β2-microglobulin (β2m) is one of the subunits of classⅠmajor histocompatibility complex (MHCⅠ) molecular, which binds non-covalently with the α-chain of the MHC-Ⅰ molecular. Based on the RT-PCR and RACE-PCR, full-length cDNA of β2m was cloned from Nile tilapia (Oreochromis niloticus). In addition, cDNA polymorphism and the mRNA expression pattern were analyzed. The results showed that the two β2m cDNA was 900 and 906 bp in length, respectively, including an open reading frame (ORF) of 351 bp encoding a polypeptide of 116 amino acids. The 5'-UTR and 3'-UTR was 68 nts and 486(492) nts respectively. The two cDNA had different genomic sequence, but they all had 3 exons and 2 introns. The putative protein of the 2 β2m had 92.3% identity. The putative amino acid sequence of β2m had 39.20%~89.8% identity with those of other teleost and mammals. The putative protein of the 2 β2ms exhibited the highest identity with zebra mbuna (Maylandia zebra) (89.80% and 84.7%, respectively). The 2 β2ms had 63.2% and 60.70% identity with catfish (Ictalurus punctatus) respectively. And β2m-2 had the lowest identity with chicken (Gallus gallus) and mouse (Mus musculus)(39.50% and 39.20%, respectively). Predictprotein online analysis found that the 2 β2m all had the typical immunoglobulin(Ig) and major histocompatibility complex protein (MHC) signature YxCxVxH. The phylogenetic tree showed that the β2ms of Nile tilapia clustered with zebra mbuna (Maylandia zebra) first, then clustered with orange-spotted grouper (Epinephelus coioides), large yellow croaker (Pseudosciaena crocea), and seabass (Dicentrarchus labrax), and then clustered with other teleost and mammals. In addition, the rainbow trout (Oncorhynchus mykiss), catfish (Ictalurus punctatus), zebrafish(Brachydanio rerio), and grass carp (Ctenopharyngodon idella) formed a subcluster. The Atlantic cod(Gadus morhua), red seabream(Parus major), and flounder (Paralichthys olivaceus) formed another subcluster. The chicken (Gallus gallus), human (Homo sapiens) and mouse (Mus musculus) formed a separate cluster. Six individuals were used to analysis the cDNA polymorphism. 56 valid sequences were obtained from 60 positive clones (10 positive clones per individuals). And 2~4 cDNA sequences per individuals were obtained. The 56 valid sequences code 6 different cDNA sequences. The 6 cDNA sequences could be divided into 2 different types, and each type had 3 subtype cDNAs. Six cDNA were named as Orni-β2m 0101, Orni-β2m 0102, Orni-β2m 0103, Orni-β2m 0201, Orni-β2m 0202 and Orni-β2m 0203 respectively. To examine the basal expression of β2m, qRT-PCR analysis was carried out in gill, brain, intestine, liver, kidney, stomach, spleen, heart, muscle and skin. To eliminate the individual variation, six fishes were sampled and analyzed separately by qRT-PCR, and their mean values were considered. The results showed that the β2m was detected in all the examined tissues. And the highest expression level of β2m was detected in kidney, spleen and heart, and higher expression in intestine and gill, whereas the lowest expression was detected in muscle and skin. The expressions level of β2m mRNA in the gill, kidney, heart and spleen of Nile tilapia increased first, and then decreased when challenged with Streptococcus agalactiae. In the infected fish gill, kidney, heart and spleen, β2m mRNA expression was increased at 1 day post infection (dpi), and touched the peak at 3 dpi. Then the expression declined at 6 dpi, and at 9 dpi the induction declined to the basal level in kidney and spleen. The results showed that the β2m may play important roles in immune response of Nile tilapia. This observation can contribute to further investigation of immune regulation of nile tilapia，and the results will give us a better understanding for the physiologyical role of β2m in fish.

Surrounding the membrane surface of lipid droplets, Perilipin1 plays important roles in the formation and maturation of lipid droplets, and regulation of fat mobilization (lipolysis). Considered as one of the important marker genes of adipocyte differentiation, many Perilipin1 studies in mammals have been carried out, while in chickens (Gallus gallus) relatively few. The objectives of the present study were to clone full length mRNA of chicken Perilipin1 gene, and analyze its subcellular location during the progression of chicken preadipocyte differentiation. We extracted total RNA of abdominal adipose tissue from chickens at 7 weeks of age. RT-PCR and rapid amplification of cDNA ends (RACE) methods were used to clone the 5'-untranslated region (5'-UTR), the coding domain sequence (CDS) and the 3'-untranslated region (3'-UTR) sequences of chicken Perilipin1 gene, and then the gene structure was analyzed. The subcellular localizations of Perilipin1 in chicken preadipocyte was examined by immunofluorescence confocal microscopy at different time points (12~120 h). Results showed that the whole mRNA length of chicken Perilipin1 gene was 2 379 bases (bp), consisted of nine exons and eight introns (with the start codon ATG located in the second exon). The CDS length of chicken Perilipin1 gene was 1 551 bp, encoding 517 amino acids, and lengths of 5'-UTR and 3' UTR were 94 and 733 bp, respectively. Sequences have been submitted to NCBI database, and assigned the serial number (Accession No. GU327532). In addition, we performed the multiple alignment for the chicken Perilipin1 CDS sequence with mouse (NM_175640) and human (NM_001145311) sequences deposited in NCBI database by the Clustalw software (http://www.genome.jp/tools/Clustalw/), and obtained the homology percentages, 38% and 41%, respectively. Therefore, sequence divergence at nucleotide level was quite large, suggesting that the role of chicken Perilipin1 in adipocye differentiation may not be fully consistent with mammals. We used immunofluorescence method combined with laser confocal microscopy to check the subcellular location of chicken Perilipin1 in the process of preadipocyte differentiation, which was induced by oleate at different time points (12, 24, 48, 72, 96 and 120 h), started on primary chicken preadipocytes cultured by the digestion method. At the first 12h of induction, few small lipid droplets were visualized in the preadipocytes, and Perilipin1 located on the surface of neonatal lipid droplets. As time passed on (24 to 48 h), more lipid droplets emerged, and Perilipin1 surrounded the lipid droplets. At late stage (72 to 120 h), several large lipid droplets formed through the fusion of small lipid droplets, and Perilipin1 convened and packaged on the surface of lipid droplets. Therefore, our results indicated that chicken Perilipin1 surrounded the membrane surface of lipid droplets during the whole process of adipocyte differentiation, and we speculated that basically, the function of chicken Perilipin1 might help in forming a protective barrier separating lipase from lipids stored in the lipid droplet. In conclusion, we have successfully cloned the full length mRNA of chicken Perilipin1 gene, and confirmed the subcellular position of Perilipin1 during chicken preadipocyte differentiation. The current study provided some molecular information for further investigating the function of chicken Perilipin1 gene.

