Pigs (Sus scrofa) have a low utilization efficiency for dietary phosphorus that is stored as phytic acid, and they excrete large amount of undigested phosphorus in the faeces, which cause serious pollution to the environment. Transgenic expression of phytase in salivary glands is a new way to improve the digestion of phytic acid and reduce the output of fecal phosphorus pollutant for pigs. This study aimed to produce and analyze transgenic pigs expressing phytase specifically in their salivary glands. A plasmid which included a expression cassette of bacteria-derived phytase gene appA (acid phosphoanhydride phosphohydrolase) was constructed. The appA gene in the plasmid was controlled by the promoter of pig parotid secretory protein (PSP) gene, which was a salivary glands specific promoter. Transgenic pig fibroblasts were produced after transfection with the plasmid treated with G418 selection. Using transgenic fibroblasts as nuclear donor cells, 14 founder (F0) pigs were generated by the somatic cell nuclear transfer technique and 8 of them were identified as transgenic pigs by PCR. The PCR showed that foreign appA particularly expressed in the salivary glands but not in the liver, kidney, heart and duodenum of F0 transgenic pigs. The digestion trial indicated that transgenic pigs had higher apparent digestibility of dry matter (P=0.108), energy (P=0.117), Ca (P=0.059) and P (P=0.092) than that of wild-type pigs, although the difference between transgenic and wild-type pigs did not had statistical significance. The fecal phosphorus output was significantly (P＜0.05) decreased by 16% in transgenic pigs, as compared to that in wild-type pigs. This suggested that transgenic expression of appA in the salivary glands improved the utilization of feed phytic acid. The appA transgene was stably transmitted from F0 transgenic pigs to their transgenic offspring as it was also detected in the genome of F1 transgenic pigs. The Southern blot analysis demonstrated that only a single copy of appA gene was inserted into the genome of transgenic pigs. The appA also specifically expressed in salivary glands but not in the brain, heart, liver, spleen, lung, kidney and pancreas tissues of F1 transgenic pigs. Among the 3 salivary glands, the appA gene expressed the highest in submaxillary gland, followed by the parotid gland and sublingual gland in F1 transgenic pigs. The transgene express pattern in F1 transgenic pigs was similar with that in F0 transgenic pigs. In this study, transgenic pigs expressing phytase specifically in the salivary glands were successfully produced, which provides scientific foundation for breeding new pig breed with low phosphorus excretion in the future.

Dermal papilla cells (DPCs) play key roles in the cyclic regulation of hair follicles (HF) by inducing the regeneration of HF and controlling the size of HF, to some extent. This study aimed to optimize the cultivation system of DPCs derived from cashmere goat (Capra hircus) skins. Meantime, miR-206 was transfected into DPC and the mRNA expression of 7 cycling-related genes were investigated, to explore its effects on HF cycling in cashmere goats. Skin samples at anagen period were collected and separated by combination of blunt dissection, neutral protease and collagenase Ⅱ to obtain single hair follicle and release dermal papilla (DP). After that five sets of cell culture medium were formulated, and immunofluorescence staining method was used to detect the expression of α-smooth muscle actin (α-SMA) and CD133 in DPCs; After transfecting miR-206 mediated by Lipofectamine?3000, the mRNA expressions of 7 genes related to hair follicle cycling were measured by qRT-PCR. Results showed that DPCs were successfully cultivated within 3 days for attachment as well as for amplification. Compared with other groups, the medium with β-estradiol (β2), insulin-transferrin-selenium (ITS), epidermal growth factor (EGF) had better attachment and amplification time, as well as cell morphology and agglutination. The results showed that the culture components for secondary hair follicle DPC (SHF-DPCs) at anagen period were dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12)(1∶1), 10% fetal bovine serum (FBS), double resistance (penicillin and streptomycin, 100 IU), β2 (0.5 μg/mL), ITS (3 μg/mL), EGF (10 μg/mL). Compared with the control group, transfecting miR-206 increased the expression of protein tyrosine phosphatase, non-receptor type 1 (PTPN1) and bone morphogenetic protein 2 (BMP2) (P＜0.01), while decreased the expression of BCL-2 assoceated athanogene 4 (BAG4), MAX dimerization protein 4 (MXD4), insulin like growth factor 1 (IGF-1) and fibroblast growth factor receptor 2 (FGFR2) (P＜0.01), and had no effect on BMP4 expression (P＞0.05). BAG4 maybe the candidate target gene of miR-206. In conclusion, The cultivation system of DPCs in cashmere goats was optimized, and miR-206 was stably overexpressed in SHF-DPCs, which affected the expression of several cycling-related genes in hair follicles, and thereby might inhibit the development of hair follicle at the anagen period. Our results will facilitate the exploration of molecular mechanism of miR-206 in the cyclic regulation of HF.

