Hspa4 gene of Rattus norvegicus (Accession No.:NM 153629) and Ubi-1 promoter of maize (Accession No:DQ141598) were respectively cloned by RT-PCR and PCR technology. The sequencing and homologous analysis results showed that Hspa4 contains 2523bps and encodes 840 amino acids and the maize ubi-1 contains 1992bps. The cloned sequences have 99.96％ and 99.55% identity with their corresponding sequences published. Based on the plant expression vector pCAMBIA1301, we successfully construed pCAMBIA1301-Ubi-Hspa4, which would be useful for improving plant tolerance to environmental stresses.

Abstract: The expressed gene in cucumber seedling leaf, which was inoculated with Colletotrichum orbiculare after Pieris rapae extract treatment and only was inoculated with C. orbiculare, were analysed by differential display reverse transcript polymerase chain reaction (DDRT-PCR). Sixty-seven differential fragments were obtained and five induced gene fragments were cloned and sequenced. Bioinformatics analysis revealed that the cDNA fragments D1 (344bp) had 95.0% homology to the photosystemIICP47 protein compound that was composed of chlorophyll a and the protein (47kD) encoded by psbB gene. It may had the transmembrane protein like CP47 and played an important role in the signal transmit of the systemic acquired resistance to anthracnose in cucumber. The other four fragments had lower homology to the gene segments registered in GenBank.

Abstract: Potato virus Y(PVY) and Potato virus X(PVX) are two of the important viruses infecting potatoes. They often simultaneously affect potatoes, resulting in 60-80% reduction. Complex RT-PCR methods were established to detect the two viruses in this paper. The result was as follow: If Oligod(T)18 was as primer in reverse transcription, PVX couldn't be detected. The reason was that the differential character of cDNA was low. To improve that of cDNA, PVX and PVY's 3' primers participated in reverse transcription not Oligod(T)18, PVX and PVY were all detected. The conclusion was that in the multiply RT-PCR, 3' primers was used in the reverse transcription, the two viruses could be all detected.

Abstract: There are the antiviral proteins in Phytolacca decandra. The study of their relationship between anatomical structure of Phytolacca decandra and proteins is of great meaning. Anatomic structures of different-aged storing roots, stems and leaves of Phytolacca decandra L. were studied by light microscope and histochemical localization. The results showed that different-aged vegetative organs have proteins except the young roots, and the protein exists in parenchyma cells. Protein content in young stems are higher and the grain size are bigger than the other organs. As to the protein content and grain size, there are no notable differences among old leaves, young leaves, old stems and old roots. No protein was found in two-year-old storing roots.

Autographa californica nucleopolyhedrovirus (AcMNPV) orf59 gene was amplified from AcMNPV genomic DNA via PCR. The PCR product was cloned into the expression vector pET-32a(+) and transformed into Escherichia coli BL21, and a fusion protein around 30kDa was expressed after induction with IPTG (isopropylthio-β-D-galactoside).Purified fusion protein was injected into New Zealand white rabbit to harvest polyclonal antibodies. Western blotting analysis of extracts of AcMNPV-infected Sf9 cells showed two specific polypeptides with apparent sizes of 38kDa and 25kDa for the Ac59 antiserum. The indirect immunofluoresecence analysis demonstrated that the Ac59 protein was distributed in cytoplasm and nucleus of infected cells at the late stage of infection. This study lays a foundation for the further research of the gene function.

Abstract:Objective To clone cDNA of N-terminal fragment of bovine bactericidal /permeability-increasing protein(BPI), to construct the procaryotic expression vectors and to express the cDNA in E.coli , to purify recombinant protein.Methods According to the sequence of Genbank, the gene which encode bovine BPI protein were amplified by RT-PCR from mRNA that were extracted from the bovine polymorphonuclear neutrophils, then the gene was inserted into expression vector pGEX-4T-1.Recombinant plasmid was transformed into E.Coli BL21, which could produce fusion protein induced with IPTG.. RESULTS A 714 bp fragment was acquired.the E.coli transformed with recombinant plasmid was induced with IPTG., A 52KD expected protein of GST-BPI fusion protein was synthesized. CONCLUSION N-terminal of BPI was successfully expressed and purified.

