Copy number variation (CNV) is an important source of genetic variation complementary to single nucleotide polymorphism (SNP). It has been shown not only relating with disease susceptibility and developmental abnormality, and also with appearance characteristics and economically important traits. Combined using the SNP and CNV will provide new research ideas for dissecting the molecular mechanism and genetic basement of complex traits of livestock and poultry. Additionally, CNV would have broad application in marker assistant selection in breeding of livestock and poultry. Genome-wide CNV discovery approaches include array comparative genome hybridization (CGH), high dense SNP chips and newly developed genome re-sequence based on high-throughput sequencing technologies. Since 2008, using hybridization- based chip and sequencing-based technologies, a series of studies have been performed to construct CNV maps in every livestock and poultry. In the present article, we summarized the detection measures to identify genome-wide CNV, methods for genotyping the detected CNV, and the considerable advances of CNV in livestock and poultry. It will provide useful information for researchers engaged in the field.

When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. Because of those cells possessing multilineage differentiation potential, dedifferentiation has become a significant research topic in recent years. Here we firstly isolated pig mature adipocytes and indentified them by GENMED staining.Then, in order to clarify the mechanism of mature adipocyte dedifferentiation, a novel ceiling culture model was developed and the mRNA expression of marker genes during dedifferentiation couse was analysized using Real-time PCR. Finally, we assessed the effect of 1 μmol/L rosiglitazone on mature adipocyte dedifferentiation. The results showed that 98.7% of isolated cells were mononuclear mature adipocytes and these adipocytes could efficiently dedifferentiate into DFAT cells under a novel ceiling culture model. During the course of dedifferentiation, the mRNA levels of adipogenic marker peroxisome proliferator activated receptor γ (PPARγ), adipocyte-type fatty acid-binding protein (aP2) and lipoprotein lipase (LPL) were up-regulated by 8, 3 and 7.5 folds, respectively. While the lipolytic marker hormone-sensitive lipase (HSL) and Adipose triglyceride lipase (ATGL) were increased about 40 and 10 folds in mRNA level, respectively. In addition, the dedifferentiation was remarkably suppressed by treatment with 1 μmol/mL rosiglitazone. These results indicate that it is mainly a lipolytic course companying with little ability of adipogenesis that mature adipocytes dedifferentiate into DFAT cells, and this will provide new theoretical reference for future research.

To clarify the function of miR-128a during the differentiation of rat (Rattus norvegicus) preadipocytes, we detected the expression level of miR-128a during adipogenesis and clarified its expression tendency during differentiation; then we constructed an over-expression adenovirus vetor of pre-miR-128a and infected rat preadipocytes. Subsequently, the expression of adipogenesis marker peroxisome proliferator-activated receptor gamma(PPARγ) and adipocyte protein 2(aP2) were analysed by Real-time PCR and Western blot in mRNA and protein level, respectively. After the adenovirus vector infecting rat preadipocytes, we detected lipids accumulation in 10th day by Oil-red O staining, observing the adipogenesis in morphology. Results showed that the expression level of miR-128a was at the minimum point in the next day of the adipocyte differentiation, and then maintained at the level of day 0. After the over-expression of miR-128a, the expression levels of adipocyte marker genes PPARγ, aP2 and the number of lipid droplets had obviously declined, compared with that in control. The results showed that miR-128a could inhibit the differentiation of pre-adipocytes in rat. We suspected that PPARγ is a potential target gene of miR-128a according to the changes of mRNA and protein expressing levels observed after the treatment of adenovirus infection in rat pre-adipocytes. Also, this study supplies the basic theory for the further research about the function of miR-128a during the differentiation of adipocytes.

