The effects of DNA concentrations(0.2~16 ng/μL), transgene contents (50.0%~0.05%,W/W), storage temperatures(22℃，4℃，－20℃) and storage periods (1，2，3 weeks and 1, 3 months) on qualitative PCR detection of transgene from transgenic rice(Oryza sativa ssp.indica) were evaluated. The result showed that all the primers can amplified targeted fragment when DNA concentration was higher than 0.4 ng/μL. For mixed samples with 1.0% and 0.1% transgene, the lowest DNA concentration which was still detectable was 1 ng/μL and 2~4 ng/μL, respectively. Sample storage at the three tested different temperatures did not affect subsequent PCR detection. And the brightness of electrophoresis strips of PCR products were gradually weakened after 3 months storage of DNA sample.

Bovine insulin-like growth factorsⅠreceptor(IGF-IR), m-calpain(CAPN Ⅱ) and uncoupling protein 3(UCP 3) loci were evaluated for associations with growth traits in Nanyang population by PCR-RFLP. Only two individuals with genotype BB were detected at IGF-IR/TaqⅠloci, allele A at IGF-IR/TaqⅠlocus and allele B at CAPN Ⅱ/HhaⅠwere dominant in the studied population which were in Hardy-Weinberg equilibrium at IGF-IR/TaqⅠand CAPN Ⅱ/HhaⅠloci and in Hardy-Weinberg disequilibrium at UCP 3/BglⅠlocus. No statistically significant differences in growth traits were observed between the genotypes at IGF-IR/TaqⅠlocus. There were statistically significant differences between the genotypes at CAPN Ⅱ/HhaⅠlocus for growth traits of six-month individuals and for heart girth and body length of different growth stages (P<0.05 or P<0.01). UCP 3/BglⅠpolymorphism had significant effects on body weight, withers heights and body length of six-month animals, and withers heights, heart girth and body length of twenty-four-month individuals (P<0.05). Individuals with genotypes AB at CAPN Ⅱ/HhaⅠand UCP 3/BglⅠloci had best performance. Therefore, polymorphisms of CAPN Ⅱ/HhaⅠ and UCP 3/BglⅠcan be potential effective markers for early selection in the breeding process of Nanyang cattle.

The genetic polymorphisms of partial sequence of DNA-dependent protein kinase gene (PRKDC) in 210 Holstein cows were detected by PCR-SSCP with five pairs of primers P1~P5. Five polymorphic sites, i.e. G34126A, C77353T, T77384G, C77406T and indel (C) at site of 77408 bp were found, and G34126A mutation caused an amino acid change of serine to asparagine. For the amplified fragment of P2, three genotypes (AA, AB and BB) were found in 210 Holstein cows, and the genotype frequency of AA, AB and BB was 0.186, 0.433 and 0.381, respectively. For the amplified fragment of P3, five genotypes (CC, CD, DD, CE and DE) were found in 210 Holstein cows, and the genotype frequency of CC, CD, DD, CE and DE was 0.786, 0.129, 0.009, 0.067 and 0.009, respectively. The relationship between polymorphisms of PRKDC gene and somatic cell score (SCS) in Holstein cows was analyzed by least squares linear model. Results showed that least squares mean of SCS for AA was significantly lower than that for AB or BB (both P<0.05), and the difference of the least squares means for SCS between AB and BB was not significant (P>0.05). AA was the most favorable genotype, BB was the most unfavorable genotype for mastitis resistance. The difference of the least squares means for SCS among CC, CD, DD, CE and DE was not significant (P>0.05). The results preliminarily indicated that allele A of G34126A polymorphic site of PRKDC gene is a potential DNA marker for improving mastitis resistance in cows.

