Abstract Ochratoxin A has been believed to be the second most harmful mycotoxin after aflatoxin and it exists widely in cereal, grain, fruit juice and beer. A large number of grains contaminated with OTA, seriously harm the health of people and animals, so removal or degradation of OTA in food and its raw materials is very important for food safety. The detoxification method which has application significance and availability should meet several requirements: The mycotoxin must be inactivated or destructed through detoxification method, making it into non-toxic complex; the fungal spores and mycelium must be destructed so that new toxins can not form; the nutritional value and the physical properties of food or feed must be maintained while it is be detoxified, and it is better to be economic and reasonable. OTA detoxification methods are generally divided into two types: one type is the effect of degradation, mainly through chemical reaction and modification of toxin, which can make the toxin non-toxic or low toxic; the other way is the removal of OTA, mainly through certain means, removing the existing OTA, and will not change its nature. At present, the research on detoxification of OTA in domestic and abroad can be divided into three kinds: physical method, chemical method and biological detoxification method, which are used different mechanisms to reduce, eliminate, adsorp, modify or decompose OTA. Physical detoxification method uses the means of cleaning dust, grinding and washing, adsorption, radiation and other means to achieve the effect of reducing or detoxifying OTA. Chemical detoxification method is mainly used to modify the toxins and other means to make it into a non-toxic material. Although the operation of physical and chemical methods are simple and the technical requirements are not high, but the efficiency of OTA detoxification is not high, and the quality of food is changed, resulting the nutrients' loss. Most important, chemical reagents residues is also a threat on food safety. In recent years, more and more researchers are paying attention to the biological detoxification of OTA with the further research of OTA detoxification. The biological detoxification of OTA is mainly by adsorption or enzymatic reaction to achieve the purpose of detoxifying by modifying toxin molecule and detoxifying toxin. The biological degradation pathway of OTA is mainly through the breaking of the amide bond which connects L-β-phenylalanine and OTα, and the OTA is hydrolyzed to the non-toxic L-β-phenylalanine and OTα. And the main detoxification enzymes that have been found include protease, OTA hydrolytic enzyme and several commercial enzymes. As one kind of proteases, the carboxypeptidase A is the protease which is the most to be found and the most widely to be used in OTA detoxifying. Meanwhile, biological detoxification of OTA has become a hot topic in the world, with its environment friendly, no damage to nutrients, high efficiency and low toxicity, high specificity and so on in the present research. This paper mainly introduces the mechanism and methods of OTA biological detoxification, including the extraction of detoxification enzyme, the mechanism of detoxification enzyme and so on.

Abstract Bone morphogenetic protein 15 (BMP15) is one member of the transforming growth factor-β (TGF-β) superfamily, also known as GDF9B for its similarity in protein structure and biological function with the growth differentiation factor 9 (GDF9), another member of the TGF-β superfamily. As an oocyte-secreted factor (OSF), BMP15 can form biologically active homodimers or heterodimers with GDF9 through non-covalent interactions, to bind to specific receptors BMPR1B and/or BMPR2 expressed on the ovarian cell membrane, playing an important role in the regulation of mammalian ovarian functions and fertility, including the promotion of follicle growth and maturation from gonadotrophin-independent stages; BMP15 gene has been identified as one of the major genes controlling ovulation rate in multiparous sheep. This review summarizes the common and differing functions of BMP15 gene between different mammalian species, aiming at providing a useful information for further study on the regulatory role of BMP15 gene on female ovarian functions. In addition, based on the rapid development of genome editing technology, prolific livestock breed is hoped to be produced in future through precise modifications on BMP15 gene.