The polymorphisms and their combination of κ–casein gene and their correlation to milk performance traits in Chinese Holstein cattle were investigated for the purpose of providing molecular maker information to facilitate the breeding efficiency of high milk protein and milk fat. PCR-RFLP、CRS-PCR and Sequencing were applied to analyze the polymorphisms of two loci in κ–casein gene exon 4 and 5 in 435 Chinese Holstein cattle. Genotypic frequencies, allelic frequencies, correlation analysis between different genotypes and genotype combinations and milk performance traits were estimated. At locus T10996A, the cows with genotype TA was very significantly higher fat rate than those with genotypes AA(P < 0.01) ; with genotype TA showed higher protein rate than those with genotypes AA(P < 0.05). At locus T12980C, the cows with genotype TC was very significantly higher fat rate than TT genotype, (P < 0.01) ; with genotype TC and CC showed higher protein rate than those with genotypes TT(P < 0.05). 8 genotype combinations were found in 435 Chinese Holstein cattle，the cows with genotype combinations TTCC had the highest milk fat percentage and the third was genotype combinations TATC. The cows with genotype combinations TATC showed highe milk protein percentage than those with genotypes AATT(P < 0.05). 【Conclusion】The correlation was detected between different genotypes and genotype combinations and fat rate and protein rate at these two loci, but there was no correlation between the polymorphisms and milk yield and fat protein ratio.

In the present study, the exon 2 of BoLA-DRB3 gene of 489 Holstein cows and 40 Holstein bulls in Hebei province was amplified and a uniform fragment of 284 bp was obtained. The 284 bp PCR product was digested with restriction endonuclease BstYⅠ, and genetic polymorphism was investigated by PCR-RFLP. Three genotypes (AA, AB and BB) were found in 489 Holstein cows by digesting the PCR product with BstYⅠ, and the frequency of AA, AB and BB genotypes was 0.078, 0.380, 0.542, respectively. Two genotypes (AB and BB) were found in 40 Holstein bulls, and the frequency of AB and BB genotypes was 0.250, 0.750, respectively. The fitnesstest showed that both Holstein cows and bulls were in Hardy-Weinberg equilibrium (cows: χ2 =0.45, P=0.799>0.05; bulls: χ2 =0.82, P=0.665>0.05). The relationship between exon 2 of BoLA-DRB3 gene and somatic cell score (SCS) in Holstein cows was analyzed by least squares linear model. Least squares mean of SCS for AA was significantly lower than that for AB (P=0.0098<0.01) or BB (P=0.00009<0.0001), least squares mean of SCS for AB was significantly lower than that for BB (P=0.0096<0.01). AA was the most favorable genotype, BB was the most unfavorable genotype for mastitis resistance. The information found in the present study is very important for improving mastitis resistance in dairy cattle by marker assisted selection.

The single nucleotide polymorphisms(SNPs) within the coding regions of CXCR1 gene in Holstein cattle and Luxi Yellow cattle and Bohai Black cattle were investigated by nested-PCR, DNA sequencing technology and CRS-PCR method. Sequencing results showed that three SNPs were identified, which they were 819(G/A),995(G/A),1008(C/T). 819(G/A) and 1008(C/T) were nonsense mutations, but 995(G/A) resulted amino acids alteration Arg422His.In Holstein cattle and Luxi Yellow cattle and Bohai Black cattle, G and G and T predominan allele were identical at 819(G/A) and 995(G/A) and 1008(C/T), which were G , G , T and the allelic frequencies were 0.63/0.73/0.71, 0.60/0.85/0.71, 0.74/0.76/0.71 respectivly. Chi-square test indicated that 995(G/A)and 1008(C/T) of Luxi Yellow cattle and all the sites of Bohai Black cattle were in accordance with the Hardy-Weinberg equilibrium (P>0.05), while all polymorphic sites were not meet Hardy-Weinberg equilibrium (P<0.05) in Chinese Holstein cattle. The value of polymorphism information content indicated that 819(G/A) and 1008(C/T)s were moderate polymorphism in three breeds（0.25＜PIC＜0.5）, while 995(G/A) were slight polymorphism in Luxi Yellow cattle and were moderate polymorphism in other two breeds.