The final and critical step in the synthesis of triglycerides is catalyzed by two acyl-coA∶diacylglycerol acyltransferase (DGAT) enzymes, i.e. DGAT1 and DGAT2. Analysis on the developmental expression of DGAT1 and DGAT2 mRNA in porcine backfat tissues is a prerequisite for the identification of genes underling fat deposition differences between Chinese indigenous fat-type and western lean-type pig breeds. The DGAT1 and DGAT2 mRNA expression in backfat tissues of Laiwu and Duroc 3-day-old piglets and adults was investigated by both RT-PCR and Real-time fluorescent quantitative RT-PCR in this study. The results indicated that, DGAT1 mRNA expression in backfat tissues was higher in adult pigs than that of 3-day-old piglets, which is 5.0-fold and 2.7-fold for Laiwu and Duroc pig, respectively. DGAT2 mRNA expression in backfat tissues was higher in adult pigs than that of 3-day-old piglets, which is 27.6-fold (P<0.01) and 4.8-fold (P<0.05) for Laiwu and Duroc pig, respectively. The increase in mRNA expression in adult backfat tissues was higher for DGAT2 than that of DGAT1. For DGAT2 expression in Duroc backfat tissues, it was higher in 3-day-old piglets, but lower in adults than that of Laiwu pigs, although the difference was not significant (P>0.05). These results suggest that DGAT2, not DGAT1, likely controls the differences in fat deposition.

The β2-adrenergic receptor (β2-AR) genes of three breeds of sheep (Ovis aries) were cloned for the first time from Suffolk, Small Tail Han and Chinese Merino (Xinjiang type) with a pair of PCR primer corresponding to the consensus sequence of mammalian β2-AR(GenBank accession No.EU707939, EU707940 and EU707941). The nucleotide sequence data showed that the coding region of the gene consisted of 1257 bp open reading frame which encoded 418 amino acid residues. The sheep β2-AR coding region nucleotide sequence was 98.4%, 88.5%, 84.2% and 80.5% identical to those of bovine, porcine, rat and human, respectively. Two copies of the nucleotide sequence coding for the second extracellular loop of sheep β2-AR were synthesized and then cloned into pET-32C (+) expression vector. After being transformed into Escherichia coli BL21 (DE3) and induced by 1 mmol/L IPTG, the recombinant fusion protein expressed in cytoplasm. SDS-PAGE and Western blotting results revealed that the molecular weight of the protein was about 26 kD, and the expression level was 40.3% of total bacterial protein.

The steroid 21-hydroxylase gene (CYP21) was studied as a candidate gene for the high prolificacy in Jining Grey goats. According to the sequence of bovine CYP21 gene, ten pairs of primers were designed to detect base variation of exons 1~10 of CYP21 gene in goat(Capra hircus) low prolificacy breeds (Angora and Inner Mongolia Cashmere goats), medium prolificacy breed (Boer goat) and high prolificacy breed (Jining Grey goat) by PCR-SSCP and PAGE. Only the products amplified by primer P10 displayed polymorphisms. For primer P10, three genotypes (AA, AB and BB) were detected in Inner Mongolia Cashmere, Boer and Jining Grey goats, and two genotypes (AA and AB) were detected in Angora goats. Sequencing revealed six nucleotide mutations (A2789C, C2791A, G2818A, G2851C, G2852T and G2854A) and one deletion mutation（c.2821_2822delCG）at exon 10 of CYP21 gene in the BB genotype compared with the AA genotype. As a result, nineteen amino acids were changed from the 426th to the 448th. The Jining Grey goats with genotype BB and AB had 0.81 (P<0.05) and 0.47 (P<0.05) kids more than those with genotype AA, and the difference of the litter size between genotypes BB and AB was non-significant (P>0.05). These results preliminarily indicate that allele B of CYP21 gene is a potential DNA marker for improving litter size in goats.

In order to study Tibetan sheep (Ovis aries) DRB3 gene polymorphism, determine its allele number, nucleotide polymorphism sites, analysis the genetic relationship and evolutionary significance of the alleles. Variation in the 500 Tibetan sheep DRB3 gene was investigated by PCR-SSCP method, followed by cloning and DNA sequencing. The results showed that there were 37 alleles were identified. There were 82 nucleotide polymorphism sites were identified in 37 sheep DRB3 gene haplotypes. Thirty five novel DRB3 exon 2 alleles were found comparison with GenBank sequences. The phylogenetic tree results showed that the 37 haplotypes of Tibetan sheep DRB3 gene exon 2 can divide into two clusters. It is showed that Tibetan sheep DRB3 gene exon 2 has high level of sequence polymorphism，Tibetan sheep DRB3 gene was differentiated into two major categories alleles from two mutant alleles at first.