To enrich studies of NAC transcription factor in upland cotton (Gossypium hirsutum) and find related NAC genes involved in stresses induced senescence, we cloned and analyzed GhNAC63, a homologous gene of JUNGBRUNNEN1, and its promoter. By qRT-PCR analysis, we explored the expression patterns of GhNAC63 in different cotton tissues, different cotyledon developmental stages, and in responses to different abiotic stresses in various culture conditions. To explore the function of GhNAC63, we constructed 35S::GhNAC63 expression vector, and transferred it into Arabidopsis through Agrobacterium LBA4404 by flower dipping method, and observed its phenotype in homozygous T4 stage. In addition, pYL156::GhNAC63 vector was constructed with pYL156-pYL192 system, and transferred into cotton to knock down GhNAC63 expression by VIGS. Gene sequence analysis showed that GhNAC63 harbored the conserved NAM domain, belonging to the NAC transcription factors, and it was conserved in the N-terminal, but variant in the C-terminal, which was consist with most of the NAC transcription factors. The full length of GhNAC63 was 870 bp, and its predicted protein contained 289 amino acid residues, with a molecular weight about 33.12 kD. Based on the genome sequence of upland cotton, GhNAC63 was located in scaffold246.1, and about 2.0 kb of its 5' UTR was cloned and analyzed. Besides basal elements, TATA-Box and CAAT- Box, a lot of stresses-related Cis-elements were contained in its promoter, such as drought-related, hot-related, phytohormone-related, and light responsive-related elements. Expression patterns analysis showed that GhNAC63 dominantly expressed in fiber, flower, stem, leaf and cotyledon, and it had the highest expression level in the middle stage of cotyledon development. Under closed culture condition, GhNAC63 was upregulated under treatments of MeJA, SA, drought and ABA in leaf, while the expression level of GhNAC63 was down-regulated under other treatments both in leaf and root, and GhNAC63 showed the most sensitive to ethylene treatment. Under open culture condition, GhNAC63 showed different expression patterns under drought, salt, abscisic acid (ABA), methyl jasmonate (MeJA) or ethylene treatments, and its expression level was highly induced by ethylene treatment. Overexpression of GhNAC63 in Arabidopsis made plants more susceptive to ethylene treatment as leaves of transgenic Arabidopsis became yellow after ethylene treatment, in addition, leaves of the transgenic plants also became wilting after drought treatment, which might indicate the function of GhNAC63 in response to ethylene or drought. On the contrary, VIGS induced gene silence led to a low expression level of GhNAC63 in cotton, with about 0.4 times decrease compared with wild type or empty vector control plants. GhNAC63 silenced seedlings can flowering just as wild type seedlings, while empty vector control plants did not bloom, thus low expression level of GhNAC63 could make cotton seedlings being stronger. In conclusion, GhNAC63 was sensitive to ethylene treatment, and it might negatively regulate ethylene or drought treatments in Arabidopsis, and it negatively regulated plant development in cotton. Promoter of GhNAC63 contained various stresses-related Cis-elements, indicating GhNAC63 might response to different stimulates. This study helps to create new breeding material which was related with stresses induced senescence, and enriches the functional studies of NAC transcription factor in upland cotton.

This research was conducted to detect the specificity of cell-cycle related and expression-elevated protein in tumor (CREPT) gene expression in different tissues and to clone CREPT gene coding sequence of the Rugao yellow chicken (Gallus domesticus). For primary understanding of biological function of CREPT gene in chicken, mostly involved with regulation of cell cycle, we constructed the eukaryotic expression vector which obtained its expression in DF-1(D fibroblast-1) cells and polyclonal antibodies to CREPT protein. The CDS zone of CREPT was amplified from the cDNA of chicken germinal ridge, protein structure anticipation and tissue expression were conducted for further understanding. The cloned chicken CREPT gene was connected into pcDNA3.1 and pEGFP-N1 eukaryotic expression vector to construct recombinant vectors pcDNA3.1-CREPT and pEGFP-CREPT, which were lately transfected to DF-1 cells to obtain polyclonal antibodies by gene-base immunization and analyze its antiserum titer. The qRT-PCR approach was used to identify the expression level of relative genes after over-expression of CREPT gene in DF-1 cells. The results of bioinformation anticipation showed that the coding length of the cloned chicken CREPT contains 978 bp sequence and codes 325 amino acids including a RPR domain (regulation of nuclear pre-mRNA domain) and a CCD (coiled-coil domain). The cloned Rugao yellow chicken CREPT CDS sequence was consistent with sequence from NCBI GenBank(G. gallus, DQ372940), the sequencing results were in agreement with the CDS sequence of the chicken (G. gallus), and the homology was 100%. Tissue transcription specificity analysis showed that transcription of CREPT was higher in testis, ovary and brain (P＜0.01), whereas the transcription of CREPT was lower in skeletal muscle (P＜0.05), intestines (P＜0.01) and spleen (P＜0.05). The recombinant vector (pcDNA3.1-CREPT and pEGFP-CREPT) was constructed. The polyclonal antiserum was prepared by genetic immunization, IFA and transfected results showed the CREPT located in cytoplasm. Best antiserum titer reached 1∶10, identified by IFA (immunogen fluorescent assay). Western blot demonstrated pcDNA3.1-CREPT eukaryotic expression vector works well in DF-1 cells and showed a high antiserum titer. qPCR detection results show that pcDNA3.1-CREPT could over-express CREPT gene in DF-1 cells (P＜0.01) and induce changes of expression of gene p15RS (P15 related gene on G1/S progression, P＜0.01), TCF4 (transcription factor 4, P＜0.05), cyclin D1 (P＜0.01) and b-catenin(P＜0.01), which play important roles in cell cycle regulations. These results indicated that the chicken CREPT was successfully cloned and specificity polyclonal antibodies were obtained. The major expression area of CREPT in DF-1 was cytoplasm. We can also conclude that over-expression of CREPT gene down-regulates the p15RS expression and up-regulates TCF4, cyclinD1 and b-catenin expression in DF-1 cells. This study provides theoretical basis for further elucidating the biological function of CREPT gene in chicken.