Plant trichomes are atrracting more and more attention for their effect on stress resistance. A F9 recombinant inbred lines population derived from the cross of wild hot pepper(Capsicum annuum L.) PM702 bearing trichomes with glabrous sweet pepper(C. annuum L.) FS871, was used for genetic analysis of trichome density on the main stem and leaves, using the mixed major gene plus polygene inheritance model. The results showed that trichome density on the main stem of pepper was controlled by two major genes and some minor polygenes(E-2-3 model). For the two genes, the additive effect was primary. The heritability of major genes for the trichome density on the main stem was 53.00%, while the heritability of polygenes was 25.30%, and the recombination rate was 0.6226. The trichome density on the leaves was controlled by two complementary major genes with additive effect and interaction plus some polygenes(E-1-7 model). The heritability of major genes of trichome density on the leaves was 50.65%, while the heritability of polygenes was 8.86%. This study provides an academic foundation for breeding highly resistant pepper varieties.

The liver plays an important role in the regulation of protein metabolism in body. The purpose of this research is to reveal the relationship between protein synthesis and secretion and liver protein metabolism in goats(Capra hircus) under different dietary patterns. Eight healthy mid-lactation goats were randomly divided into two groups, using 2×2 Latin square design with two replications. Dietary concentrate to forage ratios were 4∶6 and 6∶4. Milk samples were taken to measure milk yield and milk protein levels, and liver samples were collected by living donor liver biopsy. The differential expression proteins in the goat liver were extracted and separated by two-dimensional electrophoresis (2-DE) and MAIDI-TOF-TOF. The daily milk yield of high-concentrate group(6∶4 group)was significantly higher than that of the low-concentrate group(4∶6 group, P <0.05), but milk protein content was no significant difference between two groups (P>0.05). The results of proteomics showed that in high-concentrate group(6∶4 group), elongation factor Tu and glutamate dehydrogenase and adenosine homocysteine enzyme involved in protein metabolism had higher expression , while 26S proteasome regulatory subunit 7 and serum prealbumin involved in protein metabolism had low expression. The ability to synthesize small peptide and lactalbumin was decreased in the liver, while the catabolism of amino acids and the role of oxidative stress were enhanced. The liver metabolism had an important influence to milk protein synthesis and metabolism. It was not conducive to synthesis milk protein for lactation goats fed high-concentrate diets because the consumption of amino acids increased and the synthesis of small peptide decreased. So he raw material for milk protein synthetic was consumed too much and its mechanism will be worth further study. The results of this study provided a clue to research the liver impact of milk protein synthesis.

This experiment was designed to analyse the role of growth axis genes including growth hormore(GH), growth hormore receptor(GHR) and insulin-like growth factor 1(IGF-Ⅰ) during embryonic and post-hatch development in two duck breeds(Gaoyou ducks and Jinding ducks)(Anas platyrhynchos Domestica) with different growth rate. The expression level of GH gene in pituitary tissue and GHR and IGF-Ⅰgene in pectorale muscle and leg muscle were quantified by Real time-PCR on days 13, 17, 21, 25 and 27 of embryonic development, as well as at 7 day post-hatching. The results showed that the mRNA of all the three genes could be detected in the studied tissues in both duck breeds, and relatively similar expression profile was found in the same tissue for each gene. The expression level of GH gene in pituitary increased throughout the embryonic and post-hatch development, and the expression level in Gaoyou ducks was extremely significantly higher than that in Jinding ducks at 7 day post-hatching (P<0.01). In pectoral muscle and leg muscle, the differences of the GHR gene expression between the two breeds at the same development stage were not significant. Within the breed, the expression level of GHR gene in pectoral muscle was significantly lower than that in leg muscle on 13 and 21 embrynic development(P<0.05 or P<0.01), while the expression level of GHR gene in pectoral muscle was significantly higher than that in leg muscle at 25 and 27 embryonic age(P<0.05 or P<0.01). The expression level of IGF-Ⅰgene in pectoral muscle in Gaoyou ducks was higher than that in Jinding ducks except for 27 embryonic age, and the differences were significant on 13, 21 embryonic development and 7 day post-hatch(P<0.05 or P<0.01). The expression level of IGF-Ⅰgene in leg muscle of Gaoyou ducks was higher than that of Jinding ducks at different development stage, but the difference was not significant(P>0.05). Within the same breed, the expression level of IGF-Ⅰgene in pectoral muscle was significantly higher than that in leg muscle except for 13 embryonic age(P<0.05 or P<0.01). In conclusion, these three genes may have an important roles in the early growth and development of ducks, and they have evidently spationtemporal expression pattern, meanwhile, the expression of GH and IGF-Ⅰ genes is also affected by breed.

