This study was to express the fusion protein of the culture filter protein 10 (CFP10) and earlier secreted antigen target 6 (ESAT6) of Mycobacterium tuberculosis in the yeast Pichia pastoris system and to evaluate its potential in bovine tuberculosis (bTB) detection. The fusion fragment cfp10-esat6 was amplified by PCR and constructed into the recombinant plasmid pPICZαA-(cfp10-esat6), which was transformed into the yeast P. pastoris GS115 by high voltage electroporation. The positive recombinant yeast was induced for 3 d in the culture medium containing 1% methanol. The fusion protein of CFP10-ESAT6 (eCE) was analyzed by SDS-PAGE and then purified by nickel ions affinity chromatography column which targets the histidine (His) tag. Immunological reactivity of eCE was also appraised by Western blot. Furthermore, the application potential of eCE in bTB detection was evaluated via using it as stimulator in bovine peripheral blood interferon gamma release test in vitro (IFN-γ release test). The results showed that eCE successfully expressed in the recombinant yeast and secreted into the culture supernatant after induced by methanol. Results of Western blot demonstrated that only one specific band appeared when the fusion protein reacted with monoclonal antibodies of CFP10, ESAT6, His tag and c-Myc proteins, respectively, indicating that eCE proved an efficacious immunological reactivity. In the IFN-γ release test for 165 cow blood samples, eCE had a positive coincidence rate of 82.3% as well as a negative coincidence rate of 78.8% compared with purified protein derivative (PPD) used as stimulator in bTB detection. In summary, eCE expressed successfully in the yeast P. pastoris system and exhibited an excellent biological activity. It can be used as stimulator like PPD in IFN-γ release test for the diagnosis of bTB, as well as improving the sensitivity and specificity of the test.

Transgenic technology is one of the biotechnologies with great application prospect. The development of transgenic animals has last for 30 years. Transgenic animals and their products must be strictly evaluated for biosafety before entering into the food chain. Food safety evaluation is one of the most important parts. Based on the literatures and our reseach, this review focused on the status and development trend of food safety evaluation in transgenic animals and related products. This may contribute for the transgenic food safety evaluation system of China.

The chromosome sex determining mechanism of half-smooth tongue sole (Cynoglossus semilaevis) is heteromorphism in female gamete, and the large amount of repetitive sequences, W chromosome contains, impede the accurate assembly of W chromosome sequence, which can only be solved by physical map effectively, at present. Based on the available physical map and genetic map, physical location of sex linked markers was performed in this study using the strategies of 3D(three-dimensional)-BAC(bacterial artificial chromosome) pool construction and PCR screening. Firstly, the BAC clones of 44 384-well microtiter plates were inoculated and precultivated with new 384-well plates, and then inoculated with four 96 deep-well plates. The plate, row and column pools were obtained through pooling the BAC clone cultures of each two 384-well microtiter plates, and then DNA were extracted and the super pools were obtained by mixing some DNA of corresponding plate pools. Eventually, 3D-BAC pools including 22 BAC super pools and 440 corresponding matrix pools were constructed, which represented 4.2 genome coverage of C. semilaevis. Only 24 reactions (20 BAC matrix pools and 4 controls) were needed to complete the screening on matrix pools of a positive super pool. Then two-step PCR screening was performed on these pools with 159 sex-linked markers, and 142 markers obtained positive amplifications in the screening of super pools. Finally, a total of 100 markers were accurately located in the screening of matrix pools of these positive super pools, and they were located in 84 contigs and 20 singletons of the C. semilaevis physical map, which included a total of 1 760 clones and 21 927 consensus bands. Their physical length was 42.4 Mb, covering 5.3% of the genome of C. semilaevis. On average, 2.2 clones were used to determine a location relationship between a contig and a marker. Among these markers, 17 markers were simultaneously located in 2 or more than 2 contigs, and 11 markers were simultaneously located in contigs and singletons. In addition, 19 contigs were simultaneously located with 2 or more markers. The physical location of these sex-linked markers can promote accurate assembly of sex chromosomes sequences of C. semilaevis.

