Food allergy has been regarded as an emergent problem of public health. And walnut allergy is one of the most severe food allergies, its prevalence has risen during past decades. As favors food and important food raw material, walnut (Juglans sp.) contains some allergens, which constitute a risk for sensitized individuals, and become an issue of allergen management. Due to the prevalence and severity of walnut allergy, this review intends to provide the up-to-date information about the molecular characterization of walnut allergens, as well as the detection methods of walnut allergens in foods or foodstuffs. At present, the most popular walnut varieties are Juglans regia and J. nigra, and there are many known allergens in both species, Jug r 1, Jug r 2, Jug r 3, Jug r 4 and Jug r 5 in J. regia; Jug n 1 and Jug n 2 in J. nigra. According to their biochemical and immunological characteristics, they are classified into 2 protein superfamilies, the Prolamin superfamily, Jug r 1, Jug n 1 and Jug r 3, and the Cupin superfamily, Jug r 2, Jug n 2 and Jug r 4. The Prolamin superfamily are further divided into 3 classes: 2S albumin, nonspecific lipid transfer protein (nsLTP) and α-amylase/trypsin inhibitor, Jug r 1 and Jug n 1 belong to 2S albumin family, and Jug r 3 belongs to nsLTP. The Cupin superfamily are further divided into 2 classes according to different sedimentation coefficient, 7S globulin and 11s globulin, Jug r 2 and Jug n 2 belong to 7S globulin, Jug r 4 belongs to 11S globulin. Besides, Jug r 5 belongs to the profilins family, a kind of small class of cytosol proteins, which can bind actin, but it is also very little known. In addition, according to the difference in stability, food allergens can be divided into 2 classes, classⅠand class Ⅱ. Food allergens in class Ⅰ are usually with high stability; and food allergens in class Ⅱ is more sensitive to heat or protease treatment, less stable. To date, the vast majority of walnuts allergens belong to classⅠ, except Jug r 5, which belongs to class Ⅱ. Besides, different methods used for the detection of walnuts allergens were also summarized. On the basis of the principle of detection methods, walnut allergens detection techniques are divided into 2 classes, protein-based and DNA-based detection methods. Protein-based detection methods includes protein electrophoresis, immunology methods and mass spectrometry, so far, immunoassay methods are main analysis methods in the detection of walnut allergens. DNA-based detection methods is not completely in view of the allergen proteins, but in view of specific coding sequence of target species, including traditional PCR and RT-PCR, and in recent years, DNA-based detection methods have been widely applied in walnut allergens detection. In general, this paper summarized the physical, chemical and immune properties of different walnut allergens and related detection methods, this may help us understand the nature of walnut allergens better, design more accurate and reasonable detection methods, and this may be very helpful to ensure the safety of walnut allergic patients.

Whole genome sequencing (WGS) is a sequencing of one individual or group of species by high-throughput sequencing technology, and analyses the sequence features by bioinformatics. The aim of WGS is exploring the evolution rule and screening functional genes in genome wide. It includes de novo and re-sequencing. Because its full-information, accurate, efficient, especially effect in finding of unknown gene and unknown structural variation, it quickly surpass the previous technologies to become a major strategy in the studies of population evolution and gene discovery after the cost of sequencing great decline. Especially in domestic animals, genome-wide sequencing has widely used in chicken (Gallus gallus), pig (Sus scrofa), cattle (Bos taurus), sheep (Ovis aries), goat (Capra hircus), horse (Equus caballus), duck (Anas platyrhynchos), dog (Canis lupus), and explores the rule of animal evolution, and finds a great deal of functional genes. Furthermore, the WGS has broad application prospects in the construction of Pan-genome and breeding by whole-genome selective. Here, we introduce the characteristics and development of whole-genome sequencing, outline and discuss the application in domestic animal evolution and gene discovery, and sketch its key point and prospects.

In order to enrich the toxicity and its mechanism of ochratoxin A (OTA) in plants and explore the role of calcium in OTA toxicity to plants, this research studied the effect of calcium on the toxicity induced by OTA in Arabidopsis thaliana, by the observation of morphological changes, relative leakage rate of leaves, the content of reactive oxygen species (ROS) and malondialdehyde (MDA) under treatment with exogenous Ca2+, the calcium chelator ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA) and OTA in vitro leaves of A. thaliana. The results showed that in a certain range of concentration, under the treatment of exogenous calcium, there was no significant change in leaf morphology of A. thaliana. EGTA and OTA both led to the formation of A. thaliana leaves chlorosis and necrosis of the lesion. Ca2+ and OTA treatment together could significantly inhibit the formation of A. thaliana leaves chlorosis and necrotic lesion produced induced by OTA. EGTA treatment could aggravate those effects induced by OTA. OTA treatment increased relative leakage rate and permeability of cell membrane of A. thaliana leaves. Exogenous calcium could significantly inhibit OTA induced increase in relative leakage rate of A. thaliana leaves, 20 mmol/L Ca2+ has the strongest inhibitory effect of which the inhibition rate was 61.32%. In addition, OTA treatment brought the outbreak of ROS and the accumulation of MDA in A. thaliana leaves, and therefore caused oxidative damage. Ca2+ could effectively relieve the OTA induced A. thaliana ROS burst and reduce MDA generation; the inhibition rates were 29.7% and 71.4%, respectively. It could be concluded that OTA can induce plant toxicity in A. thaliana, calcium significantly inhibts the toxicity of OTA in A. thaliana, and plays an important role in A. thaliana's stress resistance.