Abstract Kruppel-like factor 7 (KLF7) is a factor involved in fat metabolism and its related research on growth and carcass traits in chicken (Gallus gallus) is less. In order to analyze the association between the single nucleotide polymorphism (SNPs) of KLF7 gene and carcass traits in chicken, the SNPs of the whole genome were genotyped through 600K Affymetrix Axiom Chicken SNP chip technology in Ninghai and Guangxi yellow chickens with each of 59 individuals. Within the total 38 identified SNPs, only 4 SNPs were found in exon regions of KLF7 gene. Most of the detected SNPs revealed 3 genotypes (i.e. AA, AB and BB). The χ2 test showed that the genotypic frequencies of 5 SNPs (i.e. T16936C, A18000C, C33569T, T34758C and T40474G) within Ninghai yellow chicken and 2 SNPs (i.e. A18715C and T45125A) within Guangxi yellow chicken were not in agreement with the Hardy-Weinberg equilibrium (P＜0.05), while the genotype frequencies of the other SNPs in these two chicken populations were all under the Hardy-Weinberg equilibrium (P＞0.05), which showed that there was dynamic equilibrium in two studied chicken populations. According to the classification of polymorphism information content (PIC), the polymorphism at most of the detected SNPs in the current study presented moderately in these 2 chicken breeds. Association studies between each SNP genotype and carcass traits were performed by using the GLM. The results showed that 7 SNPs (i.e. A14371G, A18000C, C28254A, C33569T, T45125A, A56295G and G59074A) of KLF7 gene in Ninghai yellow chicken and 6 SNPs (i.e. C12493G, A18000C, G30642A, A38815G, T45125A and C58906T) in Guangxi yellow chicken were significantly associated with carcass traits (P＜0.05 or P＜0.01). The 2 chicken populations possessed 2 common SNPs (i.e. A18000C and T45125A) which significantly associated with carcass traits (P＜0.05 or P＜0.01). The 2 SNPs (i.e. A18000C and T45125A) revealed 3 genotypes (i.e. AA, AB and BB) in Ninghai yellow chicken, while 2 genotypes (i.e. AB and BB) in Guangxi yellow chicken . Genotype AB of the site A18000C was significantly superior to BB in breast muscle rate in Ninghai yellow chickens (P＜0.01), while the AB genotype of T45125A was significantly superior to AA and BB in semi-eviscerated rate, eviscerated rate, abdominal fat weight and abdominal fat rate (P＜0.05 or P＜0.01). Genotype AB of the site A18000C was significantly superior to BB in breast muscle rate in Guangxi yellow chickens (P＜0.05), while the AB genotype of T45125A was significantly superior to BB in slaughter rate, semi-eviscerated rate, eviscerated weight and eviscerated rate (P＜0.05). The results suggested that KLF7 gene may be one of the candidate genes that control carcass traits, and 2 SNPs (i.e. A18000C and T45125A) could be potential candidate genetic markers for Marker-aid selection in chickens.

Abstract Kiwifruit (Actinidia chinensis) has high nutritional value and hygienical function, but softing will effect its commercial quality seriously during storage. Polygalacturonase (PG) is closely related to fruit softing which involve in degrading pectin. To explore the molecular regulation mechanism of fruit softening, the Mi liang 1 hao kiwifruit was used to study the differences of firmness, titratable acid, soluble solids and vitamin C that stored in room temperature, low temperature and treated by abscisic acid (ABA), the bioinformatics and expression patterns of polygalacturonase gene (AcPG) were also analyzed respectively. The results showed that the firmness and content of titratable acid were decreased during storage when soluble solids and vitamin C were increased reciprocally, and ABA might promote the process of kiwifruit softening, but low temperature would inhibit it. AcPG (GenBank No. KY 129958 ) was cloned with an open reading frame of 1 545 bp, which encoded 514 amino acids. Bioinformatics analysis showed that AcPG was unstable protein, had signal peptide, transmembrane structure, polygalacturonase conserved domain and phosphorylation site, and AcPG kept low homology with other species. According to qRT-PCR results, the expression of AcPG was downregulated when fruit softening during storage in room and low temperature, however, AcPG would response induction of ABA. These results indicated that cryopreservation was propitious to storage of kiwifruit, and AcPG involved in regulation of its softening. This study provides a theoretical basis for further research of fruit softing in kiwifruit by using molecular biology technology.