15-cis-ζ-carotene isomerase (Z-ISO) can isomerize 9,15,9'-tri-cis-ζ-carotene into 9,9'-di-cis-ζ-carotene, and Z-ISO gene is the last identified gene in plant carotenoid biosynthetic pathway. In order to clone the ORF of Z-ISO genes in Osmanthus fragrans and study their expression profile, ORF sequences of OfZ-ISO1 (GenBank accession: KX120175) and OfZ-ISO2 (GenBank accession: KX120176) from O. fragrans were amplified using PCR based on the sequence information of Unigene from the petal transcriptome database of O. fragrans Yanhong Gui from the Aurantiacus Group. Bioinformatics analysis indicated that both OfZ-ISO1 and OfZ-ISO2 contained 1 107 bp ORF, encoding 368 amino acid residues. Six predicted transmembrane domains were in both OfZ-ISO1 and OfZ-ISO2 proteins, as well as 3 conserved residues (H152, H268 and D296) which were essential for isomerase activity. OfZ-ISO1 and OfZ-ISO2 from O. fragrans shared high sequence similarity with ZmZ-ISO1 from Zea mays and AtZ-ISO1 from Arabidopsis thaliana. Phylogenetic analysis showed that OfZ-ISO1 and OfZ-ISO2 were gathered into one branch with Sesamum indicum Z-ISO, which revealed that OfZ-ISO1 and OfZ-ISO2 showed the closest genetic relationship with Z-ISO from S. indicum. Gene expression showed that during the flower development of O. fragrans Yanhong Gui, the expression level of OfZ-ISO1 was low in the inflorescences at bud stages, dramatically increased at linggeng stage, kept the same at initial flowering stage and greatly decreased at full flowering stage, while that of OfZ-ISO2 was low in the inflorescences at bud stages and linggeng stage, then gradually increased, and reached the maximum at full flowering stage. In the O. fragrans cultivars with different petal color, Yanhong Gui from the Aurantiacus Group showed the darkest petal color with the lowest L* value and the highest a* value and the highest total carotenoid content. In Jin Qiugui from the Luteus Group, the petal color was lighter than that of Yanhong Gui from the Aurantiacus Group and the total carotenoid content was lower than that of Yanhong Gui, and Yu Linglong from the Albus Group showed the lightest petal color with the highest L* value and the lowest a* value and the lowest total carotenoid content. Surprisingly, the expression levels of OfZ-ISO1 and OfZ-ISO2 were not the highest in Yanhong Gui with the highest total carotenoid content. Generally, the expression levels of OfZ-ISO and OfZ-ISO were the highest in Yu Linglong at linggeng stage and initial flowering stage, however, at full flowering stage, the expression levels were the same in the 3 cultivars with different petal color. The results of this study revealed that the expression levels of OfZ-ISO1 and OfZ-ISO2 during flower opening were essential to the carotenoid accumulation of O. fragrans, and total carotenoid content of cultivars with different petal color was not positively related with the expression level of OfZ-ISO1 and OfZ-ISO2. This study has important practical significance to fully reveal the mechanism of petal coloration in O. fragrans.