According to our previous research, there was an obvious relationship between the early death of chicken(Gallus gallus)-quail(Coturnix coturnix) hybrids and sex differentiation. Meanwhile, current widespread adapted methods to indentify sex differentiation stayed at RNA level, experimental steps complicated and easy to make mistakes, and RNA samples, which are needed to be measured, was quite difficult to preserved longer, but could not be simply employed. So it is necessary to find a more simple and accurate way to identify the early embryo's sexing. In this study, there were two sections for CHD (chromobox-helicase-DNA binding) gene DNA level sex determination, at first,a total of 116 chicken-quail hybrid embryos at different incubate stages (2.5~5 d) was treated as experiment group and 10 mg embryonic organs were used to DNA extraction; then the DNA extraction from blood of 60 sex-known quails (male and female were half-and-half) was regarded as control. Wpkci (W-linked protein kinase C inhibitor) for mRNA level was known as a mature method to identify sexing, and then it is used to check the result of embryos' sex determination in our research. The result showed that CHD 2550F/2718R could identify the sex of chicken-quail hybrid embryos accurately. The amplication size of male embryo tissues was 613 bp and two bands in female were 613 and 446 bp, respectively. The experimental results provide basic data for the chicken-quail hybrids sex determination mechanism.

Stearoyl-CoA desaturase(SCD) is the rate-limiting enzyme of mono-unsaturated fatty acids synthesis. To investigate the transcriptional regulation of bovine SCD gene, 4 fragment (212, 380, 416 and 760 bp) of promoter of bovine SCD were amplified from the Holstein bovine tissue by PCR using primers containing a recognition site. The potential transcriptional biding sites were predicted with bioinformatics method. Promoter fragments were cloned and ligated with a promoter luciferase reporter vector PGL3-basic. Both fragments and vectors were digested with KpnⅠ and XhoⅠ and constructed recombinant vectors pGL3-SCD1, pGL3-SCD2, pGL3-SCD3 and pGL3-SCD4.The promoter activities were determined in transiently transfected HepG2 cell treating with Insulin(10 IU/mL)and linoleic acid(120 μm) by the dual-luciferase assay system. The results indicated that binding sites for transcription factors of bovine sterol-regulated element-binding protein were present in the bovine SCD promoter gene. There was a significantly promoter activity increase with the enlargement of the SCD promoter vector length and pGL3-SCD4 showed the highest level of promoter activity. Compared with the control group, supplementing with insulin significantly increased the promoter activity of pGL3-SCD1~4 vectors in HepG2 cell (P<0.05) and the relative activity of pGL3-SCD4 was the highest of 46.93 in the four vectors. The promoter activities of pGL3-SCD1~3 were not significantly affected by linoleic acid and the activity of pGL3-SCD4 was reduced (P<0.05) with the linoleic acid treatment.These results demonstrated that the region from -398 to -742 bp of promoter pGL3-SCD4 was important for the regulation of SCD gene expression.

