In order to construct the genetic linkage map of triticale (×Triticosecale) and locate the quantitative trait locus (QTL) resistant to the stripe rust disease in triticale, F2 generation derived from triticale cross was used as the mapping population and fourteen inter-simple sequence repeats (ISSR) primers were used to amplify the F2 generation. One genetic linkage map was constructed using the Joinmap 4.0 software and QTLs related to the resistance of stripe rust was located in triticale. Main results were as follows, a) the linage map included 7 linkage groups (named LG1 ~ LG7), the total length of map was 542.9 cM, the averaged distance among markers was 5.90 cM, and there were 92 loci. b) For 184 plants of F2 population, the resistance and susceptibility to the rust disease changed continuously and the distribution frequency was nearly normal. Therefore, it was suitable to be used to locate the QTLs related to the resistance of stripe rust in triticale. c) QTL results showed that there were 6 QTLs (QDR1, QDR3, QDR4, QDR5-1, QDR5-2, QDR6) related to the resistance of stripe rust in triticale by using MapQTL6.0 software while the threshold was LOD≥2.0. Six QTLs distributed on the linkage groups of LG1, LG3, LG4, LG5 and LG6. The contribution rate of QTLs resistant to the stripe rust varied from 5.1% to 11.2%. Among 6 QTLs, QDR5-1 was the main QTL resistant to stripe rust and the additive effect varied from -0.18 to 0.27. The QTLs located in this study will provide theoretical basis for the cloning, transformation, utilization of the genes resistant to the stripe rust in triticale, and elevating the efficiency to breed new triticale varieties resistant to stripe rust disease.

Ningxia which as an important beef cattle breeding province in China has responsibility to provide superior beef meat to whole country. The purpose of that was to study on genetic polymorphism of UCP3 gene in Simmental Hybrid Cattle (Bos taurus) and analyze their association with several growth traits to find genotype with fast growth rates that provided foundation for selecting and mating. PCR-SSCP and DNA sequencing were used to detect polymorphisms, contained DNAMAM, POPGENE1.31, PIC program, SAS software to calculate allelic frequencies, genotypic frequencies, PIC value, analysis different genotype association with growth traits (body weight, body height, body length, chest girth). Five SNPs on exons of bovine UCP3 gene were identified: G5465A, C7921G, A7953G, T8018C, T8162C, and caused transversion coding from alanine to threonine, proline to arginine, cysteine to arginine, tyrosine to histidine. The result of χ2 showed that two loci were deviated from the Hardy-Weinberg equilibrium. Genetic diversity analysis showed that the PIC values of two loci were 0.372 7, 0.370 4, which represented moderate polymorphism. The result of least square analysis showed that in exon3 fragment, the individuals with GA genotype showed better body sizes values in every months than GG genotype, the mutation of 5 465 bp G→A had significant effects in body weight, height and body length, especially in the later of growth stage, weight gain showed advantages obviously. In exon6 fragment, individuals with CC genotype showed better body sizes values than TC genotype, the mutation of 8 162 bpT→C had significant influence in body height and length from birth to 18 months (P＜0.05). Furthermore, analysis showed that body measurement traits (BW: body weight; BH: body height; BL: body length) of individuals with combined genotype GACC were higher than those of the other kinds of combinations. The effect of multi-loci combinations on BW were higher than those of single-marker locus. In hybrid simmental cattle, the variance analysis showed that the different genotypes of single or combined sites are significantly or extremely associated with its growth traits, combined genotype GACC had better BW, BL than single genotypes in different months. This study concluded the loci (5 465 bp, 8 162 bp) would be suitable for marker assisted selection (MAS) of Ningxia hybrid simmental cattle and GACC genotype may be the best one for the BW trait of Ningxia simmental hybrid cattle breeding.