A cysteine proteinase inhibitor from silkworm (Bombyx mori), named Bombyx cysteine proteinase inhibitor (BCPI), demonstrated the inhibitor activity for Bombyx cysteine proteinase (BCP) and cathespin L in vitro. By proteomics tools, BCPI was also identified from silk gland of silkworm, which showed the abundant expressional level in the silk gland of Ras1CA system of transgenic silkworm. In order to explore the expression pattern and its regulation in silkworm in vivo, in this study, we cloned the gene of bcpi and investigated the spatial and temporal expression at transcriptional level and translational level. The result showed that bcpi had a length of 315 bp, encoding a protein of 105 amino acid with a signal peptide of 19 amino acid at the N-terminal and a domain of inhibitor I29 at the C-terminal. Though BCPI was a protein from silkworm with low molecular weight, it had the high similarity with N-terminal domain of the cysteine proteinase from the baculovirus of lepidopteran insect. By reverse transcription PCR (RT-PCR) and quantitative Real-time PCR (qRT-PCR), the gene demonstrated a strong expression in the haemocyte of silkworm, and had a weak expression in head and epidermis in the 3rd day of 5th instar, respectively. In addition, differentially expressional level of bcpi were analyzed during the developmental stages from 1st day of 4th instar to 10th day of pupa stage by qRT-PCR. An up-regulated expression of bcpi was found in the pre-pupal stage and late pupa stage before eclosion. The expression pattern of bcpi suggested it perhaps was involved in development and metamorphosis of silkworm. By the Western blot, the high level of BCPI was accumulated in the hemolymph of silkworm. The strongest expressional level of BCPI in hemolymph appeared in the wander stage and the stage before eclosion. The result showed the BCPI functions in the hemolymph of silkworm and regulated the activity of Bombyx cysteine proteinase in different developmental stage. In order to investigate whether expression of bcpi were affected by insect hormone, the 20-hydroxyecdysone were applied to silkworm at the 2nd day of 5th instar larval. The result showed that the up-regulated expression were induced in the silkworm by 20-hydroxyecdysone by qRT-PCR. In order to further analyzed bcpi promoter responsed to ecdysone, dual luciferase reporter assay system were applied to study the bcpi promoter with the treatment of 20-hydroxyecdysone. However, the activity of luciferase was down-regulated in the BmE cell with the treatment of 20-hydroxyecdysone when the upstream 1 500 bp region of promoter was cloned and analyzed by dual-luciferase reporter assay system. The down-regulation of bcpi promotor was also validated by gradient concentration of 20-hydroxyecdysone. We precluded that there were complicated mechanism in the regulation of expression of bcpi in silkworm. The region except upstream 1 500 bp region of bcpi may be responsible for enhancement the expression with 20-hydroxyecdysone. In conclusion, expression pattern of bcpi in different tissues and developmental stages were analyzed in this work and the relationship between expression of bcpi and 20-hydroxyecdysone was discussed. The results offer us more clues to study the expression and function of bcpi.

Placenta immunoregulating factor (PF) is composed of small polypeptides, which is extracted from the placenta. PF is valuable candidate for treatment of viral disease, immune deficiency disorder and malignant tumor. To detect the effects of sheep (Ovis aries) and cow (Bos taurus) placenta immunoregulating factor on the proliferation and migration of the human(Homo sapiens) pancreatic cancer cells-Ⅰ(PANC-Ⅰ) in vitro. PF was prepared from placenta by bienzyme hydrolysis. PANC-Ⅰ cells cultured in vitro were exposed to PF at the mass concentrations of 100~2 000 ng/mL. The effect of PF on the PANC-Ⅰ cell cycle was detected by flow cytometric method. The migration ability of PANC-Ⅰ cell was investigated with Transwell assay. The inhibition to proliferation rate of PANC-Ⅰ cells were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay. The mRNA expression levels of tumor suppressor gene (P53) and cyclin-dependent kinase inhibitor 1A (P21) were analyzed by using real-time PCR. The relative expression was determined by using the comparative 2-??Ct method. The results showed that 0.588 mg PF derived from 17.11 g sheep placenta and 0.312 mg PF derived from 0.616g cow placenta, and the quality ratio was 20 000∶1. The morphology of the PANC-Ⅰ cells was not affect by adding the PF. The cell inhibition rate of PANC-Ⅰ cells was from 9.75%~22.89% with different concentration of sheep PF and from －3.81%~23.56% with different concentration of cow PF. The most remarkable concentration of sheep PF and cow PF were 500 ng/mL and 1 000 ng/mL, respectively. At the same time, the PI and SPF of PANC-Ⅰcells was the lowest with 500 ng/mL of sheep PF, but the proliferation index (PI) and S-phase fraction (SPF) were not affected by cow PF. The migration ability of PANC-Ⅰcells was not affected by PF. The expression levels of P53 and P21 of PANC-Ⅰcells were increased and decreased respectively after the treatment of PF. The results showed that the PF had a negative effect on the proliferation of PANC-Ⅰ cells in vitro, and the different effects of sheep and cow PF on PANC-Ⅰcells may related to the composition of the PF. This study provides new ideas for the treatment of pancreatic cancer.