The Tianzhu White Yak’s SRY gene was amplified from Yak’s genome. The product was cloned to pGEM-T easy vector and sequenced by biological company. The total coding region of Tianzhu White Yak’s SRY gene is 687 bp, encoding a peptide with 229 amino acid residues. Through sequences aligned, it was discovered that there are 2 mutations in the coding region between yak and cow, causing 1 amino acid mutation in LF protein. The coding region of Yak’s SRY gene was cloned to EcoR I and Sal I sites of pET-28a(+) vector to construct an expression plasmid pET-28a/SRY. The expression plasmid was transformed to E.Coli BL21 (DE3), and induced under proper conditions then the SRY protein was highly expressed. The product of expression was identified by Western-bloting to be sure that the SRY protein of yak was expressed.

After the eukaryotic expression vector pcDNA3-NDV-F was constructed successfully, the recombinant was transfected into P815 cell by LipofectamineTM 2000, and the cells were screened with G41 8 to obtain stable clone. The total RNA was prepared from transfected cells according to the instructions of the Trizol reagent and the 1600bp fragment was obtained by RT-PCR. Westem blotting and immunofluorescence were used to detect the expression of NDV-F protein. In order to detect the stability of the transfected cell line, the cell line was freezed in the liquild nitrogen for six months. The experiments proved NDV–F gene were expressed well. These results suggested that the stable transfected P815 cell line was successfully established by Lipofectin, which provides solid foundation for further experimental studies on the function NDV nucleotide vaccine.

Abstract：pEF-neu-CHIP vector was transfected to MC3T3-E1 cells. Then cells were cultured with α-mimal essential medium supplemented with 1.0mg/ml Geneticin(G418).　The surviving (Geneticin-resistant ) cells were cloned. Finally overexpression of CHIP protein was detected by Western blot in MC3T3-E1-HA-CHIP stable cell line. MC3T3-E1 wild type cell line and CHIP-overexpressing cell lines were induced towards osteoblast with condition medium (10mM/mlglycerophosphatesodium and 50ug/ml ascorbic acid). Differentiation and mineralization of two cell lines were verified by assay of Alkaline phosphatase （ALP） activity , ALP staining, Von Kossa staining and Alizarin Red staining. The results showed that ALP activity of CHIP-overexpressing cell lines were lower than that of MC3T3-E1 wild type cell line. The same results were testified by ALP staining. Mineralization of CHIP-overexpressing cell lines were also lower than that of MC3T3-E1 wild type cell line . This result was proved by Von Kossa staining and Alizarin Red staining. All above results demonstrated that overexpression of CHIP inhibited differentiation and mineralization of MC3T3-E1 cell line.

In order to research the synergistic effect of glucose of insulin on lipogenesis in goose liver, we measured the effect of glucose of insulin on triglycerides (TG) accumulation, enzyme activity and mRNA expression of fatty acid synthase (FAS) in primary hepatocytes of Sichuan White Goose and Landes Goose. Our results were as follows: Glucose alone could induce the TG accumulation, enzyme activity and gene expression of FAS in goose primary hepatocytes of both breeds, and the induction effect was more evident when glucose of insulin cultured together. In addition, there was difference of effect of insulin and glucose on the TG accumulation, enzyme activity and gene expression of FAS in hepatocytes between the two breeds. Conclusions: Glucose alone could induce the increase of mRNA expression and enzyme activity of FAS, then resulted in the TG accumulation in hepatocyytes. Meanwhile, the induction was more evident when glucose and insulin cultured together. In addition, there was breed difference of synergistic effect of glucose of insulin on lipogenesis.