Southern rice black-streaked dwarf virus (SRBSDV), the genus Fijivirus in the family Reoviridae, is transmitted mainly by white backed planthopper (Sogatella furcifera, WBPH) in a persistent- propagative manner. SRBSDV encodes at least 6 putative structural proteins (P1, P2, P3, P4, P8 and P10) and 7 putative nonstructural proteins (P5-1, P5-2, P6, P7-1, P7-2, P9-1 and P9-2). P10 are putative major outer capsid proteins, while P9-1 can form viroplasms which is the putative sites of viral replication. In this study, to enrich the prediction system of SRBSDV, polyclonal antibodies of P10 and P9-1 were prepared. By using a Gateway prokaryotic system, firstly, part fragment of P10 (591 bp) and P9-1 (714 bp) were recombined to the pDONR221 vector, respectively. After verified by sequencing, they were recombined to the expression vector pDEST17, respectively. Then they were transformed into Escherichia coli strain Rosetta. By isopropyl-β-d-thiogalactoside (IPTG) inducing, recombinant proteins of 27 kD (for P10) and 33 kD (for P9-1) were obtained. The recombinant protein was used to immunize New Zealand white rabbits (Oryctolagus cuniculus) for preparation of polyclonal antibodies. By an indirect enzyme-linked immunosorbent assay (in-ELISA) method, the antiserum titer of P10 and P9-1 were determined to be 1∶6 400 and 1∶3 200, respectively. Western blot analysis showed that P10 and P9-1 antibodies could capture proteins of 63 and 39.9 kD in infected rice (Oryza sativa) plants, respectively, which were consistent of the actual length of the P10 and P9-1. There were no any protein captured by the 2 antibodies in healthy rice plants. Our results revealed that both antibodies could detect the infected rice plants specifically. To compare the application effect of the 2 antibodies in the detection of infected rice plants and viruliferous percent of WBPHs, immunocapture-RT-PCR (IC-RT-PCR) and dot-ELISA assays were done. In the IC-RT-PCR assays, using infected rice plants and viruliferous WBPHs as template and specific primers for P9-1 and P10, target fragments of P9-1 and P10 could obtained. In the dot-ELISA assays, a 78.7% viruliferous percent of WBPHs was obtained by using P9-1 or P10 antibodies, which showed a consistent result of the RT-PCR. Meanwhile, both the antibodies could capture the virus protein in infected rice plants by using the dot-ELISA. And there were no any blot in healthy rice plants. Our results revealed that both structural protein and nonstructural protein of SRBSDV could be used for the viral field detection, which provide support for forecast of the virus disease.