Vitamin C, also known as L-ascorbic acid (AsA), plays an important role in plant growth and development, stress response, signal transduction and so on. Its synthesis and accumulation is regulated by many biological and non-biological factors. In order to explore the mechanism of plant growth regulators regulating the accumulation of AsA in fruit, the fruits of Rosa roxburghii Tratt 'Guinong 5' were used to investigate the effects of exogenous plant growth regulators on AsA accumulation and expression of metabolite genes using the methods of incubation in vitro and spraying in vivo. The three exogenous plant growth regulators included brassinolide (BR), methyl jasmonate (MeJA) and gibberellin 3 (GA3). The results indicated that the content of AsA in the fruit of Rosa roxburghii treated by BR, including exogenous feeding and field spraying, was significantly increased, while it showed the opposite situation in the MeJA and GA3 treatment and the AsA content decreases with the lapse of time. Furthermore, different influence patterns were obtained on the expression of AsA metabolic genes by qRT-PCR analysis after three exogenous plant growth regulators treatment. The expression of genes involved in AsA biosynthesis including GDP-L-galactose phosphatase(GGP), L-galactose phosphatase(GPP), L-galactose dehydrogenase(GDH), L-galactono-1,4-lactone dehydrogenase(GalLDH), D-galacturonic acid reductase(GalUR), Myo-inositol oxygenase(MIOX), and the reduction process including Dehydroascorbate reductase(DHAR), Monodehydroascorbate reductase(MDHAR) had different degrees of upregulation when the fruits of Rosa roxburghii were treated with BR in vitro incubation, especially the effect on GGP (about 5 times after 24 h). This effect was consistent with that of the field spraying experiment. It was inferred that exogenous BR treatment could promote the accumulation of AsA in Rosa roxburghii by increasing the expression of genes of AsA synthesis and reduction process. In vitro experiment, the expression of GGP, GalLDH and DHAR was up-regulated after 24 h treatment of exogenous GA3, and the other metabolic genes showed a decreasing trend. At the same time, the expression of GDP-mannose-3′,5′-epimerase (GME), GalUR and Ascorbate peroxidase (APX) had a certain degree of upregulation after 24 h treatment of exogenous MeJA and the expression of remaining metabolic genes was different; Field spray verification experiments showed that exogenous GA3 treatment down-regulated the expression of synthetic pathway genes, meanwhile, up-regulated the expression of Ascorbate oxidase (AAO). On the other hand, the accumulation of AsA in exogenous MeJA treatment was significantly inhibited in the fruit of Rosa roxburghii. And the gene expression pattern in the synthetic pathway was different, moreover, genes in oxidation ways had no differences after MeJA treatment. In general, it did not show a consistent pattern between the expression of genes and AsA accumulation after the two exogenous plant growth regulators treatment. This study show that different exogenous growth regulators have different effects on AsA accumulation in fruits of Rosa roxburghii and would provide a theoretical basis for exploring the mechanism of plant hormones involved in AsA synthesis or accumulation.

Growth hormone (GH) has the ability to regulate the rhythm of growth. The present study was carried out to investigator the differential expression of GH, growth hormone receptor (GHR), insulin-like growth factor 1 (IGF1) and insulin-like growth factor 1 receptor (IGF1R) in different organs of Mongolia horse (Equus caballus). Four Mongolia horses with an average age of 2 years old were selected as the research object. The qRT-PCR and immunohistochemical detection were used to analyze the expression levels of GH, GHR, IGF1 and IGF1R in different organs of Mongolia horses. The results of RT-PCR results showed that the expression of GH in spleen were higher than in the lymph, heart, liver, kidney, skeletal muscle and tail muscle (P< 0.05). The expression of GHR in liver were higher than in the pancreas, tail muscle, testis, heart, skeletal muscle, lymph, kidney, spleen and lung (P< 0.05). The expression of IGF1 in liver were higher than in other organs (P<0.05). The expression of IGF1R in turn was lung, liver, spleen, testis, heart, kidney, lymph, skeletal muscle, tail muscle, pancreas (P<0.05). Unequal immunoreaction intensity was observed in different tissues by immunohistochemistry. According to the number of positive cells, the positive expression of GH protein in liver and spleen was higher than in other organs (P<0.05). It was found that the protein expression of IGF1 in liver was strongest, followed by the Spleen, kidney and testis, and that in skeletal muscle was weakest (P＜0.05). The present study showed that GH, GHR, IGF1, IGF1R had a differential expression in different tissues by studying at RNA level and protein level. This study provides a theoretical foundation and scientific basis for breeding of Mongolia horse.

Under physiological conditions, thrombomodulin (TM) can combined with thrombin into a compound, which can prevent blood coagulation and promote fibrinolysis by activating protein C; and can be absorbed by endothelial cells, where the thrombin is decomposed by intracellular lysosomes. In addition, TM can be combined with coagulation factor Xa specificity, inhibit the activation of prothrombin, so that makes the generation of thrombin decreased significantly. When pig (Sus scrofa) blood vessels expose to the human (Homo sapiens) blood after xenotransplantation, the pig TM can't combine with human thrombin, which is the reason of coagulopathy. Wuzhishan Miniature pigs are best candidate for organ xenotransplantation donor with characteristics of highly similar anatomy and physiology to human. There are three aims of this research: produce transgenetic Wuzhishan Minipigs expressing human thrombomodulin (hTM), research hTM expression patten in endothelial cells of the transgenetic pigs, and provide practical basis for solve disorders in blood coagulation after xenotransplantation. Taking the Large White Pig’s TM bacterial arti?cial chromosome (BAC) as a template, constructed a PBR322-catch vector that had homologous short arm. A 7 kb promoter of porcine TM gene was sub-cloned from BAC of Large White Pig by gap-repair method mediated by red homologous recombination system in E. Coli., positive vector pBR322-catch-pProwas identified by enzyme. hTM amplified from human cDNA and bovine Growth Hormone PolyA (bGHpA) amplified by PCR, were linked to the pBlueScriptⅡ(SK-)vector. Vector pBR322-catch-pProwas was digested by enzyme to obtain the Large White Pig’s TM promoter, then connected it with vector pBlueScriptⅡ(SK-)to construct the specific expression pBS-hTM vector containing puromycin. The Xhol Ⅰ linearized pBS-hTM was integrated into the fetal fibroblasts cell lines (WPF167 and WPF169) by electroblot. Screening cells using culture solution contained 1.0 μg/mL puromycin for 10~15 days. Two cell clones with better growth characters were used as donor cells to conduct nuclear transfer to acquire reconstructed embryo. 972 reconstructed embryos were harvested, and then transferred into 5 Wuzhishan Miniature receptor pigs. New born piglets were identified by genomic DNA PCR, and umbilical cord tissue RT-PCR and Western blot. As results, positive catch vector pBR322-catch-pPro and expression vector pBS-hTM were successfully acquired. 354 cell clones were gained after 10~15 days screening, in which 339 cell clones were positive. The reconstructed embryo had no signi?-cant differences with the normal reconstructed embryos (P＞0.05). Two receptor pigs showed pregnant by B–ultrasonic determination after 30 days and 5 piglets were born after 120 days. 4 of new born piglets were identified positive and the expression of hTM specifically on the surface of endothelial cells. As a conclusion, transgenic cloned Wuzhishan Miniature pigs were successfully achieved, which specifically expressed hTM in vascular endothelial cells. This study could provide practical basis for further in-depth study in coagulation disorders after xenotransplantation.

