The porcine SMAF1 gene was cloned by degenerate PCR, PCR products for porcine SMAF1, generated from pocine adipose tissue, covered intact open reading frame which was identified with GeneScan software and via manual inspection. The length of this cDNA sequence was 256bp (GenBank DQ191892). The sequence for pig was similar to those of human(86%)and mouse(78％). The deduced amino acid sequence of porcine SMAF1 was similar to those of human(81%), mouse(67%), rat(70%) and bovine(84%).Semiquantitative RT-PCR analysis was performed to determine the expression pattern of SMAF1 in various types of porcine tissues and the genetic difference in mRNA concentrations of SMAF1 in two genetic populations of pigs ( Meishan and Large White). Transcripts for porcine SMAF1 were abundant in adipose tissue, we observed lower levels in Large White pigs than in Meishan pigs. The successful cloning and expression analysis of porcine SMAF1 will enhance the understanding of the involvement of

Primordial germ cells (PGCs) were isolated from the genital ridges of chicken (Gallus domesticus) embryos at the 19th stage. PGCs were co-cultured with somatic cells in primary culture and subculture. Identification of PGCs was carried out by immunocytochemical methods for c-kit. In addition, PGCs were induced to differentiate into neuronal cells, epithelial and skeletal muscle cells by changing the culture conditions. The immunological characteristic of the differentiated cells could be identified by neuron specific enolase and keratin immunocytochemical staining. The above results indicated that chicken PGCs could differentiate into neuronal cells, epithelial and skeleton muscle cells in vitro.

The majority of freemartins are infertile and its karyotyping is XX/XY sex chromosome chimaerism. We have developed a new diagnostic method based on the detection of Y-chromosome DNA segments (Sry) by polymerase chain reaction (PCR) to detect infertile freemartins in cattle. In comparison with other diagnostic aids including clinical observations, erythrocyte typing, and homograft tolerance, this PCR method is more efficient, less expensive, and more practical for routine early diagnosis of the bovine freemartins.

BoLA-DRB3 is a member of the MHC (Major Histocompatibility Complex), which plays a central role in immune system and disease resistance of bovine. The exon2 of BoLA-DRB3 gene is a mainly functional region, which codes the antigen and participates in the body's immune response. In this study, The restriction polymorphism of the exon2 of BoLA-DRB3 in Luxi，Nanyang and Jinnan cattle were investigated by PCR-RFLP. 6 genotypes and 3 alleles were identified for BoLA-DRB3 locus. Statistical results showed that polymorphism sites at position 154、155、156 and 157 in Luxi catttle and Jinnan cattle fitted Hardy-Weinberg equilibrium, but not in Nanyang cattle. After sequencing, 13.70% of nuclitide mutated, which induced that 14.61％ of amino acid changed.

Based on the EST sequence of Glutathione S-transferase gene (TaGST) from Tamarix androssowii published on GenBank, a TaGST cDNA was cloned with 3’and 5’RACE techniques. The full length of TaGST is 1175 bp with ORF of 672 bp deduced 224 amino acid residues. The deduced amino acid sequence encoded by TaGST is homologous with that of known plant GST and the highest identity is 48% (GST10 of Glycine max). To characterize its function, the TaGST expression vector was constructed with pET-28a(+) and transformed into E. coli BL21. The SDS-PAGE result showed that the TaGST protein was expressed and the molecular weight was about 26 kD which is similar to the caculated one’s. The enzyme activity assay of the TaGST indicated that TaGST could catalyze the substrate reaction of CDNB with GSH. This confirmed that the TaGST did encode a functional GST protein.

Based on the EST sequence of Glutathione S-transferase gene (TaGST) from Tamarix androssowii published on GenBank, a TaGST cDNA was cloned with 3’and 5’RACE techniques. The full length of TaGST is 1175 bp with ORF of 672 bp deduced 224 amino acid residues. The deduced amino acid sequence encoded by TaGST is homologous with that of known plant GST and the highest identity is 48% (GST10 of Glycine max). To characterize its function, the TaGST expression vector was constructed with pET-28a(+) and transformed into E. coli BL21. The SDS-PAGE result showed that the TaGST protein was expressed and the molecular weight was about 26 kD which is similar to the caculated one’s. The enzyme activity assay of the TaGST indicated that TaGST could catalyze the substrate reaction of CDNB with GSH. This confirmed that the TaGST did encode a functional GST protein.