Toll-like receptors (TLRs) are highly conservative protein regulators participating in innate immune response. This test aimed to clone and analyse TLR4 gene fragment and distribution in organs of experimental animal Mongolian Gerbil (Meriones unguiculatus). Conservative region of TLR4 gene which was compared among rat (Rattus norvegicus), mouse (Mus musculus) and hamster (Mesocricetus auratus) was chosen to design the primers, and Mongolian Gerbil TLR4 was cloned. After sequencing, the sequences were further analyzed and compared homology with DNASTAR Meglign software between Mongolian Gerbil and other different species of animals. Semi-quantitative PCR and immunohistochemical assay were used to determine the expression levels of TLR4 mRNA and protein in heart, liver, spleen, lung and kidney of normal Mongolian Gerbil, respectively. Results demonstrated that TLR4 gene fragment of Mongolian Gerbil (GenBank accession No. KX137123) was successfully cloned and showed more than 87.4% homologous with other mouse genus, and the highest for 89.1% with hamster. Homology with human (Homo sapiens) and swine (Sus scrofa) showed 69.5% and 73.1%, respectively. These results showed that the kinship of TLR4 gene might be relatively closer between Mongolian Gerbil and hamster, and farther with human and swine. Semi-quantitative PCR showed that TLR4 differentially expressed in 5 different tissues, and the expression was the highest in lung but the lowest in liver. Immunohistochemical assay further confirmed the semi-quantitative PCR result in protein level. Positive reaction products mainly distributed in lung and spleen in Mongolian Gerbil. High abundance of TLR4 proteins distributed mainly in alveolar bronchial epithelial cells of lung. TLR4 was located mainly in marginal zone around splenic nodule and with little on nodule region of spleen. Sporadic TLR4 proteins could be found in kidney. But no observant reaction products was detected in heart and liver definitely. This study will be a pioneer test for further research on innate immunity of Mongolian Gerbil which performs excellent infection model for some specific pathogens.

As a recently discovered muscle factor, human(Homo sapiens) Irisin is an excretive polypeptide fragment whose precursor is fibronectin type Ⅲ domain-containing protein5(FNDC5). Irisin can transfer the signal of skeletal muscle and keep the relationship between skeletal muscle and the peripheral tissues. Irisin will certainly be a very promising active factor, and become the new targets for prevention and control of metabolic disease and its complications. Therefore, exploiting a method that can be used to mass produce and easily purify human Irisin recombinant protein is very significant. To achieve the prokaryotic expression of human Irisin, the FNDC5 cDNA was obtained from the muscles, then the restriction enzyme cutting site NdeⅠ and XhoⅠ were introduced to human Irisin gene by the specific primers amplification. The gene sequence was digested by NdeⅠ and XhoⅠ and cloned into prokaryotic expression vector, pET-30a that digested by NdeⅠ and XhoⅠ, to form pET-30a-Irisin. The recombinant plasmid was transformed to Escherichia coli Rosetta (DE3) pLysS and was screened by kanamycin to form Rosetta-pET-30a-Irisin recombination strain. Then the expression of interest protein was induced with 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol/L isopropyl β-D-1- thiogalactopyranoside(IPTG), and the recombinant protein detected by sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE), the gommures was stained by Coomassie brilliant blue for 4 hours and discolored for 3 hours to analyze the expression of recombinant proteins. In order to realize the soluble expression of recombinant proteins, the Rosetta-pET-30a-Irisin recombination strain was induced with 0.1 mmol/L IPTG at 18 ℃. And the recombination strains induced with IPTG were broken by ultrasonication, the soluble recombinant proteins in cracked supernatant were purified by Ni-sepharose, then detected and identified by Western blot. The DNA sequence analysis showed, that the sequence of cloned human Irisin was in accord with that published by NCBI, and Escherichia coli recombinant expression vector pET-30a-Irisin had been successfully constructed. The results of SDS-PAGE and Western blot indicated that, the Escherichia coli recombination strain Rosetta-pET-30a-Irisin could realize the dissoluble expression of human Irisin induced with 0.1 mmol/L IPTG at 18 ℃. The 14 kD recombinant protein was purified by Ni-sepharose, and the recovery rate was about 2 mg/L. A soluble expression system for human Irisin protein and high purity of human Irisin protein was successfully constructed and obtained. And the system was simple operation and low cost. This study provides theoretical references for the study of structure, function and application of human Irisin.

DNA methylation is a hotspot in epigenetic research. This study was aimed to explore whether the force-feeding could change the levels of DNA methylation and gene expression related to lipid metabolism in liver of Landes(Anser anser), and to investigate the relationship between DNA methylation and gene expression. The levels of DNA methylation of stearovl-CoA desaturase 5 gene (SCD5), sterol regulatory element-binding protein 2 gene (SREBP2) and elongase of very long chain fatty acids 6 gene (ELOVL6) were analyzed with pyrosequencing method and quantified by qRT-PCR. The upstream promoter sequence of SCD5 (about 2 800 bp) was downloaded from University of California Santa Cruz (UCSC) website, there were 2 CpG islands, and the percentage of cytosine, G+C and CpG island were respectively reached of 22.7%, 50.06% and 3.81% after analyzing from the 406~1 220 bp CpG island (full-length fragment of 815 bp). The upstream promoter sequence of SREBP2 (about 2 248 bp) was also downloaded from UCSC website, there were 2 CpG islands, and the percentage of cytosine, G+C and CpG island were respectively reached of 24.87%, 56.61% and 5.29% after analyzing from the 1~965 bp CpG island (full-length fragment of 965 bp). The upstream promoter sequence of ELOVL6 (about 2 248 bp) was downloaded from UCSC website, there was 1 CpG island, and the percentage of cytosine, G+C and CpG island were respectively reached of 37.14%, 55.71% and 4.30% after analyzing from the 1 426~1 775 bp CpG island (full-length fragment of 350 bp). A total of 23 CpGs related DNA methylation were detected by using pyrosequencing method after amplified the promoter sequence of CpG island of SCD5, SREBP2 and ELOVL6 (each gene had two fragments of 37~55 bp). Results showed that the levels of DNA methylation of the 2 fragments of SCD5, SREBP2 and ELOVL6 in force-feeding group (FFG) and control group(CG) were respectivlely higher than 20%, belongs to high methylation status. The levels of DNA methylation of CG were greater than FFG. Significant difference were found on DNA methylation in the first fragment of SCD5 and the first fragment of SREBP2 between FFG and CG(P＜0.05), markedly significant difference (P＜0.01) in the second fragment of SREBP2, but no significant difference(P＞0.05) in the second fragment of SCD5 and the first fragment and the second fragment of ELOVL6 between FFG and CG. The mRNA expression of mRNA of SCD5 and ELOVL6 in FFG were greater than those of CG, and there relative gene expression of SREBP2 in FFG was lower than that of CG (P＜0.05). In conclusion, force-feeding decreased the expression of DNA methylation of SCD5, SREBP2 and ELOVL6, increased the mRNA expression of SCD5 and ELOVL6 in liver of goose, but decreased the mRNA expression of SREBP2, which indicated that the methylation of SCD5 and ELOVL6 played a negative regulation effect on the mRNA expression of SCD5 and ELOVL6. The result will provide a reference to the research method of DNA methylation on epigenetics and an important molecular genetics basis on breeding high quality and high yield goose fatty liver, it will also provide theoretical basis for researching the metabolic diseases such as obesity and fatty liver.