Synthetic hexaploid wheat (SHW) is known to be an excellent vehicle for transferring genetic variations especially many traits present in the D genome of Aegilops tauschii Cosson accessions (2n=2x=14, DD) to cultivated wheat (Triticum aestivum)(2n=6x=42, AABBDD) for improving its quality. The objective of this study was to identify 102 simple sequence repeat (SSR) markers associated with 4 important agronomic traits (plant height (PH), top internode length (TIL), spike number per plant (SNPP) and spike length (SL)) in a natural population (86 SHWs and 42 common wheats). A total of 660 alleles were identified; the number of alleles per locus varied from 3 to 11 with an average of 4.6. The polymorphism information content (PIC) value ranged from 0.247 7 to 0.897 1 with an average of 0.723 5. The maximum and minimum values of PIC values were WMS174 and GWM601, respectively. BARC241, GWM498a, GWM495, GWM359b, GWM273a and GWM508 had relative larger PIC values. The tested materials consisted of three subpopulations by the population structure analysis and cluster analysis, the two results were basically the same. There were slight differences, these may have been due to their complex genetic backgrounds. Additionally, the result of cluster analysis using unweighted pair-group with arithmetic mean (UPGMA) was in accordance with the model based analysis, except for V023 and V072; these were observed to cluster in another subgroup. However, there was a clear distinction between SHWs and common wheats, indicating that the genetic background of most SHW varieties varies greatly compared to common wheat varieties. Association analysis between traits and simple sequence repeat (SSR) markers was performed using the generalized linear model (GLM) approach. 20 and 17 loci were found to be associated with the above-mentioned traits in 2015 and 2016, respectively (P＜0.01). In 2015, 10 loci were found to be associated with more than one trait. WMC702 was associated with both SNPP and SL; WMS408 was related with PH, TIL and SL; the explanation rate ranged from 0.039 8 to 0.247 2; the maximum and minimum rates were for GWM-111, and WMS-174, respectively. In 2016, 12 loci were detected to be associated with more than one trait. GWM570b had significant association with both TIL and SL; GWM41 were associated with PH and TIL. The explanation rates range from 0.063 9 to 0.191 3, with the maximum and minimum values observed for GWM508 and WMS408, respectively. These tested significant markers may broaden our knowledge for how to harness genetic diversity and provide resource for further wheat molecular marker assisted breeding.

Hvsusiba2 is a transcription factor acting on the upstream of starch synthesis pathway, and be recognized as a key regulator for barley (Hordeum vulgare) starch acumulation. In order to improve the rice (Oryza sotiva) grain starch content and understand how Hvsusiba2 works in rice, the Hvsusiba2 were introduced , which be constructed under down-stream of a specific expression promoter pSBEIIb from barley via agrobacterium-mediated transformation. Twenty-eight independent transformed lines were obtained. After self-crossing and screen, two homozygous lines with single-copy integrated were selected for investigation. By using qRT-PCR, genes involved in starch synthesis transcript level in 2 lines rice and non-transgenic control were detected. The results showed Hvsusiba2 was efficiently transcription in rice, along with 14 of genes involved in starch synthesis transcript were significantly altered. Among them 3 involved in amylopectin synthesis genes including starch branching enzyme gene (OssbeIIb), as well as isoamylase gene (Osiso1) and sucrose transporter gene (Ossut1) were elevated to above 2 time higher in the transgenic lines compared with the wild-type control. In additions 2 sucrose synthase genes (Ossus2, Ossus3), sucrose transporters gene (Ossut5) as well as UDP-glucose pyrophosphorylase gene (Osugp1) and starch branching enzyme gene (OssbeI) transcription were enhanced, leading to seeds starch content increased from 77.73% to 86.49% and 85.15% in 2 lines of Hvsusiba2 rice, respectively. Our data indicate that manipulation of starch in rice grain is possible using transcription factor. The results provide a theoretical basis for improving the content of rice endosperm starch by molecular method.

