Melanoeortin receptor-4 gene (MC4R) belongs to the super family of G protein-coupled receptors that is implicated in the control of feeding behavior and energy homeostasis in mammals. This study aimed to explore the association of single nucleotide polymorphism (SNP) of MC4R gene with Tibetan sheep (Ovis aries) growth traits, so as to search for molecular markers related with growth traits, DNA sequencing method was applied to detect and genotype polymorphisms within MC4R gene in Tibetan sheep populations, and analyzed the associations of polymorphisms and combined genotypes with growth traits of Tibetan sheep. Results showed that 4 SNPs in MC4R gene were detected, including g.1803G＞A in exon 1, g.1261T＞C in intron 2 and two mutants (g.1803G＞A and g.1918C＞T) in 3'-untranslated region (3'UTR). Association analysis in 555 Tibetan sheep population indicated that, at g.880G＞A locus, individuals with genotype AA had larger higher body weight, body height, body length and chest circumference compared with the genotype GG (P＜0.01 or P＜0.05). At g.1261T＞C locus, the body weight of animals with TC genotype was significantly higher (P＜0.05) than that of animals with TT genotype. Moreover, the g.1803G＞A locus was significantly affected body weight (P＜0.01), and GG was the preferable genotype. The optical combined genotype of MC4R was diplotype H13H7 (GTGC-ATAC), which was more effective on growth traits than other combined genotypes (P＜0.05). These results suggested that the single nucleotide polymorphisms and combined genotypes of MC4R gene affected the growth traits of Tibetan sheep, and they could be used as candidate genes for breeding Tibetan sheep with better growth traits.

MiRNA is a non-coding small RNA in eukaryotic organisms. It plays an important role in the development of animals and plants. However, the molecular mechanism of rice resistance to high temperature participated by miRNA has seldom been studied. As an important staple food, rice (Oryza sativa) frequently suffers from heat stress during the growing season. Clarifying plant heat stress response and tolerance mechanism at miRNA level is the basis to breed varieties with a suitable heat stress (HS) tolerance. In previous study, six libraries (double for 0, 12 and 24 h heat treatment) constructed from heat tolerant variety Oryza sativa ssp. indica cv. HT54, were sequenced by next-generation sequencing (NGS) technique. Those study found that the miR396 family member and their putative target genes might play pivotal roles as regulator reacting to heat stress. In this study, the expression of all 8 miR396 family members and 12 predicted target genes were detected in seedlings of Oryza sativa spp. japonica cv. Nipponbare with 48 ℃ heat stress for 6 different time points. The results indicated that all 8 miR396 family members and 12 predicted target genes showed different reaction to heat stress. The expression level of miR396a, miR396e and miR396f increased at high temperature stress at each time points, the expression level increased up to 10 fold (in miR396a and miR396e), even 100-fold (in miR396f); while miR396b, miR396c, miR396d, miR396g and miR396h decreased at 1.5, 3, 6 h, but increased at 12, 24 h. Correspondingly, the OsGRF family showed irregular upward or descending expression with high temperature stress. The target gene OsGRF2 decreased up to 8~10-fold. OsGRF2 had a high negative correlation with the expression of miR396a, miR396e and miR396f, indicating that OsGRF2 was the target gene for miR396a, miR396e and miR396f. These results suggest that miR396 would play a key role in rice heat tolerance.

Somatic incompatibility (SI) commonly occurs among intraspecific heterokaryons of basidiomycetes with different genotype, preventing plasmogamy and exchange of genetic information among them, controlled by SI loci. It is unclear whether the SI loci expression in the monokaryon strains. Dikaryon strains DK13×3 was bred by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, the transcriptomes of the 3 strains were obtained. 76 unigenes in the transcriptomes involved in SI, and categorized of heterokaryon incompatibility gene (het) het-e, het-6, tol (tolerant), NACHT (nucleoside-triphosphatase domain)/WD40 (ligand-binding domain; approximately 40 amino acids terminating with tryptophan and aspartic acid residues) gene homologues, respectively. 61.8% of the unigenes were all expressed in the 3 strains, 23.7% were expressed only in MK13 and DK13×3, 11.8% were expressed only in MK3 and DK13×3, 1.3% were expressed only in DK13×3 or only in MK13 and MK3. Four of these unigenes were selected and their expression levels were verified by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The quantitative RT-PCR results were consistent with RNA-Seq. These results showed that almost all unigenes involved in SI were expressed in the dikaryon strains and more than 70% were expressed in the 2 monokaryons strains, suggesting that these genes may be involved in self/non-recognition in ho-ho crosses and he-ho crosses in basidiomycetes. This study suggests that somatic incompatibility genes in basidiomycetes may affect the success rate of hybridization and the fertility of hybrid progeny.