Sec14p, found in yeast (Saccharomyces cerevisiae) and containing a typical conserved domain SEC14, is as research object possessing the same function with phosphatidylinositol transfer protein. Sec14p phosphatidylinositol transfer protein exists widely in eukaryotic cells and may be involved in a variety of biological activities, such as membrane trafficking, plasma membrane development, fatty acid metabolism, yeast microspore formation and the growth of plant root hair. However, it's relationship with plant environmental stresses have not been reported. In order to study the function and mechanism of Sec14 superfamily in wheat (Triticum aestivum) and further enrich the database of wheat genome, in silico cloning technique was used to clone TaSEC14p-5 gene (GenBank No. KU639968) from the genome of wheat cultivar Jinghua 9. The analysis of nucleotide sequence indicated that TaSEC14p-5 gene sequence contained a 984 bp intact ORF, which encoded a peptide of 327 amino acids. The predicted molecular weight and isoelctric point of TaSEC14p-5 were 37.1 kD and 7.70, respectively. The amino acids analysis indicated that the predicted protein sequence contained a typical SEC14 superfamily domain and a CRAL_TRIO_N binding domain in plant. Evolution and clustering analysis revealed that TaSEC14p-5 protein shared 96.45% and 88.38% sequence similarities with AeSEC14p (Aegilops tauschii) and Os02g0200000 (Oryza sativa) phosphatidylinositol transfer protein. The expression pattern analysis, carried out by qRT-PCR, indicated that TaSEC14p-5 was constitutively expressed in various tissues of wheat at the booting stage, with higher level in stem, and then in stamen, but less in root. Meanwhile, it was induced by abscisic (ABA) (200 μmol/L), drought (PEG6000), high-salt (250 mmol/L NaCl) and cold (4 ℃) stresses, but under high salinity it was strongly up-regulated. This research aimed at identificating a novel wheat Sec14p phosphatidylinositol transfer protein gene (TaSEC14p-5) relative transcript level in the above mentioned 4 stress treatments. The conclusion was that the novel TaSEC14p-5 gene was an excellent candidate gene, because it was involved in a variety of signal transduction pathway adverse treatment. Otherwise, the gene of interest provides a new research direction for improving resistance and other molecular mechanism in wheat.

Fibroblast growth factor 18 (Fgf18) is a secreted signaling molecule and involved in regulating hair growth cycle of mouse (Mus musculus) and cashmere goat (Capra hircus). Primers attached with XbaⅠ and SacⅠ restriction enzymes were designed to clone Fgf18 CDS sequence, and then the PCR product was ligated to the downstream of ultra-high sulfur keratin (UHS) promoter. For application in somatic cell nuclear transfer (SCNT), enhanced green fluorescent protein gene (EGFP) and neomycin gene (Neo) promoted by cytomegalovirus (CMV) promoter were added into the recombinant plasmid. Four restriction enzymes (SacⅠ, BglⅡ, SphⅠ and EcoT14Ⅰ) digestion results showed that the follicle-specific expression vector of Fgf18 gene was successfully constructed, named as pUHS-Fgf18. Three positive transgenic mice were obtained by using pronuclear microinjection, and the integration efficiency was 5.8%. RT-PCR and Western blot results showed that the expression levels of Fgf18 were significantly increased in the skin of transgenic mice, compared to controls (P＜0.05). In order to verify the tissue-specific expression, the expression levels of Fgf18 mRNA were also detected in transgenic mice skin, liver, kidney, heart, testis, skeletal muscle, spleen, brain, lung, stomach and small intestine tissues by using RT-PCR. The results showed that the expression levels of Fgf18 mRNA were significantly increased in transgenic mouse skin (P＜0.05) and there were no significant difference in other tissues between transgenic mouse and controls (P＞0.05). Above mentioned research indicated that pUSH-Fgf18 vector can specifically express Fgf18 in skin. Then RT-PCR was used to detect the copy number of Fgf18 in transgenic mice. The result showed that they were ranging from 8~210, and no copy was detected in controls. This study successfully established a transgenic mouse model, which can serve as a favorable tool to study the function and mechanism of Fgf18 in hair growth cycle, and will provide a theoretical basis to produce Fgf18 gene modified cashmere goats.

