As a famous local pig breed in China, Taihu pig (Sus scrofa) has many characteristics such as high fecundity, resistance to rough feeding, good disease resistance and good flesh quality. It has been an important genetic resource for molecular breeding and genome selection. Based on the bioinformatics techniques such as pig quantitative trait locus (QTL) database, gene ontology (GO) database and ingenuity pathway analysis (IPA) database, this study mined the genes related to growth traits in pigs. In order to verify the accuracy of the candidate genes obtained, this study uploaded the candidate genes to the IPA database for online analysis. The results indicated that the candidate genes obtained were mainly enriched in 10 gene networks, and they were mainly enriched in gene expression, body development, cell growth, embryonic development, bone and muscle system development and other functions. Many candidate genes related to pig growth traits have been widely studied and reported. All of the above indicated that the candidate genes of pig growth trait obtained had a certain degree of confidence. This study based on the sequencing data in genotyping by genome reducing and sequencing (GGRS) platform, detected the SNPs of 6 Taihu pig breeds and screened out specific SNPs which had interspecific differences with western breeds. Then through the analyses of genetic correlation, SNP specificity, SNP location and other information, this study predicted the sites where can genetically improve the local breeds of Taihu by gene editing. In order to screen out the best single guide RNA (sgRNA) near the gene editing site and complete the gene editing, this study prepared a script based on Perl language to analyze the whole pig genome, and found out the protospacer adjacent motif (PAM) and sgRNA target sites in pig genome. In the pig genome, a total of 127 353 502 PAM sites were detected, with an average of one PAM site per 20 bp, and these PAM sites were evenly distributed within the chromosome. A total of five PAM types were observed in this study, including TGG, AGG, GGG, CGG and NGG. The frequency of NGG was very low, only 0.0082%, probably due to low quality sequences. The other four PAM sites accounted for 34.52%, 32.61%, 26.43% and 6.43%, respectively. At the same time, this study screened out the best sgRNA for CRISPR/Cas9 gene editing by the off-target analysis of sgRNA in pig genome. 428 candidate genes for growth traits in pigs were got, and the number of specific SNPs related to growth of the pig breeds Erhualian, Fengjing, Jiaxing, Mizhu, Mmeishan, Shawutou, Smeishan were 412、282、372、414、393、279、458, respectively. Next, based on the off-target analysis, four optimal sgRNAs were provided for each growth-related SNP site. This study provides the data foundation for subsequent efficient and accurate gene editing.

Long non-coding RNAs (lncRNAs) regulate the transcription of peroxisome proliferator-activated receptor gamma gene (PPARγ) in mammals, thereby affecting the proliferation and differentiation of adipocytes. In order to explore if lncRNA plays regulatory role on PPARγ in chickens (Gallus gallus), this study analyzed public chicken RNA-seq data by bioinformatics methods, and found that in intron 1 of chicken PPARγ, a sense transcription produces a 1 483 bp lncRNA (named PLNC). Subsequent analysis using qRT-PCR revealed that the expression levels of both PLNC and PPARγ transcript 1 (cPPARγ1) were relatively high in chicken adipose tissue at 1, 4 and 7 weeks of age. The preadipocytes and mature adipocytes in 14-day-old abdominal adipose tissue of Arbor Acres (AA) chickens were directly isolated. The expression levels of PLNC and PPARγ and chicken PPARγ transcript 1 (cPPARγ1) in preadipocytes were significantly higher than that in mature adipocytes. During the differentiation of chicken preadipocytes, expression trend of PLNC, total PPARγ and cPPARγ1 were similar to each other. Transfection experiment simultaneously with PLNC interference RNA and cPPARγ1 promoter reporter gene showed that inhibiting PLNC could significantly improve the activity of renilla luciferase. After treatment with different concentrations of rosiglitazone on preadipocytes for 24 h, with the concentration rising up, the expression levels of PLNC and cPPARγ1 elevated first. When cPPARγ1 expression reached the plateau, PLNC expression decreased. Taken together, this study found that PLNC was involved in differentiation of chicken adipocytes, potentially through competitively regulating the promoter activity and transcription level of cPPARγ1. This study provides new clues of the molecular regulation mechanism of chicken adipose tissue growth and development, and also insights on the treatment of excessive fat deposition, and therapy for obesity and metabolic related diseases.