Pi2 is a broad-spectrum rice blast resistance gene to the physiological races of rice blast fungus and has great application value in rice blast resistance breeding. But the wide application of the gene in rice marker-assisted selection (MAS) breeding process is seriously hindered because the operation of the developed molecular markers in the marker assisted selection of the gene is complex and difficult to achieve high throughput and accurate detection. High resolution melting analysis (HRM) is a recently developed PCR-based DNA polymorphism detection technology that is easy to operate and with high resolution, low cost, and accurate results. In this study, we took sequence alignment analysis of the genomic sequence of Pi2 gene with that of Pi9, Piz-t, PigmR and PigmS, and found 6 specific single nucleotide polymorphism (SNP) loci in Pi2 gene. Then we sequenced the DNA flanking the 6 specific SNPs of Pi2 gene in 12 rice varieties. Sequence alignment showed that they were divided into 6 categories. Based on these differences, 6 pairs of primers were designed for genotyping the Pi2 gene in different rice varieties based on the HRM analysis. Primer pair Pi2-HRMF1/R3 was found to be able to classify 12 rice accessions into 6 types with high stability and reliability, which were in good agreement with the gene sequence. Through the HRM-detection of Pi2 in 23 rice sterile line and maintainer line materials, we found that none of them contained the gene. Therefore, the Pi2 gene can be introduced into these sterile lines and maintainer lines via MAS breeding by using the Pi2-HRMF1/R3 primer pair combined with the HRM technology, in order to enhance the blast disease resistance of hybrid varieties which are derived from these sterile lines and maintainer lines. When the Pi2-HRMF1/R3 primer pair was used to analyze the segregating populations from the crosses between Huazhan, which carries the Pi2 gene, and four other recurrent parents which do not have the Pi2 gene, and found that the marker could well distinguish the Pi2 donor parent from the recurrent parents as well as the heterozygous genotype in the Pi2 gene locus in the offspring. Compared with the previously reported Pi2 gene specific molecular markers, the molecular marker developed in this study is more efficient in detection and easier in operation. Therefore, the Pi2 gene specific molecular marker combined with the HRM technology in this study can be used for efficient and accurate identification of the Pi2 gene in genetic resources and for high throughput screen of the descendants for improved resistance to blast disease based on Pi2. The method will serve as an efficient and accurate technical support to molecular breeding in a large scale in the future.

Under starvation, lots of genes involved in energy metabolism of fat will change in order to adapt to environmental changes for farm animal and human (Homo sapiens). In this study, in order to investigate the regulation role of miR-378 for fat metabolism under short-term (24 h) starvation, wild type (WT) mice and miR-378 knockout (KO) mice (Mus musculus) were used to investigate the fat metabolism difference between normal feeding and high-fat induction conditions. The tissues of brown fat, inguinal fat, gonadal fat, perirenal fat and subcutaneous fat of two months and high-fat induced two months of mice were collected. To explore the effect of miR-378 KO on fat decomposition and fat synthesis under starvation, decomposition related genes were detected by qRT-PCR. The results suggested that the expression of Pparγ was decreased, the expression of hormone sensitive lipase (HSL), long-chain acyl-Coenzyme A dehydrogenase (Acsl1) and acyl-Col synthetase long-chain family member 1 (Acadl) were increased, and also the expression of miR-378 was increased in fat. The expression of fat decomposition maker genes in miR-378 KO were decreased compared with WT. In conclusion, the results demonstrated that miR-378 could promote the decomposition of fat and miR-378 KO inhibit the decomposition under starvation. This study suggests that miR-378 could be a regulatory factor on affecting the fat decomposition in mice, would provide an reference for studying human and other animals fat decomposition.

In wheat (Triticum aestivum), the cytosolic glyceraldehyde-3-phosphate dehydrogenase gene TaGAPC5 was induced under abiotic stresses. Sequence analysis showed that the TaGAPC5 gene promoter region contained three W-box cis-elements. In this study, PCR-mediated mutagenesis method was performed to introduce -640 site mutagenesis, -640 and -700 both sites mutagenesis, and -640 and -700 and -997 three sites mutagenesis in TaGAPC5 gene promoter region. Then, recombinants of the three mutagenesis sequences with pC0390-GUS vector were constructed, respectively, and transformed into Nicotiana tabacum by Agrobacterium-mediated approach. The transformed tobaccos growth under normal conditions and were treated with 100 nmol/L abscisic acid (ABA) and 20% PEG8000, respectively. Histochemical staining results showed that the three mutations of TaGAPC5 promoter all had promoter activity. The results of GUS enzyme activity in tobacco leaves revealed that under normal growth condition, compared with the TaGAPC5 gene promoter without mutagenesis，-640 alone site mutant didn't affect the activity of the promoter, the activity of mutant at -640 and -700 and -997 three sites was decreased significantly(P＜0.01). What's more, under the 100 nmol/L ABA treatment, -640 site mutant, -640 and -700 both sites mutant, and -640 and -700 and -997 three sites mutant all significantly reduced the activity of the TaGAPC5 gene promoter (P＜0.01); under 20% PEG8000 treatment, the activities of -640 alone site mutant and the -640, -700, -997 mutant were both decreased more significantly (P＜0.01). Taken together, the results proved that -640, -700, -997 three W-box sites in TaGAPC5 gene promoter region had a great role in promoter activity, particularlly in 100 nmol/L ABA、20% PEG8000 conditions, and suggested that the W-box cis-elements of TaGAPC5 gene promoter may participate in the regulation of TaGAPC5 gene expression under abiotic stress. Thus, the results of this study could provide important clues for regulatory mechanism of TaGAPC5 gene under abiotic stress in wheat.