To study the related traits changes of milling quality and appearance quality of rice (Oryza sativa) under drought stress environmental conditions, explore QTL which controlled rice quality related traits stably existing under drought conditions, and analyze the interaction effects between environment and QTLs, this study investigated a recombinant inbred lines (RILs) population deriving from the across between upland cultivar Xiaobaijingzi and rice cultivar Kongyu131 and 2 parents as experimental material, which consisted of 207 individual high-generation (F8, F9) RILs group. Repeated tests under drought stress and normal irrigation were conducted to study correlated analysis of 4 quality traits, including brown rice rate (BRR), milled rice rate (MRR), head rice rate (HMRR) and chalky rice rate (CG). Furthermore, QTL mapping were researched. The genetic map that was built based on F6 RIL population, which derived from Xiaobaijingzi and Kongyu 131, was used for QTL mapping. The genetic map contained 104 markers, covering the rice genome 1 295.03 cM, and average genetic distance between markers was 12.45 cM. The results showed that there were statistically significant correlations between BRR and MRR, HMRR in 2 environments. CG negatively correlated with MRR, but significantly negatively correlated with HMRR in 2 environments. The genetic of traits expressed as the quantitative characters with the continuous distribution in 2 environments. Totally, 24 additive QTLs and 9 pairs of epistatic interactions QTLs were detected for 4 traits in 2014 and 2015, which distributed on chromosome except 10 and 12. Five additive QTL (qBRR11a, qMRR11a, qHMRR6a, qCG6a and qCG6c) were detected and stably expressed under drought stress environment in 2 years survey. Three additive QTLs and 4 pairs of epistatic QTLs were detected significant environmental interaction effects. The expressions of each traits, which were affected slightly by the water environment, were mainly additive genetic effects. Hence, the specific QTLs under drought stress which were and discovered can lay a foundation for genetic improvement of grain quality.

The feet weight trait of chicken (Gallus gallus) is one of the most important economic traits in the chicken industry. In order to reveal the genetic basis of feet weight trait and scan molecular markers which can be used to improve the feet weight trait, the feet weight trait was measured after Jinghai yellow chicken in core group being slaughtered. Genome-wide association study was carried to detect SNPs that was associated with feet weight trait by using specific-locus amplified fragment sequencing technology. Chicken reference genome was analyzed using the prediction software based on the gene characteristics to choose the appropriate restriction enzyme. Then a series of procedures were put into effect to create database to do the sequencing by IlluminaHiSeq2000. A total of 103 680 specific-locus amplified fragment sequencing technology (SLAFs) were found with an average read depth of 5.46 in the research population. Of these, 88 135 were found to be polymorphic (85%). The distribution of the SLAFs was even throughout the whole genome. After SNPs were detected among the defined SLAF fragments, 90 929 SNPs were identified after quality control measures. Then the SNPs that associated with chicken's feet weight trait were used to do enome level SNP classification and analysis method (GWAS) analysis, 16 SNPs that were related to feet weight were detected. 2 SNPs of rs475641139 and rs475548810 reached 5% Bonferroni genome-wide significance (P＜5.5E-07) and the rest of 14 SNPs reached "suggestive" genome-wide significance (P＜1.1E-05). At last, 9 potential functional genes were got after screening every important SNPs' ambient 1 Mb window areas (SNP location right and left 0.5 Mb). The cellular component, molecular function and biological process were analyzed by using Gene Ontology (GO) database. Furthermore, all 13 SNPs were distributed in the region of 72.8~77.9 Mb on chromosome 4. Nine genes of dihydropteridine deductase gene (QDPR), LIM domain-binding protein 2 gene (LDB2), family with sequence similarity 184 member B gene (FAM184B), recombination signal binding protein for immunoglobulin kappa J region gene (RBPJ), fibroblast growth factor-binding protein 2 gene (FGFBP2), F-box and leucine-rich repeat protein 5 gene (FBXL5), cholecystokinin receptor type A gene (CCK1R), WD repeat containing protein 1 gene (WDR1) and tubby like protein 4 gene (TULP4) and 72.8~77.9 Mb region on chromosome 4 were important candidate genes and region affecting feet weight trait in Jinghai yellow chcken. This study can provide the theory basis for the feet weight trait's marker assisted selection.