Seed and pod traits are closely related to seed yield of soybean (Glycine max). The present study used the interval complete interval mapping method (ICIM) to analyze the recombinant inbred line (RIL) population phenotypic data across two-year, and obtained 7 quantitative trait loci (QTL) related to the seed and pod traits. 154 original QTLs related to seed and pod trait of soybean from database and references were analyzed. 23 Meta-QTL intervals were obtained by Meta-analysis. Moreover, the Overview method was used to optimize these QTLs based on statistical analysis. After the Overview analysis, 13 valid QTL regions were obtained, and 5 QTL regions were narrowed down to 0.5 Mb. Furthermore, genetic annotation for the Meta-QTL interval and 687 genes were obtained, and 11 genes were selected as candidate genes. 11 candidate genes participated in the process of plant growth and fruit maturation. The experimental method of this study and results are important for QTL mapping associated with seed and pod traits and assisted molecular breeding of soybeans.

Diacylglycerol acyltransferase (DGAT) widely existing in organisms plays an important role in the synthesis of triacylglycerol. In peanuts (Arachis hypogaea), two copies of AhDGAT3 gene (AhDGAT3A and AhDGAT3B) exist in peanut genome, and have been cloned in our previous research. In order to study the functions of the acyltransferase during lipid accumulation, and explore the breeding strategy of increasing peanut oil content and optimizing the fatty acids composition, two types of plant overexpression vectors driven separately by Cauliflower mosaic virus (CaMV) 35S constitutive promoter and the seed-specific promoter from soybean agglutinin Lectin gene were constructed. Through peanut genetic transformation and successive screening for the transgenic plants of T0~T4 generation by sequencing the PCR products of target gene, the homozygous transgenic lines carrying the 4 constructs stably in genetics were developed. On the major agronomic characters, such as flowering times, the quantity of branches and disease resistance, there were no significant differences between the transgenic lines and the control. The detail oil contents and fatty acid compositions of the transgenic seeds were determined by near infrared spectrum (NIR) and gas chromatography (GC). It was found that compared with FH1, the contents of the protein and the oil in transgenic seeds were similar; however, the contents of C18 fatty acids change significantly in comparison with the other fatty acids, stearic acid increased by 11.84%~22.95%, oleic acid increased by 5.13%~21.26%, while linoleic acid decreased by 3.87%~31.18%, and correspondingly, the ratio of oleic acid and linoleic acid (O/L) increased by 5.01%~76.19% compared to the control. The results suggested that AhDGAT3 maybe prefer to oleoyl as the substrate and cause more oleic acid accumulating in transgenic peanut seeds. In this work, it was also found that the improvement of O/L is higher in transgenic peanuts with AhDGAT3B than that in AhDGAT3A overexpression lines, inferring that AhDGAT3B had stronger biological functions. In addition, the changes of O/L in transgenic peanut seeds with different promoter were varied greatly, because the CaMV 35S promoter has higher expression level than Lectin promoter. This research may provide the theoretical foundation for the improvement of peanut oil quality.

Dabry's sturgeon (Acipenser dabryanus) is a national-level protected animal with important ecological value and of germplasm resources value. Molecular biology techniques such as real-time quantitative real-time PCR (qRT-PCR) play an important role in the protection and germplasm development process of Dabry's sturgeon. The selection of internal reference genes in qRT-PCR technology is related to the accuracy of the test results. In order to screen the potential reference gene of Dabry's sturgeon, Three potential internal reference genes, namely β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor 1α (EF1-α), were obtained by homologous cloning combined with local Blast method. Mega 5.05, DNAstar 7.1, CPHmodels 3.2, and ClustalW were used to analyze the biological information of this three genes. The qRT-PCR was used to detect the three gene Cq value in the brain, liver, kidney, pancreas, spleen, esophagus, stomach, pyloric caeca, duodenum,intestinum valula, rectum, muscle, gill and skin of Dabry's sturgeon. The expression stability of the three genes in 14 tissues was evaluated using geNorm, NormFinder and Bestkeeper. The results showed that the full-length CDS of potential qRT-PCR reference genes β-actin, GAPDH and EF1-α were 1125, 999 and 1383 bp, encoding 375, 333 and 461 amino acids, respectively. Bioinformatics analysis showed that the amino acid sequence and protein three-dimensional structure deduced by the three genes were highly consistent with other species. The amplification efficiencies of β-actin, GAPDH and EF1-α qRT-PCR primer, was 92.2%, 93.0%, and 92.7%, respectively; the melting temperature (Tm) of the amplification products were 84°C, 83.5°C, and 85°C, respectively. Comprehensive geNorm, NormFinder and Bestkeeper evaluation results showed that the stability of the three reference genes was EF1-α>β-actin>GAPDH in 14 tissues and EF1-α and β-actin can be used as reference genes for Dabry's sturgeon qRT-PCR study. In this experiment, we obtained the full-length CDS of three potential reference genes β-actin, GAPDH and EF1-α, and confirmed that EF1-α and β-actin are suitable for the qRT-PCR study of Dabry's sturgeon. This study provides basic data for the application of qRT-PCR technology in Dabry's sturgeon research.

