymorphisms of porcine thyroid-stimulating hormone β subunit gene (TSH-β) and investigate its associations with porcine carcass and meat quality traits. Three SNP were identified by resequencing the entire genomic sequence of porcine TSH-β gene: A-175G located in the 5' untranscribed region, non-synonymous mutation G+52A located in the coding region and G+570A located in the intron. These three loci were genotyped in total of 548 pigs which including 24 Pietrain, 28 Landrace, 30 Duroc, 57 Yorkshire, 49 Jinhua pigs, 33 Chalu black pigs, 41 Jiaxing black pigs and 286 F2 animals of a cross of Pietrain and Jinhua pigs by PCR-RFLP and tetra-primer ARMS-PCR methods. Association study was carried out in the F2 population. Considerable genetic diversity of porcine TSH-β gene (Shannon I = 0.48) and gene differentiation among breeds(Fst=0.5389) were observed. The prevailing allele of locus A-175G in the four foreign breeds and Chalu black pigs was A, while it was G in Jinhua and Jiaxing black pigs. And association analysis showed that animals with AA genotype were significantly higher (P<0.05) than individuals with GG genotype in carcass length, daily gain, ham weight and ham muscle weight. The polymorphism of locus G+52A was mainly observed in Jinhua pig, and it was significantly associated with intramuscular fat content. The polymorphism of locus G+570A was only observed in Jinhua and Jiaxing black pigs, and it seemed to be fixed for G allele in the other breeds, association analysis revealed that the carcass length and ham muscle weight were significantly higher in pigs with GG genotype than that with AA genotype. The results suggested that TSH-β can be the functional candidate gene for the corresponding QTLs located on Sus scrofa chromosome 4.

The full open reading frame (ORF) of eukaryotic translation initiation factor 4E (eIF4E) was obtained from silkworm(Bombyx mori) pupal cDNA library and was named BmeIF4E (GenBank accession No. DN443192). In order to study the biological function of BmeIF4E, the ORF of this gene was cloned and the procaryotic expression recombinant plasmid pET-28a(+)-BmeIF4E was constructed to express His-BmeIF4E fusion protein in Escherichia coli BL21(DE3). Because the rBmeIF4E in E.coli was soluble, the protein was purified with metal-chelating affinity chromatography directly. rBmeIF4E was used to generate anti-BmeIF4E polyclonal antibodies, to determine the subcellular localization of BmeIF4E. Immunostaining indicated that BmeIF4E protein could be found in both the cytoplasm and nucleus. The gene expression features in different organizations and different developmental stages were analyzed using Real-time RT-PCR and Western blotting analyses. The results showed that BmeIF4E were expressed in all tissues and the expression levels were highest in malpighian tubes, followed by epidermis, fat body, spiracle, ovary, gut and head, and were lowest in the silk glad. Additionally, BmeIF4E expression began during the egg stage, increased during the larvae stage and pupa stage, reached a peak during the moth stage. Furthermore, RNA interference technology was applied to silence the target mRNA and the cell viability was assessed by Western blot and MTT assay. After 72 h of RNAi, the MTT assay indicated that when BmeIF4E was knocked down in Bm5 cells, the cell viability was decreased significantly. BmeIF4E may play an important role in the whole life cycle of Bombyx mori as a valuable house-keeping gene.

ΔFosB, a truncated form of FosB, which impacts on fat and calcium deposition, drug addiction and other aspects, has extensive biological functions. The objective of this study was to silence the expression of ΔFosB using the RNA interference by recombinant adenovirus, and provided a material to investigate the relative functions of ΔFosB. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-492/572) which targeting two different sites of ΔFosB mRNA and then oligonucleotides were cloned into shuttle vector pENTR/CMV-GFP/U6. After detection of interference efficiency, we recombined a pENTR/CMV-GFP/U6-572 and adenovirus backbone vector pAD/PL-DEST, to produce recombinant vector pAD/PL-DEST/CMV- GFP/U6-572. The fifth generation recombinant adenovirus particles (AD-ΔFosB-572) were produced and further amplified by transfecting HEK-293 cells. The titer of adenovirus reached 1.58 × 109 PFU/mL determined by TCID50 assays. Western blot and Real-time Quantitative PCR indicated that AD-ΔFosB-572 had better interference efficiency by infecting goat mammary gland epithelial primary cells after 24, 48 and 72 h, in which mRNA expression levels of ΔFosB gene were reduced 45%, 73% and 81%， respectively. Western blot also showed that the expression of ΔFosB protein was reduced by infecting the cells with AD-ΔFosB-572. So AD-ΔFosB-572 has been proved that it has significant interference effect on expression of ΔFosB and can be used for subsequent studies of gene function.