To obtain scientific data for the healthful aquaculture of crisp grass carp(Ctenopharyngodon idellus), intestinal microflora composition, serum enzyme and growth performance of grass carp at different stages of feeding broad bean(Vicia faba) were examined to assess the physiological and biochemical changes during the transformation from grass carp to crisp grass carp. During the different crisping stages (30, 60, 90 and 120 d) and the last stage of crisp grass carp taking artificial feed for 30 d(the 150 d), the changes in the composition of intestinal microflora, activity of serum enzyme and growth performance were analyzed. The results showed that, on 30 d, there was no significant difference of average weight between broad bean group and control group (P＞0.05); 60~150 d, the average weight of grass carp feeding broad bean were lower than that of control group (P＜0.05, P＜0.01). On 30 and 60 d, the head kidney somatic index(HKBI) and hepatosomatic index(HSI) of broad bean group were not significantly different compared with the control group (P＞0.05), which all lower than those of the control group (P＜0.05 or P＜0.01) of 90~120 d; There was no significant difference of HSI between broad bean group and control group (P＞0.05) of 150 d. The activities of serum alkaline phosphatase, lysozyme and complement 3(C3)(except that of 30 d) of broad bean group were all lower than those of control group, but the activities of 3 enzymes rised after feeding crisp grass carp with artificial diet. Compared with the control group, the diversity of intestinal microflora of broad bean group decreased; The number of probiotics species including Cetobacterium, Bacillus and Eubacterium was diminishing and the number of opportunistic pathogens including Aeromonas veronii, A. sobria and Vibrio anguillarum increased in broad bean group. In addition, Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria were the dominant intestinal microflora of both groups. The results demonstrated that, with the increase of crisping time, broad bean gradually changed the composition of intestinal microflora and inhibited the activities of 3 serum enzymes and growth performance of grass carp; Composition of intestinal microflora and enzymes activities were found to be a recovering trend after feeding crisp grass carp with artificial diet for 30 d. It is presumed that the inhibited factors for the growth and immunity of crisp grass carp were probably the anti-nutritional substances from broad bean. This research will provide scientific data for the healthful aquaculture of crisp grass carp.

MYB gene family are important transcription factors involving in plant responses to biotic and abiotic stresses by controlling the expression of diverse functional genes. Utilizing genomic sequences of diploid Gossypium raimondii, a new cotton MYB transcription factor gene GhMYB11 (GenBank accession No. HQ234875.1) was cloned from upland cotton(G. hirsutum) cultivar Lumianyan 32. The full length of GhMYB11 cDNA was 1 001 bp coding an open reading frame (ORF) of 828 bp. There were 11 SNPs (single nucleotide polymorphism) between the ORF sequences of GhMYB11 and G. raimondii 001G177100.1, of which 7 SNPs caused amino acid alternation. Phylogenetic analysis showed that GhMYB11 was highly similar (E value=7e-83 and 1e-90, respectively) to AtMYB13/14, which were 2 important factors involving in ABA synthesis and signal transduction in Arabidopsis thaliana, with 2 highly conservative MYB DNA binding domains R2R3 and a transactivation domain. Real-time quantitative PCR analysis revealed that after Verticillium dahliae strain VD8 inoculated, GhMYB11 expression of cotton seedlings very significantly up-regulated by 14.49 times(P＜0.01) in cotton leaves and by 5~6 times significantly(P＜0.05) after 10% PEG6000, 150 mmol/L NaCl and 200 mmol/L H2O2 treated, respectively. The result suggested that GhMYB11 maight play a key role in cotton responding to biotic and abiotic stresses and could be treated as an important candidate gene in genetic improvement for stress resistance of upland cotton cultivars. This work provides basic materials for cotton stress tolerance enhancement through genetic engineering.