Metallothioneins (MTs) are rich in cysteine (Cys), playing an important role in detoxification, scavenging oxygen free radicals, fruit development and maturation and other processes. The MT gene VvMT2 (GenBank No. EU280163) was screened from cDNA library of Thompson Seedless (Vitis vinifera). And the full-length genomic DNA and promoter sequences were cloned by PCR. The experiment aimed at its coding structure and properties of VvMT2 gene and revealing the expression pattern of the gene in different stages of seed development and under different plant growth regulator treatments. The results indicated that the full-length of VvMT2 gene was 998 bp with 3 exons and 2 introns. The full-length cDNA was 527 bp and encoded 79 amino acids which contained 14 cysteines. VvMT2 gene had a Metallothio_2 structural sequence. The predicted molecular weight of VvMT2 was 7.71 kD and isoelectric point was 4.67, which was an acidic protein. There was a strong hydrophobic region in the middle. It had no signal peptide and was located outside the cell. The obtained VvMT2 promoter region sequence was 783 bp, which contained 12 upstream regulatory elements especially essential elements for transcription and stress response and hormone response elements. In the beginning of seed development, the expression level of VvMT2 gene in Thompsolln Seedless was higher than that in Pinot Noir significantly, and both were opposite in the later stage of seed development. The expression level of VvMT2 gene in Thompson Seedless was higher in 25 and 35 d after flowering. Soon after, it decreased rapidly and was below the full flowering level. The expression level of VvMT2 gene in Pinot Noir responded earlier (20 d after flowering), and significantly increased at the later period. On the other hand, the expression level of VvMT2 genes in grape leaves was rapidly reduced after spraying with plant growth regulators. And it returned to pre-spray level within 24 h. With the treatment of methyl jasmonate, the expression level of the gene in leaves did not increase. However it increased 12 h after ethylene treatment. And it was higher than that of the control significantly 24 h after ethylene treatment. With abscisic acid treatment, the gene expression recovered and increased fastest. The study provide theoretical basis for further function study of VvMT2 gene in hormone regulation and seed development process.

Arabidopsis thaliana Ovate Family Proteins (AtOFPs) is a novel plant-specific transcription factor. As one of important member of the OFPs, AtOFP1 is the earliest and most studied transcriptional repressor. It has been proved that AtOFP1 can directly regulate the transcriptional activity of AtGA20ox, a key gene of GA synthase, and inhibit cell elongation. 3-Amino-acid loop extension homeodomain protein (TALE) plays a central role in plant development. It has a potential interaction with AtOFP1, and existing studies have shown that 9 OFPs and TALE proteins have a close functional association with their homeodomains. The literature showed that the ovate family proteins have potential interactions with TALE transcription factor, and identified 13 transcription factors that interact with AtOFP1 by the way of a screening library which include KNAT5. KNAT5 is expressed in the lateral root primordium and may participate in the occurrence of lateral roots, which has an important influence on plant growth and development. In order to further verify the interaction between AtOFP1 and KNAT5 and the effects of the interactions on Arabidopsis thaliana, we first verified the interaction between AtOFP1 and AtKNAT5 by yeast two-hybrid, then their biocompatibility was further verified by bimolecular fluorescence complementation (BiFC). In addition, the knat5 homozygous mutant was screened, and the gene expression of OFP1 and KNAT5 in Col-0, knat5 mutant, ofp1 mutant and HAOFP1 overexpression plant was analyzed by qRT-PCR to compare the expression pattern. Similar expression patterns in the roots and leaves of the plants were found. The existed synergistic expression in roots and leaves of Arabidopsis thaliana could affect the number of lateral roots and root length, which play an important role in plant growth and development. The present study lays the foundation for further exploring the mechanism of AtOFP1-KNAT5 interaction and its regulation function in plant growth and development.