The physicochemical properties of most food allergens confer stability to the proteolytic in the digestive tract. Stability of simulated gastric fluid (SGF) is an important parameter for the estimation of food allergenicity. To study the digestive stabilities of a major fish allergen — parvalbumin (PV) and non-allergenic proteins from the muscle of carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix) in SGF and simulated intestinal fluid (SIF), three kinds of proteinases including pepsin, trypsin, and chymotrypsin from porcine (Sus scrofa) were used to simulate digestive proteinases from humans (Homo sapiens). Two runs of trichloroacetic acid precipitation and gel filtration chromatography were used to purify the fish PV. Results were evaluated via Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) and Western blot. The results showed that PVs of carp and silver carp with molecular weight of approximate 10 kD was obtained. Similar results were obtained in SGF assays of purified PVs of carp and silver carp. The original PV band with pepsin was almost completely degraded within 60 min, and some stable peptide fragments were observed. Both trypsin and chymotrypsin could not effectively degrade PV in 240 min. In the SGF digestion on non-allergenic fish sarcoplasmic proteins were rapidly degraded within a short period of time, whereas the digestion time of PV was prolonged. Western blot analysis indicated that the polyclonal antibody against silver carp PV could specifically detect the PV and its degraded products. In conclusion, results indicated that fish PV was more resistant to proteinase digestion than non-allergenic proteins, and pepsin treatment was more effective than trypsin and chymotrypsin in reducing hypersensitivity. This study provides a theoretical reference for the development of hypoallergenic fish products in the future.

Laminin-beta-1 (LAMB1) gene is the member of the laminin family, and plays an important role in biological processes. To analyze the genetic effects associated with the wool traits of candidate genes LAMB1, based on DNA pools with the re-sequencing technology to screened 5 single nucleotide polymorphisms (SNPs) data of LAMB1 gene exon regions, combining with single strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP) technology to type the genotype, the genetic effects of LAMB1 gene 5 SNPs on wool traits of totally 418 sheep (Ovis aries) in fine-wool sheep at Xinjiang Kunes farm were analyzed by the GLM of SAS 8.1. Linkage disequilibrium (LD) was used by HaploView 4.2. The results showed this population genetic variations of fine-wool sheep was in the medium level, there were 5 non-synonymous mutation SNPs. AA and GA genotype in the identification of the body weight and body weight after shearingk of the individual value was extremely significant higher than GG genotype on SNP2 (P＜0.01). CC, TT and TC genotypes in the staple length of the individual value was extremely significant on SNP4(P＜0.01). CC genotype in the body weight after shearingk of the individual value was extremely significant higher than TT and TC genotype on SNP4 (P＜0.01). While, each genotypes in the part of wool traits on other SNPs were significance (P＜0.05). SNP1 and SNP3, SNP2 and SNP3 were in the linkage disequilibrium state, LAMB1 gene might be used as one of a potential and valuable candidate molecular marker on wool traits in fine-wool sheep. The study revealed the molecular genetic features of LAMB1 gene and evaluated the genetic relationship in fine-wool sheep. The results provide molecular genetic basis for efficient breeding of fine-wool sheep breeds, and efficient conservation and utilization the germplasm resources.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily, and there are 3 subtypes including α, β/δ and γ, and all PPARs members play a crucial role in cell growth and differentiation. In order to investigate their distinct functions in the process of skeletal muscle development, PPARs promoter sequences in duck (Anas platyrhynchos) were cloned using PCR and were analyzed using several bioinformatics tools. The mRNA expression profiles of PPARs and their common transcription factors in the breast and leg muscle of duck during embryonic stage were detected using qRT-PCR. The PCR results showed that PPARα, PPARβ and PPARγ promoter sequences of duck were 2 526, 1 631 and 2 942 bp,respectively, and GenBank accession were KX845431, KX845432 and KX845433, respectively. Their homology reached 99%, 97% and 97% with the predicted sequences of duck genome in NCBI, respectively. All PPARs promoters in duck shared higher homology compared with chicken (Gallus gallus) near the translation initiation sites through analyzing the cloned promoter sequences. The typical cis acting elements (CAAT-box and TATA-box) had been predicted to exist in the promoter region of PPARs, and common binding sites of transcription factors (specificity protein 1 (Sp1), nuclear factor kappa B (NF-κB) and CCAAT/enhancer binding protein alpha (C/EBP-α)) were predicted to exist in all duck PPARs members. Moreover, PPARα and PPARγ shared 9 common transcription factors, and PPARβ shared 8 with PPARγ, while PPARα and PPARβ shared the least number of common transcription factor. The qRT-PCR results showed that PPARα, PPARβ and PPARγ expressed in breast and leg muscle tissues of duck. Clustering results showed that the expression patterns of PPARβ and PPARγ were more similar compared with PPARα in leg muscle. It could be seen that the functions of the 2 genes were more similar in the process of duck skeletal muscle development. Besides, in breast muscle, the expression pattern of transcription factor NF-κB and PPARβ gene was consistent. PPARs members all participated in the regulation of skeletal muscle development, and the function of PPARβ might be larger; the function of PPARβ and PPARγ in skeletal muscle development might be similar. In addition, the NF-κB might control the expression of PPARβ in skeletal muscle. The results provide theoretical basis for the expression and function identification of PPARs gene.