Lenok (Brachymystax lenok), which was a famous endemic fish in China, had undergone a dramatic decline for the impacts of human and environment. In this paper, the genetic diversity of the seventy-two individuals, which from the three wild populations of Mudanjiang River (MD), Yalujiang River (YL) and Wusuli River (WSL) respectively, were analyzed using the AFLP method. In total, 541 polymorphic loci of 559 were amplified with 12 primer pairs and the percentage of polymorphic loci was 96.78 %. The Shannon information index of the MD, YL and WSL populations was 0.3988±0.2913, 0.3254±0.3037, 0.2125±0.2862, respectively. And the Nei's gene diversity index was 0.2737±0.2062, 0.2229±0.2129, 0.1446±0.1985, respectively. The average Ht was 0.3512±0.0.0208 and Hs was 0.2137±0.0152. Among the three populations the average genetic distance (Dst) was 0.1375. The gene differentiation coefficient (Gst) of 0.3914 indicated that there was genetic differentiation of a certain degree among the populations. The genetic diversity was 60.85% within population and 39.15% among the population. The gene flow index (Nm) was 0.7776. The AMOVA analysis indicated that the average Fst was 0.55336. 44.84% of variance was among populations and 55.16% within populations. The MD group showed the highest polymorphism ratio and the lowest in WSL group.

Two pairs of primers were designed according to the published ORF2 gene sequence of PCV2, and ORF2 genes was amplified from PCV2 strain isolated from Henan province .The PCR products was cloned into pMD18-T easy vector and the recombinant plasmids named pMD18- ORF2 were obtained. The sequence of the cloned PCR product was analyzed and compared with other PCV2 isolate in GenBank. The results indicated that the homology of their nucleotide sequence was 92.4% to 99.6% ,and ORF2 gene displayed 90.6％to 97.9％identity based on amino acids with other PCV2 strains. Meanwhile, a fragment of ORF2 gene with a size of 587bp was amplified by PCR from pMD18-T-ORF2 of PCV2 and cloned into pET-32a vector .The recombinant plasmid was successfully expressed in Escherichia Coli BL21 by induction of IPTG. The molecular weight of fused recombinant protein was 40KD by SDS-PAGE and could be recognized by antiserum against PCV2 by Western-Blot.

Paraoxonase is a HDL-associated esterase, and is proposed to play a key role in lipid metabolism and the onset of cardiovascular diseases. The objectives of the present study were to determine associations between these polymorphisms of PON1 gene and growth and carcass traits. For this purpose, genotyping was performed on males of 18 Angus, 23 Jinnan cattle, 20 Limousin, 28 Luxi cattle, 26 Qinchuan cattle, 29 Simmental, 29 Charolais. In the association studies traits of interest were analyzed using the RFLP-PCR and GLM procedure of SAS 9.1 and least square means of the genotypes were compared by the Tukey’ s test. Association of PON1/ EcoRⅤ genotypes with body weight, average daily gain, rib eye area and tenderness were significant (p < 0.05), and AG genotype was significant difference with others in average daily gain and tenderness (p < 0.05), with AA genotype may have higher rib eye area (p < 0.05). The association analysis of the PON1/ AluⅠ polymorphism showed significant associations between genotypes and growth: body weight and carcass traits net meat weight and tenderness (p < 0.05). AA genotype was considered more favorable than others in all growth and production traits. There were significant differences among breeds for all associated interests traits and difference between beef breeds and native breeds are obviously in all aspects. And these results may be useful for the breeding selection.

PCR-SSCP method was used to identify the polymorphism sites in exon 2 domain of ADSL gene and 5’-flanking domain of GARS-AIRS-GART gene. Association between the founded polymorphism sites as well as pyramiding genotypes and Inosine Monophosphate (IMP ) contents in muscles were tested by least square analysis. The reproduction performance of individuals with different pyramiding genotypes were also analyzed. The results showed the founded polymorphism sites C3484T and C-179T had significant correlation with IMP contents in muscles. The TTs were two advantageous genotypes. Their additive effects for ADSL gene and GARS-AIRS-GART gene were respectively 0.1710 mg/g and 0.128 mg/g, and the dominant effects were -0.0250 mg/g and 0.031 mg/g. The pyramiding genotype TTTT had higher IMP contents than other pyramiding genotypes. The effects of two advantageous TTs in pyramiding genotype TTTT were still significant on IMP contents, however, their interaction effect was not significant (P>0.05). The average weights of 90 days and egg reproduction in pyramiding TTTT populations had no variation.