Cremastra appendiculata is a perennial rare medicinal plant. Under natural conditions, it's hard to reproduce sexually, and the vegetative reproduction cannot effectively branch development to increase the number of pseudobulb due to apical inhibition of newborn pseudobulb. These factors lead to a very low breeding coefficient. Therefore, to explore internal mechanism of pseudobulbs growth in cluster, the pseudobulbs of C. appendiculata were treated by decapitation, auxin transport inhibitors N-1-naphthylphthalamic acid (NPA) and 2, 3, 5-triiodibenzoic acid (TIBA). In this way, endogenous plant hormone levels might be changed to promote the growth of lateral buds of C. appendiculata. After decapitation and application of auxin transport inhibitors (NPA and TIBA) treated the pseudobulb of C. appendiculata for 6 days, the results showed that auxin levels in the lateral buds were significantly reduced (P＜0.05), and cytokinin levels were obviously increased (P＜0.05). The results of qRT-PCR showed that cytokinin levels increased because of rapidly up-regulated key genes of cytokinin synthetase (isopentenyl transferase, IPT) after treatments. After the decapitation, NPA and TIBA treating for 40 d, the germination rate of the biennial pseudobulbs in pseudobulbs string were increased from 0% to 95%, 85% and 90%, respectively, and the triennial pseudobulbs were increased from 0% to 15%, 76% and 75%, respectively. For the treatments of decapitation, NPA and TIBA, the number of seedlings per plant could reach to 2.20, 3.13 and 3.05 times of the control after 90 d, respectively. The application of zeatin promoted the germination of lateral buds of C. appendiculata in a certain extent (biennial: 51%; triennial: 35%), but did not increase the rate of seedling emergence, which indicated that cytokinin promoted the germination of lateral buds, but could not promote to continued growth. The change of auxin levels was necessary for the continued growth of lateral buds. The results of this study indicated that auxin might regulate transcriptional level of IPT gene to control cytokinin level, and indirectly regulated lateral buds development and growth of C. appendiculata. This research increases the propagation coefficient of C. appendiculata, and provides a reference for the apical dominance theory of underground stems.

Heme oxygenase 2 (HMOX2) is a constitutive subfamily member of heme oxygenase family, and HMOX2 plays an important role in the metabolic regulation of hypoxia stress. This study was to investigate the role of HMOX2 in the adaptation mechanism to high altitude hypoxia, the HMOX2 gene CDS of Tibetan sheep (Ovis aries) was cloned, reverse transcription PCR (RT-PCR), bioinformatics analysis and the comparative analysis of the blood-gas between Tibetan sheep and Hu sheep (control) was carried out. Quantitative real-time PCR (qRT-PCR) was used to analyze the expression differences of HMOX2 gene in different tissues of Tibetan sheep and Hu sheep. The results showed that the CDS region of Tibetan sheep HMOX2 gene (GenBank No. MH358397) was 1 005 bp in length and encoded 344 amino acids, forming a transmembrane hydrophilic non-secreted protein. The results showed that there were 3 mutations in HMOX2 gene, which were G485C, T486G and A500T, respectively, but there were two mutations in G485C and T486G compared with Hu sheep, which did cause 162th cysteine acid into serine, and A500T mutation caused 167th glutamine acid into leucine. The deduced amino acid sequence of Tibetan sheep HMOX2 shared 99%, 99%,99% ,97%, 97%, 84% and 80% identities with those of Ovis aries, Capra hircu, Pantholops, Bos indicus, Bos taurus, Sus scrofa, Homo sapiens. The results of blood-gas analysis showed that the P(O2), P(CO2), HCO3- and SpO2 of Tibetan sheep were significantly lower than Hu sheep (P＜0.01); HGB and HCT of Tibetan sheep were significantly higher than those of Hu sheep (P＜0.01); qRT-PCR results showed that HMOX2 mRNA were expressed in the different organizations, the expression of gene was detected in Tibetan sheep and Hu sheep. The expressions in the lung and heart were significant different (P＜0.01), and the expression in liver, skeletal muscle and fat of Tibetan sheep were significantly higher than the Hu sheep (P＜0.05). The correlation between HMOX2 gene expression and hemoglobin content in Tibetan sheep and Hu sheep showed that there was a significant positive correlation between HMOX2 gene expression and hemoglobin with a correlation coefficient of 0.535 (P＜0.05). There was a significant negative correlation between gene expression and hemoglobin, with a correlation coefficient of -0.097 (P＞0.05). Thus, hypoxia-specific expression of HMOX2 gene can regulate the metabolism of hemoglobin downstream of the hypoxia metabolism pathway, thereby weakening the damage of cells caused by hypoxia stress. This study results provide basic data for the molecular mechanism of Tibetan sheep's adaptation to hypoxia.