XY355 promoter, a petal-specific promoter, was amplified from the genome of rapes (Brassica napus) by PCR. Then the XY355 promoter was connected with a maize anthocyanin regulatory gene Lc to construct an expression vector pXY60.The vector was introduced to the Agrobacterium tumefaciens strain LBA4404 by freeze-thaw method .The explants of leaf discs from tobaccos (Nicotiana tabaccum L.) and petunias (Petunia hybrida) were transformed by LBA4404 respectively. The results of the PCR analysis among these regenerated plants indicated that Lc gene was integrated into the genomes of transgenic tobaccos and petunias respectively. The observation of the phenotypes showed the flower color of transgenic tobaccos was changed from light red to red and that of transgenic petunias was changed from white to light purple.

The objective of this study was to identify and exploit quantitative loci (QTLs) associated with grain quality from common wild rice. Using single-point analysis and chromosomal segment substitution method, a total of 16 QTLs for 5 traits related to grain quality, including percent of brown rice, percent of head rice, percent of chalky grain, white core area and length : width ratio, were detected using an introgression line population of Chinese common wild rice (O. rufipogon Griff.) from Yuanjiang, Yunnan province, in the background of Indica cultivar (O.sativa L.) Teqing. For 18 of the QTLs detected in this study, the O.rufipogon-derived allele contributed a desirable effect in a Teqing background. QTLs influencing 3 traits of grain quality were found near RM289 on chromosome 5 and these alleles from common wild rice increased grain length : width ratio, decreased percent of chalky grain. One QTL for percent of chalky grain and white core area, explaining 14% and 9% of the phenotypic variance respectively, was detected near RM152 on chromosome 8 and the O.rufipogon-derived allele decreased percent of chalky grain and white core area. The results presented here may be useful in improvement of rice grain quality by marker-assisted selection. It was also suggested that there is enormous potential of improving grain quality of cultivated rice utilizing the favorable genes of wild species in rice breeding program.

Four kinds of processed rice products were extracted of genomic DNA by the following three methods: classic phenol/chloroform extraction method, CTAB precipitation extraction method and guanidine hydrochloride extraction method. The total DNA quantity can be extracted from different quantity of each processed rice product and the starting material (g) was 120 mg, 800 g and 2000 mg. The quality and quantity of extracted DNA were assayed by PCR with the primer pair SPS-F/SPS-R. Results indicated that all of extracted genomic DNA by the CTAB precipitation extraction method can be amplified by PCR with exception of 0.12 g starting material. So we concluded that the CTAB precipitation extraction method has the highest efficiency of extracting genomic DNA and has least inhibiting information on PCR.

In this experiment, we hope to establish a method to detect Mycoplasma in th live vaccine by PCR-ELISA, and to inquire the optimum experiment conditions and superiority combination of PCR and ELISA. According to the 16s rRNA gene sequence published in GenBank, which include the sequences of 1 chicken Mycoplasmas (M. gallisepticum) and 3 swine Mycoplasmas (M. hyopneumoniae, M. hyosynoviae and M. flocculare), The PCR primers and the probe labled by Dig and Biotin was designed by the software DNAstar and Primer 5.0. At the same time, the genome DNA of Mycoplasma was extracted and purified. Then the system and condition of PCR-ELISA was optimized. At last, this method was evaluated through comparing with PCR-electrophoretic method in priactice. This method was applied to detect the samples of 30 batches Newcastle Disease live vaccines(I strain), 30 batches Newcastle Disease live vaccines (La Sota Strain) and 30 batches Swine Fever live vaccine which were bought from market randomly. The results showed that the rate of Mycoplsma contamination from different vaccines by PCR-ELISA was 36.5%. It was 11.2% higher than the result detected by PCR.The method of PCR-ELISA has the character of simlification, speediness and reliability for qualitive and quantifictional analysis of Mycoplasmas. It is hopeful to be used in practice as a detection kit for Mycoplasmas contamination in live vaccine.

Abstract: The cDNA encoding mannanase without the native secretion was cloned by RT-PCR using RNA of the Trichoderma reesei as template. The secreted expression plasmid pPIC9K-man1 of Pichia pastoris was constructed and digested with SacⅠ and transformed into pichia pastoris GS115 by PEG . The positive transformants that integrated man1 gene in their genomes were obtained by PCR technique and determining Mut phenotype. After screen, the recombinant P. pastoris strain RMAN23 was obtained and fermented in large scale 5L fermenter. The recombinant mannanase activity could reach to 470IU/mL .The properties of the recombinant mannanase were characterized.