Ascorbate peroxidase (APX) is one of the important enzymes which can remove the reactive oxygen species (ROS) in the plant. In this study, APX activity in sugarcane (Saccharum officinarum) smut resistant variety (Yacheng05-179) was significantly higher (P＜0.05) than that in the susceptible one (Liucheng03-182) after inoculated with Sporisorium scitamineum within 48 h. ScAPX (GenBank accession No. KJ7565501) was separated from sugarcane with cloning and RT-PCR technologies. The bioinformatic analysis showed that the total length of ScAPX gene was 1 171 bp, which contained a complete open reading frame (1 038 bp) and encoded 345 amino acids. The ScAPX contained no signal peptide and belonged to nonsecretory protein which was likely located in the matrix of mitochondrial (91.1%) and chloroplast (88.7%). The results of tissue specificity analysis showed that the highest expression level of ScAPX was in stem skin which was 19.7 times of that in leaves. The transcript of ScAPX increased under the stresses of salicylic acid (SA), methyl jasmonate (MeJA), hydrogen peroxide (H2O2), abscisic acid (ABA), sodium chloride (NaCl) and polyethylene glycol (PEG). The peak of the ScAPX transcript under the stresses of SA, MeJA and H2O2 was accumulated during the initial period, which was earlierly found than that under ABA, NaCl and PEG stresses, and then it declined gradually. The expression pattern of ScAPX in response to ABA, NaCl and PEG treatments was not clear after 24 h, while the transcripts were kept in the peak at 24 h. Though the gene expression under the exogenous stresses were different, it was undoubted that ScAPX was positive response to the external stress. This study provides the basis for further functional analysis and application of this gene in the future.

Cytochrome P450s are one of the largest enzyme protein families in higher plants and widely involve in secondary metabolism and stress tolerance. In this study, a CYP81 homologous gene FtP450-R4(GenBank Accession No. KM271986) was obtained from Fagopyrum tataricum. The full-length of FtP450-R4 cDNA contained a 5'-UTR (49 bp), a 3'-UTR (194 bp) and an ORF of 1 527 bp. It encoded a protein of 508 amino acid residues. Bioinformatics analysis showed that the FtP450-R4 was located in endoplasmic reticulum by the 4~24 amino acid residues in the N-terminal, and it shared the homology of 44%~46% with F3'H, I2'H, and other plants CYP450 proteins. Sequence multi-alignment showed that FtP450-R4 contained the classic motifs and conserved regions of CYP450s, but excluded the feature motifs of GGEK in F3′H. Phylogenetic tree showed that FtP450-R4, Arabidopsis thaliana CYP81 families and I2′H were gathered in a large cluster, suggesting that FtP450-R4 might involve in flavonoids hydroxylation or stress response. FtP450-R4 could be significantly induced in cotyledons by UV-B, cold and drought stress, but not in hypocotyls. The recombinant protein of FtP450-R4 expressed in a soluble form in Escherichia coli strain BL21(DE3) and activity identification showed that it could perform enzyme catalysis using reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) and kaempferol as substrates. Totally, this work can make better understand for the function of CYP450s and provides fundamental information for the flavonoids regulation mechanism in F. tataricum.

To understand the genetic diversity of barley(Hordeum vulgare) and identify SSR markers associated with resistance to barley leaf stripe, 100 pairs of SSR markers were used to detect the diversity of 86 barley entries. A total of 269 alleles were detected with an average alleles per locus of 2.69 and a range of 2~5. The allele frequency varied from 0.012 to 0.988, and the Shannon's index varied from 0.063 to 1.385. The genetic similarity ranged from 0.542 to 0.965. The polymorphism information content (PIC) value ranged from 0.023 to 0.737, with an average of 0.357. The barley entries were divided into 5 subgroups based on their population structure. Five markers were found to associate with barley leaf stripe resistance under general linear model(GLM)program, and the rate of phenotypic variation explained ranged from 6.20% to 11.15%. Two SSR markers, TACMD and MGB317, showed a significant association with resistance to barley leaf stripe (P＜0.01), which explained 9.24% and 9.45% of the phenotypic variation. Three markers were associated with resistance to barley leaf stripe under mixed linear model(MLM) programe, the rate of phenotype variation explained ranged from 4.57% to 17.63%. This study provides some theoretical basis to barley leaf stripe resistant breeding.