The Toll like receptor family (TLRs) is an important type of pattern recognition receptor (PRRs), which has been widely studied in recent years. TLRs activates cascade reaction in the process of signaling pathway by identifying the invasion of pathogenic microorganisms, inducing tissue or organism to produce cytokines and inflammatory factors, which plays an extremely important role in the host against pathogen infection. The purpose of this study was to explore the correlation between the overexpression of goat (Capra hircus) TLR4 gene in mouse macrophages and the expression of TLR2 gene. In this experiment, the whole length of CDS of goat TLR4 gene was cloned by reverse transcription PCR (RT-PCR) amplification combined with T cloning method. The pEGFP-N1-TLR4 eukaryotic expression vector was transiently transfected to mouse macrophages (Ana-1) with lipofectamine3000TM after identification the eukaryotic expression vector by connection, transformation and double enzyme digestion. The total RNA was extracted after transfection, and TLR4 and TLR2 gene mRNA in transfected cell Ana-1 were detected by qRT-PCR and the differential expression was analyzed by the software of SPSS 18.0. Results showed that the TLR4 gene CDS region was 2 537 bp, 3 mutation sites (T426C, C1254T, G1515A) in the CDS region of TLR4 gene were found, and 3 mutations did not cause changes in amino acids, which belonged to synonymous mutations. The eukaryotic expression vector of pEGFP-N1-TLR4 were successfully transfected and expressed in the mouse macrophages, the qRT-PCR test showed that the expression of TLR4 and TLR2 was positive group＞negative group＞control group (blank), in control group, there was no significant difference in mRNA expression between TLR4 and TLR2 gene (P＞0.05), while mRNA expression in TLR4 positive group was significantly higher than that in TLR2 positive group (0.01＜P＜0.05). The expression level of TLR2 gene mRNA in the positive group was also significantly higher than that in the blank group (0.01＜P＜0.05). The results showed that the overexpression of TLR4 could promote the TLR2 gene. This study provide a basis for further study of the interaction between TLR4 and TLR2 genes and their intracellular expression mechanism.

The apolipoprotein family plays an important role in the transport and metabolism of lipids and cholesterol in the body. The apolipoprotein B (ApoB) plays an important role in lipid metabolism. Previously it was found that the genotypes of chicken (Gallus gallus) ApoB g.-112A>G (single nucleotide polymorphism, SNP) were significantly associated with abdominal fat weight and abdominal fat percentage in chickens. To determine whether the ApoB gene g.-112A>G is a functional SNP, we conducted the following research work: Association analysis between different genotypes and plasma biochemical markers, body weight and abdominal fat traits; The mRNA expression levels of the 3 ApoB genotypes in the liver and small intestine of chickens were detected and compared; The luciferase reporter gene vector containing different alleles in the promoter region of chicken ApoB gene was constructed and transfected into chicken fibroblast cell (DF1) and human hepatoma (HepG2) cells respectively; The possible mechanism of action of the ApoB gene g.-112A>G was predicted and analyzed by on-line bioinformatics software. It was found that the A allele could change the TBP and C/EBPα transcription factor binding sites in the chicken ApoB gene promoter region. It could be assumed that the g.-112A>G could influence the promoter activity of ApoB gene, which in turn affected gene expression. To confirm this hypothesis, the effect of pCMV-TBP and pCMV-C/EBPα on the activity of the dual-luciferase reporter gene of different alleles was analyzed in DF1 and HepG2 cells. The major results as follows: (1) Association analysis showed that genotypes of g.-112A>G in ApoB gene were significantly associated with high-density lipoprotein (HDL) (P＜0.01), and significantly associated with HDL/low-density lipoprotein (LDL) and Body Weight (BW) (P＜0.05), and closely associated with Triglyceride (TG)(P=0.0549), Total Cholesterol (TC)(P=0.0926) and Abdominal Fat Weight (AFW) (P=0.0805). Multiple comparisons showed that HDL, HDL/LDL, and BW were significantly higher in individuals with GG genotype than those with AG and AA genotypes (P＜0.05). (2) qRT-PCR results showed that the ApoB gene expression level of AG genotype was significantly lower than that of AA genotype and GG genotype in both chicken liver and small intestine (P＜0.05). (3) Bioinformatics predictions revealed that the A allele could change the TATA-box binding protein (TBP) and CCAAT/enhancer binding protein alpha (C/EBPα) transcription factor binding sites in chicken ApoB gene promoter region. (4) The study successfully constructed reporter gene vectors containing different alleles of the chicken ApoB gene g.-112A>G, and reporter gene activity analysis indicated that there were significant differences in the reporter gene activity of the A and G alleles (P＜0.05), and the reporter gene activity of the A allele was significantly higher than the G allele. (5) TBP eukaryotic expression vector pCMV-TBP was successfully constructed. pCMV-TBP and pCMV-C/EBPα (kept in the lab) were co-transfected with reporter vectors of different alleles. The results showed that transcription factors TBP and C/EBPα promoted luciferase activity of the A allele more strongly than that of the G allele (P＜0.05). The above results suggested that ApoB gene g.-112A>G might be a functional SNP locus, and it could be preliminarily identified as a functional molecular marker for quality chicken breeding. The present study provides experimental data for further exploration of the function of ApoB gene in fat development.