The recombinant expression of linoleite isomerase using Escherichia coli BL21as host cell was studied. A pair of primers were designed and synthesized according to the linoleic isomerase(gLI) gene sequence published by Genbank. Linoleic isomerase gene was amplified by PCR from genome of L.reuteriPYR8. Recombinant pET-gLI was constructed after the gLI gene was sequenced. BL21(DE3) was induced with 1.0mmol/L IPTG, SDS-PAGE showed recombinant protein was about 67 KD. After inducted and purified, the gLI can converts linolenic acid to CLA in phosphate buffer(pH7.3) 30℃ activity enzyme 1377U, which gave a foundation for biological activity and machnism。

The porcine SMAF1 gene was cloned by degenerate PCR, PCR products for porcine SMAF1, generated from pocine adipose tissue, covered intact open reading frame which was identified with GeneScan software and via manual inspection. The length of this cDNA sequence was 256bp (GenBank DQ191892). The sequence for pig was similar to those of human(86%)and mouse(78％). The deduced amino acid sequence of porcine SMAF1 was similar to those of human(81%), mouse(67%), rat(70%) and bovine(84%).Semiquantitative RT-PCR analysis was performed to determine the expression pattern of SMAF1 in various types of porcine tissues and the genetic difference in mRNA concentrations of SMAF1 in two genetic populations of pigs ( Meishan and Large White). Transcripts for porcine SMAF1 were abundant in adipose tissue, we observed lower levels in Large White pigs than in Meishan pigs. The successful cloning and expression analysis of porcine SMAF1 will enhance the understanding of the involvement of

Primordial germ cells (PGCs) were isolated from the genital ridges of chicken (Gallus domesticus) embryos at the 19th stage. PGCs were co-cultured with somatic cells in primary culture and subculture. Identification of PGCs was carried out by immunocytochemical methods for c-kit. In addition, PGCs were induced to differentiate into neuronal cells, epithelial and skeletal muscle cells by changing the culture conditions. The immunological characteristic of the differentiated cells could be identified by neuron specific enolase and keratin immunocytochemical staining. The above results indicated that chicken PGCs could differentiate into neuronal cells, epithelial and skeleton muscle cells in vitro.

This study was carried out to investigated the effects of methionine and methionylmethionine on cultured bovine mammary epithelial cells. The methionine-modified medium consisted of 0-100 μg/ml of methionine. The expression of casein αs1 gene was increased with adding level of methionine from 0 to 60 μg/ml, and then decreased. The estimated optimal adding level of methionine was at 57 μg/ml. Expression of casein αs1 gene was much higher when 60 μg/ml methionylmethionine was added instead of methionine (P<0.01). These results indicated that mammary epithelial cells can utilize methionylmethionine for milk protein synthesis with a higher efficiency than methionine.

The majority of freemartins are infertile and its karyotyping is XX/XY sex chromosome chimaerism. We have developed a new diagnostic method based on the detection of Y-chromosome DNA segments (Sry) by polymerase chain reaction (PCR) to detect infertile freemartins in cattle. In comparison with other diagnostic aids including clinical observations, erythrocyte typing, and homograft tolerance, this PCR method is more efficient, less expensive, and more practical for routine early diagnosis of the bovine freemartins.

Analysis of a radiation hybrid panel indicated that porcine TLR4 gene is closely linked to the marker SW1957 (15 cR, LOD score 15.16) which is located on porcine chromosome 1 q2.9-q2.13. Using RT-PCR analysis, porcine TLR4 transcripts were detected in testicle, grey matter, skeletal muscle, white matter, kidney, heart, small intestine, thymus, lymph node, spleen, lung and liver, and the hightest lever expression in lung. These study will facilitate the understanding of porcine TLR4 as a disease resistance/susceptibility candidate gene.

In order to clarify the structure of duck major histocompatibility complex (MHC ) class I, duck MHC class I light chain cDNA (Anpl -b2m) was cloned from a duck cDNA library by Full RACE PCR.. Subsequently, the mature protein of 2m gene was expressed solubility in pMAL-p2X /E. coli TB1 system. The expressed MBP-2m fusion proteins were purified, cleaved by the Factor Xa protease and Western-blot analysis. The purified MBP-2m, and MBP were analyzed by circular dichroism (CD) spectrum. The duck MHC class I light chain (Anpl -b2m) is 792bp length, containing 18bp 5'-UT and 414bp 3'-UT; The mature peptides of the Anpl-b2m encoded 119 amino acids, including 20 aa of signal peptide and 99 aa of mature protein. According to the sequences analysis, two cysteines are involved in the Ig-folding of the molecule within the 25 and 80 sites, and the ¨YTCRVDH〃 arround the cysteine at the 80 site is similar to the YxCxVxH Ig-motif character. The Anpl -b2m shares a 30.3%-65.9% aa homology with the chicken, fish, frog, human and mouse. Analysis by circular dichroism (CD) spectrum of purified Anpl-b2m protein revealed that the Anpl-b2m protein displayed typical sheet structure and the contents of 冄-helix,-sheet, turn, and random coil were 0aa, 50 aa, 23aa, 26 aa, respectively. The 3D of Anpl-b2m proteins by homology modeling were similar as the 3 human b2mˇs.