Codon usage bias has been documented in a wide diversity of species. The relative contributions of mutation and various forms of natural selection on codon usage bias are different. Plant specific WRKY transcription factors play important roles in transcriptional regulation and signal transduction. However, systematical analysis on codon usage bias of Medicago truncatula WRKY (MtWRKY) genes has not been reported. In the present study, a systematic examination of codon usage for MtWRKY genes was carried out. The results showed that all MtWRKY genes were below the high effective number of codons(ENC) standard curve, suggesting that other factors independent of nucleotide composition had affected codon usage bias. Neutrality plots (GC12 vs. GC3) were used to analyze the relationships among the 3 codon positions. There was a significant positive correlation (r=0.34, P＜0.01) between GC12 and GC3 codons of MtWRKY genes, indicating that GC mutational bias led to similar GC content in all codon positions. Moreover, GC content in MtWRKY genes showed a wide range of GC3s values (0.2~0.5), indicating that mutational pressure was the main factor in shaping codon usage. The CT contents were higher than that of GA on the 3rd position of codons according to parity rule 2 analysis. GC or AT were used disproportionately, with C and T used more frequently than G and A in the 3rd position of codon in MtWRKY genes. This result indicated that natural selection contributed to MtWRKY codon usage bias, but mutational bias was the major influence on codon usage. Correspondence between frequency of optimal codons (Fop) and GC content analysis showed that there was a significant positive correction between Fop and GC content of exon (r=0.57, P＜0.01) and weak positive correction between Fop and GC content of intron (r=0.09, P＞0.05). There was a significant positive correction between Fop and length of exon (r=0.28, P＜0.05), and a negative correction between Fop and length of intron (r=-0.01, P＞0.05). These results indicated that mutation contributed to codon usage bias of exon, while natural selection was the main factor that shaped the intron sequences. This study identified 4 optimal codons, on which the 3rd position exclusively used G or C. All together, these results provide important information for codon optimization on transgenic studies of WRKY genes in the future.

Mitofusin 2(Mfn2) participates in apoptosis and cell cycle regulation, which also has an important role in oocyte and embryo development. In order to determine Mfn2 gene effect on mouse(Mus musculus) oocytes during in vitro maturation(IVM) in the present study, firstly, the Mfn2 gene was cloned from mouse ovary by RT-PCR, and an eukaryotic expression vector pMfn2-Venus was constructed. pMfn2-Venus was transfected into 293T cells(virus pakge cell) after mediation by Lipofectamine 2000, identified by fluorescence microscopy observation and Real-time PCR. Then, the cRNA of Mfn2-Venus transcriped in vitro was microinjected into mouse oocytes, and the expression and location was observed under fluorescence microscopy. Finally, the oocytes which were injected Mfn2-Venus cRNA were collected for mature cultivation and the rate of germinal vesicle break down(GVBD) and first polarbody(PB1) were calculated, respectively. The results showed that the recombinant vector pMfn2-Venus was successfully constructed. After transfection or microinjection, the fusion protein could express efficiently and localize accurately both in 293T cells and mouse oocytes. This greatly facilitated the further study of Mfn2, especially its role on oocytes maturation and early embryo development. The rate of GVBD and PB1 were reduced significantly(P＜0.0001, P＜0.05) compared with the that of control group. In conclusion, these data demonstrated that Mfn2 gene had effect on mouse oocytes maturation, it provides a new direction in the research of in vitro maturation of oocytes and a platform for the study of the Mfn2 gene in the process of meiosis.

microRNA (miRNA) is a class of endogenous and noncoding RNA, which involves in many physiological processes including cell growth, development, differentiation, proliferation and apoptosis. In order to study the regulation effect of miR-let-7a on growth and development in thyroid, we identified the thyroid tissue and culuture cells in vitro of Yorkshire pig(Sus scrofa) by immunofluorescence technique and detected the expression of miR-let-7a in various growth stages in thyroid of Yorkshire pig by quantitative Real-time PCR(qRT-PCR). To study the effect of miR-let-7a on apoptosis of thyroid cells, miR-let-7a transfected mimics cells were detected to determine the differences of thyroid cell apoptosis rates between the experimental and control groups with Flow Cytometry, and predicted that the target genes of miR-let-7a were related to cell apoptosis. The results showed that thyroglobulin (TG) in thyroid tissue and cells glowed green fluorescence after identification which proved thyroid cells in vitro normal function. In the embryonic period , the expression of miR-let-7a increased gradually with the rise of embryo age. In the growth period, the expression reached the minimum at the 60th day and then increased, but after the 90th day it decreased again and lasted to the 120th day and then increased slowly. Compared with control group, the apoptosis rate of transfected group significantly increased (P＜0.05). In conclusion, suggested that the changes in miR-let-7a expression might affect the regulation on thyroid cell apoptosis directly or indirectly. The study provides useful clues for further research on the functional mechanism of miR-let-7a in pigs.