This paper was to obtain goat(Capra hircus) GST-Myc fusion protein by means of molecular cloning, prokaryotic expression and protein purification. The c-Myc cDNA of goat was amplified by RT-PCR from intestine tissues, and plasmid pMD18-T-Myc was constructed by T-A cloning. DNA sequencing showed that the cDNA was 1 320 bp，with a complete open reading frame that encoded 439 amino acids, sharing higher nucleotide homology(99.2%) with that of sheep(Ovis aries). The cDNA fragment was subcloned to pGEX-KG, and recombinant plasmid pGEX-KG-Myc was constructed, which was verified by restriction endonuclease analysis and DNA sequencing. The recombinant plasmid was transformed into E.coli BL21, and GST-Myc fusion protein was expressed with induction of IPTG, and then purified with Glutathione-Sepharose 4B argrose. Illustrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), the purified fusion protein was about 75 kD, a clear, and single band, which was expected. Western blot assay revealed that fusion protein could be identified by GST antibody. In conclution, we have cloned goat c-Myc gene, obtained GST-Myc fusion protein with higher purity, which will lead to preparation of polyclonal or monoclonal anti-Sox2 antibody, and further to application in identification of goat iPS cells (induced pluripotent stem cells).

There are two major methods to introduce exogenous substances like foreign DNA into the chicken embryo. One is microinjection of the foreign DNA into the subgerminal cavity of fresh fertilized eggs before incubation. The other is microinjection of the foreign DNA into the yolk sac vein vessel after the fertilized egg incubated for 72 h. Workload, technical difficulty, effectiveness, and the hatchability of recipient chickens were listed as criteria to evaluate the two methods. The results indicated that the former way was easier to conduct but with a lower hatchability of 9.5%. The foreign DNA could be found in the recipient embryos of different ages and the chicks after hatching; The latter way was relatively difficult but with a higher hatchability of 35.7%. The foreign DNA could be found in the recipient embryos of different ages and the chicks after hatching, also without integration. Which way to be selected depends on the aim of the experiment, action time of the exogenous substances and the sample size.

The genetic structure in the Bian chicken(Gallus gallus) of two generations(generations 0 and 1) was analysed using 21 microsatellite markers. The effect of conservation for the Bian chicken was evaluated through comparing the difference in genetic diversity of the two generations. The results showed that a total of 122 alleles were detected in the two generations, of which, 102 were detected in the two generations, and 19 alleles were unique to generation 0. The average polymorphism information content(PIC) of generations 0 and 1 was 0.5473 and 0.5437, respectively, and the average expected heterozygote frequency(He) of the two generations was 0.5967 and 0.6009, respectively. The inbreeding coefficient was 0.233 in generation 0 and 0.134 in generation 1. The genetic diversity of the Bian chicken was very high. Through selection and breeding for one generation, the PIC and He were maintained, while the inbreeding coefficient(Fis) was somewhat decreased. It indicated that the conserved method was feasible. The conservation method not only conserves the genetic diversity of the Bian chicken, but also avoides inbreeding depression.

The mRNA expression of muscle development and energy metabolic related genes were investigated by inhibiting janus protein-tyrosine kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signal pathway using AG490. The tentative male Kunming mice (Mus musculus) were treated as intraperitoneal injection of AG490 for two weeks. After execution, the leg muscled was surgically removed and total RNA was extracted. Real-time quantitative PCR was used to examine the mRNA expression of the key factors in JAK2/STAT3 signal pathway JAK2 and STAT3, the growth relative genes of skeletal muscle myogenic determination（MyoD）and myogenic factor 5 (Myf5）, and energy metabolic relative genes Liver X receptors（LXRα）and Uncoupling proteins (UCP3）. Compared with control, the body weight of mice were significantly decreased (P＜0.05); the body temperature was stable; the expression of JAK2 and STAT3 mRNA were significantly decreased（P＜0.05）; the expression level of LXRα, UCP3, MyoD5 and Myf5 mRNA were decreased extremely significant(P <0.01). JAK2/STAT3 signal pathway may regulate the growth of skeletal muscle and energy metabolism by decreasing the mRNA expression levels of the growth of skeletal muscle development and energy metabolic relative genes.