BoLA-DRB3 is a member of the MHC (Major Histocompatibility Complex), which plays a central role in immune system and disease resistance of bovine. The exon2 of BoLA-DRB3 gene is a mainly functional region, which codes the antigen and participates in the body's immune response. In this study, The restriction polymorphism of the exon2 of BoLA-DRB3 in Luxi，Nanyang and Jinnan cattle were investigated by PCR-RFLP. 6 genotypes and 3 alleles were identified for BoLA-DRB3 locus. Statistical results showed that polymorphism sites at position 154、155、156 and 157 in Luxi catttle and Jinnan cattle fitted Hardy-Weinberg equilibrium, but not in Nanyang cattle. After sequencing, 13.70% of nuclitide mutated, which induced that 14.61％ of amino acid changed.

Two binary vectors containing tomato presystemin gene pNAR304（UbiI5’+Prosystemin+NOS3’）and pNAR305 (UbiI5’+Prosystemin+NOS3’ + PinⅡ5’+PinⅡ+PinⅡ3’) were constructed and were introduced into rice cvs. Xiushui63, Hejiang19 and Nipponbare by Agrobacterium-mediated transformation. From tests of hygromycin resistance, PCR and Southern blot, 14 independent transgenic plants were confirmed. Northern blot analysis indicated that both presystemin gene and PinⅡ gene could transcribed in these transgenic plants. But in the rice insect resistance bioassays to stripe stem borer (Chilo suppressalis) and brown plant hopper (Nilaparvata luguns stal), insect resistance of those plants transformed by prosystemin gene alone（pNAR304） showed no improvement than control plants and insect resistance of transgenic plants carrying both prosystemin gene and PinⅡgene（pNAR305）exhibited significant increase than control plants, but their insect resistance level was similar to those transgenic plants only could not regulate the expression of PinⅡgene in transgenic rice. From above experiment results, we think that there may be no signal pathway for tomato systemin in the rice and, probably, signal transmission of injury in rice were other pathway different from dicotyledonous systemin.

In order to clarify the structure of duck major histocompatibility complex (MHC ) class I, duck MHC class I light chain cDNA (Anpl -b2m) was cloned from a duck cDNA library by Full RACE PCR.. Subsequently, the mature protein of 2m gene was expressed solubility in pMAL-p2X /E. coli TB1 system. The expressed MBP-2m fusion proteins were purified, cleaved by the Factor Xa protease and Western-blot analysis. The purified MBP-2m, and MBP were analyzed by circular dichroism (CD) spectrum. The duck MHC class I light chain (Anpl -b2m) is 792bp length, containing 18bp 5'-UT and 414bp 3'-UT; The mature peptides of the Anpl-b2m encoded 119 amino acids, including 20 aa of signal peptide and 99 aa of mature protein. According to the sequences analysis, two cysteines are involved in the Ig-folding of the molecule within the 25 and 80 sites, and the ¨YTCRVDH〃 arround the cysteine at the 80 site is similar to the YxCxVxH Ig-motif character. The Anpl -b2m shares a 30.3%-65.9% aa homology with the chicken, fish, frog, human and mouse. Analysis by circular dichroism (CD) spectrum of purified Anpl-b2m protein revealed that the Anpl-b2m protein displayed typical sheet structure and the contents of 冄-helix,-sheet, turn, and random coil were 0aa, 50 aa, 23aa, 26 aa, respectively. The 3D of Anpl-b2m proteins by homology modeling were similar as the 3 human b2mˇs.

Abstract: The cDNA encoding mannanase without the native secretion was cloned by RT-PCR using RNA of the Trichoderma reesei as template. The secreted expression plasmid pPIC9K-man1 of Pichia pastoris was constructed and digested with SacⅠ and transformed into pichia pastoris GS115 by PEG . The positive transformants that integrated man1 gene in their genomes were obtained by PCR technique and determining Mut phenotype. After screen, the recombinant P. pastoris strain RMAN23 was obtained and fermented in large scale 5L fermenter. The recombinant mannanase activity could reach to 470IU/mL .The properties of the recombinant mannanase were characterized.