As a traditional medicine, Tripterygium wilfordii, which the main active ingredients are terpenes and alkaloids, has a long history and wide range of applications in agriculture and medicine. The WRKY transcription factor is a plant-specific transcription factor and involves in plant growth, development, aging, biotic and abiotic stress and secondary metabolites biosynthesis regulation. From the hairy roots of T. wilfordii, This study cloned a WRKY transcription factor and named it as TwWRKY1 (GenBank No. GAVZ01022264.1) using rapid-amplification of cDNA ends (RACE) technology. The full-length of the gene is 962 bp, the open reading frame is 537 bp and encodes 178 amino acids. The analysis of bioinformatics showed TwWRKY1 contains a typical WRKY conserved domain, zinc finger structure was C2H2, which belonged to Class Ⅱ of WRKY family; the phylogenetic analysis showed that the TwWRKY1 has a 83% parental relationship with Coptis jiponica CjWRKY1. Analysis of TwWRKY1 expression in different tissues showed that the gene constitutively expressed in roots, stems and leaves and the expression level was the highest in young leaves and was the least in natural root using qRT-PCR. The expression of TwWRKY1 was induced by MeJA(methyl-jasmonic acid) and SA(salicylic acid), TwWRKY1 was induced to increase expression by MeJA in 6 hours, SA had no apparent induction in 9 hours and TwWRKY1 was induced to increase expression by SA after 12 hours. The addition of MeJA could induce the biosynthesis of triptolide, wilforgine and wilforine in T. wilfordii hairy roots using high-performance liquid chromatography (HPLC), which could apparently promote wilforgine and wilforine content and have a little effect on triptolide. The content change of triptolide, wilforgine and wilforine would be increased in early stage and decreased in late stage. The expression level of TwWRKY1 and the content change of wilforgine had the same fluctuation after addition of MeJA, it was supposed that transcription factor TwWRKY1 could regulate the secondary metabolite biosynthesis. The results will provide the scientific basis for researching the molecular characterication of TwWRKY1 gene and understanding the regulation mechanism of secondary metabolite biosynthesis pathway in T. wilfordii.

The determination and evaluation of carcass performance and meat quality traits are the foundation of the development and utilization of pig (Sus scrofa) germplasm resources, and the red muscle fibers and white muscle fibers encoded by MyHC genes are positively correlated with the tenderness and juiciness of pork. In order to facilitate the rational utilization and promotion of newly cultivated lean-type Rizhao large white (RZW) pigs, 34 fattening RZW pigs were slaughtered to detect carcass performance and meat quality. The traits including carcass performance, meat quality and nutrient content traits and so on were measured and compared with these of Yimeng black (YM), Large white (LW) and Yorkshire Guanzhong black (YG) pigs. Futher, the expression level of myosin heavy chain genes MYH1, MYH2, and MYH4 were also examined in leg muscle and dorsal muscle of RZW and Meishan (MS) pigs. The results showed that the 75.43% of slaughter rate, 32.79% of leg hip ratio and 67.32% of lean meat rate of RZW pigs have no significant difference with LW pigs, but significantly higher than local varieties YM and YG pigs. RZW pigs showed significant heterosis and genetic complementarity on carcass performance. But on meat quality traits, meat color scored 4.4 points and the intramuscular fat content 1.54%, have improved compared with LW pigs. However, the ability of fat deposition is significantly weaker than that of YM pigs, and the meat is less tasty. In nutrients, RZW pigs had rich in amino acids. The total amino acids and essential amino acids content are Extremely higher than that of YM , LW and YG pigs. However, The overall fatty acid content of RZW pigs was significantly lower than that of the other three breeds. The results also showed the expression of MYH1 and MYH2 in the leg muscle of RZW pigs was significantly higher than that of the back muscle, and the expression of MYH2 was significantly higher than that of Meishan pigs. In addition, it is interesting the expression of three types of MyHC genes in dorsal muscle of RZW pigs was lower than that in MS pigs. Thus, the new breed RZW pigs had good performance of meat production and rich nutrition, but meat quality needs to be further improved. This study provided the foundation for the reasonable continuous breeding, marketing and utilization of RZW pigs. And the results of MyHC genes expression in RZW and MS pigs might provide the theoretical basis on meat quality molecular mechanism.