To characterize the ω-gliadin gene family of Shaan 253, six pieces of complete coding regions were amplified from this wheat (Tricicum aestivum) variety using primer pairs designed according to the known ω-gliadin gene sequences. The amplified DNA fragments were separated and recovered from agarose gel, subsequently ligated into pMD19-T vector, and then transformed into Escherichia coli strain DH5α. Five positive clones were sequenced. And five ω-gliadin gene sequences were obtained, designated as Gli-S253-1, Gli-S253-2, Gli-S253-3, Gli-S253-4 and Gli-S253-5, respectively and the corresponding GenBank accession numbers were GQ423428, GQ423429, GQ423430, GQ423431and GQ423432, respectively. Sequence analysis showed that: (1) There were four main types of ω-gliadin gene silence(Q→*, I→*, R→* and W→*), of which the first type was dominant(about 66.6%); (2) Two kinds of coding glutamine (Q) were the most likely to nonsense mutations (CAA→TAA and CAG→TAG); (3) Gene silence occurred mainly in the triplet code of the first base (accounting for 77.8%), mutation frequency of the second base was about 33.3%, while the third one has not been reported; there was a rare silence mutation (ATA→TAA) in GQ423428, while two loci involved in variation in a triplet code at a time; (4) The number of gene silence mutation was up to six in GQ423428 and GQ423429, including four sites (349, 373, 478 and 592) which occurred in all of the five genes. (5) From the structure of mature protein, nonsense mutations appeared mainly in the repeat region, rather than the signal peptide or the N-terminus of ω-gliadin; (6) Phylogenetic analysis indicated that ω-gliadin/secalin of tribe Triticeae was divided into three branches. The results from this study indicated that five pieces of ω-gliadin genes were successfully separated from Shaan 253, which could expand the genetic variation for wheat quality improvement, and even provide molecular assists during the conventional wheat breeding.

To identify and tag new resistance genes to stripe rust in a translocation line M8657-1 (commom wheat(Triticum aestivum) translocated with Leymus mollis (Trin.) Hara), the yellow rust races CYR30, CYR31, SU11-4 and SU11-11 were seleceted as inoculum. Genetic analysis of resistance to stripe rust was made in the progeny population of M8657-1/Mingxian169 at seedling stage. The results showed that one recessive gene conferred resistance to stripe rust race CYR30 and SU11-11, respectively; Two dominant karyoplasm genes conferred resistance to CYR31; And the resistance to SU11-4 was controlled by one dominant gene which was temporarily designated as YrElm1-4. The F2 population inoculated with SU11-4 were used for SSR analysis. SSR markers, as well as 320 pair primers, combined with bulked segregation analysis(BSA) revealed that three SSR markers,Xgwm636, Xwmc522 and Xwmc453, located on the short arm of chromosome 2A were linked to YrElm1-4. And YrElm1-4 was deduced to locate on the chromosome 2AS based on the three markers location. So the three SSR markers can be used in molecular-asisted breeding.

Fifty three rhizobial strains isolated from root nodules of Kummerowia striata in part of northwest China were studied by a series of analysis on C, N nutrient utilization, resistance to antibiotics, colorant and hydroxybenzene, endurance to salt and alkali. At the same time, 16S rDNA PCR-RFLP and phylogeny analysis were used to study the genetic diversity of these strains. The results showed that there were great diversity among these strains, 12.5% of strains could resist 300 μg/mL chloramphenicol, 58.9%of strains could resist 2% NaCl, 38% of strains could resist 700 mg/L hydroxybenzene. The analysis of 16S rDNA PCR-RFLP revealed that these tested rhizobial strains belonged to Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Agrobacterium, respectively. Apparently, the great genetic diversity at the level of species and genus exists among Kummerowia striata rhizobia in northwest China.

To investigate the effect of PCR amplification with nano-silver on DNA synthesis and its mechanism, the effect of PCR amplification with nano-silver on DNA synthesis was obtained by the observation of different sizes(38, 61, 83 and 130 nm) and different concentrations(5, 10, 25, 50, 100 and 250 μg/mL) of nano-silver particles in polymerase chain reactions (PCRs). and the UV-visible spectroscopy was used to analyze its molecular mechanism. The results showed that when PCRs were exposed to four sizes of nano-silver particles respectively within a dose range, the effect of PCR amplification with 38 and 61 nm nano-silver groups was much stronger than that of 83 and 130 nm nano-silver groups. When PCRs were exposed to different concentrations of nano-silver particles, we found that nano-silver could inhibit the DNA synthesis obviously when the dose ≥10 μg/mL, and its inhibition showed a significant dose - effect relationship. Integrated agarose gel electrophoresis and UV-visible spectrum, we claim that the adsorption of polymerase, primers and templates by nano-silver is the main reason for inhibition of DNA synthesis. In addition, a small amount of Ag+ released by sliver nanoparticles is another key factor for inhibition of the DNA synthesis.