Putian black duck (Anas anas domesticus) is the sole native variety in black-feather laying duck, which is rare waterfowl resource in China. The microphthalmia-associated transcription factor (MITF), the most important transcription factor of melanocytes, could be induced by α-MSH (melanocyte-stimulating hormone) and bind to the cAMP responsive element binding protein (CREB) to activate MITF gene expression in melanocytes which plays a key role in the synthesis of melanin. To investigate the relationship between the expression of the MITF gene and regularity of eumelanin deposition in different tissues of Putian black duck, the MITF gene was firstly cloned and analyzed from the skin of Putian black duck with nested PCR and sequenced (GenBank No. MG516571). MITF gene expression was detected in various tissues of Putian black ducks via real-time quantitative PCR, and deposition of eumelanin in the tissues was measured by UV spectrophotometry. The results showed that the length of MITF gene from Putian black duck was 1 341 bp, which contained the complete CDS sequence (12~1 334 bp) and encoded 440 amino acids. The amino acid homology compared with Anas platyrhynchos was 100%, and it was above 95% compared with chicken (Gallus gallus) and quail (Coturnix japonica). The genetic relationship of MITF between Putian black duck and Anas platyrhynchos was the closest, followed with Anser cygnoides, Silky flow, Gallus gallus, and relationship compared with pig (Sus scrofa), sheep (Ovis aries), and other mammals was the farthest. The mRNA expression of MITF gene in Putian black duck skin tissue was the highest in the tissues (skin＞kidney＞gizzard＞liver＞muscle) (P＜0.01). The deposition of eumelanin was basically consistent with the expression of MITF gene in each tissue of Putian black duck. The expression of MITF gene had significant positive correlation with the production of eumelanin in the liver tissues of Putian black duck (P＜0.05). In summary, the expression of MITF gene had tissue specificity in Putian black duck, which might have positive regulation on the deposition of eumelanin and could be used as a candidate gene for the regulation of melanin deposition. The study could provide basic material for the selection of melanin trait in Putian black duck.

The uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier family, which play important roles in several physiological processes, including thermogenesis, reactive oxygen species generation, lipid metabolism and insulin secretion. Although the roles of UCP3 gene are well understood in mammals, little is known about it in pigeon (Columba livia). In this study, ten white king pigeons were selected as the research object. The full-length cDNA sequence of UCP3 gene was cloned from spleen by rapid amplification of cDNA ends (RACE) and analyzed using bioinformatics tools. The distribution and expression level of pigeon UCP3 in different tissues were determined by qRT-PCR. The results showed that the full-length of pigeon UCP3 (GenBank No. KU166860.1) cDNA was of 1 413 bp including a 924 bp open reading frame (ORF), 75 bp 5'UTR and 414 bp 3'UTR. The ORF encoded polypeptides of 307 amino acids. Structure prediction showed that there were three mitochondrial carrier motifs in the pigeon UCP3 protein. The deduced amino acid sequence of pigeon UCP3 shared identity of 90%~93% with other birds, 72%~75% with mammals, 70% with Danio rerio, and 69% with Xenopus laevis. The phylogenetic relationship analysis indicated that birds, mammals, fish and amphibians each formed a branch. And in the bird branch, Columba livia was closely related with Anas platyrhynchos. These results were similar to the biological classification of different species. The results of qRT-PCR showed that pigeon UCP3 were expressed in all 6 tissues examined. The UCP3 mRNA expression levels in lung were highest, which were significantly higher than that in other tissues (P＜0.05), followed by spleen and kidney, and relative lower expression was detected in muscle and heart, and the lowest expression was found in liver. The results of this work may provide a theoretical basis for the further study of its function in poultry.