Abstract: Hepatitis B surface antigen gene was cloned from Hepatitis B virus DNA by using PCR reaction, then it was inserted into pBin438 and the plant expression vector pBHB was constructed. Transformed crowtoe by agrobacterium were analyzed for the integration of the transgene by PCR. It indicated the exterior gene had been integrated into the crowtoe.

XY355 promoter, a petal-specific promoter, was amplified from the genome of rapes (Brassica napus) by PCR. Then the XY355 promoter was connected with a maize anthocyanin regulatory gene Lc to construct an expression vector pXY60.The vector was introduced to the Agrobacterium tumefaciens strain LBA4404 by freeze-thaw method .The explants of leaf discs from tobaccos (Nicotiana tabaccum L.) and petunias (Petunia hybrida) were transformed by LBA4404 respectively. The results of the PCR analysis among these regenerated plants indicated that Lc gene was integrated into the genomes of transgenic tobaccos and petunias respectively. The observation of the phenotypes showed the flower color of transgenic tobaccos was changed from light red to red and that of transgenic petunias was changed from white to light purple.

Getting the chitinase catalytic domain of Streptomyces roseoflavus by PCR, The catalytic domain was then connected with S. lividans chiC fragment which contains three domains (cellulose binding domain, chitin-binding domain, signal peptide domain). The fragment was cloned into pET23b(+). Then the recombinant plasmid pLCH was transformed into E. coli JM109(DE3). The transformat was induced expressing with IPTG. Its chitinase activity was be found on the chitin culture medium.

Two binary vectors containing tomato presystemin gene pNAR304（UbiI5’+Prosystemin+NOS3’）and pNAR305 (UbiI5’+Prosystemin+NOS3’ + PinⅡ5’+PinⅡ+PinⅡ3’) were constructed and were introduced into rice cvs. Xiushui63, Hejiang19 and Nipponbare by Agrobacterium-mediated transformation. From tests of hygromycin resistance, PCR and Southern blot, 14 independent transgenic plants were confirmed. Northern blot analysis indicated that both presystemin gene and PinⅡ gene could transcribed in these transgenic plants. But in the rice insect resistance bioassays to stripe stem borer (Chilo suppressalis) and brown plant hopper (Nilaparvata luguns stal), insect resistance of those plants transformed by prosystemin gene alone（pNAR304） showed no improvement than control plants and insect resistance of transgenic plants carrying both prosystemin gene and PinⅡgene（pNAR305）exhibited significant increase than control plants, but their insect resistance level was similar to those transgenic plants only could not regulate the expression of PinⅡgene in transgenic rice. From above experiment results, we think that there may be no signal pathway for tomato systemin in the rice and, probably, signal transmission of injury in rice were other pathway different from dicotyledonous systemin.

The objective of this study was to identify and exploit quantitative loci (QTLs) associated with grain quality from common wild rice. Using single-point analysis and chromosomal segment substitution method, a total of 16 QTLs for 5 traits related to grain quality, including percent of brown rice, percent of head rice, percent of chalky grain, white core area and length : width ratio, were detected using an introgression line population of Chinese common wild rice (O. rufipogon Griff.) from Yuanjiang, Yunnan province, in the background of Indica cultivar (O.sativa L.) Teqing. For 18 of the QTLs detected in this study, the O.rufipogon-derived allele contributed a desirable effect in a Teqing background. QTLs influencing 3 traits of grain quality were found near RM289 on chromosome 5 and these alleles from common wild rice increased grain length : width ratio, decreased percent of chalky grain. One QTL for percent of chalky grain and white core area, explaining 14% and 9% of the phenotypic variance respectively, was detected near RM152 on chromosome 8 and the O.rufipogon-derived allele decreased percent of chalky grain and white core area. The results presented here may be useful in improvement of rice grain quality by marker-assisted selection. It was also suggested that there is enormous potential of improving grain quality of cultivated rice utilizing the favorable genes of wild species in rice breeding program.

Four kinds of processed rice products were extracted of genomic DNA by the following three methods: classic phenol/chloroform extraction method, CTAB precipitation extraction method and guanidine hydrochloride extraction method. The total DNA quantity can be extracted from different quantity of each processed rice product and the starting material (g) was 120 mg, 800 g and 2000 mg. The quality and quantity of extracted DNA were assayed by PCR with the primer pair SPS-F/SPS-R. Results indicated that all of extracted genomic DNA by the CTAB precipitation extraction method can be amplified by PCR with exception of 0.12 g starting material. So we concluded that the CTAB precipitation extraction method has the highest efficiency of extracting genomic DNA and has least inhibiting information on PCR.