Real-time fluorescence quantitative PCR was applied to investigate the developmental expression patterns of thyroglobulin (TG) and thyroid peroxidase (TPO) genes from the thyroid tissue of castrated and uncastrated Jinhua pigs at the ages of 60, 90 and 120 days. Results showed that the expression levels of TG and TPO mRNA in castrated Jinhua pigs at 90 days was significantly higher than that of other days (P<0.05), while the expression level of TG in uncastrated pigs at 120 days was significant lower than those at 60 and 90 days (P<0.05), but no significant difference in TPO expression level among ages (P>0.05). The comparison of TG and TPO mRNA between castrated and uncastrated pigs was different significantly at 60, 90, and 120 days (P<0.05). The results suggested that there may have been a positive relationship of the expression levels of the TPO and TG mRNA with the weight gain and intramuscular fat content in pig after castration.

Using real-time PCR technique, the expression of DNA methylation associated genes(DNA methyltransferase gene: Dnmt1 and Dnmt3a) in porcine GV- and MⅡ-oocytes, 2-cell to morula embryos derived from in vitro fertilization(IVF) and intracytoplasmic sperm injection(ICSI) were analyzed, and the expression of embryonic anti-apoptotic gene(B cell lymphoma/leumia-2, Bcl-2) was examined as well. The results showed that Dnmt1 behaved high expression in the oocytes and could sharply decrease in both IVF and ICSI group during early stage of embryonic development. Compared with IVF embryos, ICSI embryos behaved similar expression pattern, which was higher only at 4-cell stage than that from IVF group. The expression of Dnmt3a started to decrease during maturation in vitro and maintained a low level at 2- to 8-cell stage and then began to increase. The expression level of Dnmt3a in ICSI embryos after 8-cell stage was lower than that of IVF embryos. With the development of embryos, the expression level of Bcl-2 mRNA in either IVF or ICSI embryos could gradually decrease, the trend of which was more evident in ICSI embryos, indicating that the ability of embryonic anti-apoptosis decreased. It may be the main cause impacting the development of embryos.

The objectives of the present research are to investigate whether embryo culture media had prefere- nces to oxygen tension during the in vitro development of mouse early embryos, and to explore feasibility of using physical lung air to support the in vitro development of mouse embryos, and to evaluate effect of well of the well(WOW) culture on in vitro pre-implantational deveopment of mouse embryos. The results were: First, cleavage rate and blastocyst rate were not significantly different between medium CZB and mKSOM on the use of three gas phases: 4%CO2＋16%O2＋78%N2＋2%H2O (lung air), 5%CO2＋5%O2＋90%N2 (5%O2, low oxygen) and 5%CO2＋20%O2＋75%N2(20%O2, high oxygen) (P＞0.05), but mean total cell numbers per blastocyst cultured in CZB medium were higher than that in mKSOM when the lung air was used(P＜0.05). Second, based on mKSOM medium, the blastocyst rate(22.6%) in 5%O2 gas phase was notably higher than that in other two gas phase(P＜0.05). Third, as for CZB medium, blastocyst rate was not different significantly among three gas environments (P＞0.05). Fourth, both the blastocyst rate （74.6±5.1%） and the mean total cell numbers per blastocyst（76±2） cultured in WOW system were obviously higher than that in the group culture system(38.2±6.6% and 58±4). Taken together, these results indicate that the mKSOM medium and the CZB medium have their corresponding preferences to oxygen tension during in vitro culture of mouse embryos, and the lung air was reaffirmed to be able to effectively support in vitro pre-implantation development of mouse embryos, and WOW culture system can apparently enhance the in vitro developmental competence and blastocyst quality of mouse embryos.