WRKY is a class of transcription factors related to plant stress-tolerance, which improve plant resistance by activating expression of a series of stress response gene downstream. In this study, we screened a stress-induced WRKY transcription factor gene GhWRKY2 from Gossypium hirsutum based on the gene chip, it belonged to WRKYⅡ subgroup. We obtained the full length of GhWRKY2 cDNA by RT-PCR technology, and made sequence analysis and structure-function prediction by bioinformatics software. According to the sequence homology of GhWRKY2 protein, a multiple sequence alignment and phylogenetic tree construction of homologous species was made by MEGA7. Amino acid the physical and chemical properties was analysised by ProtParam and Motif scan; protein sequence was analysised by SMART online tools; the structure and tertiary structure of GhWRKY2 gene were analyzed by SWISS-MODEL online tool; the structure and tertiary structure of GhWRKY2 gene were analyzed by SWISS-MODEL online tool. The results showed that the GhWRKY2 contained a 942 bp open reading frame encoding a 313 amino acids residues with a predicted molecular mass of 34.22 kD and an isoelectric point of 8.76. The transient expression of GhWAKY2-GFP into Arabidopsis protoplasts showed that the GhWRKY2 was located in the nucleus. Moreover, it was found to have transcriptional activation activity. The clone of stress response GhWRKY2 gene, lay the foundation for further confirming the biological function of resistance at the molecular level, and revealing mechanism of abiotic stress resistance in cotton, and providing new candidate genes for stress tolerance breeding in cotton.

Abstract Cellulose binding proteins (CBP) are cell wall-related effectors secreted by plant-parasitic nematodes during parasitism. In this study, a new cellulose binding protein gene Me-cbp-1 (GenBank No: KU350655) was cloned from the root-knot nematode Meloidogyne enterolobii by rapid-amplification of cDNA ends (RACE) based on transcriptome sequencing analysis. The full-length of Me-cbp-1 cDNA was 809 bp, including a 627 bp open reading frame (ORF). The cDNA contained of 43 bp 5'-untranslated region (UTR) and 139 bp of 3'-UTR. The ORF encodes a deduced protein of 208 amino acids with a calculated molecular weight of 22.2 kD and a pI of 4.0. The predicted protein Me-cbp-1 had a putative signal peptide for secretion and a cellulose binding domain. Homology analysis showed that Me-cbp-1 shared 88%~91% identities with cellulose binding proteins from root-knot nematodes M. incogntia, M. javanica and M. arenaria. In situ hybridization analysis showed that the transcript of Me-cbp-1 accumulated exclusively within cells of subventral gland on the J2s of M. enterolobii. To illustrate the importance of Me-cbp-1 in the parasitism of M. enterolobii, gene expression was knocked down using RNAi. Knocking down the expression of Me-cbp-1 in pre-parasitic nematodes significantly reduced the penetration rate of plants by more than 26%. The proportion of nematodes inside the roots relative to those transferred to the roots was 26.8% for the nematodes treated with Me-cbp-1 dsRNA, whereas 36.4% of the nematodes from the gfp dsRNA treatment were found inside the roots 10 days after inoculation. It was indicated that an important role of Me-cbp-1 in the early stages of invasion and migration of J2s in the root. These above results are useful for deeper elucidating the molecular mechanism of parasitism and pathogenesis of M. enterolobii.

Abstract Bacterial biofilm is the leading cause of bacterial drug resistance increases. Staphylococcus epidermidis is an opportunistic pathogen, once it formed the biofilm, it will adhere to the surfaces of medical device and make the diseases recurrent and difficult to cure. Quorum sensing system has been recently regarded as the research target to suppress the bacterial biofilm formation. S-ribosyl homocysteine lyase (LuxS) is the key enzyme for the synthesis of autoinducer-2 (AI-2), they constitute the AI-2/LuxS quorum sensing system in bacteria. In this study, the biofilm formed in the early stage was chosen as the experiment subject, then antisense locked nucleic acid (LNA) technology was used to block the expression of LuxS gene in S. epidermidis AI-2/LuxS quorum sensing system, the inhibitory effect of 2 antisense locked oligonucleotides named LNA81 and LNA352 was compared by the transcription level of LuxS gene, Using real-time PCR measured the relative expression quantity of biofilm formation related genes, including sigma factor B (sigB), intercellular adhesion AB (icaAB), staphylococal accessory regulator A (sarA), accumulation-associated protein (aap), fibrinogen-binding protein (fbe) and autolysin E (atlE). AI-2 signaling molecule activity was measured by Vibrio harveyi bioluminescence assay, and the formation and characteristic parameters of biofilm were analyzed by confocal laser scanning microscope (CLSM) and image structure analyzer (ISA) software. Results showed that between the 2 antisense locked oligonucleotides, LNA81 and LNA352, 100 nmol/L LNA81 mixed with 2.5 μL RNAiMAX transfection reagent exhibited better inhibition in expression of LuxS through targeting LuxS mRNA, it could get a 43% inhibition ratio in LuxS transcription at 6 h. When the transcription of LuxS was inhibited partially, the transcript level of gene sigB and icaAB increased, sarA, aap, fbe and atlE decreased. The activity of AI-2 signaling molecule was decreased in 9 h compared to control group, at the time of 6 h its activity got a minimum, and after 9 h its activity was closed to the control group, this tendency was consistent with the transcript level of gene LuxS which was inhibited to a minimum at 6 h and its inhibition ratio became unsteady after 6 h. Finally, the ability of biofilm formation was decreased, the structure of biofilm was more simple than control group. This study is focus on the regulating effect of AI-2/LuxS quorum sensing system on the early biofilm formation, antisense strategy was used to target LuxS mRNA, which is the critical gene for the formation of AI-2, it not only has the ablity of quorum sensing system but also plays an important role in sulfur metabolism and glycometabolism in bacteria. It can be seen that AI-2 signaling molecule activity and expression of genes involved in biofilm formation were affected when the LuxS gene expression was partial blocked. The biofilm formation is a dynamic process, it is affected by various factors, the changes of above factors were finally resulting in inhibition of the biofilm formation. These results will provide a theoretical basis for the further study on AI-2/LuxS quorum sensing system and its regulatory effects on the biofilm formation in S. epidermidis, they also can provide more potential targets and therapeutic schedule to prevent S. epidermidis infection.