Drought is one of the vital factors that affect the apple (Malus × domestica) production in China, while the ability of drought resistance of stock directly affects the scion growth and development, fruit yield and quality. In this study, seven apple dwarfing stocks from SH series (SH1, SH6, SH40), M series (M26, M9-T337) and G series (GX, CG24), grafted on base rootstock Malus hupehensis var. pingyiensis respectively, were used for evaluation on drought resistance. After 40 d drought stress, growth and physiology indices were analyzed comparatively, and the ability of drought tolerance was ranked by comprehensive evaluation of subordinate function value (SF). Those results showed that the trunk growth rate (TGR) for GX, CG24 and M9-T337 was worst restricted, whereas SH6 performed the highest TGR both in control and water stress plants, and the later up to 80.6% contrast to the control. Besides, GX and M26 got the largest drop on transpiration rate (Tr) than others under drought stress, which decreased 50% than that of control. Net photosynthetic rate (Pn) in M9-T337 and SH6 was affected non-significantly under drought stress, whereas the GX showed the lowest Pn than other stocks. For the detection on leaf relative conductivity, the significant elevations under drought stress occurred in GX and CG24, and SH6 showed the most stability than others. Results on leaf water potential suggested that SH series dwarfing stocks had higher water potential whether in well-watered or drought conditions, whereas GX suffered series drop. Collectively, based on comprehensive evaluation of SF for above five indexes detected, drought resistance evaluation were conducted and ranked as follows: SH6＞SH40＞M9-T337＞SH1＞M26＞CG24＞GX. In following comparing analysis between SH6 and GX, four antioxidases including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) showed higher activity levels in SH6 than those of GX, and response actively with drought stress process was observed in SH6 but none in GX. In addition, cluster analysis of gene expression levels in SH6 and GX samples showed that 12 genes were divided into two groups. The first group, including 6 genes (DREB1A, DREB2A, DREB2B, DREB2C, DREB6 and ZAT10) expressed at higher levels in GX samples, whereas the other group expressed mainly in SH6 samples, from which 4 genes (SOD1, APX1, CAT1 and DHAR2) were belong to antioxidase families, suggesting higher levels of antioxidant enzyme activities may contribute to drought resistance. This study provides a theoretical reference for the application of apple dwarfing stock and drought-resistant breeding.

Transcriptome sequencing is a rapid and efficient way to obtain gene expression information. Betula luminifera is one of the superior woody species in China. In order to excavate the genes involved in its wood formation, the transcriptome sequencing, sequencing data assembly, and functional annotation and classification of the sequences were conducted by using the stems and leaves as materials in this study. Then the full-length cDNAs of S-adenosylmethionine synthase (SAMS) gene were isolated by RACE technique. The sequence feature and phylogenetic tree were analyzed by using bioinformatics software, such as DNAStar, MAGA7.0, and the expression pattern was detected by fluorescence quantitative RCR. The results showed that a total of 1.9 Gbp sequencing data was obtained, and were assembled into 54 577 Unigenes. By BLAST comparison against the NR (non-redundant protein database) and the Swiss-Prot protein sequence database, 65.8% (35 920) of Unigenes were annotated, which corresponded to 24 482 Unique protein accessions. Among these annotated Unigenes, 7 954 and 9 997 were assigned to Gene Ontology (GO) classes and Clusters of Orthologous Groups (COG), respectively. Based on transcriptomes sequencing, BlSAMS1, BlSAMS2 and BlSAMS3 were cloned, with the cDNA length of 1 578 bp, 1 555 bp and 1 266 bp, respectively. The molecular weights of the corresponding encoded proteins were 42.9, 43.1 and 42.7 kD, each of which contained typical conservative domains of SAMS protein. Phylogenetic tree showed that BlSAMS1 was grouped with Catharanthus roseus SAMS2, and BlSAMS2, Malus xiaojinensis SAMS, and Vitis vinifera SAMS4 were in the same clade. BlSAMS3 was clustered into a clade with Arabidopsis thaliana SAMS3 and Pinus pinaster SAMS1, which were involved in lignin biosynthesis. The expression levels of BlSAMS1 were relatively high in leaf samples. The BlSAMS2 and BlSAMS3 were predominantly expressed in stem and xylem, and their expression levels in stem increased as the lignification progressed. It could be speculated that the BlSAMS3 played an important role in lignin biosynthesis of B. luminifera wood. Transcriptome information and the BlSAMSs expression patterns could lay a foundation for further dissection on the molecular mechanism of B. luminifera wood formation.