One hundred and thirty seven SSR (simple sequence repeat) and EST（expressed sequence tag） markers were used to analyze the genotype of 93 individuals of BC1F1 generation which cross from Barbless carp (Cyprinus pellegrini) and Hebao-cold tolerance red carp(Cyprinus carpio wuyuanensis). Using Windows Map Manager 2.0 software analyzed the quantitative trait related to feed conversion efficiency by single marker regression analysis. Results showed that eight SSR markers ( HLJ133, HLJ360, HLJ380, HLJ562, HLJ740, HLJ852, HLJ930 and HLJ1420) and two EST markers ( HLJE318 and HLJE417 ) were discovered related to common crap's feed conversion ratio. The phenotype variation was explained by these loci, ranged from 4% to 11%. The genotypes of these correlative loci were determined by ANOVA analysis. In loci HLJ360、HLJ380 and HLJ562 respectively, genotypes 212/212, 236/224 and 220/220 are positive correlation to feed conversion efficiency trait. These functional markers and genotypes which related to common crap's feed conversion may be used for the QTL mapping and marker assisted selection in common carp.

A stable line of transgenic Tanichthys albonubes containing red fluorescent protein(RFP) gene was produced previously and RFP gene is proved transmitting in a Mendelian fashion. For further reveals the integration characteristics of RFP gene in transgenic fish, the number of construct copies, the arrangement of constructs and the nearby sequence of foreign gene inserted were detected using PCR, Southern blotting and genomic walking. The results showed that the integration pattern of exogenous gene was single locus, multi-copy (2 or 3) and head-to-tail multimers. Both fragments of 1.0 and 1.2 kb were amplified by genomic walking from 5'-and 3'-flanking sequence, respectively. Sequence analysis showed that 5'-flanking sequence existed in wild Tanichthys albonubes genome sequence, and there was no homologous sequence in the GenBank database, the 3'-flanking sequence was 99% identical to that of phage P1 partial genome, but this sequence could not be detected in wild Tanichthys albonubes genome. These result provide a evaluation basis for the genetic stability of RFP transgenic T. albonubes.

A pair of primers were designed by referring to species close to mink(Mustela. vison), the complete mitochondrial cytochrome b gene(Cytb) sequences of six mink breeds and Zuojia mink were determined using cloning, sequenceing and assembling. Sequence analysis used DNAstar v6.13 and DnaSP4.0 revealed that Cytb Gene of minks were 1 140 bp in length, the composition of nucleotides A, G, T and C was 29.8%, 13.0%, 30.3%and 26.9%, respectively. Twelve variable sites were detected, and they all were transition. Multiple sequences alignments by ClustalX1.8 showed that they were high similarity in Cytb gene among minks. Combined with homologous sequences of 14 species in the Mustela retrieved from GenBank, the phylogenetic trees based on nucleotide and amino acid sequences were constructed by Neighbor-joining method, respectively. Two trees had same topologies. The results indicated that minks in China had closer evolutionary relationships with American minks than with European mink(M.lutreola). The divergence time between American mink and European mink might have started at Miocene.

PCR-RFLP and sequencing of rDNA ITS regions of six geographic populations in genus Helicotylenchus were made. Four species, Helicotylenchus microcephalus, H. digonicus, H. dihystera, and H. pteracerus, were identified.Both the sequence length and digestion patterns were the same in the three populations of H. digonicus. The digestion patterns varied in different four specices, which was identified as H. microcephalus, H. digonicus, H. dihystera and H. pteracerus, respectively based on the morphological method. The combination of six enzymes, AluⅠ, AvaⅠ, HaeⅢ, HinfⅠ, MspⅠ and RsaⅠ, could be used effectively to identify and classify this four Helicotylenchus species. This is the first report about the molecular data of the nematode genus in China.

Erwinia amylovora causes fire blight of pear, apple, and many of the Rosaceae family. The cyclic adenosine monphosphate (cAMP) receptor protein(CRP), which is encoded by crp gene, plays a crucial role in expression of the pectinolysis genes and regulation of the pathogenicity in Erwinia chrysanthemi. In this study, the homolog of crp gene, designated Eacrp, was identified and cloned in Erwinia amylovora. A crp disruption mutant EaΔcrp was successfully generated by homologous recombination. Complement strain EaΔcrp (pME-crp) was also constructed. Then crp gene was characterized to virulence, hypersensitive response(HR), extracellular polysaccharide (EPS), motility and other phenotypes of the phytopathogens Erwinia amylovora. The results showed that the crp mutant EaΔcrp displayed the reduced virulence, EPS production and growth in vitro and motility. However, compared to wild type strain NCPPB1665(Ea1665), the induction of HR in nonhost tobacco, peroxide resistance, sedimentation, biofilm synthesis and AI-2 production of the crp mutant EaΔcrp was not affected. These findings demonstrated that crp gene in Erwinia amylovora is involved in EPS production, growth, and motility and play an important role in virulence.