Abstract Domestic goats (Capra hircus) are evolved from wild goats, and there are good adaptability, wide distribution and abundant breed resource for goats in China. Natural and artificial selection for long time may lead to differences in polymorphism and differentiation between goats of South and North of China. To study genetic diversity and origin as well as differentiation of goats in South and Northwest of China, D-loop region of 69 individuals belonging to 5 goat breeds distributing in South-China were sequenced and analyzed combing with 33 sequences from 3 goat breeds distributing in Northwest of China. The results showed that content of A+T and G+C in South and Northwest were 60.1%, 39.9% and 60.0%, 40.0%, respectively, indicating no apparent difference in base composition between the 2 areas. The average haplotype diversity (Hd) and nucleotide diversity (π) of goats in South China were 0.954±0.005 and 0.019 57±0.002 10, respectively, while they were 0.919±0.106 and 0.019 08±0.001 15 for goats in Northwest; Fs of all data sets were large negative value (P＜0.05) while values of sum of squared deviation (SSD) and raggedness had no significant difference (P＞0.05), which indicated nucleotide mismatch distribution had not deviated from population expansion model, and at 13th、37th、61st nucleotide, there was a peak for each site; NJ (neighbor-joining) tree showed 2 lineages: A and B, and all goats of South and Northwest distributed in the 2 lineages. Furthermore, lineage B divided into 2 sub-clade, B1 and B2, implying persistently differentiation. Media-joining network showed the same result to the NJ tree, and further pointed out H1 was the highest frequency haplotype appearing. When it comes to genetic distance, it was 0.022 and 0.019 within population for goats of South and Northwest. These results suggested that genetic diversity of goats in South area was slightly higher than in Northwest, although a little lower than that in past several years; events of population expansion had arisen for the whole haplotype group as well as A and B lineages; C. aegagrus was the wild ancestor for domestic goats of South and Northwest of China referred in this study. This research offers theoretical support for further understanding origin, differentiation and genetic diversity of goats which distributed in South and Northwest of China, and provide theoretical basis for protecting native breeds.

Abstract Mycoplasma bovis (Mb) is an important pathogenic mycoplasma, which can cause a variety of diseases if cow (Bos taurus) infected with the Mb. Fructose bisphosphate aldolase (FBA) is one of the pivotal enzymes that involves in the p rocess of glycolysis pathway, which provided the substrate for the dehydrogenation of glyceraldehyde 3-phosphate, and widely exists in the living nature. In order to study the distribution of FBA in Mb cells, In this study, a pair of specific primers were designed according to fba gene sequence of Mb PG45 reported in GenBank. Then the fba of Mb Wuwei stain was amplified by PCR, and it was cloned into pMD19-T vector. After finished sequencing and comparing in the NCBI with other strains of Mb isolating from China, the phylogenetic trees were constructed applying with DNA Star software. Based on the sequencing and gene optimization, the prokaryotic expression vector pET-fba was constructed and transformed into Escherichia coli Transetta (DE3). Then, The target protein was induced by isopropyl β-D-1-thiogalactopyr-Anoside (IPTG). After that, we purified recombinant protein rMbFBA using Ni-NTA His Bind to immunize New Zealand rabbit (Oryctolagus cuniculus), Which aimed at preparing the polyclonal antibodies against rMbFBA. Subsequently, the antibody lever was detected by enzyme linked immunosorbent assay (ELISA). What's more, subcellular localization of FBA in Mb was performed using Western bolt. In conclusion, The results showed that the open reading frame of fba gene contains 876 nucleotides (GenBank No. KX828563), encoding a protein with 292 amino acids protein. However, there were 2 TGA codes in the sequence, which encodes trptophan in mycoplasma, but it is translated as the terminator coden in Escherichia coli. So the overlap PCR was used to make the 2 TGA termination coden mutations successfully mutate into synonymous coden TGG. The phylogenetic tree analysis from fba gene sequence of Mb Wuwei strain has high homology with other Mb strains separating from China, which indicated that the fba gene is highly conserved. Flowing that, the optimized gene was successfully expressed in Escherichia coli Transetta(DE3) when induced with final concentration of 1 mmol/L IPTG for 10 h at 20 ℃, after detected by the technology of sodium dodecyl sulfonate-polyacrylamide geleletrophoresis (SDS-PAGE), the result display that the relative molecular weight was approximately 34 kD, which existed mainly in the form of soluble protein. However, the negative control in this non target strip. The ELISA titer of antiserum was approximately up to 1∶25 600. The results of Western bolt confirmed that the prokaryotic expression products of fba gene of Mb Wuwei strain has good immunogenicity, and the FBA of Mb distributed in both cytoplasm and the membrance equally. All the results of this research lay a foundation data for further study on the biological function of FBA of Mb.