Deleted in azoospermia-like gene (Dazl) is required for differentiation of male animal germ cells. However, the regulation mechanisms of Dazl gene are not exactly the same in broad variety of animals or in the same animal at different developmental stages. In order to investigate the expression patterns, cellular localization and regulatory mechanisms involving with spermatogenesis of the Dazl gene in sheep (Ovis aries)testes at different developmental stages, testis, heart, liver, spleen, lung, kidney and muscle tissues were collected from Small-tail Han sheep at 0, 2, 5, 12 and 24 months of age. The Dazl mRNA expression patterns in 0, 2, 5, 12, 24-month-old sheep testes and multiple somatic tissues, including heart, liver, spleen, lung, kidney and muscle of 12-month-old sheep were detected by quantitative real time PCR (qRT-PCR), and the Dazl protein expression and cellular localization in testes at different developmental stages were observed and measured by Western blot and immunohistochemistry, respectively. The qRT-PCR and western blot results revealed that Dazl genes were expressed in sheep testes throughout development at mRNA and protein levels, and their expression patterns were similar basically. The Dazl mRNA and protein were at a lower expression level in pre-pubertal sheep testes (0-, 2- and 5-month-old), and the expression of the Dazl mRNA and protein in 12-month-old were significantly higher than those in 0-, 2- and 5-month-old (P＜0.01), whereas their expression levels were decreased in 24-month-old sheep testis. The qRT-PCR results in various tissues at 12 months of age, including testis, heart, liver, spleen, lung, kidney and muscle tissues, showed that the Dazl mRNA was predominantly expressed in testis, and low-expressed in heart, whereas not expressed in other tissues. The immunohistochemical staining in testes at different developmental stages showed that the Dazl protein was localized to Leydig cells in pre-pubertal (0-,2 -and 5-month-old) sheep testes, while it was localized to Leydig cells, primary spermatocytes, secondary spermatocytes and sperm cells in 12- and 24-month-old sheep testes. These results suggested that the Dazl gene was highly expressed in testes, playing vital roles in the proliferation of Leydig cells in various age of sheep testes, spermatocytes and sperm cells in post-pubertal sheep testes. It is inferred that the Dazl gene is involved in the regulation of postnatal ovine spermatogenesis via direct effect (modulating the maturation and meiosis of spermatocytes) and indirect effect (modulating the proliferation of Leydig cells). The observation provides a scientific basis and reference for further research on the regulatory mechanism of Dazl gene during spermatogenesis.

Claudin3 (CLDN3) regulates the movement of small molecules across the sertoli cell (SC) tight junctions (TJ) and creates physical barrier for preleptotene / leptotene spermatocytes. In order to explore biological effects of CLDN3 on yak SC, this study used reverse transcription polymerase chain reaction (RT-PCR) to clone CLDN3 cDNA of yak (Bos grunniens) and used bioinformatics method to analyze it's biological characteristics. The different concentrations of follicle-stimulating hormone (FSH) (0, 10, 25, 50, 75, 100 ng/mL) were added into culture medium during yak sertoli cells (SCs) cultured in vitro, and the levels of CLDN3 mRNA and protein were evaluated by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting (WB). What's more, the location of CLDN3 protein on SC was detected by immunofluorescence. The results showed that yak SC exhibited bipolar corpuscula in nucleus after Feulgen staining and had a positive reaction of Fasl by the immunocytochemical identification. The CLDN3 cDNA of yak was successfully cloned, and it contained ORF of 1 074 bp length (GenBank No. YK411920) and 660 bp CDS, encoding 219 amino acids. The sequence alignment showed that the homology between yak and other species were relatively high (≥90%). CLDN3 protein could be detected in nucleus, cytoplasm and plasma membrane of SC from immunofluorescence analysis. qRT-PCR detection showed that the 10, 25, 50, 75 and 100 ng/mL FSH treatment groups could improve relative expression of SC CLDN3 mRNA. The CLDN3 mRNA expression level both 25 and 50 ng/mL groups were significantly higher than other groups (P＜0.01). The CLDN3 mRNA of 25 ng/mL group was the highest expression level (P＜0.01). The difference between 10 ng/mL and control group was not significant (P＞0.05). 75 and 100 ng/mL were significantly different from the control group (P＜0.05). 25 ng/mL FSH group of CLDN3 protein was the highest of the groups (P＜0.01), and the expression of CLDN3 protein in 100 ng/mL FSH was lower than that in the control group (P＞0.05). In summary, CLDN3 amino acids sequence was conserved in the different genus. FSH may be as an important hormone during the formation of TJ in testis of yak. The expression both of CLDN3 mRNA and CLDN3 protein relied on concentration of FSH in which the optimum FSH concentration was 25 ng/mL (P＜0.01). The research could provide the basis for revealing the biological effect of yak CLDN3 during sperm migration.