In this experiment, we hope to establish a method to detect Mycoplasma in th live vaccine by PCR-ELISA, and to inquire the optimum experiment conditions and superiority combination of PCR and ELISA. According to the 16s rRNA gene sequence published in GenBank, which include the sequences of 1 chicken Mycoplasmas (M. gallisepticum) and 3 swine Mycoplasmas (M. hyopneumoniae, M. hyosynoviae and M. flocculare), The PCR primers and the probe labled by Dig and Biotin was designed by the software DNAstar and Primer 5.0. At the same time, the genome DNA of Mycoplasma was extracted and purified. Then the system and condition of PCR-ELISA was optimized. At last, this method was evaluated through comparing with PCR-electrophoretic method in priactice. This method was applied to detect the samples of 30 batches Newcastle Disease live vaccines(I strain), 30 batches Newcastle Disease live vaccines (La Sota Strain) and 30 batches Swine Fever live vaccine which were bought from market randomly. The results showed that the rate of Mycoplsma contamination from different vaccines by PCR-ELISA was 36.5%. It was 11.2% higher than the result detected by PCR.The method of PCR-ELISA has the character of simlification, speediness and reliability for qualitive and quantifictional analysis of Mycoplasmas. It is hopeful to be used in practice as a detection kit for Mycoplasmas contamination in live vaccine.

Abstract: The S1 gene of IBV was cloned into expression vector pGEX-6p-1 by gene recombination technology. E. coli transformed by the recombinant plasmid containing IBV S1gene was induced by IPTG. after the induced A E. coli were lysed, fusion proteins in pGEX-IBV-S1A and pGEX-IBV-S1B were revealed by SDS-PAGE. And, obvious bands in pGEX-IBV-S1A and pGEX-IBV-S1B were showed by Western-blot. The result demonstrated partial antigenicity of the expression products. GST fusion proteins of A and B were purified using Glutathione Sepharose 4B. Rabbits were vaccinated with the purified GST fusion proteins. The antibody and virus were incubated in 37℃ incubator for 30 minutes, and then TOC experiment was made. The result showed that the antibody induced by segment A could inhibit the partial activity of IBV with SN endpoint of 1:18 while the antibody induced by segment B could completely inhibit the activity of virus with SN endpoint of 1:53. It is proved that the immunogenicity of segment B was better than that of segments A.

SPF chickens infected with chicken reticuloendotheliosis virus(REV) induced immunosuppression, Levels of IFN-gamma（IFN-γ） in chicken spleen lymphocytes were measured by double mAb sandwich ELISA. The results showed that chickens grew tardily and appeared the typical symptom of immunosuppression such as severe atrophy in central immune organs. IFN-γ ELISA demonstrated significant increased IFN-γ production in spleen lymphocytes at 7 days post-infection (dpi) compared to the age-matched controls. Until 15 dpi, the IFN-γ levels was the highest and then decreased, but showed markedly higher than the controls. This is the first report of IFN-γ production in SPF chickens infected with REV measured by directly quantitative method.

The recombinant expression of linoleite isomerase using Escherichia coli BL21as host cell was studied. A pair of primers were designed and synthesized according to the linoleic isomerase(gLI) gene sequence published by Genbank. Linoleic isomerase gene was amplified by PCR from genome of L.reuteriPYR8. Recombinant pET-gLI was constructed after the gLI gene was sequenced. BL21(DE3) was induced with 1.0mmol/L IPTG, SDS-PAGE showed recombinant protein was about 67 KD. After inducted and purified, the gLI can converts linolenic acid to CLA in phosphate buffer(pH7.3) 30℃ activity enzyme 1377U, which gave a foundation for biological activity and machnism。

This study was carried out to investigated the effects of methionine and methionylmethionine on cultured bovine mammary epithelial cells. The methionine-modified medium consisted of 0-100 μg/ml of methionine. The expression of casein αs1 gene was increased with adding level of methionine from 0 to 60 μg/ml, and then decreased. The estimated optimal adding level of methionine was at 57 μg/ml. Expression of casein αs1 gene was much higher when 60 μg/ml methionylmethionine was added instead of methionine (P<0.01). These results indicated that mammary epithelial cells can utilize methionylmethionine for milk protein synthesis with a higher efficiency than methionine.