Polo-like kinase 1 (Plk1) has been determined to have a variety of roles in the precise regulation of centrosome duplication and separation, spindle assembly, chromosome detachment, however, much less is known about its expression and function during the meiotic progression in oocytes. The present study was conducted to investigate the dynamic expression and the possible roles of Plk1 in the metaphase Ⅰ (MⅠ) stage spindle formation in porcine oocytes. Immunofluorescent staining coupled with confocal microscopy imaging techniques was used to examined the dynamic changes of Plk1. Then, the specific inhibitor GSK461364 was used to explore the potential functions of Plk1 during the meiotic maturation of porcine oocytes. Results showed that Plk1 was observed at germinal vesicle breakdown (GVBD), MⅠ and anaphase I and telophase I (ATI) stage, with a similar distribution of α-tubulin location. GSK461364 treatment resulted in the failure of polar body extrusion in pig oocytes in a concentration-dependent manner, and the first polar body emission rate was significantly decreased (39.37+5.46)% VS (81.10+7.69)% (P＜0.01) when the concentration of GSK461364 reached 0.3 mol/L, which accompanied by misaligned chromosomes. Additional confocal microscopy results showed that GSK461364 treatment resulted in a significantly higher percentage of the treated oocytes with aberrant spindles during the first meiotic division. This study show that distribution of Plk1 is closely associated with the α-tubulin expression, and the regulation is related to its effects on spindle formation during the first meiotic division in porcine oocytes. The results of this study will provide an experimental basis for further understanding the regulation mechanism of oocyte meiosis and further improve the in vitro maturation efficiency of porcine oocytes.

Porcine (Sus scrofa) β-defensin 1 (PBD1) is a cationic antimicrobial peptide with 3 pairs of disulfide bonds , and has very strong antibacterial activity, and thus could be a good candidate as a bactericidal agent for pigs. Two pairs of specific primers (P1/P2 and P3/P4) were designed and synthesized according to the nucleotide sequence of the full porcine β-defensin 1 (PBD1) gene published in GenBank (No. NM213838). The fragment containing the full PBD1 gene was amplified by P1/P2 and reverse transcription polymerase chain reaction (RT-PCR) from total RNA extracted from porcine tongue tissue samples, PCR product was cloned into pMD18-T vector for sequencing. The result revealed that the nucleotide sequence of PBD1 gene was consisted of 310 bp in length. The PBD1 gene of mature protein was amplified using primers P3/P4. A prokaryotic plasmid of pET28a-SUMO-PBD1 was obtained by subcloning the encoding region of the mature peptide of PBD1 gene into pET28a-SUMO vector and transformed Escherichia coli Rosetta (DE3) competent cell. The expressed products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot test after optimizing the various expression conditions such as concentration of isopropy-β-D-thiogalactoside (IPTG) and its cultured time and temperature. Fusion protein SUMO-PBD1 was presented as a form of soluble and its expression was highest when it was induced by addition of IPTG to a final concentration of 0.7 mmol/L for 6 h at 20 ℃. The recombinant PBD1(about 4.3 kD) was successfully obtained by small ubiquitin-like modifier (SUMO) protease digestion and purified. This study will lay a foundation for further study of the antimicrobial activity of defensins.

Fatty acids synthase (FASN) is a key enzyme for the synthesis of fatty acids in animal tissues. In order to study the regulation mechanism of FASN gene in dairy cows (Bos taurus), three different length fragment of FASN promoter was cloned from the genomic DNA by PCR and the transcription factor binding sites were predicted using bioinformatics software. After transfected with sterol regulatory element binding protein1 (SREBP1) eukaryotic expression vector and FASN promoter vectors in L02 hepatocytes cell line from human (Homo sapiens), expression of nuclear SREBP1 protein was investigated using western blotting. The promoter activity and mRNA expression of FASN gene were investigated using double luciferase system and qRT-PCR.The results indicated that the pGL3-FSAN1,pGL3-FASN2 and pGL3-FASN3 promoter fragments were 177, 255 and 399 bp, respectively. Bioinformatics analysis of sequence revealed that the 399 bp of FASN promoter sequence contains several binding site for transcription factors, including specificity protein1(SP1), CCAAT-enhancer-binding protein(C/EBP), and nuclear factor Y (NF-Y). The sequence of pGL3-FASN2 and pGL3-FASN3 promoter contains the SREBP1 transcription factor binding site. Expression of nuclear SREBP1 protein was promoted by transfected with pcDNA3.1-SREBP1 vector.After transfected with pGL3-FSAN1, pGL3-FASN2 and pGL3-FASN3 vector, and co-transfected with pcDNA3.1 and pcDNA3.1-SREBP1, the activity of pGL3-FASN1 promoter was not affect by the pcDNA3.1 treatment. Compared with pGL3-FASN1 promoter activity, the activity of pGL3-FASN2 and pGL3-FASN3 promoters increased significantly by the pcDNA3.1-SREBP1 treatment (P＜0.01). Compared with control group, FASN mRNA expression was significantly increased by 1.67-fold (P＜0.05) after transfected with SREBP1 vector. The present study reveals that expression of FASN gene is regulated via nuclear SREBP1 protein and provide basic information for the transcriptional regulation of FASN gene in dairy cows.