Abstract Porcine contagious pleuropneumonia (PCP) is a respiratory disease caused by Actinobacillus pleuropneumoniae (APP) infection, which is one of the 5 major epidemics in the world's pig industry. Based on APP OmpP2 sequences in GenBank, the primers were designed. APP OmpP2 was amplified by PCR from APP genome, sequenced and submitted to NCBI (Access No. KM357903). Bioinformatics analysis showed that the APP OmpP2 gene was composed of 1 131 bp, encoding a 376 amino acid protein. This protein was a porin protein with high homology among different APP serotypes. APP OmpP2 gene was subcloned into pET32a (+) to construct recombinant plasmid pET-OmpP2. rOmpP2 expression in Escherichia coli BL21 was induced by isopropyl β-D-1- thiogalactopyranoside (IPTG), and determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) and Western blot. The results showed that rOmpP2 with 42 kD was detected. Animal experiment was conducted by BALB/c mice (Mus musculus). The serum immunoglobulin G (IgG) level was detected by enzyme-linked immuno sorbent assay (ELISA). The results showed that survival rate of infected mice with aluminium adjuvant+50 μg rOmpP2, aluminium adjuvant+30 μg rOmpP2 and aluminium adjuvant+10 μg rOmpP2 were 50%, 30% and 10%, respectively, and all mice died in adjuvant group and control group. The surviving mice slowly returned to normal health status. Mice that were subcutaneously immunized with rOmpP2 showed an partial protection against the challenge with APP. This study provides basic data for screening new molecular vaccines of porcine contagious pleuropneumonia.

Abstract Fatty acyl acyl carrier thioesterase A (FATA) plays an important role in the process of fatty acid synthesis metabolism. The expression pattern of FATA of Jatropha curcas (JcFATA) and its promoter all has not been reported. To study the expression pattern of JcFATA gene, semi-quantitative RT-PCR and qRT-PCR were applied to detect the expression specificity of JcFATA gene. The results showed that JcFATA gene could express in multiple tissues of J. curcas, especially highly expressed in roots, flowers and leaves. In addition, in order to analyze the expression pattern of JcFATA gene promoter (PJcFATA), JcFATA gene promoter was cloned firstly and inserted into a binary expression vector pCAMBIA1300G, and then the fusion expression vector of β-glucuronidase (GUS) gene drived by JcFATA gene promoter (p1300G-PJcFATA-GUS) was constructed successfully. The fusion expression vector was introduced into Arabidopsis thaliana wild type by Agrobacterium-mediated method. After effective selection for hygromycin B resistance, the resistant plants were detected by PCR and GUS histochemical analysis. PCR detection results showed that JcFATA gene promoter had been transferred into the genome of A. thaliana (JGD No. Jcr4S00539). Results of GUS histochemical staining of tissues in different development periods of A. thaliana revealed that GUS activity displayed obviously tissue specificity, with the deeper staining in roots, flowers and leaves. All above results paved the way to further function study of JcFATA gene and JcFATA gene promoter.

Abstract Chicken (Gallus gallus) and duck (Anas platyrhynchos) are major hosts of Newcastal virus (NDV) disease with distinct response to infection and in which host innate immune response maybe play an important role. In order to compare the host innate immune response of chicken and duck and ensure their role of the difference in pathogenicity infected with duck-origin NDV, the message RNA expression levels of pattern recognition receptors (PRRs), cytokines, antiviral proteins and major histocompatibility complex (MHC) molecules genes in both chicken and duck peripheral bloods isolated at different times were examined by using the quantitative real-time polymerase chain reaction method. Gene expression profiles showed that duck-origin isolates (SD03) replicated more efficiently and had a significantly higher titer in chicken spleens compared with duck spleens at each time point; toll-like receptor 7 (TLR-7), interleukin 1β (IL-1β), IL-2, IL-6, IL-8, interferon β (IFN-β), MHCⅠ and MHCⅡ exhibited distinct expression patterns and different levels in chicken and duck peripheral blood; Furthermore, the expression of all examined genes, except IL-2, were greater in chicken peripheral blood. Results showed that distinct expression patterns of toll-like receptors, cytokines and antiviral proteins and the effect of difference in pathogenicity associated with innate immune response of chicken and duck to NDV infection. Meanwhile, the results are also benefit to better understand the viral pathogenesis to avian species.