Analysis of a radiation hybrid panel indicated that porcine TLR4 gene is closely linked to the marker SW1957 (15 cR, LOD score 15.16) which is located on porcine chromosome 1 q2.9-q2.13. Using RT-PCR analysis, porcine TLR4 transcripts were detected in testicle, grey matter, skeletal muscle, white matter, kidney, heart, small intestine, thymus, lymph node, spleen, lung and liver, and the hightest lever expression in lung. These study will facilitate the understanding of porcine TLR4 as a disease resistance/susceptibility candidate gene.

Abstract: The S1 gene of IBV was cloned into expression vector pGEX-6p-1 by gene recombination technology. E. coli transformed by the recombinant plasmid containing IBV S1gene was induced by IPTG. after the induced A E. coli were lysed, fusion proteins in pGEX-IBV-S1A and pGEX-IBV-S1B were revealed by SDS-PAGE. And, obvious bands in pGEX-IBV-S1A and pGEX-IBV-S1B were showed by Western-blot. The result demonstrated partial antigenicity of the expression products. GST fusion proteins of A and B were purified using Glutathione Sepharose 4B. Rabbits were vaccinated with the purified GST fusion proteins. The antibody and virus were incubated in 37℃ incubator for 30 minutes, and then TOC experiment was made. The result showed that the antibody induced by segment A could inhibit the partial activity of IBV with SN endpoint of 1:18 while the antibody induced by segment B could completely inhibit the activity of virus with SN endpoint of 1:53. It is proved that the immunogenicity of segment B was better than that of segments A.

SPF chickens infected with chicken reticuloendotheliosis virus(REV) induced immunosuppression, Levels of IFN-gamma（IFN-γ） in chicken spleen lymphocytes were measured by double mAb sandwich ELISA. The results showed that chickens grew tardily and appeared the typical symptom of immunosuppression such as severe atrophy in central immune organs. IFN-γ ELISA demonstrated significant increased IFN-γ production in spleen lymphocytes at 7 days post-infection (dpi) compared to the age-matched controls. Until 15 dpi, the IFN-γ levels was the highest and then decreased, but showed markedly higher than the controls. This is the first report of IFN-γ production in SPF chickens infected with REV measured by directly quantitative method.

Abstract: Hepatitis B surface antigen gene was cloned from Hepatitis B virus DNA by using PCR reaction, then it was inserted into pBin438 and the plant expression vector pBHB was constructed. Transformed crowtoe by agrobacterium were analyzed for the integration of the transgene by PCR. It indicated the exterior gene had been integrated into the crowtoe.

Getting the chitinase catalytic domain of Streptomyces roseoflavus by PCR, The catalytic domain was then connected with S. lividans chiC fragment which contains three domains (cellulose binding domain, chitin-binding domain, signal peptide domain). The fragment was cloned into pET23b(+). Then the recombinant plasmid pLCH was transformed into E. coli JM109(DE3). The transformat was induced expressing with IPTG. Its chitinase activity was be found on the chitin culture medium.

The induced herbivore-resistant genes are classified into three categories, which are defense genes, defense compound synthesis genes and signal pathway genes. The herbivore-resistant genes are regulated by plant hormones such as jasmonic acid, salicylic acid, aspirin, abscisic acid and ethylene, and other signals. The traffic of these signals in plant consists of a complex signal transduction network of plant defense against herbivores. The study of induced herbivore-resistant genes will provide new ways to control insect pests.

The induced herbivore-resistant genes are classified into three categories, which are defense genes, defense compound synthesis genes and signal pathway genes. The herbivore-resistant genes are regulated by plant hormones such as jasmonic acid, salicylic acid, aspirin, abscisic acid and ethylene, and other signals. The traffic of these signals in plant consists of a complex signal transduction network of plant defense against herbivores. The study of induced herbivore-resistant genes will provide new ways to control insect pests.

In our previous study, we had successfully obtained normal mice derived from oocytes following intracytoplasmic fresh sperm injection (ICSI). In the current study, we tested the developmental potency of ICSI embryos produced using freeze-dried sperm. The sperm of B6D2F1 mice were firstly freeze-dried in Tris-HCl-EDTA（pH8.2）solution for 4 h, then were individually injected into mature KM oocytes. Six hours later, 83.0% of fertilized eggs shown two pronuclei and extruded the 2nd polar body. The development rates of these fertilized embryos at the 2-cell (92.0% vs 99.5%), 4-cell (52.7% vs 97.2%), morulae (36.6% vs 86.3%) and blastocyst stages (21.4% vs 68.7%), were significantly (p<0.01) lower than that of the embryos fertilized by fresh sperm injection. After transferred to pseudo-pregnant KM female mice, 63.0% (17/27) of the 2-cell embryos were implanted on D10 and 21.7% (13/60) of them developed to term. Eleven pups developed into normal and fertile adult. Here we reported that the first birth of mouse offspring following ICSI using freeze-dried sperm in China. It was valid to store mammalian sperm using freeze-dried procedure.