In order to study the regulatory mechanism of microRNA(miRNA) in Turpan black sheep(Ovis aries) estrous cycle and cultivate sheep with high fecundity, three sheep were selected for collecting ovaries during follicular and luteal phase with surgery in this study. The miRNA data were obtained with high throughout sequencing technique and analyzed with bioinformatic method. The expression profile and the differential expression of miRNAs of Turpan black sheep ovaries in luteal and follicular phase were constructed and investigated. The target genes of differentially expressed miRNAs in two phases were screened for Gene Ontology(GO) and Kyoto Encyclopedia Of Genes And Genomes (KEGG). Three differential miRNAs were randomly selected for validation of expression level by the real-time quantitative PCR(qRT-PCR). The results showed that 139 known miRNAs were expressed in luteal and follicular ovaries of Turpan black sheep, including 126 common miRNAs in two phases, 3 miRNAs specific for luteal phase and 10 miRNAs specific for follicular phase. There were 19 miRNAs with significantly differential expression between two phages. The GO and KEGG results indicated that these miRNAs were enriched in five pathways: Antigen processing and presentation, Protein processing in endoplasmicreticulum, Proteasome, Lysosome and Leishmaniasis. The qRT-PCR results illustrated that the expression levels of the three differentially expressed miRNAs were consistent with the sequencing results. The expression profile and the differential expression of miRNAs of Turpan black sheep ovaries in luteal and follicular phase and the related signal pathways of the predicted target genes were obtained in this study, which provides a data basis for further function study of miRNA. The differentially expressed miRNAs may regulate the reproductive activities of Turpan black sheep through multiple pathways related with immunity and protein processing from result of GO and KEGG analysis.

The Galectin-1(GAL-1) gene in the deer antler stem cells is found to be highly expressed during antler regeneration period, indicating that it may be an important regulator for antler regeneration. To study the expression and possible biological function of GAL-1 in antler stem cells, antlerogenic periosteum and pedicle periosteum(potentiated pedicle periosteum and dormant pedicle periosteum) and facial periosteum of sika deer (Cervus nippon) were used as experimental materials. In this study, the GAL-1 gene CDS region of sika deer was cloned and bioinformatics analysis of GAL-1 was carried out using software or website. The relative expression of GAL-1 in the antler stem cells was detected by using Western-blot and qRT-PCR technique. The results showed that the GAL-1 ORF was 408 bp in length and encoded 134 amino acids. The relative molecular mass was 14.69 kD and the amino acid sequence was close to those of bovine (Bos taurus)and Ovis aries. GAL-1 was mainly distributed in the interstitial substance. It does not have transmembrane regions and glycosylation sites, and there may be two phosphorylation sites. The secondary structure of the GAL-1 protein was mainly β-sheets and random coil, and the tertiary structure was similar to that of the bovine. Expression level of GAL-1 was higher in the facial periosteum. In this study, the structural characteristics of GAL-1 protein was predicted, the expression level of GAL-1 protein in antler stem cells was detected and would provide the basic data for the further study of antler regeneration.

Based on the Molecular genetics to study the causes of the attack behavior of Muscovy ducks (Cairna moschata), the experiment adopted the software of The Medial Recorder 2.0 to record the behaviors of 120 Muscovy ducks in good growth for 1 month (24 h each day). The use of instantaneous observation and continuous observation screening: Muscovy ducks (experimental group), which performed aggressive behavior, and Muscovy duck (normal group), which did not show aggressive behavior. For the selected control and experimental Muscovy ducks, the experiment adopted the Illumina HiSeq 2000 high flux transcriptome sequencing technology to screen the differentially expressed genes in the hypothalamus of Muscovy ducks which were divided into an experimental group and a control group (repeated 3 times in each group). Making use of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to screened differentially expressed genes. The differentially expressed genes were enriched in gene ontology and KEGG pathway. The results showed that in the hypothalamus of Muscovy ducks which had the attack behavior, there were 626 differentially expressed genes annotated into the GO database, in which 137 genes were up-regulated and 489 genes were down-regulated. There were 26 differentially expressed genes involved in regulating the attack behavior, in which 4 genes were up-regulated and 22 genes were down-regulated. The results of KEGG annotation showed that the differentially expressed genes were enriched in six pathways, such as Phagosome, Lysosome, Salmonella infection, MAPK signaling pathway, Progesterone mediated oocyte maturation, mTOR signaling pathway and so on. The results also showed that in the Regulation of action cytoskeleton and the Neuroactive ligand receptor interaction, the enrichment of the differentially expressed genes was not significant, but the enrichment was a very large degree. In these pathways, the differentially expressed genes about the attack behavior of Muscovy ducks were screened. By the annotation of GO and KEGG database, 33 candidate genes were initially screened out and their regulatory mechanisms were associated with Muscovy duck attack, the ERBB signaling pathway may be the key pathway for the adjustment of the duck attack behavior. This experiment provides a theoretical basis for the molecular genetics study of the attack behavior of Muscovy ducks.