It has been clear that silkworm (Bombyx mori) 30K protein in larval hemolymph has the highest apoptosis inhibition activities. As one of the member of 30K apolipoproteins family, Bm30Kc6 has been reported to have obvious anti-apoptotic activity, but the anti-apoptotic mechanism was still not very clear. In this study, the silkworm BmN cells were infected with recombinant virus Bacmid-Bm30Kc6 constructed by our lab and the expressed proteins were purified by a Ni-NTA agarose affinity filler. SDS-PAGE and Western blot results showed that the Bm30Kc6 proteins were highly purified. Then BmN cells were treated with different concentrations of hydrogen peroxide (0.1, 1, 5 and 10 mmol/L) to induce cell apoptosis, the results showed that a final concentration of 5 mmol/L H2O2 incubated with BmN cells for 4 h could be successfully induced cell apoptosis. Based on this cell damage model, the anti-apoptotic mechanism of Bm30Kc6 was studied. Firstly, cell death detection ELISA kit was used to evaluate the cell apoptosis, the results showed that 5 μg/mL Bm30Kc6 could remarkably slow down DNA fragmentation in BmN cells. Then cell proliferation ELISA kit was used to test the cell viability, the results showed that Bm30Kc6 could also enhance the viability of BmN cells obviously. Moreover, 8-isoprostane EIA kit was used to test the concentration of 8-isoprostane inside BmN cells, the results showed that H2O2 treatment could enhance the formation of 8-isoprostane obviously, however, Bm30Kc6 protein could decrease the concentration of 8-isoprostane obviously. Finally, Western blot results showed that Bm30Kc6 could inhibited the H2O2-induced cytochrome c released from mitochondria to cytoplasm inside BmN cells. In conclusion, Bm30Kc6 may inhibit BmN cell apoptosis by decreasing the level of oxidative stress inside cells and inhibit cell apoptosis by blocking the mitochondrial signaling pathway. This study will provide basic information for further study on the anti-apototic mechanism of Bm30Kc6 portein.

In order to obtain a Brucella vaccine candidate which can distinguish between natural infection and attenuated vaccine, this paper studied the phosphoglucomutase gene (pgm) influence on Brucella M5-90 vaccine strain virulence and immune evaluation. The recombinant plasmid pGEM-7zf-Δpgm was constructed, and which was electroporated into Brucella melitensis M5-90 competent cells, screening for Brucella vaccine strain M5-90 pgm gene deletion mutant strains (Δpgm) and to detect the M5-90 Δpgm strain genetic stability, and immunogenicity. The test results showed that the reverse mutation did not occur within 15 passages; the Δpgm antibody content and the parent strain M5-90 group had no significant difference; Δpgm had the ability to produce the IL-2 was stronger than that of M5-90, the ability to produce INF-γ was lower than the M5-90; the rose bengal plate agglutination test and tube agglutination test of this gene deletion strains was not agglutinated; the results of Western blot confirmed that the Δpgm could not induce the mice to produce anti-pgm protein antibody. The Δpgm constructed in this study, had good genetic stability and immunogenicity, provided basic data for the next step to more in-depth develop Brucella gene-deleted vaccine.

In order to investigate the molecular evolutionary characteristics of Porcine circovirus type 2 (PCV2) in Zhejiang and surrounding areas. One hundred and eighty field samples with mixed tissues(spleen, lung and lymph node) from suspect PCV2-infected pigs during 2007~2012 were detected for the PCV2 by the established PCR method, and T-A clone and sequencing were used to get the complete genome of PCV2. Seventy-four cases out of 180 diseased pigs from these herds were PCV2 positive. PCV2-positve rate between 2007 and 2012 (41.7%, 52.0%, 44.1%, 37.0%, 40.5% and 33.3%, respectively) remained in the same level that during 2004~2006. Forty-five PCV2 complete genomes belonged to subgroups 1A (23.9%), 1B (21.7%), 1C (47.8%) and Group 2 (0%), respectively. Group 1 was still the dominant genotype of PCV2 in Zhejiang and surrounding areas. However, as the prevalence of PCV2 in this region, virus belonged to subgroup 1C appeared, increased and gradually dominated. The Cap variability of PCV2 isolates was analyzed in this study, and the result contributed to better grasp the homology of the strains in the detected area.