Glutamine (Gln) can egulate expressions of several inflammatory cytokines while it is not clear if it can attenuate inflammatory injury induced by lipopolysaccharide (LPS). The sustentacular cells (SCs) of Bos taurus on their third generation were cultured with various concentrations (0.5, 1, 2, 4 and 8 mmol/L) of Gln for 12 h and the survival rates of the cells were measured using CCK-8 kit 4 h after changing medium. The cell survival rates were 90.81% treated with 2 mmol/L of Gln. The readily cultured SCs were randomly divided into 4 groups including control group (blank), LPS group (cocultured with 0.1 μg/mL LPS for 12 h), Gln group (cocultured with 2 mmol/L Gln for 12 h) and LPS+Gln group (treated with 0.1 μg/mL LPS for 4 h after cocultured with 2 mmol/L Gln for 12 h). Heat shock protein72 (HSP72), Interkeukin receptor-associated kinase-M (IRAK-M), Toll-interacting protein (Tollip) and Zinc finger protein (A20)mRNA and protein expressions were detected by using qRT-PCR and Western blot, respectively. Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interleukin-13 (IL-13) and Interleukin-17 (IL-17) protein expression were measured by using Enzyme linked immunosorbent assay (ELISA). The results showed that Gln could induce SCs HSP72, IRAK-M, Tollip and A20 expression. The HSP72, IRAK-M, Tollip and A20 levels were significantly (P＜0.05) increased compared to blank control group while the above indicators were all significantly (P＜0.05) decreased in LPS+Gln group compared with LPS group. Similarly, IL-1β, IL-6, IL-8, IL-10, IL-13 and IL-17 expressions were significantly (P＜0.05) increased in LPS group compared to the blank control group while they were all significantly (P＜0.05) decreased in LPS+Gln group compared with LPS group. The above results demonstrated that LPS can induce expressions of inflammatory cytokines and negative feedback regulatory factors including HSP72, IRAK-M, Tollip and A20. Gln can inhibite inflammatory cytokines induced by LPS and stimulate anti-inflammatory cytokines and thereby attenuates inflammatory injury.

Myostatin (MSTN) is a member of the transforming growth factor β (TGF-β) superfamily, which plays a key role in muscle development. Numerous studies have shown that knockout of MSTN results in a widespread increase in skeletal muscle mass, and a significant reduction in fat accumulation. However, the mechanisms involved in fat deposition process yielded by MSTN gene suppression has not been understood. In the present study, we constructed 5 two-way expression antisense RNA vectors pMSTN-CMV-CAG-antiMSTN, then transfected bovine fetal fibroblast cells, and screened the best antisense RNA vector which significantly disturbed the MSTN expression in protein level with Western blot. The vectors were then confected to a bovine muscle satellite cells and the effect of MSTN knockdown to genes associated with adipogenesis such as fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase-1 (SCD-1), genes associated with lipolysis such as carnitine palmitoyltransferase 1b (CPT-1B), acyl-CoA oxidase(ACOX1) and enoyl-CoA hydratase 1 (ECHDC1), and fat transport protein (FATP) gene were investigated. The results showed that genes associated with adipogenesis and lipolysis were all up-regulated except the expression of ECHDC1 when MSTN knockdown in muscle satellite cells. The expression of gene associated with fat transport has no significant change. The results suggested that MSTN knockdown could enhance both adipogenesis and lipolysis, so the reduced fat mass resulted from MSTN knockdown was due to the increased lipolysis rather than inhibition of adipogenesis. And the expression of CPT-1B was up-regulated in the highest level among the genes investigated, this result suggested that the effect of MSTN to fat was mainly by the process of β oxidation, because CPT-1B was a main factor of β oxidation. This study provides a theoretical basis for the decreased fat content resulted from MSTN reduction, and makes a preliminary study for the effect of MSTN on fat deposition.