Pharyngeal tonsil (PhaT) belongs to mucosal-associated lymphoid tissue and plays an important role in mucosal immunity of respiratory tract. IgA (immunoglobulin A) and IgG (immunoglobulin G), which are 2 important effector molecules of mucosal immunity, have extensive immunological function. This research aims to study the changes of histological structure characteristics of pharyngeal tonsils during the development of yak (Bos grunniens), and the distributive characteristics and changing regularities of IgA and IgG antibody secreting cells (ASCs) in pharyngeal tonsils. In this study, histology method was used to observe and compare the histological structure characteristics of pharyngeal tonsils between newborn and adult yak. The expression of IgA and IgG in pharyngeal tonsils of newborn and adult yak was detected by immunohistochemistry (IHC). Histological results showed that pharyngeal tonsilar epithelium was roughly divided into 2 types, namely non-reticular epithelium and reticular epithelium. Non-reticular epithelium referred to pseudostratified columnar epithelium with a large number of alcian blue-periodic acid schiff (AB-PAS) positive goblet cells and non-keratinized stratified squamous epithelium. However, the reticular epithelium overlying the pharyngeal tonsil was the lymphoepithelium that was frequently infiltrated by lymphocyte. The lamina propria of yak pharyngeal tonsils was composed of lymphoid follicles and diffuse lymphoid tissue. A large number of high endothelial veins and lymphatic vessels could be found in the diffuse lymphoid tissue. After comparing with the pharyngeal tonsils of newborn and adult yak, only a several primary lymphoid follicles in the newborn yak's pharyngeal tonsils was found. However, not only primary lymphoid follicles, but also secondary lymphoid follicles can be found in adult yak's pharyngeal tonsils. In addition, the statistics of the number of lymphoid follicles showed that in the same unit field of view (100×), the number of lymphoid follicles was approximately (2±0.6019) in newborn yak, while was approximately (13±0.4079) in adult yak, which was much higher than that of the newborn(P＜0.01). The immunohistochemical results showed that IgA and IgG ASCs were distributed in the subepithelial region of non-reticular epithelium, the reticular epithelium region, inter-follicles area, lymphoid follicles and glandular compartments in the 2 groups, and the distribution of IgA and IgG ASCs was the highest in the subepithelial region of non-reticular epithelium, respectively. In addition, the average number of IgG ASCs per unit area(0.023 mm2) were higher than that of IgA ASCs in both 2 groups, and there were significant difference in the adult group(P＜0.01), but no difference in the newborn group (P＞0.05). The results indicated that the primary lymphoid follicles of yak pharyngeal tonsils have been formed in the newborn stage; IgA and IgG ASCs played an important immunoprotective effect on the mucosal surface through the non-reticular epithelial; IgG may be dominant in the immune defense of yak pharyngeal tonsils due to there was a preponderance of IgG ASCs in 2 groups. This study provides morphological and immunological information for the mucosal immunity and the pathogenesis of related diseases.

CCAAT/enhancer binding protein alpha gene (CEBPA) plays an important role in lipogenesis, growth, and development, and sine oculis homeobox homolog 1 gene (Six1) plays a regulatory role in skeletal muscle development and muscle fiber type transformation, and both of the two genes are closely related to bone development. The single nucleotide polymorphisms (SNPs) of the exons of CEBPA gene and Six1 gene were detected by direct sequencing in this study. The purpose of this study was to uncover the effects of CEBPA gene and Six1 gene on the carcass traits and body size traits of Wuliang Mountain black-bone chicken (Gallus gallus). The results showed that three SNPs (i.e. 74 C＞G, 552 G＞A and 936 C＞T) were found in CEBPA gene, and two SNPs (i.e. 375 G＞A and 564 G＞A) were found in Six1 gene. All SNPs except two SNPs (i.e. 936 C＞T of CEBPA gene and 375 G＞A of Six1 gene) exhibited three genotypes, and the predominant alleles were non-mutation allele. The chi-square test showed that allelic frequency and genotype frequency of two SNPs (i.e. 552 G＞A of CEBPA gene and 564 G＞A of Six1 gene) didn't agree with the Hardy-Weinberg equilibrium (P＞0.05), while others did. As for polymorphic information content, all SNPs were in a low polymorphism. The mutation (C→G) of 74 bp in CEBPA gene caused the change of amino acids (i.e. Threonine → Serine), while others didn't. Moreover, there was no strong linkage disequilibrium(LD) among each gene's respective SNPs. In the group of Wuliang Mountain black-bone chicken, 5 haplotypes and 7 diplotypes were formed on CEBPA gene, and 4 haplotypes and 5 diplotypes were formed on Six1 gene. Association studies of genotypes of SNPs and diplotypes with carcass traits and body size traits were performed by using the GLM. The results showed that four SNPs (i.e. 74 C＞G and 936 C＞T of CEBPA gene, 375 G＞A and 564 G＞A of Six1 gene) were significantly or highly correlated with carcass traits and body size traits (P＜0.05 or P＜0.01). 74 C＞G of CEBPA gene was significantly correlated with the percentage of half-eviscerated yield, the percentage of eviscerated yield, body slope length, breast width, breast depth and fossil bone length (P＜0.05). 936 C＞T of CEBPA gene was significantly correlated with live weight, dressed weight, half-eviscerated weight, eviscerated weight, breast muscle weight, body slope length, pelvis width and shank length (P＜0.05 or P＜0.01). The percentage of half-eviscerated yield and eviscerated yield of diplotype H1H1 and H4H4, breast muscle weight, pelvis width and shank length of diplotype H1H2, body slope length of diplotype H1H4 and H4H4, breast width, breast depth and fossil bone length of diplotype H4H4 were significantly higher than other partial diplotypes, respectively (P＜0.05 or P＜0.01). 375 G＞A of Six1 gene was significantly correlated with breast depth and pelvis width (P＜0.05 or P＜0.01), while it was not significantly associated with carcass traits. 564 G＞A of Six1 gene was significantly correlated with breast muscle weight, the percentage of breast muscle, breast angel, shank length and shank circumference (P＜0.05 or P＜0.01). The breast muscle weight and the percentage of breast muscle of H6H6, H6H7 and H6H8 diplotypes, dressed percentage of H6H7 diplotype, eviscerated weight, breast depth and pelvis of H6H8 diplotype, breast angel, shank length and shank circumference of H7H7 diplotype were significantly higher than other partial diplotypes, respectively (P＜0.05 or P＜0.01). The present research suggested that CEBPA gene and Six1 gene had significant genotype effects on the carcass traits and body size traits of Wuliang Mountain black-bone chicken and could be potential candidate genes for molecular marker-aid selection in Wuliang Mountain black-bone chicken and other chicken breeds.