Endo-N-acetyl-β-glucosaminidase (Endo/ENGase, EC3.2.1.96) is a kind of glycohydrolase which recognizes and cleaves the β-1,4-glycosidic bonds of N,N′-acetylglucosamine in core structure of N-linked glycans. It is widely used in analyzing N-glycosylation of protein. In order to characterize the enzymatic properties of endo-β-N-acetylglucosaminidase from Enterococcus faecalis, the endo-N-acetyl-β-glucosaminidase gene endoEf of Enterococcus faecalis was cloned by PCR and the prokaryotic expression vector pET-28a-endoEf was constructed using seamless cloning technology. The catalytic properties and pivotal catalytic residues of EndoEf were analyzed on the basis of recombinant expression and purification. The results showed that EndoEf was efficiently expressed in Escherichia coli BL21 Star (DE3) in soluble form, and that the target protein was successfully purified from bacteria solution by one step Co2+ affinity chromatography with a yield of 202.1 mg per liter. The specific activity of EndoEf was measured as 1.0×104 U/mg using ribonuclease B (RNaseB) as substrate. EndoEf belonged to the glycoside hydrolase family 18 (GH18) which contained conservative motifs DXDXE. In addition, both natural or denatured RNaseB and ovalbumin (Ova) were able to be hydrolyzed by EndoEf. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) further confirmed the hydrolysis on N-linked sugar chains in RNaseB. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight of EndoEf was 30.2 kD. Enzymatic properties analysis showed that the optimum temperature and pH range of EndoEf were 40 ℃ and 5.0~7.0, and EndoEf was relatively more stable at 40~50 ℃ and pH 7.0~9.0. EndoEf had salt-tolerance up to 1 mol/L NaCl, and remained full activity under either condition of 100 mmol/L DTT, 2% SDS and Triton X-100. Three mutants D125N, D127N and E129Q of EndoEf were constructed by site-directed mutagenesis method. Activity analysis showed that E129Q was almost inactive, and D127N lost most of its activity, while D125N had no significant change in activity. It could be concluded that the glutamic acid at position 129 was essential for catalytic reactions. The establishment of recombinant expression system and analysis of enzyme properties may lay a foundation for the practical application of EndoEf on glycoprotein study.

n-3 polyunsaturated fatty acids exerts multiple physiology functions, which has to be provided from foods in mammals. The fatty acid desaturase-1 gene (fat-1) encoding an n-3 fatty-acid desaturase converts n-6 to n-3 fatty acids, which plays an important role in improving meat quality. To understand the fatty acid composition of fat-1 transgenic sheep (Ovis aries). In the present study, sheep fetal fibroblasts were isolated and transfected with fat-1 overexpression plasmid. It was carried out that nuclear transfer and embryo implantation using the positive monoclonal and untransfected cells. Transgenic fat-1 sheep were obtained successfully. 32 fatty acids in 5 tissues were investigated by gas chromatograph attached to mass spectrometer for fatty acid determinator (GC-MS-QP2010). Reconstructed embryos (411 for untransfected fibroblast and 441 for fat-1 transgenic cell) at the 1-2 cell stage were transferred to the oviducts of 99 synchronism recipient sheep (47 for control and 52 for positive cell). 8 individuals were got, including 5 positive fat-1 transgenic and 3 control animals. 32 fatty acids were divided 3 categories: saturated (C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0, C21:0, C22:0, C23:0, C24:0), monounsaturated (C14:1, C15:1, C16:1, C17:1, C18:1n-9t, C18:1n-9c, C20:1n-9, C22:1n-9, C24:1n-9), polyunsaturated fatty acids (C18:2n-6t, C18:2n-6c, C18:3n-6, C18:3n-3, C20:2, C20:3n-6, C20:4n-6, C20:3n-3, C20:5n-3, C22:2n-6, C22:6n-3). The percentages of saturated, monounsaturated and polyunsaturated fatty acids were not significantly different between transgenic and control groups in brain and gluteus tissues. However, in transgenic sheep, saturated fatty acid decreased in liver, heart and testis tissues, monounsaturated fatty acids rose in liver and testis tissues, and monounsaturated fatty acids reduced in heart, polyunsaturated fatty acids decreased in liver tissue, but increased in heart and testis tissues. The content changes of monounsaturated fatty acid C18:1n-9c and saturated fatty acid C16:0, C18:0 determined the change trends on monounsaturated and saturated fatty acids in transgenic sheep. These 3 fatty acids were not significantly different between transgenic and control animals in brain and gluteus tissues. Furthermore, there were not significant difference in total polyunsaturated fatty acids in brain and gluteus tissues between transgenic and control groups. Looking into the 11 polyunsaturated fatty acids in brain and gluteus tissues, the results indicated that the percentage content of C20:2, C20:3n-3 and C20:4n-6 decreased, but C20:5n-3 and C22:6n-3 increased in fat-1 transgenic group. fat-1 decreased polyunsaturated fatty acids in liver tissue, and increased them in heart and testis tissues, which was mainly determined by C20:2 (eicosadienoic acid, EDA), C20:3n-3 (eicosatrienoic acid, ESA), C20:4n-6 (arachidonic acid, ARA), C20:5n-3 (eicosapentaenoic acid, ETE) and C22:6n-3 (docosahexaenoic acid, DHA) fatty acids. Both EPA and DHA increased in 5 tissues in transgenic sheep, especially EPA. The contents of C20:3n-3 and C20:4n-6 were decreased, and the contents of C20:5n-3 and C22:6n-3 are increased by fat-1, thus the ratio of n-3/n-6 fatty acids was increased. The present study clarifies the compositions and characterizations of fatty acids in fat-1 transgenic sheep, which provides theoretical evidence for exploiting high-quality sheep, improving the structure of fatty acid in human diet and reducing the risk of obesity.