Cell death-inducing DFFA-like effector c (CIDEC) is related closly to adipose differentiation and promoting fat deposition. In this study, sheep (Ovis aires) CIDEC cDNA was cloned by PCR and analyzed by bioinformatics. Expression profiles of CIDEC in different tissues of sheep were detected by semi-quantitative RT-PCR. The expression levels of CIDEC in tail fat tissue after persistent starvation in Altay sheep was determined by Real-time quantitative PCR (qRT-PCR). The result showed that the coding sequences (CDS) of sheep CIDEC gene was 714 bp and encoded 237 amino acids (GenBank accession No.KM199684). Bioinformatic analysis indicated that multiple phosphorylation and protein kinase C sites existed in the secondary structure of sheep CIDEC protein. The RT-PCR results confirmed that CIDEC mRNA expressed in adipose tissue only, and the expression level was much higher in tail fat than that in esenteric fat. After persistent starvation and fasting for 4 weeks, the expression of CIDEC in tail fat of Altay sheep decreased sharply, its level was very significantly lower than that in normal food-intake sheep (P＜0.01). Taken together, these results demonstrated that CIDEC might play an important role in the process of tail fat deposition. In conclusion, this work serves as the basis for further study on CIDEC in the tail fat of sheep.

Protein phosphatase 3 catalytic A (PPP3CA) is a major isozyme of PPP3C in skeletal muscle and plays an important role in myofiber differentiation. In the present study, expression of PPP3CA mRNA was quantified by absolute quantitative RT-PCR in the pectorale and leg muscle tissues from Jinding ducks and Gaoyou ducks (Anas platyrhynchos domestica) differing in growth rates on days 13, 17, 21, 25, 27 of embryonic development and 7 days post-hatching (PH). The results showed that there was a significant variation with the age of ducks in the PPP3CA mRNA expression profile of the same muscle tissue in 2 duck breeds, but there was no significant breed and sex effect. In both pectoral and leg muscles, the expression peak of PPP3CA mRNA appeared at 13 embryonic day (E13 d) and the PPP3CA mRNA expression level at 7 d PH was very significantly higher than that at E27 d (before hatching) (P＜0.01). In pectoral muscles, the expression of PPP3CA mRNA was firstly declined from E13 d to E21 d, then increased from E21 d to 7 d PH, and the lowest point appeared at E21 d which was very significantly lower than that at other embryonic age or day-old (P＜0.01). In leg muscles, the expression of PPP3CA mRNA was significantly declined from E13 d to E17 d, then significantly increased to E21 d, then decreased gradually to E27 d, and finally increased to 7 d PH; the lowest point appeared at E27 d which was very significantly lower than that at other embryonic ages or day-old (P＜0.01), and the expression level was not significantly different among E17 d, E25 d and 7 d PH (P＞0.05). The correlation analysis showed that varying degrees of linear correlation were found between the expression of PPP3CA mRNA in leg muscles and myofiber types, diameter, cross-sectional area and density the data were detected in former studies in 2 duck breeds; the expression levels of PPP3CA mRNA in pectoral and leg muscles of 2 duck breeds were significantly positively correlated with IGF-Ⅰ mRNA expression which was detected by us in former studies (P＜0.05 or P＜0.01). These results suggested that PPP3 might have potential functions in controlling myofiber phenotype and development during embryonic and early post-hatch development in ducks and there was a synergistic effect between PPP3 and IGF-Ⅰ which might be involved in the regulation of growth development of duck skeletal muscle. The above results provide some valuable clues for understanding the role of PPP3 in the early development of muscles in ducks and a theoretical basis for improving the meat quality in ducks.