The intestinal tract of a vertebrate is a complex ecosystem that often harbors a complex assemblages of bacterial community. During the intestinal microbiota and the host, the microbial community has become an integral component of the host. Among its many important functions, the intestinal microbiota can convert feedstuffs into microbial biomass and fermentation end products that can be utilized by the animal host. The composition of the intestinal bacterial community is determined in part by dietary preferences and host life histories. The eel (Anguilla anguilla) is the major culture species in China, feeding with dough feed largely based on eel feed is a common practice. α-starch is an important component of eel feed ingredients. In order to study the eel intestine bacteria utilization of α-starch and in vitro metabolic characteristics, the α-amylase producing abilities of strains isolated from eel intestine. The results indicated that there were α-amylase producing strains in eel intestine. It showed that the microbial communinty colonized in the eel intestinal tract may played an important role in the nutrient digestion and absorption. 4 α-amylase producing strains were isolated, which accounted for 12.9% of the total strains populations in the eel intestinal tracts. Among them, the strain MJ18 had the highest ability to utilize α-strach. The study was designed to identify strain MJ18 by morphology and molecular methods. Strain MJ18 shared following characteristcs: Gram negative, 1.2 μm×2 μm in size, short rod, flagellum, motile. The result of sequencing the 16S rRNA gene revealed that strain MJ18 homology higher than 99% with Edwardsiella tarda strain KC570942.1. According to the characteristics of morphological features and 16S rRNA sequences analysis, the strain MJ18 was identified as Edwardsiella tard. Kinetic studies showed that the michaelis constant of α-amylase (Km) was 4.64 mg/mL, and maximum velocity (Vmax) was 45.45 mg/(min·mL). Biolog-AN system with 95 kinds of carbon source was used to exploit the metabolism feature of strain MJ18. The changes of absorbance values of strain MJ18 in 95 kinds of carbon sources were determined in different time. Results showed that strain MJ18 utilized carbon sources selectively, and the number of positive wells was 9 kinds. Dextrin, maltotriose and pyruvic acid were the principal intermediates in α-starch metabolism. The 3 cabon sources utilization ratio were more than 3%, which were higher than other carbon sources. Further analyses showed average well color development (AWCD) values were at satationary phase after 96 h cultivation. Significant differences in different sole carbon utilization characterized as AWCD value were found. The nucleotides utilization capacity was the highest, followed by organic acid and carbohydrates, and the lowest at amino acides. With the extension of the culture time, the utilization of carbohydrates, organic acid and nucleotides at strain MJ18 improved, but the utilization of amino acides did not change. This study screened and identified a α-amylase producing strain MJ18 which was isolated from the intestinal tracts of eel, which can provide new insights into potential contribution of intestine bacteria to α-starch metabolism in eel.

As we all known, the availability of thyroid hormone within the hypothalamus is a key determinant of seasonal transitions and they convert between bioactive and inactivating type through catalyzing by type Ⅱ iodothyronine deiodinase(DIO2) and type Ⅲ iodothyronine deiodinase(DIO3). So far, many previous studies has revealed that DIO2 and DIO3 genes might regulate seasonal reproduction in rodents (Rodentia) and sheep (Ovis aries), but it is not clear in regulation of goat seasonality. To determine the roles of the 2 genes in goat (Capra hircus) seasonal reproduction, their spatial expression profiles and the differences of expression levels in hypothalamic-pituitary-gonadal axis were analyzed between Jining Grey goat (year-round estrus) and Liaoning Cashmere goat (seasonal estrus) by using RT-PCR and Real-time quantitative PCR methods, respectively. The results showed that (1) DIO2 mRNA expressed in almost all tissues studied, while DIO3 mRNA was mainly found in goat hypothalamus, pituitary, cerebellum, hippocampus, pons, and it was also determined in ovary, adrenal gland and slightly expression in goat spleen, both gene were with similar tissue profiles between two goat breeds except a small quantity of DIO3 expression in cerebral cortex in Liaoning cashmere goat. (2) DIO2 was found in all of 4 tissues detected which related to reproductive regulation and in Jining Grey goat, it expressed higher in pituitary than other tissues (P＜0.05). For Liaoning Cashmere goat, the expressions were significant different among 4 tissues except between hypothalamus and pituitary. DIO2 mRNA expression was higher in pituitary for Jining Grey than Liaoning Cashmere goat (P＜0.05), while that was lower in uterus than Liaoning Cashmere goat (P＜0.05) and without obvious difference in ovary and hypothalamus between 2 breeds even it appeared higher levels in Jining Grey goat (P＞0.05). (3) In Jining Grey goat, the expression of DIO3 was the highest (P＜0.05) in hypothalamus among 4 tissues studied and uterus was detected no DIO3 mRNA. As for Liaoning Cashmere goat, DIO3 expressed the highest in ovary (P＜0.05), then hypothalamus and pituitary, at last lowest in uterus. The level of DIO3 mRNA was lower in ovary and uterus of Jining Grey compared to Liaoning Cashmere goat (P＜0.05), while its expression in hypothalamus was opposite (P＜0.05) and there was no difference in pituitary between these 2 breeds (P＞0.05). The results hinted that DIO2 and DIO3 genes might be related to regulation of seasonal reproduction in goats, which provides reference for preliminarily revealing the molecular regulatory mechanism in goat reproductive seasonality.