Phosphate solubilizing bacteria (PSB) can increase available phosphorous in soil and improve yields in several crop species like rice (Oryza sativa), maize (Zea mays), potato (Solanum tuberosum), sunflowers (Helianthus annuus) etc. In previous studies, many phosphate solubilizing bacteria had been isolated and widely used in agricultural production. However, the colonization of phosphate solubilizing bacteria in plant rhizosphere have not been completely characterized. Phosphate solubilizing bacteria WY4 is a bacterium isolated from the soil of Brassica chinensis rhizosphere soil. WY4 is a gram-positive bacterium which is tested by Gram staining and its cell morphology is rod shape. The sequence analysis of 16S rRNA gene in the NCBI database using a BlastN analysis showed 99% similarity to Bacillus megaterium strain. To evaluate colonization in soil and Chinese cabbage rhizosphere by this microorganism, generated WY4-GFP, a green fluorescent protein (GFP) labeled derivative of WY4, and inoculated the marked strain into Chinese cabbage seedlings. Compared with the original strain, GFP-labeled had a smaller influence on the growth and phosphate solubilizing activity. While, the results demonstrated that WY4-GFP could survive in the natural soil to achieve 105 CFU/g (wet soil) at 21 days; WY4-GFP increased the plant height, root length, leaf numbers, fresh weight and dry weight of Chinese cabbage plants. Phosphorus could promote root growth of Chinese cabbage, WY4 could increase content of available phosphorus as PSB, so the root system was developed after application of WY4: The root volume that reached 1.72 mL was significantly higher than water treatment (0.38 mL). After the treatments of WY4-GFP and WY4, the fresh weight of the plant reached 17.48 and 17.52 g/plant respectively. There was no significant difference between WY4 and WY4-GFP in growth promotion. The results further indicated that WY4-GFP flourishing colonizes in Chinese cabbage plants and rhizosphere soils, colonization of WY4-GFP analysis in nature soil or rhizosphere soils showed that the population of WY4-GFP decreased gradually as the extension of time. At 0~10 d, The number of WY4-GFP decreased very fast (from 13.32~3.47×106 CFU/g), but after 20~30 d incubation in Chinese cabbage rhizosphere soils, the number of WY4-GFP were basically stabled at 104 CFU/g. Furthermore, the results indicated that Bacillus megaterium WY4 displayed good colonization ability in the rhizosphere of Chinese cabbage. Plentiful WY4-GFP colonized in different growth period (5, 10 and 20 d) of the root tips and was detected in meristernatic zone. Meanwhile, less WY4-GFP appeared to the root elongation zone, lateral root hair and hypocotyls catheter, but numerous labeled bacteria existed in intercellular spaces of Chinese cabbage roots. Taken together, these results indicated that WY4-GFP invaded Chinese cabbage through the root tips or intercellular spaces, and they could be transported into the hypocotyls catheter by the vascular bundle system of roots. Preliminary study on the mechanism of phosphorus solubilizing bacteria promoting the growth of Chinese cabbage showed the good application prospects of PSB. The results provide important information for further study on the relationship between phosphate solubilizing bacteria and plant.

The bone morphogenetic protein receptor ⅠB (BMPR-ⅠB) gene may play important roles in breeding process of mammals, but its molecular hereditary mechanism is seldom studied in goat (Capra hircus). In order to reveal the effect of BMPR-ⅠB on reproductive traits of Qianbei Ma goat(Ovis linnaeus), direct sequencing combined with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)technique were used to detect the single nucleotide polymorphisms in exon 9 of BMPR-ⅠB. And the relative expression of BMPR-ⅠB mRNA was quantified in different tissues by qRT-PCR. The result showed that one SNP locus-C40G was found in exon 9 of BMPR-ⅠB in Qianbei Ma goat, and it belonged to synonymous mutations, three genotypes CC, CG and GG were detected by SSCP analysis, those goat with CG genotype achieved significant difference level compared to CC and CG genotypes individuals for litter size (P＜0.05). The qRT-PCR result showed that BMPR-ⅠB expressed in detected all 5 tissues for single lamb group and more than double lambs group of Qianbei Ma goat, and the expression trend was consistent in the 2 groups, high expression in the ovaries but low in fallopian tubes. Moreover, the mRNA expression of BMPR-ⅠB in ovaries of 2 groups achieved extremely significant difference level (P＜0.01), and no significant difference were found among other tissues (P＞0.05). Sequence alignment of BMPR-ⅠB nucleotide among species revealed that BMPR-ⅠB of Qianbei Ma goat had similarity of 99.6%, 98.8%, 99.2%, 97.4%, 97.8%, 99.0%, 98.0% and 99.6% with Boer, Bos taurus, Capra hircus, Homo sapiens, Mus musculus, Ovis aries, Sus scrofa, Yunling Black goat. These results indicated that C40G mutation of BMPR-ⅠB may have influence on fertility of Qianbei Ma goat. On the basis of the traditional breeding method, the marker-assisted selection of BMPR-ⅠB can be conducted to further provide theoretical foundation for the prolificacy improvement of Guizhou local goat breeds.