The genetic diversity of 39 indigenous Chinese goat breeds(Capra hircus) and 1 exotic breed was investigated using 9 microsatellite DNA markers recommended by the Food and Agriculture Organization of the United Nations (FAO) and the International Society for Animal Genetics (ISAG) through fluorescence PCR. The mean expected heterozygosity across loci within populations ranged from 0.4346 to 0.6951. Genetic differentiation measures were moderate, with a mean FST of 0.115 and GST of 0.112. Percentages of variation among and within populations were 13.3% and 86.7%, respectively. The result of phylogenetic trees (UPGMA) analysis, principal component analysis and Bayesian clustering analyses were similar. Forty goat breeds were divided into 5 clusters. Boer was an individual cluster and the Chinese goat breeds subdivision in 4 clusters, the southwest China cluster, the south China cluster, the central China cluster and the north China cluster. Results indicate that the Chinese goat genetic diversity was rich and the genetic variation existed mainly within breed, and has great potential in breeding.

This study was conducted to investigate factors influencing intracytoplasmic sperm injection(ICSI) of x-sorted frozen-thawed sperm in dairy cow so as to establish a preliminary procedure for ICSI in dairy cow. In experiment 1, two different sperm states (motile sperm and immotile sperm) were used in ICSI, the higher cleavage and blastocyst rate of ICSI zygotes were obtained from the motile sperm (86.4% and 28.1%) than that those from the immotile sperm (82.3% and 25.7%), but there was no significant different (P>0.05). In experiment 2, three different concentrations (5%, 8% and 10%) of pdyvinyl pyrrolidone(PVP) in the sperm injection vehicle solution were compared. There was no effect of the three PVP concentrations on cleavage rate after sperm injection, but regular cleavage rate and blastocyst rate were descended with increasing concentration of PVP, regular cleavage rate (78.4% and 69.1%) and blastocyst rate (29.7% and 20.4%) were significantly higher with 5% than 10% PVP(P< 0.05). In experiment 3, two positions (6 and 12 o'clock) of the first polar body (PB) located in oocytes at ICSI were examined. There was no significant difference in the cleavage rate (P>0.05) between the two positions of the PB, but regular cleavage rate (76.4% and 66.7%) and blastocyst rate(28.6% and 19.0%) were significantly improved at 6 o'clock of the polar body(P<0.05). The result indicated that the motile sperm be used in ICSI and the sperm was treated in the manipulation medium with 5% PVP, and the oocytes should be injected with the polar body at 6 o'clock, can improve the in vitro development of the ICSI zygotes in dairy cow.

To prepare artificial antigen of 19-nortestosterone(19-NT) and identify its immunological traits, succinic anhydride was employed to synthesis 19-NT-17-succinic anhydride ester, which was proved by(+)ESI-MS spectrum and 1H-NMR spectrogram. The 17β-19-NT ester hapten was conjugated to bovine serum albumin(BSA) as immunogen by the EDC method, and to ovalbumin(OVA) as detection antigen by mixed- anhydride technology, respectively. The results of infrared spectra(IR) and UV-visible spectra indicated that the artificial antigen was synthesized successfully and the conjugation ratio of NT-BSA was 18∶1. Balb/C mice(Mus muscalus) immunized by NT-BSA were introduced to obtain NT pAb, whose antibody titer for indirect ELISA were 1∶25 600, after the concentrations were optimized by the checkerboard method. Sensitive icELISAs were selected, showing the average IC50 value of 27.3 ng/mL, the detection range of 3.3~225.0 ng/mL, and the lowest detection limits of 1.6 ng/mL, respectively. The antisera cross-reacted with epi-nortestosterone was about 69%, but not higher than 0.05% to other hormones. The produced antibody show high sensitivity and excellent specificity, lay a potential foundation for preparing NT-Kit and rapid test strip.

Pigeon is a kind of monomorphic bird, and also a typical monogamous bird, this situation challenges the couple and human-assisted breeding of pigeon(Columbalivia Gmelin). Using feathers of pigeon as DNA sources , a PCR test was used to amplify two homologous fragments of the CHD-W(380 bp) gene, unique in female, and the CHD-Z(450 bp) gene, occurring in the two sexes. The results showed that high-quality genomic DNA was extracted from feathers of pigeon, and there was a single CHD-Z band in males but females had both, CHD-Z and CHD-W band. This sexing technique is objective and non-invasive and can be useful in the management of pigeon production.