Nucleotide binding and oligomerization domain 1 gene (NOD1) plays important role in fish innate immunity. In order to obtain the SNPs associated with the disease resistance in Nile tilapia (Oreochromis niloticus), genome sequence NOD1 (GenBank No. MF479261) was cloned. Through cloning sequencing or direct sequencing of PCR products, 44 SNPs were obtained from 39 individuals of 20 Nile tilapia parents family. By directly sequencing or SNaPshot method, 83 individuals from susceptible group (SG) and 83 individuals from resistance group (RG) of offspring were used to analyze polymorphisms and genetic parameter. Popgen 32 was used to calculate the genetic parameter and polymorphisms of the NOD1 SNPs in susceptible group and resistance group. The results showed that 24 SNPs were genotyped successfully. The polymorphism information content (PIC) value of the 24 SNPs of NOD1 gene ranged from 0.09 to 0.37, which suggested that all 24 SNPs were low or moderate polymorphism. The correlation between the NOD1 SNPs and the trait of S.agalactiae resistance was analyzed. The results showed that SNP5 (G-2284C) was significantly associated with S. agalactiae susceptible trait (P＜0.05), SNP13 (G-8956C) were significantly associated with S. agalactiae resistant trait (P＜0.05). SNP12 (G-8955C) was significantly associated with S. agalactiae resistant/susceptible trait (P＜0.05). The linkage disequilibrium analysis and prediction of haplotypes showed that all the 24 NOD1 SNPs formed 5 haplotype blocks and 16 haplotypes. The haplotypes H4-2 (AC) was significantly associated with S. agalactiae resistant trait. These results indicate that the SNP site SNP5, SNP12, SNP13 and the haplotypes H4-2 of NOD1 gene can be candidate molecular markers for molecular marker assisted selection. This study provides data base for breeding resistant strains of Nile tilapia.

The change of Bacillus thuringiensis (Bt) receptor protein expression is one of the key factors affecting the resistance of target insects to Bt, but the mechanism of its expression regulation is not clear. MicroRNAs (miRNAs), as a class of non-coding RNAs, could be paired with target mRNA to regulate gene expression at post-transcriptional level, and thus participate in the regulation of insect growth, development and stress response. To investigate the role of microRNA-31 (miR-31) in the production of resistance of Asian corn borer (ACB, Ostrinia furnacalis) to pesticidal crystal protein (Cry1Ab), the expression of miR-31 in different tissues of Cry1Ab resistant strain of ACB (ACB-AbR) and Bt sensitive strain (ACB-BtS) was analyzed. It was found that the expression of miR-31 in the epidermis of ACB-AbR was significantly higher than that in the epidermis of ACB-BtS, but decreased significantly in the midgut of ACB-BtS and ACB-AbR, and the expression of miR-31 in the midgut of ACB-AbR was lower than that in the midgut of ACB-AbR. Then, the sensitivity of Sf9 cells to Cry1Ab was analyzed after the expression of miR-31 was changed. It was found that the increased expression of miR-31 resulted in an increase of the sensitivity of Sf9 to Cry1Ab, whereas the decrease in the expression of miR-31 resulted in a decrease of the sensitivity of Sf9 to Cry1Ab. These results suggested that the decrease of miR-31 expression in midgut of ACB-AbR may be one of the reasons leading to its resistance to Cry1Ab. This study provides basic data for further analysis of the mechanism of Bt resistance in ACB.