Wheat(Triticum aestivum L.) as the main food crop, studying its utilization of heterosis and male sterility is the matter that has been tried to overcome in the world. To deeply research the mechanism of the physiological male sterility of wheat, the relationship between the ubiquitination of histones and the physiological male sterility was studied, which based on the research front of histone modifications. Used physiologically male sterility induced with chemical hybridizing agent SQ-1 and corresponding normal lines of complete fertility as experimental materials in wheat, investigated the rate of the physiological male sterility as well as their pistil fertility. The histones were extracted from the two lines' flowers. The ubiquitination levels of the histones were monitored with Western blot, and then cloned the cDNA functional sequence of RAD6, which was identified as an ubiquitin-conjugating enzyme of histone H2B. Bioinformatics technique was applied to analyze RAD6 such as sequence multiplication, constructed a phylogenetic tree, forecasted secondary structure and 3D structure of RAD6. Lastly, qRT-PCR was performed to analyze the expression of the RAD6 gene. The results showed that the chemical hybridizing agent SQ-1 had a sound effect on mile-killing. The ubiquitination level of histone H3 was almost the same between the male sterile and fertile lines. During mononuclear period, the ubiquitination of histone H2A was seemed to be existing in fertility, but disappearing in the sterility ones; the ubiquitination level of histone H2B in physiological male sterile wheat was seemed to be significantly less than that in fertile ones, meanwhile, the expression of the RAD6 gene in sterility wheats was apparently suppressed. These data indicated that SQ-1 distinctly restrained the ubiquitination levels of histones H2A, H2B as well as the expression of the RAD6 gene in the mononuclear period, which may be influencing the translation of some key genes and evently result in physiological male sterility in wheat. Thus, it can be seen that there is a close relationship between the ubiquitination of the histones on chromosomes and the physiological male sterility induced by the chemical hybridizing agent SQ-1 in wheat. The research supplies scientific evidence for the ubiquitination of histones involve in the wheat physiological male sterility, meanwhile, it establishes the solid epigenetics study foundation for wheat physiological male sterility mechanism.

Doxycyline(DOX)-inducible expression systems provide strict control over transgene expression spatially as well as temporally to avoid non-specific expression. Moreover DOX has no cytotoxic effects to the cells and animals, so it is used widely in transgenic research. Ptet-on system is more sensitive to DOX and yields lower background expression than that of the other inducible systems. For realizing the controllable expression of exogenous growth hormone (GH) gene at a specific time and space, we constructed inducible expression vector about tetracycline (TET-ON) system, and did a further study for testing its inducible activity at cellular level. The results showed that: (1) we have successfully constructed vector about pTRE-GH12; (2) we have harvested GH cells which had high inducible expression efficiency; (3)after induced by using DOX in the positive cells, we could get detection of the expression of exogenous GH, QRT-PCR results showed that the induction activity after using DOX was 264.481 times as before; there were no change in control group cells using or no using DOX , the differences between experimental group and control group were significantly different (P<0.01), so as the induction of DOX before and after in experimental group (P<0.01); (4)the results of Western blot were consisted with the result of QRT-PCR , the quantity of GH protein between the experimental group and control group cell showed significant differences (P<0.01), so as the experimental group before and after using DOX (P<0.01). All of our research results indicated: we have realized controllable expression of targeted gene pig growth hormone (pGH) by using tetracycline (TET-ON) inducible expression system, so as to provide a technological basis for preparing transgenic animals which can express GH inducible later.

Because Bt Cry 1Aa, Cry 1Ab and Cry 1Ac share high sequence identity(82%~90%), it is difficult to prepare Bt Cry1Ab monoclonal antibody that has highly specific by using conventional method. In order to prepare monoclonal antibody against Bt Cry1Ab, we acquired the amino acid sequence of Bt Cry1Ab protein from NCBI. According to the antigenicity, hydrophilicity and accessibility analyzed results by ANTHEPORT and DNAStar computer software, the specific peptide of Bt Cry1Ab was synthesized by a chemical process. The Bt Cry1Ab peptide was linked with carrier keyhole limpet hemocyanin(KHL), and then used for immunized mouse as antigen. Twenty-two strains of hybridoma, which produced monoclonal antibodies to the peptide, had been obtained by cell fusion technique. One hybridoma strain(3A10), which was specific to natural Bt Cry1Ab selected by indirect ELISA. The result of isotype analysis showed that the monoclonal antibody of 3A10 hybridoma strain belong to IgG1 subclass and the light chain was κ chain. The hybridoma chromosome number was 89~108. The monoclonal antibody of hybridoma ascites titers to synthetic peptide was 1∶1×107 and to natural Bt Cry1Ab protein was 1∶1×104. The purified MAB titers to synthetic peptide was 1∶1×108 and to natural Bt Cry1Ab protein was 1∶2×104. The relative affinity of the monoclonal antibody was 0.5 μg/mL. The monoclonal antibody of hybridoma strain could react specifically with the Bt Cry1Ab protein without reacting with Bt Cry1Ac and Bt Cry1Aa proteins by ELISA. The lowest detectable value of the monoclonal antibody to Bt Cry1Ab was 10 ng/mL by indirect ELISA detection. The monoclonal antibody of hybridoma strain can distinguish transgenic Bt CrylAb cotton and general cotton(Gossypium hirsutum L.) by ELISA, and it can recognize specifically Bt Cry1Ab protein in cotton.