To develop a Real-time fluoresecent quantitative PCR assay for the detection of Actinobacillus pleuropneumoniae(APP) and apply the developed assay to clinical samples. According to the conserved 391bp neucleotide sequences in apxⅣA gene of APP strains, reported in GenBank, a set of primer pairs were designed to establish APP specie-specific Real-time PCR and standard curve occurred after amplification using gradient diluted plasmid as template. After developed, the method was used to quantitatively detect the copies of APP in lung tissues both collected from disease or artificially infected pigs. Data obtained demonstrated that the newly developed method was highly sensitive, specie-specific, repeatable and applicable for clinical diagnosis. The method established in this research has potential to pave the way for early and rapid detection of APP or quantitative analysis for the infection degree of APP.

BALB/c mice were immunized by purified Bovine viral diarrhea virus-1（BVDV1）and Bovine viral diarrhea virus-2 （BVDV2）. Murine myeloma SP2/0 cells were fused with the splenocytes of the immunized mice. Two indirect ELISA with BVDV1 and BVDV2 as coated antigen separately were used to screen antibody-producing hybridomas. Three McAbs against BVDV were obtained, one of them showed positive reaction only to BVDV1 named BV1; another showed positive reaction only to BVDV2 named BV2; the third one could react with both BVDV1 and BVDV2 named BV12. Cross reaction tests suggested that both BV1 and BV2 were specific to BVDV1 and BVDV2 respectively, BV12 was also reactive with Hog chorera virus (HCV) besides BVDV1 and BVDV2. The ascites titers of BV1、BV2、BV12 were 105、106、105 respectively, and all of them had neutralization activity, the highest neutralization titer was 1：741455. The subtypes of BV1、BV2、BV12 were IgG1、IgG2a、IgG2a respectively, the light chain of all McAbs was ????chain. The chromosome configuration of the three hybridoma cells were102、99、100 respectively.

Based on the sequence of RAPD marker OPS03-1354 linked to anthracnose (Sphaceloma ampelinum) resistance gene, two oligomers, R1 (5'-CAGAGGTCCCTAGCTAAT-3') and R2 (5'-ACAATCACCCAACTC CTC-3'), were designed and synthesized, and used as special primer for PCR analysis among the parents and 51 F1 individuals from interspecific cross combination Vitis pseudoreticulata Baihe-35-1×V. vinifera Carignane. A DNA band about 1 100 bp could be amplified among resistant plants using primer R2, and was present in resistant plants of 339 F1 individuals from interspecific cross combination Guangxi-1 (V.pseudoreticulata)×Jingkejing (V.vinifera) and of 47 lines (varieties) of grape resources, including Chinese wild Vitis, American wild Vitis and European grape, by PCR analysis. The result showed that the DNA band about 1 100 bp was a sequence-characterized amplified region (SCAR) marker linked to anthracnose-resistance gene in grape. The plants with the SCAR marker were the carrier and expression of anthracnose-resistance gene. Cloning and sequencing of the SCAR marker were carried out. The length of the SCAR marker was actually 1 110 bp and it was named SCS03-1110. This research reveals that the oligomer R2 (5'-ACAATCACCCAACTCCTC-3') as a DNA probe can be used to identify anthracnose-resistance gene of grape germplasm resources and hybrids from anthracnose-resistance breeding program by PCR assay.

A survey was made into the European database on field trials of genetically modified crops during 1991~2008 in order to reveal the characteristcs and trend in research and development of genetically modified plants. Results indicated that totally 2 352 field trials have been undertaken in 22 European member states during the last 18 years, with 83 species of crops involved．The most frequent trials have been found on four crop species, i.e., corn （Zea mays L.）, oilseed rape （Brassica napus L.）, potato （Solanum tuberosum L.） and beet（Beta vulgaris L.), amounting for 74% of the total trials. Four member states, France, Spain, Italy and the United Kingdom in order are most frequent in the trials．In addition, a comparison in the research and development characteristics and trend of genetically modified plants of the European union was made with the United States, Canada and among the European member states. A reference view was provided to the development of genetically modified crops in China.