Sugarcane mosaic virus (SCMV), one of the most important maize viruses, causes annually serious yield loss in maize (Zea mays) industry. Establishment of sensitive and specific virus detection technique is critical for preventing and controlling SCMV. For this purpose, 8 hybridoma lines (12A10, 10B11, 6A1, 19F3, 15C12, 22A2, 21H3, and 21C12) secreting monoclonal antibodies (MAbs) against SCMV were obtained using the purified virions of SCMV Yunnan isolate (SCMV-YN) as the immunogen and the hybridoma technology. With the obtained hybridomas, the ascitic fluids containing MAbs were produced. Using an indirect-enzyme-linked immunosorbent assay (indirect-ELISA), the titers of 8 ascitic fluids containing MAbs were detected to be 10-6 or 10-7, and all 8 MAbs belonged to IgG1, κ light chain. Western blot assay demonstrated that 3 MAbs (22A2, 21H3 and 6A1) recognized common epitopes on capsid protein (CP) of SCMV Beijing isolates (SCMV-BJ) and SCMV-YN. MAbs 12A10, 15C12, 10B11, 21C12 and 19F3 could only recognize the specific epitopes on SCMV-YN. The dot enzyme-linked immunosorbent assay (dot-ELISA) was established by these prepared MAbs as the first antibody. Results of the specificity analyses of the developed dot-ELISAs indicated that 3 MAbs (22A2, 21H3 and 6A1) and their dot-ELISAs had a positive response when detected SCMV-BJ and SCMV-YN isolates, while other prepared 5 MAbs (12A10, 19F3, 10B11, 21C12 and 15C12) and their dot-ELISAs only had a positive response with SCMV-YN isolate, manifesting that those 5 MAbs could be used to detect and identify SCMV-YN isolate. Sensitivity analyses indicated that the dot-ELISAs based respectively on MAbs 15C12, 10B11, 21H3 or 6A1 were the most sensitive, and their sensitivities were up to 1∶10 240 (W/V, g/mL) dilution for SCMV-infected maize leaf crude extract. Detection sensitivity of the MAbs 19F3 or 12A10 was 1∶5 120 dilution, and 22A2 or 21C12 was 1∶2 560 dilution. The anti-SCMV MAbs and the developed dot-ELISA serological detection assay in present study will provide technology support for detecting and diagnosing SCMV, identifying viral strains, breeding for disease resistance and establishing scientific prevention and control for this viral disease.

Myoblast is capable of self-renewal and polymorphism, and plays an important role in the regeneration and repair of skeletal muscle. In order to establish a method for isolation and purification, culture and identification of dairy goat (Capra hircus) myoblasts in vitro, and to understand the identification of myoblast features of proliferation and myogenesis, and to provide experimental materials for the regulation mechanism of muscle development in dairy goats, the samples were taken from skeletal muscle of Xinong Saanen dairy goat kids. Cells were disassociated with collagenase I and trypsin following with purification of cell using differential adhesion approach. qRT-PCR and immunofluorescence staining were conducted to further identify purified cells. Cell morphological results showed that purified cells were in spindle shape and maintained a stable status of growth. Myogenic regulatory factors, myogenic differentiation factor (MyoD) and myogenic regulatory factor 5 (Myf5), and desmin were positively expressed in purified cells (percentage of purified cell ＞ 95%) as observed from the results of qRT-PCR and immunofluorescence staining. Purified myoblasts were able to fuse into myotubes after induced differentiation. Myosin heavy chain (MYHC), a specific marker of differentiated myotubes, was positively expressed. This study establish a method of isolation and culture of dairy goat myoblasts in vitro. Myoblast line with high purity is successfully obtained, which is able to be used as an experimental material in studies of regulation mechanism of muscle development in dairy goats.

Platelet-derived growth factor-D (PDGF-D) is a new member of PDGF family, it can regulate many biological processes, including angiogenesis, tissue fibrosis, tumorigenesis and lipid metabolism. It was found that PDGF-D is closely related to the sheep (Ovis aries) tail fat deposition and tail type, this gene could be used as candidate gene for tail type selection. PDGF-D can specifically bind to and activate its cognate receptor PDGFRβ (platelet-derived growth factor receptor β), then regulate many cellular processes, including cell proliferation, differentiation and apoptosis. This review introduced the discovery, structure, expression, physiological function and signal transduction pathway of PDGF-D gene, proposed the research emphasis of PDGF-D gene in the future. This article provides a valuable reference for PDGF-D gene research.

Insects are the most animals on both number and diverse. They are the bearers of plant pollination, important biological resources (such as medicinal purposes). On the other hand, insects are the propagators of diseases, the enemy of agriculture, forestry, and crop production. The proliferation of insect populations not only depends on developed neurosensory systems and a series of response mechanisms, but also on strong reproductive capacities. Insects have a complex reproductive structure and a variety of reproductive methods. As a neurotransmitter, neuromodulator, and neurohormone, biogenic amine is an important factor correlated with many physiological events such as reproductive development and reproductive behavior in insects. This article reviews the effects of biogenic amines on insect reproduction from the aspects of reproductive status, germ cell and organ development, mating behavior and oviposition behavior. It also describes the current status and bottlenecks of study for the impact of biogenic amines on insect reproduction, and looks forward to development in the future.