Rice (Oryza sativa) is one of the most important food crops in the world. Among the factors harmful to rice production, weeds results in the most severe yield loss. To control weeds by herbicide is convenience, fast and low cost, that becomes the first choice in weeding. To cultivate transgenic non-selective herbicide resistant crops can decrease the kind, dosage and residual of herbicide, and prompt the productive efficiency of land, that plays an important role in commercial crop production. The Mat (methionine sulfone N-acetyltransferase) gene from bacterium Nocardia sp. AB2253 is a novel glufosinate resistant gene reported in 2009, which may be useful to develop herbicide resistant crops. However, the previous studies showed that the original Mat gene was not powerful enough in transgenic rice to provide high level of glufosinate resistance. In this study, the optimized Mat# gene according to codon bias in rice was transformed into an indica rice cultivar 9K (O. sativa ssp. indica) by use of Agrobacterium-mediated method. PCR analysis and Southern blot analysis of transgenic plants showed that the target gene Mat# had been integrated into rice genome with two copies. Glufosinate spraying on T2 generation plants at seedling stage showed that the T2 generation plants were resistant to glufosinate with a tolerance concentration of 1 000 mg/L at least. Progeny segregation statistics showed that the ratio of plant resistant and sensitive to glufosinate in T2 generation was 3.56∶1, which was conform to Mendelian inheritance by Chi-square test. Germination of T3 generation seeds under glufosinate stress showed that the T3 generation plants were resistant to glufosinate with a tolerance concentration of 600 mg/L at least, which indicated the resistance level of transgenic plants with Mat# gene was not lower than that of 9KA2 with Bar gene transformed. The investigation of agronomic traits showed that there was no significant differences in main agronomic traits between the T2 generation plants and non-transgenic controls, which indicated the introduction of alien gene did not have an obvious impact on main agronomic traits. Enzyme activity assay showed that the average activity of N-acetyltransferase in leaf was about 6.6 times of that in non-transgenic control. These results suggestted that the optimized Mat# gene provided higher glufosinate resistance than the original version, which can be used as a selection marker and new herbicide resistant gene resource in transgenic crop breeding.

Sex determining region Y-box 9 (SOX9) plays a particularly important role in the process of the individual development of mammals, and widely exists in various mammalian tissues. SOX9 gene mainly expresses on mammalian testicular tissues and is involved in the regulation of proliferation and differentiation of testicular cells. In this study, SOX9 expression was detected by qRT-PCR in various 6-month-old the Small Tail Han sheep (Ovis aries) tissues such as heart, liver, spleen, lung, hypothalamus, pituitary, testis and so on. Furthermore, mRNA expression and cellular localization of SOX9 were checked by qRT-PCR, Western blot and immunohistochemistry in testis during different development stages such as 0, 2, 6, 12, and 24-month-old. The qRT-PCR results showed that SOX9 was expressed in all the tissues detected, which expressed in pituitary with highest quantity, normal quantity in hypothalamus and testis, weak expression in muscle tissues (longissimus dorsi and biceps femoris), and mRNA expression from high to low in turn was: pituitary, hypothalamus, testis, liver, lung, heart, kidney, pineal gland, skin, biceps femoris, longissimus dorsi, spleen. At different developmental stages of testis, the mRNA expression of SOX9 were firstly decreased and then increased with month increasing, in which SOX9 expression in 6-month-old sheep testis was significantly higher than 0, 2-month-old (P＜0.05) while that was remarkably lower than 12, 24-month-old (P＜0.05). Expression trend of SOX9 protein was in accordance with SOX9 mRNA. The Western blot showed that SOX9 protein was lowly expressed in 0, 2, 6-month-old sheep testis, but its expression was higher and had increasing trend in 12, 24-month-old sheep testis. In addition, the immunohistochemical staining revealed that immunoreactivity was located in a small numbers of sustentacular cells and interstitial cells in 0, 2-month-old testis, while positive reaction was located in sustentacular cells, partial interstitial cells, even slight numbers of primary spermatocytes and secondary spermatocytes in 6, 12, 24-month-old testis. This study demonstrated that the SOX9 gene was widely expressed in sheep various tissues, and it plays an important role in the hypothalamic-pituitary-testicular axis, and it has a close relationship with the process of proliferation and differentiation of testicular cells.

The non-structural protein gene (NS) of influenza A viruses is separated into 2 alleles, A and B, with less than 70% sequence homology between each other. The NS1 is one of the major virulent factors that are able to antagonize the host immune system. In this study, on the basis of a systemic phylogenetic analysis, the NS1 protein of 4 Avian influenza viruses (AIV) strains (A/duck/Hunan/S1256/12(H3N8), A/environment/Hunan/S4484/11(H12N7), A/duck/Guangdong/07/00(H5N1) and A/duck/Shanghai/08/01(H5N1)) with NS gene of allele B prokaryotically expressed and were purified, and their thermal stability were further examined. Viral RNA (vRNA) was extracted from virus-infected allantoic fluid, followed by cDNA synthesis by reverse transcription PCR with the Uni12 primer. The full-length of NS1 fragments were then amplified with specific primers and cloned into the pGEX6P-1 vector for expression in the BL21 bacterial strain. Since the wild-type NS1 proteins were unstable and precipitated during purification, we determined to generate truncation mutants of NS1 protein containing amino acids 1M-202A as well as two mutations, R38A/K41A. The truncation mutant constructs expressed in BL21 bacterial strain, and progressively purified by glutathione-sepharose 4B affinity chromatography, cation exchange chromatography, and Hitrap Superdex75 column chromatography. The phylogenetic analysis revealed that the NS1 protein of all 4 viruses belonged to allele B. The expression and purification condition of NS1 protein was sequentially optimized from wild type, full length mutant to truncated mutant. High quality of purified NS1 protein with purity over 90% was finally obtained from the truncated mutant. In addition, the NS1 protein of A/duck/Hunan/S1256/12(H3N8) exhibited the highest heat stability. This study provides a foundation for further studying the structure and biological function of the allele B NS1 protein.