Rye(Secale cereale), which contains excellent resistance and stress tolerance genes to against biotic and abiotic stress, is an important germplasm for wheat (Triticum aestivum) improvement. To enlarge the germlplasm resources for wheat breeding, the crosses between common wheat cv. W770B (2n=6x=42, AABBDD) and rye (Secale cereale; 2n=2x=14, RR) via wide hybridization was conducted, and several derived lines with great characteristics were obtained after a long-time screening. Then, the toothpicks infected with Rhizoctonia cerealis which cultivated on freshly prepared potato-dextrose agar (PDA) were put in the leaf sheaths of these materials at the five-leaf stage of wheat plants. The plants after inoculation were cultivated in greenhouse for five weeks, and then identified the occurrence degrees and calculated the disease indexes. Derived line 7-1,with a sharp eyespot disease index of 32.8% was selected and identified by toothpick method through several years for improving the accuracy of the identification. In order to figure out the genetic constitution of 7-1, morphological method, cytogenetical method, genomic in situ hybridization (GISH), sequence characterized amplified region(SCAR) markers, simple sequence repeat(SSR) markers and gliadin analysis were conducted in this study. The results of cytogenetics observation of root tip cells and pollen mother cells showed that the number and structure of chromosome of 7-1 were stable and the configuration of 7-1 was 2n=42=21Ⅱ. GISH analyses using rye genomic DNA as probe indicated that 7-1 contained two chromosome arms of rye. The analysis of rye genome SCAR markers showed that D15 and P13LF/R can amplify 900 bp around and 850 bp around rye specific bands both in rye and 7-1. And the analysis of 1RS SCAR markers of rye showed that ω-sec-p1/ω-sec-p2, ω-sec-p3/ω-sec-p4 and IB-267 amplified 1 100 bp around, 450 bp around and 300 bp around rye target bands both in rye and 7-1. Both analysis of rye genome SCAR markers and rye chromosome SCAR markers showed that 7-1 possessed the 1RS chromosome arm of rye. SSR primers on the long arm and short arm of seven wheat homoeologous groups were screened, but only Xgwm264 and Xgwm11, which derived from 1BS didn't amplify corresponding bands. On the contray, the remainging primers from other chromosomes all amplified bands. The analysis of SSR markes showed 7-1 lacked the 1BS chromosome arm of common wheat. Gliadin Analysis showed that secalin characteristic bands were amplified in 7-1. Consequently, 1BS of wheat was substituted by 1RS of rye and the derivative line was confirmed as a 1BL/1RS translocation line. In this study, 7-1 was identified as the 1BL/1RS translocation line with the resistance to sharp eyespot, broadened materials for wheat breeding, and could be provided as a new germplasm resource for sharp eyespot in future.

The low efficiency for recombined RNA viruses is the limiting factor in the study of plant RNA virus-based expression vectors. To enhance the accumulation levels of recombinant proteins in plants, we explored the application potential and effect of addition of multiple introns into the viral vector in plant virus RNA-based expression system. According to the characteristics of introns in higher plant, 11 introns were selected from the Arabidopsis thaliana genome. The intron sequences were synthesized in vitro and the intron-containing viral vectors were constructed based pTBSV-G, a Tomato bushy stunt virus (TBSV)-based vector. The NetPlantGene server was employed to predict coding and non-coding sequence features in synthetic viral vectors in vitro. And then reverse transcription PCR (RT-PCR) and Western blots were used to verify whether the introns in the viral vector could be correctly identified and processed by the nuclear pre-mRNA processing machinery in vivo. The expression levels of green fluorescent protein gene (gfp) in inoculated Nicotiana excelsiana leaves were examined by Western blots and enzyme linked immunosorbent assay (ELISA) respectively. Those data were gathered to evaluate the validity and regularity of introducing introns in the coding region of the viral genome. The NetPlantGene software could accurately identify all introns inserted into the viral coding region. The result indicated that it is a useful tool for prediction of coding and non-coding sequence features in designed viral vector before experimentally inoculated to plant. The results of RT-PCR showed that the sizes of amplification products in control group were larger than those in the experimental group and it was consistent with the theoretical prediction. Western blots analysis showed that the tagged proteins (P19, P33 and P92) could be detected by using the respective antibody. The results of RT-PCR and Western blots indicated the artificial introns in the viral vector can be correctly processed in the nucleus of the host cell. Compared with pTBSV-G, the GFP accumulation levels increase of about 2 fold when the viral vectors with 2 introns or 11 introns were inoculated. However, the viral vector with 9 introns does not significantly enhance the accumulation of GFP. In conclusion, the additions of introns in viral vector were able to increase the accumulation of recombinant proteins in plants. Nevertheless, the enhancement was not associated with the numbers of intron, whereas the position of introns might play a more important role on the accumulation of recombinant proteins in plants. The use of introns in plant virus RNA-based vectors will provide the basis for applications in molecular virology studies in the near future.