Hepatocyte nuclear factor 1α (HNF1α) plays an important role in the carbohydrate and lipid metabolism by regulating numerous related genes in liver on mammals, but there are few studies in poultry. To obtain the chicken (Gallus gallus) HNF1α protein, the CDS fragment of chicken HNF1α was amplified by PCR from the plasmid pcDNA3.1-HNF1α constructed previously in our lab. After the fragment being cloned into the site between NdeⅠ and XhoⅠ of the pET30a, which protein fused 6-His at its C-terminus, the BL21-pET30a-HNF1α recombinant expression strain was successfully constructed through the recombinant expression plasmid (pET30a-HNF1α) being transformed into BL21 (DE3). To induce the HNF1α recombinant protein, the BL21-pET30a-HNF1α was cultured in Luria-Bertani (LB) medium with different concentrations of isopropyl-β-D-thiogalactoside (IPTG) (such as 0.1, 0.2, 0.4, 0.8 mmol/L). The analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and Western blot showed that the recombinant protein was more highly expressed in BL21 at 37 ℃ for 4 h with 0.1 mmol/L IPTG than others. And it's molecular weight was about 69 kD, which was consistent with molecular weight by theoretical calculation. The results showed that the recombinant protein was mainly expressed with the form of inclusion body although the inducible temperature was dropped to 15 ℃. Then after these inclusion bodies were completely denatured with 8 mol/L urea, the HNF1α recombinant proteins were adsorbed onto the Ni2+-IDA resin by 6-His tag fused at its C- terminal, and these proteins were refolded in situ by the urea being phase out to zero in the washing process. Finally, the refolded proteins were eluted by the elution buffer with 300 mmol/L iminazole. By the SDS-PAGE gel analysis, the results showed that the chicken HNF1α recombinant protein near 95% purity was obtained from the denaturing solution using this one-step method combined with Ni2+ affinity chromatography and solid-fluid renaturation in situ. These results lay a foundation for the further study of the structure and function of the HNF1α, simultaneously pave the way for the preparation of the corresponding antibody.

Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme for the synthesis of monounsaturated fatty acids in hepatocytes, and plays an important regulation role in the composition of fatty acids in the body. In order to explore the genetic regulation mechanism of SCD1 gene on the fat deposition of poultry, 21 days embryo duck (Anas platyrhynchos) hepatocytes were extracted by the method of type Ⅳ collagenaseand. The pEGFP-N3-SCD1 eukaryotic over-expression vector was constructed and transferred into the hepatocytes. The over-expression of SCD1 gene in duck hepatocytes was measured by HIS labeling assay kit to understand the effect of SCD1 on lipid metabolism. The experimental results showed that initial separation of hepatocytes was good, and the cells were basically covered with culture plates until 96 h, The recombinant plasmid pEGFP-N3-SCD1 and green fluorescence could over- expression stably in the primary hepatocytes of the ducks. The correlation between over-expression and lipid index showed that the expression of SCD1 was positively correlated with total cholesterol (TC) and low density lipoprotein (LDL) (P＜0.01), The content of high density lipoprotein (HDL) was significantly negative correlation (P＜0.01) but triglyceride (TG) (P＞0.05). The results showed that the recombinant plasmid pEGFP-N3-SCD1 could be successfully isolated and purified from the liver extraction by using type Ⅳ collagenase. Over-expression of SCD1 gene had a regulatory function for lipid metabolism of duck primary hepatocytes and it is useful for the future study about the genetic regulation mechanism of SCD1 on duck fat deposition.

Visual microarray technology based on the traditional microarray technology, has a great significance for the detection of clinical disease detection and diagnosis. In this study, the specific primers were designed, according to the conservative gene nucleprotein (NP) of Avian influenza virus (AIV), cDNA was obtained from RT-PCR. The recombinant plasmid of AIV-NP was successfully constructed. Meanwhile, fusion (F) gene of Newcastle disease virus (NDV), nucleocapsid (N) gene of Infectious bronchitis virus (IBV) and thymidine kinase (TK) gene of Infectious laryngotracheitis virus (ILTV) were successfully recovered from the conserved bacteria. The oligonucleotide probes were designed on the basis of target gene sequences. The visual microarray was prepared and hybridized to the PCR products labeled biotin. The parameters of the visual microarray were optimized. The results showed that 25 μmol/L of oligonucleotide probes, 1 h of hybridization time, 50 ℃ of hybridization temperature, 2 000 times diluted of 1.0 mg/mL Streptavidin HRP Conjugate and 5 min of diaminobenzidine (DAB) staining time, were the optimal condition for microarray detection. The 96 clinical samples were simultaneously detected by the visual microarray and PCR/RT-PCR, the results showed that the detection rate of two methods was consensus. The visual microarray of AIV-NDV-IBV-ILTV developed in this study is a fast, accurately and high-throughput detection methods, which could provide a new technology for chicken disease clinical diagnosis.