SKIP (ski-interacting protein) is a kind of metabolism related protein, playing an important role in regulating resistance to abiotic stress. In this study, the SbSKIP gene of Sorghum bicolor was cloned, whose cDNA encoding region included 1 485 bp and encoded 494 amino acids. Phylogenetic tree analysis showed that the SbSKIP gene of sorghum had the highest homology with Aegilops tauschii, and the second was Arabidopsis thaliana. According to phylogenetic tree analysis data, the amino acid sequence of SbSKIP had 52% similarity with Aegilops tauschii and 42% similarity with Populus trichocarpa (XP_002324443.2), Gossypium arboreum (KHG23995.1), Glycine max(ACY79501.1), Arabidopsis thaliana (NP_565151.1), respectively. Quantitative PCR analysis result showed that under the condition of simulated drought (16.5% PEG 6000), SbSKIP gene expression in leaves and roots did not changed significantly in 0 h, but it increased along with processing time. After 16 h of drought treatment, the expression significantly increased more than twice. And gene expression of sorghum leaf area was obviously higher than that of roots. We had constructed plant expression vector pSH-SbSKIP and transferred the vector to tabacco (Nicotiana tabacum) through leaf disc method transformation. And after resistance selection and PCR identification, we got a total of 25 strains of transgenic plants and harvested T0 generation seeds. TP4-4, TP4-9, TP4-13 had a high expression level of SbSKIP among transgenic plant. And the 3 different T0 plant strains TP4-4, TP4-9 and TP4-13 were used to analyze the seed germination and seedling drought tolerance at 0, 150 and 300 mmol/L mannitol simulated drought conditions, respectively. Results showed that the seed germination rate of transgenic plants were over 60% higher than that of wild type. As well as the root growth of wild type was obviously suppressed at the seedling stage under the 300 mmol/L mannitol treatment for 10 d. Under the natural condition with 20% PEG 6000, the seedlings which Long out of the 5~6 leaves were treated for 14 d. Results showed that the bottom of transgenic tobacco leaves margin slightly yellowed, and the bottom of wild type tobacco leaves mostly yellowed and even showed signs of wilting. And the average activity of super-oxide dismutase (SOD) of transgenic plants was 91.2% of that in the wild type, and H2O2, malondialdehyde (MDA) respectively than that of the wild type reduces the 42% and 32.7%, the content of soluble sugar was 1.1 times than that of wild type. Preliminary identification of SbSKIP gene expression to improve the drought tolerance of tobacco plants, and provides the basis for the further study SbSKIP gene function and the gene cloning and functional analysis and provide basic data for the creation of transgenic drought resistant materials.

The aim of this study is to verify whether involved in mammalian embryonic development by Somatic cell. This paper discusses the effects of luteinizing hormone (LH) and 17β-estradiol (E2) on oviductin (Ovn) by oviduct epithelial cells. We culture yak (Bos grunniens) oviduct epithelial cells in vitro with different concentration LH and E2 (10, 25, 50, 100 and 200 ng/mL). The relative expression level of Ovn gene and protein in different groups were detected by qRT-PCR and immunofluorescence, and location of ovn protein was detected by immunofluorescence after 72 h. The result obtained in this study was as follows that: (1) LH improved the relative levels of Ovn mRNA; These improvements were highest in the 10 ng/mL LH group (P＜0.05), followed by 25, 50, 100 and 200 ng/mL groups, the relative levels of Ovn mRNA were the lowest in control group (0 ng/mL LH); E2 improved the relative levels of Ovn mRNA; These improvements were the highest in the 25 ng/mL E2 group (P＜0.05), followed by 50, 10, 100 and 200 ng/mL groups, the relative levels of Ovn mRNA were the lowest in control group (0 ng/mL E2). (2) Detection by immunofluorescence showed that Ovn protein could be observed in yak cumulus cells cytoplasm as well as within nuclear membrane, and the expression of nuclear membrane was higher than the cytoplasm. LH and E2 could improve the relative expression levels of Ovn protein in yak oviduct epithelial cells; The improvements were the highest in the 10 ng/mL LH group (P＜0.05). However, the Ovn protein expression was decreased when LH concentration over 25 ng /mL. The relative levels of Ovn protein were the lowest in control group (0 ng/mL LH); The effective range of E2 (10~25 ng/mL), and the highest Ovn protein copy numbers at 25 ng/mL(P＜0.05), and reduced protein expression during all others. However, the Ovn protein expression was decreased when E2 concentration over 50 ng/mL. The relative levels of Ovn protein were the lowest in control group (0 ng/mL E2). These results suggestted that LH and E2 had significant effect on the secretion of Ovn in the oviduct epithelial cells of the yak. The optimal action concentration of LH was 10 ng/mL and the E2 was 25 ng/mL. This study provides basic information for the mechanism of LH and E2 on Ovn secretion of epithelial cells of oviduct, and also provides theoretical foundation to regulate embryonic development of yak with related hormones.