Cytochrome P450 enzymes (CYPs) are involved in many physiological functions in insects, such as the metabolism of signal molecules, adaption to host plants and insecticide resistance. Species of Lepidoptera, which include the most disruptive agricultural pests, rank only secondly to those of Coleoptera. On basis of the draft genome sequence of lepidopteran insect, Manduca sexta L., a genome-wide analysis of CYPs was performed and 110 CYP-related sequences were obtained, which could be classified into 29 families according to standard nomenclature, a lot of which were tandemly arranged on chromosomes. We furtherly compared the CYP-related sequences with those of monarch butterfly (Danaus plexippus L.) and the model insect silkworm (Bombyx mori L.). The result revealed that there were 12 pairs of orthologs in CYP2 and mitochondrial clans, whereas many CYPs in CYP3 and CYP4 clans were present with species-speci?c expansions. There were more orthologous CYP pairs among lepidopteran insects in comparsion with those between lepidopteran insects and the other insect orders. Many orthologous groups of paralogous genes could be also distinguished from the phylogenetic tree in spite that a high frequency of species-specific expansion, relatively few different CYP families and subfamiles were represented among different lepidopteran insects. Extensive synteny of CYPs was also maintained in the different lepidopteran insect genomes. The insights obtained from comparative genomic analysis will greatly facilitate researches on all lepidopteran CYPs, and in particularly on selective targets for innovative pest management.

Swine transmissble gastroenteritis(TGE) is a kind of hyperinfected viral disease in pigs (Sus scrofa) induced by Swine transmissble gastroenteritis virus (TGEV), and leads to death of large numbers of the pigs. The present study aimed to evaluate the effect of Polygonum cillinerve polysaccharide (PCP) on inhibition of TGEV. Based on the detection results of TCID50 of TGEV and maximum cytotoxic concentration (TC0) of PCP on in vitro growth of swine testicular (ST) cells, the PCP was pro-seeded, co-seeded or post-seeded with TGEV in ST cells, then the anti-virus adsorption, inactivation and inhibition of the proliferation of PCP were analyzed by 3- (4,5- Dimethylthiazol-2- yl)-2,5- diphenyltetrazolium bromide (MTT) method and quantitative Real-time PCR (qRT-PCR). The detection of in vitro viral infection indicated that the value of TCID50 on TGEV was 10-6.25. After 72 h in vitro culture, ST cells were well-grown without the cytotoxic response when treated them with PCP at concentration of lower than 20 μg/mL compared with the blank controls, which determined the TC0 of PCP was 20 μg/mL. According to the results of MTT, under the maximum safe concentration (20 μg/mL) of PCP, the anti-adsorption effect of PCP on TGEV in vitro was the best, the inhibition rate was 85.2%, the inhibition rate on in vitro virus proliferation was 78.4%, while the inhibition rate of direct inactivation to TGEV was 74.4%, and the inhibition rate was positively correlated with the concentration. Moreover, qRT-PCR detection indicated that 3 administration methods of PCP could significantly reduce relative TGEV N mRNA expression at the transcriptional level (P＜0.01) compared with the virus control, and with a dose-dependent characteristic. The results suggested that all 3 administration methods of PCP could inhibit the proliferation of TGEV at ST cells in vitro, and the prevention effect of PCP was the best. This work demonstrated the in vitro anti-virus effect of PCP on TGE, and can provide some basis on resource exploitation and multiple utilization of Polygonum cillinerve.

Research results of miniature pig inbred cultivation and application, which were achieved by the scientists from Institute of Animal Sciences of Chinese Academy of Agricultural Sciences (CAAS). By comprehensive measures of "inbreeding", improving the level of nutrition, estrus synchronization, and cage rearing technology et al., which lasted 15 years, they bred international's first inbred pigs with two Wuzhishan mini-pig(WZSP)(Sus scrofa) as progenitor, gradually overcame three stages problems as high offspring malformation rate, high weakling piglet rate and low survival rate. This article describes the breeding process, inbred line identification, germplasm characteristics of the new genetic resources, and their development and utilization et al. The results fill the gaps for pig inbred line in domestic and foreign research, and provide a new vision for animal inbreeding theory and applied research in large mammals inbred line.