Senescence, regarded as the last stage of tobacco leaf development, seriously affects the accumulation of the photosynthetic products along with degradation of chlorophyll and lipid, resulting in the decrease of the yield and the quality of the crop. Transgenic technology is an important technical means to cultivate new varieties with high yield and resistance, which is helpful to increase the yield of the unit area of the tobacco (Nicotiana tabacum). During the growth of tobacco, the delay of senescence can increase the amount of photosynthetic products, which could increase the yield of crop. After harvesting, senescence delay of the leaves can keep the freshness of tobacco, so as to solve the problem of storage and transportation. Therefore, research on leaf senescence regulation has a very important significance in agricultural production. In this work, the Agrobacterium-mediated transformation method were adopted to introduce BAS1 (phyB activation-tagged suppressor 1) gene driven by the SAG12 promoter into tobacco and 45 SAG12-BAS1 trangenic plants were obtained according to the result of resistance screening and PCR identification. Among these transgenic plants, eight transgenic tobacco plants showed that their leaves senility were delayed. Leaf chlorophyll content, protective enzyme activity and plant growth phase were observed and measured on the wild type and transgenic SAG12-BAS1 tobacco leaves during leaf senescence. The results showed that the chlorophyll content of transgenic SAG12-BAS1 tobacco plants was higher than that of the wild type from the top to the base. The results of content analysis of super-oxide dismutase (SOD) and malondialdehyde (MDA) investigated between the wild type and transgenic SAG12-BAS1 tobacco demonstrated that SOD activity in transgenic SAG12-BAS1 tobacco plants was 31.98 % higher than that of wild type and the content of MDA was 48.28% lower than that of the wild type. The conclusion that the senescence of transgenic tobacco delayed for 10~15 d than that of the wild type was drawn through the observation of tobacco growth development process. Besides, the result of cytokinin content detected between the wild type and transgenic tobacco at the beginning of leaf senescence showed that the cytokinin content of the wild type was 40.2% lower than that of transgenic plants. The above results indicated that transgenic SAG12-BAS1 tobacco have delayed the senescence process and that may relate with the increase of cytokinin content and protective enzyme activity and the initiation of senescence promoter. This study provides a theoretical basis for the further study of the function of SAG12-BAS1 gene, and it also provides the basis for the development of genetically modified anti-aging materials.

Bone morphogenetic protein 15 (BMP15) is an important regulation factor of reproductive traits in sheep (Ovis aries), however, the specific mechanism now is not certain. This study aimed to investigate the relationships between the expression levels of BMP15 mRNA and the mutations of BMP15 gene and fecundity in different sheep breeds. The expression profilings in tissues and the expression levels in ovary were determined between high fecundity breed (Small Tail Han sheep) and low fecundity breeds (Tan sheep and Sunite sheep) by using RT-PCR and qRT-PCR methods, respectively. The 3 new mutations in ovine BMP15 gene were detected in 16 sheep populations of different fecundity by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing methods, individually. The results showed that BMP15 mRNA was detected in sheep ovary, oviduct, uterus, spleen, duodenum, skeletal muscle and subcutaneous fat, and the BMP15 mRNA of ovary was higher than that of other tissues; The BMP15 expression levels were different in ovary of 3 breeds, and Small Tail Han sheep (high fecundity breed) was significant lower than that of Tan sheep and Sunite sheep (low fecundity breeds) (P＜0.05), Tan sheep and Sunite sheep had no significant difference in ovary (P＞0.05); The new mutations of FecXGr, FecXO and G971A were not detected in all of the 16 sheep populations. The results implied that the BMP15 mRNA expression level was high in ovary and the ovulation performance and the litter size of sheep were negative correlation with the expression level of BMP15 in ovary. This study will provide a reference for revealing the functional mechanism of the regulatory factors of fecundity in sheep.

The barley (Hordeum vulgare) net blotch is one of the most usual barley diseases, caused by Pyrenophora teres. The barley net blotch is becoming so popular, the major reasons is that our country is short of germplasm resources of resisting barley net blotch. In order to increase the diversity of barley net blotch resistance in cultivated barley, the genetic diversity of barley was analysed, and the reaction to P. teres of 89 barley materials at seedling stage was evaluated. Eventually, identified SSR markers associated with resistance to barley net blotch in barley with an association mapping approach. The results showed that 89 barley entries were analyzed by using 70 pairs of SSR markers which were used to detect the genetic diversity among cultivars. A total of 302 alleles were detected, the average each pairs had 4.31 and the arrange of allele number was 2~8. The allele frequency varied from 0.141 2~0.916 7, the genetics diversity ranged from 0.103 9~0.894 4, and the genetics similarity ranged from 0.520~0.873. The polymorphism information content (PIC) value ranged from 0.098 5~0.884 6 with an average of 0.537. Barley entries were divided into 2 subgroups based on their population structure, group one had 22 cultivars, group two had 67 cultivars, the genetic similarity of the all group was from 0.635~0.895. The genetic similarity of MEI 41/I and MEI 43/I was the highest, it was 0.895. There was the large difference among barley materials, and disease index ranged from 31.67~96.32, and there were no immune or high resistant varieties, just 2 resistant-middle resistant (R-M) varities with the disease index of 31.67 and 33.56, one cultivar (ZD02732407) was moderate resistant (MR), disease index was 45.22%, and then there were 14 middle resistant-middle susceptible (MS-S) varieties, 50 middle susceptible-susceptible (MS-S) varieties and 7 high susceptible (S) varieties. The association analysis result showed that 5 markers were found to associate with barley net blotch resistance under general linear model (GLM) program, these QTLs were located on chromosomes 1H, 3H, 5H and 6H, and the rate of phenotypic variation explained ranged from 7.2%~22.4%. Bmac29 and Bmag0613 showed significant association with resistance to barley net blotch (P＜0.01), which explained 10.7% and 22.4% of the phenotypic variation. This study demonstrates that AM is an effective technique for identifying and mapping QTL for barley net blotch resistance in a natural population and then this study also provides some helpful basis to barley net blotch resistant breeding.