Abstract Tail-interacting protein 47 gene (TIP47), known as mannose 6-phosphate receptors binding protein 1 (M6PRBP1), is a member of the (perilipin/ADRP/TIP47, PAT) family, which plays a crucial regulatory role during lipid droplets (LD) biogenesis and secretion process. Milk fat, one of the important nutrients in goat (Capra hircus) milk, is especially rich in short and medium chain fatty acids, which can be absorbed by human (Homo sapiens) body easily. At the same time, the polyunsaturated fatty acids in goat milk have a good effect on anti-oxidation. However, the role of TIP47 on fatty acid metabolism in goat mammary gland is poorly understood. Thus, studying the function of the TIP47 on formation of the milk LD has a great significance to regulate the nutritional value of goat milk. RT- PCR was used to clone TIP47 CDS. The Block-iT short hairpin RNA (shRNA) interference system was used to design specific shRNAs according to the CDS of TIP47 mRNA and 293A cell line was used to package and amplify the adenovirus in this experiment. Then using qRT-PCR to detect the relative expression level of genes and oil red O was used to identify the changes of lipid droplets in intracellular. The full-length 1 314 bp CDS of TIP47 (GenBank No. HQ846827) was cloned onto pDsRed1-C1vector which could express the red fluoresence protein. Three pairs of pENTR/CMV-GFP/U6-494/828/888 and pDsRed1-C1-TIP47 were co-transfected into 293A cells to filter out the most interference efficient shRNA. The results showed that shRNA-888 could markedly reduce the red fluorescence, so shRNA-888 was considered to inhibit the expression of TIP47 significantly. Then shRNA-888 was packaged and amplified high-titer virus Ad-shRNA-888 into 293A cells. The titer of the Ad-shRNA-888 was 108 U/mL and the optimal multiplicity of infection (MOI) was 200 in goat mammary epithelial cells. Compared with the control, the expression of TIP47 and adipose triglyceride lipase (ATGL) mRNA were downregulated by 61% (P＜0.01) and 15% respectively, and expression of fatty acid synthase (FASN) and hormone-sensitive lipase (HSL) mRNA were upregulated to 1.77 (P＜0.05) and 2.8 (P＜0.01) folds but no noticeable changes for the mRNA expression of stearoyl-CoA desaturase (SCD) when treated mammary epithelial cells with the Ad-shRNA-888 after 72 h. And the result of oil red O staining showed that 100 μmol/L oleic acid could significantly increase the accumulation of lipid droplets in mammary epithelial cells, however the intracellular accumulation of lipid droplets had a downward trend after treating with the Ad-shRNA-888 but did not fully suppress the formation of intracellular lipid droplets. Taken together, the qRT-PCR results indicated that Ad-shRNA-888 could effectively downregulate the expression of TIP47, which contributed to changing fatty acid metabolism related genes significantly in goat mammary gland epithelial cells, including FASN and HSL. Moreover, the result of oil red O staining suggested that there might be other genes involved in the process of the formation of lipid droplets in a way of coordinated control. Here, our study demonstrated that TIP47 played a vital role in fatty acid metabolism during the dairy goat lactation process, and provided the basic data for further research in regulating fatty acid metabolism.

Abstract Coix (Coix lachryma-jobi) is a kind of medicine and food crop, while drought is the most main factor limiting its growth. To explore the applicative effects of uniconazole (S3307) on coix seedlings under drought stress, the growth parameters, anti-oxidant enzyme activities, chlorophyll content and chloroplast ultrastructure in Yiliao No. 5 under drought stress were investigated through nutrient solution cultivation in greenhouse. The results showed that the stem diameter and ground biomass were significantly improved with 9 mg/L S3307 under drought stress (P＜0.05). Compared with the S0 (drought group), stem diameter were increased for 16.19%, and the above-ground and under-ground biomass increased for 19.56% and 22.75%, respectively. The activities of peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) were significantly enhanced (P＜0.05), while POD and CAT activities of roots were higher than those of leafs. Total chlorophyll content in the S3 (9 mg/L S3307 group) was the highest in all the treatments, which was increased by 7.35% compared to S0 (drought treatment group) (P＜0.05). Meanwhile, S3307 addition could improve chloroplast and root cell ultrastructure and lighten the damage to grana ultrastructure under drought stress, which was consistent with increasing chlorophyll content. All the analysis showed that 9 mg/L S3307 concentration was effective on alleviating the injury of coix seedlings under drought stress. This study can clarify physiological mechanism of coix drought resistance and provide a theoretical basis for S3307 in coix production and practice of drought resistance.