Genetically modified (GM) soybean (Glycine Max) event SHZD32-1 was developed by Chinese scientists with independent intellectual property rights, and the event has herbicide resistance by translating glyphosate resistant gene G10-EPSPS into cultivated variety 'Zhongdou 32 (Glycine Max)'. At present, GM soybean event SHZD32-1 has entered the stage of environmental testing and has a broad application prospect. So far, there are little reports about detection methods for GM soybean event SHZD32-1. In order to protect and improve the implementation of laws and regulations for GM soybean event SHZD32-1, the specific primer pairs and probes were designed based on the flanking sequences between the soybean genome and inserted exogenous fragment of SHZD32-1. Conventional PCR and qRT-PCR detection methods had been developed in this study. Specificity detection of conventional PCR and qRT-PCR found that all mixture GM samples DNA could not obtain the positive results except containing the SHZD32-1 genomic DNA, indicating the methods were good for specificity. Conventional PCR could detect 0.1% of GM SHZD32-1 event, and limit of detection (LOD) of qRT-PCR method could reach 21 copies of GM SHZD32-1 genomic DNA. The results were obtained by using the conventional PCR and qRT-PCR method for 60 repetitions under the LOD concentration. Therefore, the two methods had high specificity, good sensitivity, and strong stability. This research established GM SHZD32-1 specific PCR detection method is an important content of molecular characteristics of the evaluation of GMO, could be used to GMO safety regulation and provide technical support in China.

Quantitative Real-time PCR (qRT-PCR) is a commonly-used method for studying gene expression currently, in which choosing appropriate reference genes for specific materials or particular conditions is a prerequisite for accurately analyzing relative expressions of target genes. This study took the roots, stems, leaves and bamboo shoots of Phyllostachys edulis as experimental materials and analyzed the expressions of 5 kinds of widely used reference genes - glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), heat shock protein (HSP), 18S ribosomal RNA (18S rRNA) and eukaryotic initiation factor 4α (eIF-4α) in different tissues or organs by the aid of the qRT-PCR. Three programs including GeNorm, NormFinder and BestKeeper were used to determine the expression stability of these reference genes. The results showed that the most stable reference genes of P. edulis had many differences in the various experimental conditions. After the comparative analysis using GeNorm, NormFinder and BestKeeper, eIF-4α had the highest expression stability and was viewed as the corrective reference gene when the genes expression in different leaves of P. edulis were analyzed and compared by qRT-PCR. When the differences of genes expression were compared in different parts of bamboo shoots, ACT or 18S rRNA could be chosen as the corrective reference gene in different parts of bamboo shoots. In order to compare the differences of gene expression among the roots, stems and leaves of P. edulis, the expression stability of 18S rRNA was the best on GeNorm and BestKeeper and was second on NormFinder. Therefore, 18S rRNA could be chosen as corrective reference gene in different tissues of P. edulis. These findings were inconsistent with those of previous studies, which ACT was used as the only one reference gene. This study suggested that suitable reference genes should be selected on the basis of specific requirements, experiment conditions, and the characteristics of experimental material in practical applications. The results of this study would provide a theoretical basis for selecting the appropriate reference gene of P. edulis in the analysis of qRT-PCR.

Flow cytometry has been widely used in chromosome ploidy identification and genome size estimation in plants. This research is to establish a flow cytometry method suitable for jujube (Ziziphus jujuba) in order to provide technological supports for jujube ploidy breeding and genome analysis. Chinese jujube 'Linyilizao' (diploid) and its autotetraploid 'Chenguang' were employed as materials to systematically compare the ploidy detection effects of cell lysis solution types, maturity level of leaves, preserved way of leaves, and dosage of propidium iodide (PI). The results showed that cell lysis solution of Tris-MgCl2, woody plant buffer (WPB) and general purpose buffer (GPB) had clear peak when the 'Linyilizao' was as the test material. The GPB and Tris-MgCl2 had clear peak, while WPB not showed peak when the 'Chenguang' was as the test material. In general, cell lysis solution of Tris-MgCl2 showed better results than cell lysis solution of WPB and GPB. So the cell lysis solution of Tris-MgCl2 was the best for cell lysis and WPB was the worst. Young leaves showed better result than mature leaves, and senescent leaves could not make clear peak. With the increase of preserved period of leaves, the testing effects slightly decreased under 4 ℃ and -20 ℃. There was no significant difference among the test effects of leaves preserved 1~3 d at 4 ℃ or 3~6 months at -20 ℃. And the leaves preserved 7 d at 4 ℃ or 12 months at -20 ℃ could still be used for ploidy identification. In consideration of cost and detection effect of different dosages of PI, 30 μL PI was optimal. By using the above established method for ploidy identification, the ratio of fluorescence intensity of triploid to diploid in Chinese jujube and sour jujube was in the range of 1.43~1.60. In addition, the ratio of fluorescence intensity of tetraploid to diploid was in the range of 1.70~2.11, while the ratio of hexaploid to diploid was 2.90~3.27. The genome size for Chinese jujube 'Dongzao' was estimated as 449.94±3.60 Mb by this method, which just has 1.33% deviation compared with the result of actual genome sequencing. Meanwhile, the genome size of sour jujube 'Xianxiansuanzao' (Z. acidojujuba), Indian jujube 'Tainan1' (Z. mauritiana) and wild diploid Indian jujube were 404.25±2.33, 1 349.73±8.22 and 462.97±8.72 Mb, respectively. In this study, a method of flow cytometry suitable for Ziziphus plants ploidy identification and genome size estimation was established. The new method is simple, accurate, labor-saving and very fast, and a detection of over 200 samples could be completed by 1 people within 1 day. It could also be used for other plants including pear (Pyrus bretschneideri), grape (Vitis vinifera), poplar (Populus trichocarpa) and cabbage (Brassica rapa). The results of this study can provide technical support for Zizyphus plant on ploidy breeding and omics study, and also a reference for other plants.