Follicle-stimulating hormone (FSH) is a glycoprotein and gonadotropin hormone synthesized and secreted by animal anterior pituitary basophils. In males, FSH participates in spermatogenesis. In this study, weight analysis, histopathology analysis, qRT-PCR analysis and methylation analysis were performed to evaluate the influence of Erhualian FSH high-level expression on the testes of F1 Erhualian FSH transgenic (TG) boars and their wild-type (WT) male full/half sibs at the age of 15 d (n=6) and adulthood (n=10). Six suckling piglets including 3 TG and 3 full-sib WT individuals and 10 adult boars including 5 TG and 5 half-sib WT individuals were slaughtered. Absolute weight and relative weight analysis of the testes were performed. There were no significant differences in the testes absolute weight of suckling piglets (P＞0.05) and adult boars (P＞0.05). There were also no significant differences in the testes/body weight ratio of suckling piglets (P＞0.05) and adult boars (P＞0.05). Testes absolute weight and testes/body weight ratio analysis showed that Erhualian FSH high-level expression had no influence on the testes weight. Paraffin sections of the testes samples were stained with hematoxylin-eosin (H&E) and observed with a BA410 microscope (Motic, China). Gonocyte cells per unit area (about 171 100 μm2) for suckling piglets and intersitial cells per unit area (about 39 000 μm2) for adult boars were calculated. Area and perimeter per seminiferous tubule were also calculated using Image-Pro Plus 6.0 software. Only a significant increase were observed in gonocyte cells per unit area of 3 TG suckling piglets compared with that of 3 full-sib WT suckling piglets (P＜0.001). Analysis of HE-stained paraffin sections of testes indicated that Erhualian FSH high-level expression could increase gonocyte number and had no effect on the organizational structure of the testes of Large White boars. The expression levels of genes related to reproduction in the testes were detected by qRT-PCR. No significant difference (P＞0.05) was found in the expression levels of follicle-stimulating hormone (FSHR), luteinizing hormone receptor gene(LHR), and androgen receptor gene (AR) between TG boars and their WT male full/half sibs at the age of 15 d and adulthood, demonstrating that Erhualian FSH high-level expression had no influence on the expression levels of genes related to reproduction in the testes. Meanwhile, the methylation levels of FSHβ and FSHR in the testes were detected. There was no significant difference (P＞0.05) in the methylation levels of FSHβ and FSHR between adult TG and WT boars, revealing that Erhualian FSH high-level expression had no effect on the methylation levels of FSHβ and FSHR in the testes of Large White boars. The results in this study showed that high-level expression of Erhualian FSH could increase gonocyte number to improve spermatogenesis ability of Large White boars and also provides references for spermatogenesis process in human studies.

Peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) has been a hot studying spot gene in meat quality traits of livestock because of its prominent role in regulation of muscle fiber type switching. To study the expression profile of PGC-1α gene in 2 phenotypically distinct muscles between 2 chicken breeds at early growing stage, qRT-PCR and Western blot were used to detect the mRNA and protein expression of PGC-1α gene at 0, 1, 3, 5, 7 and 9 weeks in soleus and extensor digitorum longus between Qingyuan Partridge chicken (slow-growing chicken breed) and Recessive White chicken (fast-growing chicken breed). The results showed that the expression of PGC-1α mRNA of the 2 muscles declined firstly, then increased following by decreasing, and finally increased again. The expression peak of PGC-1α mRNA of all the muscles appeared at 0 week on which the expression levels were significantly higher than that at other weeks (P＜0.05), and the lowest expression in soleus in 2 chicken breeds appeared on 3 week, and the lowest expression in extensor digitorum longus in 2 chicken breeds appeared on 7 week. While the expression of PGC-1α protein of the 2 muscles firstly gradually increased from 0 week to 5 week, then declined from 5 week to 7 week, and increased again from 7 week to 9 week. The expression peak of PGC-1α protein in extensor digitorum longus in Recessive White chicken appeared at 5 week. Although the expression trends of mRNA and protein of PGC-1α gene of the 2 muscles were not identical during early postnatal growth, they were highly muscle phenotype and breed specific. In general, the expression levels of mRNA and protein of PGC-1α gene in soleus were higher than those in extensor digitorum longus of both chicken breeds at the same weeks of age, and the expression of PGC-1α mRNA in 2 muscles was extremely significant difference at 0 week (P＜0.01). The expression levels of mRNA and protein of PGC-1α in Qingyuan Partridge chicken were higher than those in Recessive White chicken at the same weeks of age, and the expression of PGC-1α mRNA in 2 muscles was extremely significant difference at 0 week (P＜0.01). These results indicated that the expression level of PGC-1α might concern with the content of slow muscle fiber in skeletal muscle in chicken, and its function might be closely related to the chicken meat quality. There search provides some valuable clues for understanding the role of PGC-1α in the skeletal muscles in chickens and a theoretical basis for improving the meat quality in chickens.