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major pathogens causing reproductive and respiratory symptoms in pigs (Sus scrofa), and N protein is the main structure protein and antigen protein of the virus. Therefore, this experiment was aimed to study the immunogenicity of N protein of North American genotype Porcine reproductive and respiratory syndrome virus (NA-PRRSV). The gene was amplified from the RNA of NA-PRRSV QH-08 strain by RT-PCR, whose product was approximately 372 bp. The product was cloned into pET30a(+)vector, RT-PCR, digestion and sequencing were used to identify positive plasmid. The identified recombined plasmid was expressed by Escherichia coli BL21 (DE3) and was induced by 1 mmol/L isopropy-β-D-thiogalactoside (IPTG) at 37 ℃. The recombinant N protein was purified by Ni-NTA and refolding. The expressed recombinant N proteins was used as an immunogen for immunization of New Zealand white rabbits (Oryctolagus cuniculus) by subcutaneous injection in several sites on back. The specificity of the serum antibody was determined by Western blot. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDA-PAGE) showed that the recombined protein was successfully expressed in E. coli BL21 (DE3) with a relative molecular weight of 24 kD. The results of Western blot showed that this recombined N protein was specifically reacted with NA-PRRSV positive pig serum; No cross reacted with Porcine parvovirus type 1 (PPV1) and PPV2 positive pig serum. The prepared lepus antisera was specifically reacted with recombined N protein. The recombinant vector pET30a-NA-PRRSV-ORF7 was successfully constructed, and the recombined N protein with excellent immunogenicity was successfully expressed in E. coli which provided a theoretical basis for the diagnosis of NA-PRRSV.

Abstract Nannochloropsis is a kind of economic microalgae with great development potential and its large-scale farming mainly depends on outdoor raceway pond. Low temperature becomes one of main limiting factors which affect the yield and quality of Nannochloropsis. Salicylic acid (SA) can enhance the low temperature resistance of many plants. This study transferred N. oceanica cells (25 ℃, mid-logarithmic phase) to low temperature (15 ℃) stress for 96 h. According to the difference of final concentrations of salicylic acid and cultivation temperature, the experiment was divided into 5 groups ("25 ℃+SA0", "15 ℃+SA0", "15 ℃+SA5", "15 ℃+SA15" and "15 ℃+SA25"). The cell density, soluble sugar, soluble protein, malondialdehyde (MDA) content, fatty acid composition and transcript levels of 3 fatty acid desaturase genes (Δ5 desaturase, Δ6 desaturase and Δ12 desaturase) were determined of 5 time points (0, 24, 48, 72 and 96 h). The results showed that growth of N. oceanica was inhibited under low temperature stress, while exogenous salicylic acid could promote its growth even though not exceed the group under normal cultivation temperature (25 ℃). Soluble sugar, soluble protein and MDA contents were compared between groups "15 ℃+SA0", "15 ℃+SA5" and "15 ℃+SA15". The results showed that SA could significantly increase its soluble sugar and soluble protein content, decline its MDA content, greatly augment the percentage of unsaturated fatty acids. The best effect emerged under the condition of 15 mg/L salicylic acid. The transcript levels of 3 fatty acid desaturase gene were also analyzed of groups "15 ℃+SA0" and "15 ℃+SA15". The results indicated that the transcript levels of Δ5 desaturase and Δ6 desaturase were decreased in N. oceanica under low temperature stress, while exogenous SA could ease the effect of low temperature stress and enhance the transcript levels of Δ5 desaturase and Δ6 desaturase which corresponds to an increase in the percentage of unsaturated fatty acids. The transcript level of Δ12 desaturase was decreased, while exogenous SA did not significantly promote its transcript. Thus it could be seen that SA could increase intracellular osmotic adjusting materials, reduce peroxidation of membrane lipid, increase the content of unsaturated fatty acid in order to maintain membrane fluidity, eventually relieve the damage of low temperature stress to N. oceanica and improve its low temperature resistance. It is the first time to explore the effect of salicylic acid on low temperature resistance of N. oceanica. The study, with better theoretical significance and economic value, will provide a theoretical basis for optimizing microalgae cultivation and stress resistance.

Abstract With the increasing of the global commercial production of genetically modified products, the detection of transgenic lines has gradually become the development trend of the detection of genetically modified components in internal and international. To carry out the regulations of genetically modified organisms in China's inward and outward, environmental release and labeling, and achieve effective management of genetically modified soybean (Glycine max), the loop-mediated isothermal amplification (LAMP) primers were designed according to the flanking sequence of genetically modified soybean DAS-81419-2, DP356043-5, A2704-12, FG72, BPS-CV127-9 and MON89788, separately. Through specificity, sensitivity and limit of detection testing, an LAMP method for the inspection of genetically modified soybean DAS-81419-2, DP356043-5, A2704-12, FG72, BPS-CV127-9 and MON89788 was developed. The results showed that this method could detect genetically modified soybean DAS-81419-2, DP356043-5, A2704-12, FG72, BPS-CV127-9 and MON89788 specifically and stably with a low detection limit of 0.1% (W/W). Since the LAMP detection could be finished within 30 min and the results could be directly detected according to the color change produced by SYBR Green Ⅰ dye with the naked eye. This method allowed rapid and accurate detection of genetically modified products on the inspection agency and relevant law enforcement departments, and non-essential of complex and expensive equipment.