Viral diseases cause adverse effects on the yield and quality of lilies grown in China. However, we still can't detect and eliminate these viruses effectively. The viral detection methods used these days are either time-consuming and expensive or sometime provide false-negative results occasionally. On the other hand, difficulties in operation mechanisms and low virus-free rate are the major constraints restricting the development of virus elimination techniques. Addressing these limitations, we carried out this study to establish effective ways to detect and eliminate three major viruses in lilies namely, Cucumber mosaic virus(CMV), Lily symptomless virus(LSV) and Lily mottle virus(LMoV). In vitro plantlets of Lilium longiflorum cv. Raizan 1 were used as subject material. Specific primer pairs were designed according to the gene sequence of the three viruses and 18S rRNA, and used for multiplex RT-PCR. Several reaction components (dNTPs, Mg2+ and Taq DNA polymerase), annealing temperature and cycle numbers were optimized to conduct multiplex RT-PCR effectively. Among couple of used reaction systems, the most effective and precise reaction system was as follow: dNTPs(2.5 mmol/L) 1.5 μL, Mg2+(25 mmol/L) 1.5 μL, Buffer 1.875 μL, cDNA 1 μL, Taq DNA polymerase 0.2 μL and each primer 0.5 μL, adding H2O to a final volume of 25 μL. The optimal reaction procedure wasas follow: 94℃ denaturation temperature for 5 min;94℃ for 30 s,52.4℃ annealing temperature for 30 s,extension phase with 72℃ for 30 s,30 PCR cycles;72℃ for 10 min,and kept at 4℃ to terminate the reaction. Thus, we established an efficient method to detect the three viruses simultaneously. The 18S rRNA was used as an internal control to avoid false-negative during the procedure. Heat treatment (38℃ for 14 h, 32℃ for 10 h, all in dark) combining with bulblet culture could eliminate more than 90% of the CMV in lilies, but remains ineffective in eliminating LMoV or LSV. After heat treatment, shoot tips were cut with a different size range of ≤1 mm, 1~2 mm and ≥2 mm respectively and culture in light or dark conditions. The results illustrated that culturing shoot tips of ≤1 mm size under light condition was the most effective way to eliminate all the three viral strains. High virus -free rate of 93.3% was observed in this method. Additionally, short culture cycles, easy handling and enhanced virus-elimination rate makes it an ideal method for producing virus-free lilies.

A specific, sensitive and rapid TaqMan fluorescence quantitative PCR was established for testing Rabbit Bordetella bronchiseptica. In present study, a pair of primers and probes were designed from target gene of virulence factors CyaA of Bordetella bronchiseptica. Amplified PCR product was cloned and sequenced, the results showed that the homology was 100% compared with the reference sequence published in GenBank. The positive recombinant plasmids were served as quantitative detection of standards to establish standard curve. The detectable quantity of Bordetella bronchiseptica genomic DNA was 0.32 pg, which was 25 times sensitivity compared with common PCR, there was no cross reaction with common clinical bacteria by TaqMan fluorescence quantitative PCR. The 45 suspected samples were detected by TaqMan fluorescence quantitative PCR or routine PCR. The positive detection rates were 75.5% and 66.7%, respectively, the coincidence rate was 88.2%. The results showed that the TaqMan fluorescent quantitative Rabbit Bordetella bronchiseptica detection method was specificity, sensitivity and reproducibility. The present study provides an effective sensitive and reproducible method to diagnose and control Rabbit Bordetella bronchiseptica in further research.