Kale (Brassica oleracea var. acephala) belongs to the Brassica genus of Cruciferea. Ornamental kale has colorful leaves under cold temperature, and is a good all-purpose plant material from late autumn to early spring due to its beautiful and long lasting foliage and edible traits. As the strong heterosis, the commodity is basically an F1 hybrid. Based on the years of work with the double haploid breeding of ornamental kale, this study summarized our achievements in double haploid (DH) plant production via microspore culture and its application in breeding. The materials used included the round leaf types (cabbage type), the curly leaf type, and the feathered leaf type. Microspore donor plants were 103 different genotypes, which came from commercial varieties, F2~F3 selfed progenies, and new hybrid combinations. Microspore embryoids and derived plants were obtained from 77.7% and 55.3% of the 103 genotypes, respectively. The average number of embryoids per petri dish (3×105 microspores) varied from 0 to 314, and much higher number of embryoids were formed from donor plant hybridized between DH lines. Non synchronous developments of the embryoids were observed in general, and the plant formation frequencies varied from 0 to 51.3%. F1 hybrids owned significant higher embryogenesis capacity than progenies of themselves, but plants possessing of relatively higher embryoid formation capacity in F2 ~F3 population also existed. There were haploidy, diploidy, triploidy, tetraploidy and mixed-ploidy in the regenerated plants, and the frequency of doubled haploid (DH) in average was about 42.7%. The phenotype diversities were rich in DHs derived from F1 hybrids, while the diversities were relatively low in DHs derived from the F3 donor plants. Multi-traits, such as leaf color, blade profile, plant type, etc., were rapidly fixed and multilevel displayed in the DHs, also the recessive gene controlling traits were contemporary expressed. Compared with the conventional breeding, more than half of the time could be saved using DH breeding system for a new F1 hybrid release in ornamental kale. This research provides a relative complete reference for double haploid breeding not only for kale but also for other crops.

Influenza A viruses (IAVs) can infect poultry and many mammals including human (Homo sapiens), causing significant economic losses, and seriously threatening human health. Influenza vaccination is the most effective measure to prevent the occurrence of influenza. However, the effects of different immunization strategies on the immune responses following vaccination remains obscure. In this study, two antigenically distinct H9N2 subtypes of avian influenza viruses, A/chicken/Hubei/YK524/2015 and A/chicken/Shandong/LX830/2014, referred to as YK524 and LX830 were selected, respectively. The effects of homologous immunization and heterologous immunization and virus inactivation on antibody production were investigated. The viruses were purified by sucrose gradient centrifugation. Female BALB/C mice (Mus musculus) of 6~7 week old were immunized with purified Influenza A virus. Haemagglutinin inhibition titer in sera was measured regularly. The results showed that the antibody level produced by live virus antigen was higher than that induced by inactivated virus antigen in mice, with better cross reactivity. It suggested that live viruses were more likely to stimulate the body to produce antibodies with broad cross reactivity. In addition, heterologous immunity stimulated the body to produce more cross-reactive antibodies. Mice with higher antibody titer were selected to prepare monoclonal antibodies against influenza virus using hybridoma technique. Monoclonal antibodies were prepared by fusion of splenocytes and mouse myeloma Sp2/0 cells. Indirect immunofluorescence assay was performed to screen the antibody secreting clones. Following 3 round of sub-cloning, 3 hybridoma cell lines capable of secreting antibodies against influenza virus, namely H9-1, H9-2 and H9-3, respectively, were obtained. All three antibodies showed hemagglutination inhibition and neutralization activity against LX830 and another H9N2 influenza virus Mink/SD/14, although the titer of H9-1 and H9-2 were much higher than H9-3. This results suggested that the antigen recognition epitopes of these three antibodies were located at the haemagglutinin(HA) receptor binding site. All three antibodies could not bind specifically to YK524, none of them displayed hemagglutination inhibitory and neutralization activity towards YK524. We presume that this might be due to the key amino acids difference in the hemagglutinin between LX830 and YK524. This study discussed the way to stimulate the body to produce wider spectrum of antibody response, and would provide experimental basis for the development of influenza vaccine and for the optimal immune strategy design. The monoclonal antibody prepared in this study could be used for further diagnosis of influenza virus.