In the present work，floral aberrances appeared in some of CBF1 transformed tobacco plants. Comparisons of wild type plants, the main phenotype changes of transformed plants were aberrant petals, smaller flowers and shorter androeciums. Furthermore, the ratio of pollen germination was decreased and the capsule number was reduced. Using genomic DNA of each aberrant plant as a template, TAIL-PCR was performed with five arbitary degenerate (AD) primers pairing with 3 nested specific primers designed toward outside in pBI121 plasmid. After 3-step PCR reactions, the flanking sequence of each T-DNA inserting site was obtained. The PCR products were sequenced directly if it is longer than 300 bp. These sequences were searched against GenBank database using blast n or searched against pBI121 sequence using bl2seq. Analysis of flanking sequences demonstrated that they shared the same T-DNA inserting site. It was suggested that the aberrance of the transformed tobacco did not result from different sites of T-DNA insertion.

In our previous study, we had successfully obtained normal mice derived from oocytes following intracytoplasmic fresh sperm injection (ICSI). In the current study, we tested the developmental potency of ICSI embryos produced using freeze-dried sperm. The sperm of B6D2F1 mice were firstly freeze-dried in Tris-HCl-EDTA（pH8.2）solution for 4 h, then were individually injected into mature KM oocytes. Six hours later, 83.0% of fertilized eggs shown two pronuclei and extruded the 2nd polar body. The development rates of these fertilized embryos at the 2-cell (92.0% vs 99.5%), 4-cell (52.7% vs 97.2%), morulae (36.6% vs 86.3%) and blastocyst stages (21.4% vs 68.7%), were significantly (p<0.01) lower than that of the embryos fertilized by fresh sperm injection. After transferred to pseudo-pregnant KM female mice, 63.0% (17/27) of the 2-cell embryos were implanted on D10 and 21.7% (13/60) of them developed to term. Eleven pups developed into normal and fertile adult. Here we reported that the first birth of mouse offspring following ICSI using freeze-dried sperm in China. It was valid to store mammalian sperm using freeze-dried procedure.

In the present work，floral aberrances appeared in some of CBF1 transformed tobacco plants. Comparisons of wild type plants, the main phenotype changes of transformed plants were aberrant petals, smaller flowers and shorter androeciums. Furthermore, the ratio of pollen germination was decreased and the capsule number was reduced. Using genomic DNA of each aberrant plant as a template, TAIL-PCR was performed with five arbitary degenerate (AD) primers pairing with 3 nested specific primers designed toward outside in pBI121 plasmid. After 3-step PCR reactions, the flanking sequence of each T-DNA inserting site was obtained. The PCR products were sequenced directly if it is longer than 300 bp. These sequences were searched against GenBank database using blast n or searched against pBI121 sequence using bl2seq. Analysis of flanking sequences demonstrated that they shared the same T-DNA inserting site. It was suggested that the aberrance of the transformed tobacco did not result from different sites of T-DNA insertion.

Transgenic cotton expressing the Cry1Ac toxin from Bacillus thuringiensis has been widely planted in China since 1997, which reached to 70% of the total cotton area in 2006.The monitoring results on environmental impacts of Bt cotton commercialization indicated that the target pests, cotton bollworm and pink bollworm were effectively controlled while the mirids evolved to be key pests in cotton system. There were no significant changes in resistance gene frequency of field populations of cotton bollworm, but a shift toward tolerance increase was apparent in the intensive planting area of Bt cotton, indicating that potential resistance risk from the target pest has become a major threat for sustainable planting of Bt cotton. In consideration of the factors associating with the resistance evolution, a risk management strategy is discussed in this paper.

Transgenic cotton expressing the Cry1Ac toxin from Bacillus thuringiensis has been widely planted in China since 1997, which reached to 70% of the total cotton area in 2006.The monitoring results on environmental impacts of Bt cotton commercialization indicated that the target pests, cotton bollworm and pink bollworm were effectively controlled while the mirids evolved to be key pests in cotton system. There were no significant changes in resistance gene frequency of field populations of cotton bollworm, but a shift toward tolerance increase was apparent in the intensive planting area of Bt cotton, indicating that potential resistance risk from the target pest has become a major threat for sustainable planting of Bt cotton. In consideration of the factors associating with the resistance evolution, a risk management strategy is discussed in this paper.