There are two genotypes (ⅠandⅡ) of Duck reovirus (DRV) in clinical infection ducks (Anas platyrhynchos domestica). To establish a multiplex one-step RT-PCR method for the simultaneous detection and differentiation of DRV genotypeⅠ(classical DRV, C-DRV) and genotype Ⅱ (novel DRV, N-DRV) from clinical samples, two sets of primers were designed for a RT-PCR assay system according to S1 and S4 non-structural protein encoding sequences of two genotypes viruses. The amplification conditions, including primer concentration, annealing temperature and cycle times, were optimized. The results showed that the most effective and precise reaction system was as follow: PrimeScript 1 Step Enzyme Mix 1 μL, 2×1 Step Buffer 12.5 μL, each primer (20 μmol/L) 1 μL, RNA 1.5 μL, adding RNase Free dH2O to a final volume of 25 μL; The optimal reaction procedure was as follow: Reverse transcription for 30 min at 50 ℃; Pre-denaturation for 2 min at 94 ℃; Denaturation temperature for 2 min at 94 ℃, annealing temperature for 30 s at 55 ℃, extension for 30 s at 72 ℃, total of 30 cycles. These genotype-specific primer pairs could amplify 249 and 505 bp PCR products corresponding to C-DRV and N-DRV, respectively, and 2 fragments of 249 and 505 bp were simultaneously amplified from the mixed RNA sample of C-DRV and N-DRV. The assay exhibited high specificity as all negative controls and other duck pathogens, such as the Duck tembusu virus (DTMUV), Duck plague virus (DPV), Newcastle disease virus (NDV), Muscovy duck parvovirus (MDPV), Duck hepatitis A virus (DHAV-1) and H9N2 Avian influenza virus (AIV). The sensitivity of this method was determined to be 0.47 50% tissue culture infective dose (TCID50) for C-DRV and 0.62 TCID50 for N-DRV. Virus RNAs extracted from different generations and different times were detected by the developed multiplex one-step RT-PCR. The result showed that the method was found to be highly reproducible. In addition, RT-PCR assays of DRV diseased fowl samples (total 239) collected from different regions of Zhejiang province during 2011~2015 were taken. The detected results showed that the positive rates of C-DRV and N-DRV were 2.5% (6/239) and 31.4% (75/239), respectively. Six C-DRV positive samples were all Muscovy duck. In 75 N-DRV positive samples, there were 54 Muscovy duck (Cairna moschata) samples (positive rate 72%), 5 goose (Anser anser) samples (positive rate 6.7%) and 16 other breed duck samples (positive rate 21.3%). The results indicated that N-DRV was the currently prevalence genotype in Zhejiang province. P10 and P18 sequence of positive tissues were analyzed to confirm the accuracy of the test results. In addition, the multiplex one-step RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for C-DRV and N-DRV infections in duck, and the assay can provide effective technical support for molecular epidemiological survey of DRV.

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus. It mainly infects piglets and causes severe enteritis accompanied by diarrhea and vomiting and other symptoms. The symptoms are symptomatically indistinguishable from those that caused by Porcine transmissible gastroenteritis virus (TGEV). In clinic the co-infection of PDCoV and TGEV is common, which causes huge economic losses to the swine industry. In order to control and prevent effectively these diseases, development of rapid detection methods of PDCoV and TGEV is the primary task. Thus, according to the analysis of PDCoV and TGEV N gene sequences in GenBank, two pairs of primers targeting the N gene of PDCoV and TGEV were designed respectively. By optimizing the PCR reaction conditions, a duplex RT-PCR detection method was established for the simultaneous detection of PDCoV and TGEV, and the specificity, sensitivity and repeatability of the method were studied. The results showed that the duplex RT-PCR had high sensitivity and specificity with a limited detection of 3.14×102 copies/μL for PDCoV and 3.68×103 copies/μL for TGEV, respectively, and no specific amplifications from other virus, such as Porcine epidemic diarrhea virus (PEDV), Porcine bocavirus (PBoV), Porcine respiratory and reproductive syndrome virus (PRRSV) or Porcine pseudorabies virus (PRV). 252 clinical samples tested by the duplex RT-PCR showed that the positive rates of PDCoV and TGEV were 12.30% and 4.76%, respectively, co-infection rate of PDCoV and TGEV was 0.79%. The date showed that the duplex RT-PCR method established in this study could be used for the clinical detection of PDCoV and TGEV, which provides the theoretical basis information and technical support for further applied to clinical differential diagnosis and epidemiological investigation of TGEV and PDCoV.