This work focus on clearing active segments of chicken (Gallus gallus) major histocompatibility complex (MHC) class Ⅱmolecules binding with invariant chain (Ii) for research of mechanism of MHC and Ii action in presenting antigen peptides. To this end 3 DNA segments (Sβ1, β1 and β2TC) were cloned from cDNA of B-LB gene, which was kept in our laboratory, and inserted into prokaryotic or eukaryotic expression plasmids respectively. Then these recombinant plasmids were respectively transfected or co-transfected with Ii into engineering bacteria, Rosetta (DE3). The expression products or complexes (B-LB segment and Ii) were purified by affinity chromatography and identified by pull-down and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, the expression and localization of B-LB segments and Ii in the eukaryotic cells were observed too. The results showed that first the 3 recombinant plasmids (pGEX-4T-1-B-LB-Sβ1, pGEX-4T-1-B-LB-β1 and pGEX-4T-1-B-LB-β2TC) could well express, and the interest proteins (B-LB-Sβ1, B-LB-β1 and B-LB-β2TC) also well be purified by an affinity chromatography. Secondly the segment B-LB-Sβ1 or B-LB-β1 rather than B-LB-β2TC bound His/Ii, which adsorbed to Ni-column, forming complexes by the co-transfection and followed pull-down, because in the Western blot the 3 interest proteins could be recognized by specific antibody in the co-transfected products, but only B-LB-Sβ1 and B-LB-β1 were found as the complexes (His/Ii and B-LB-Sβ1, His/Ii and B-LB-β1), which were dissociated from Ni-column in pull-down. Finally it was proved that B-LB-Sβ1 and B-LB-β1 could express and localize in the endometrial system of 293T cells, which kept the function such as whole MHC molecule, but the B-LB-β2TC lost this function. All of above results first time provide an evidence of binding of active segments of MHC class Ⅱmolecules with Ii in vitro.

Necrostatin-1 (Nec-1) is a small molecule inhibitor of necroptosis which specifically target the receptor-interacting protein 1 (RIP1) kinase, and has been extensively used in disease models to investigate the role of RIP1 in cell death and inflammation. RIP1 is a crucial adaptor kinase involved in the activation of NF-κB, production of reactive oxygen species (ROS) and phosphorylation of mitogen activated protein kinases (MAPKs), which all play important roles in apoptotic signaling. It has been proved that the vaccine strain Mycobacterium bovis BCG can elicit robust protective immunity and induce apoptosis of macrophages. However, the effect of Nec-1 on the apoptosis of macrophages induced by BCG remains unknown. In this study, we investigated the role of Nec-1 on apoptosis induced by BCG in a macrophage cell line RAW264.7.Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cell apoptosis rate was detected by Annexin V-FITC/PI double staining. Mitochondrial membrane potential (MMP) was assessed by Jc-1 dye. Caspase-3 activity in the cell extract sampled was measured by Caspase-3 Colorimetric Assay Kit. Furthermore, the mRNA level of RIP1, RIP3, Bax and Bcl-2 were tested with RT-PCR, and the expression level of Bax, Bcl-2, Caspase-3, RIP1 and RIP3 were analyzed by western blotting. The results showed that Nec-1 significantly increase MMP and reduce apoptosis rate of RAW264.7 infected with BCG. RT-PCR and western blotting confirmed that the mRNA and protein expression of Bcl-2 was increased, while mRNA and protein levels of RIP1, RIP3 and BAX was decreased. We also found that expression and activity of caspase-3 was decreased. Taken together, these findings indicated that Nec-1 could inhibit BCG-induced macrophage apoptosis by increasing the level of Bcl-2 and decreasing expression of the pro-apoptotic protein BAX, RIP1 and RIP3. This finding may thus provide an insight into the underlying mechanism of alveolar macrophage cell death in response to mycobacterial infection.

ATP-binding cassette (ABC) transport protein is one kind of super family transporter, which exists in almost prokaryotic and eukaryotic cells, and plays an important role in physiological activities. In this paper, the structure characteristics of ABC transport protein were detailedly introduced, and divided into 8 subfamilies from A~H based on phylogenetic analysis of conservative regions. Besides, the latest researches progress of ABC transport proteins on enhancing resistance of plants and fungi to heavy metals were also introduced and interpreted. Meanwhile, the probable mechanism of it on how improving host resistance to heavy metals was summarized and analyzed from 3 points, which were vacuole sequestration, direct efflux and mitochondrial ABC transporter. Moreover, the viewpoint of ABC transport protein preference was proposed, according to the law of choosing and transporting metal ion. At the end, the research direction and potential application of ABC transport protein were simply discussed. Thus, it would be reliable reference for further research of ABC transporter protein.

Brassica napus (AACC, 2n=38) is the most widespread oil crop in China. As single nucleotide polymorphism (SNP) is the most important molecular marker technology, it is extremely important to study on SNP in understanding the forward genetics. In the past five years, the genome sequences of B. napus and its ancestry species, named Brassica rapa and Brassica oleracea, had been successfully assembled and released that could enable efficient study regarding SNP and its role in the genetic study of oilseed rape. This review summarized the biological characteristics and the techniques developed for studying SNP, highlighting the pivotal role of SNP in linkage map construction and association mapping in rapeseed. Moreover, it further signifies the importance of SNP in terms of germplasms' population structure as well as evolution analysis and marker associated breeding, which can provide researchers a better understanding regarding the value of SNP study in B. napus.