In this paper, microsatellite method was used to explore the genetic diversity and genetic relationship of 6 mainly domesticated wild ducks and Shaoxing partridge duck (Anas platyrhynchos domestic). 11 microsatellite loci were tested in the 7 species of duck, which were Common mallard (A. platyrhynchos), Xihu mallard (A. platyrhynchos), Fenghua mallard(A. platyrhynchos), Spot-billed duck (A. poecilorhyncha), Northern pintail (A. acuta), Red-crested pochard (Netta rufina), and Shaoxing partridge duck, totally 329 samples. The results showed that 141 effective alleles (Ne) were detected totally, the average effective allele per locus was 12.821, the average expected heterozygosity (He) was 0.856, and the mean polymorphic information content (PIC) was 0.845. All loci were moderately or highly polymorphic loci and were suitable for assessment of duck genetic diversity. High genetic diversity was detected in the 7 populations, and their polymorphism information contents were between 0.310 and 0.960. F- statistic analysis showed that Fst of all sites were between 0.047 and 0.469, the average Fst was 0.171, which indicated that there was a large genetic variation among populations. Nei's genetic distance (Ds) analysis showed that the genetic distances among Common mallard, Xihu mallard, Fenghua mallard and Shaoxing partridge duck were closely, and genetic distances from them to Common mallard, A. acuta and Netta rufina were far, simultaneously, the genetic distances among these three varieties were far as well. Phylogenetic tree analysis based on Nei's genetic distance revealed that N. rufina was clustered into one group separately. Moreover, the genetic distance between Shaoxing partridge duck and Common mallard was closer than the distance between Shaoxing partridge duck and Common mallard. It indicated that Shaoxing partridge duck is more likely to originate from Common mallard.The analysis of genetic diversity and relationship among 6 wild duck breeds and Shaoxing partridge duck further improve the genetic structure of wild duck and domestic.

How to confirm the functions of the genes and their regulation mechanisms by the massive information such as genome, transcriptome and the metebolomics is one of the core tasks in the field of life science. In present study, to make use the biological information data published in databases of Arabidopsis thaliana, the trichome related functional genes were screened based on the correlation between the difference of leaf trichome density variations and expression level differences of relevant genes among 31 Arabidopsis populations, which was established by linear correlation analysis model. All 1 747 genes were screened as their gene expression level was related to leaf trichome density from totally 33 554 genes. The number of negatively related genes were 195, and positively related genes were 1 552. The positively correlated genes were performed for function enrichment analysis. The results showed that the cluster terms mainly concentrated in the 4 aspects, including stress response, cellular component organization, biological regulation and metabolic processes. There were the most hierarchical clustering terms in the stress responses respect, including immune response and innate immune response, stress response, defense response, wound response, response to biological stimuli, fungal defense reaction, bacterial response, chemical stimulation of the response, organic material response, jasmonic acid response to stimuli, in response to carbohydrate stimulation and chitin response cluster terms. There were metabolism of glucosinolates, indole derivatives synthesis and metabolism, indole glucosinolates metabolism, protein phosphorylation and phosphate metabolism cluster terms about the metabolic process. There were cell wall modification and cell wall depolymerization cluster terms about the cellular component organization, and there were signal transduction, stress response regulation and defense response regulation cluster terms about biological regulation. In particular, there were genes directly related to the development of trichome and genes directly related to the secretion of the defense metabolites of trichome in the screened genes. Such as MYB23, a transcription factor which could induce the initiation of leaf trichome, DIS1 (distorted trichomes 1) which had an important influence on the development of trichome, and CYP81F2 (cytochrome P450 family 81 subfamily F polypeptide 2) and MYB51 which were both involved in the synthesis of indole glucosinolates. And the expression level of the 4 genes mentioned above all had a significant positive correlation with the density of the leaf trichome. Comparison of the function enrichment results of screened genes in the present research with the previous functions studies on leaf trichome proved practicability of the present method. Therefore, this method may provide a reference to screen target functional genes related to quantitative traits not only in A. thaliana but also in other species.

The Japanese flounder (Paralichthys olivaceus) culture industry faces unprecedented crisis, due to the high density of factory farming, water pollution and other reasons. In the research, 57 families were established in April 2015, based on stock with disease-resistance and fast-growing families followed by our previous study, including 11 F3 families and 46 F4 families, F4 families were firstly established in China. Disease resistance was investigated using 46 families, tail number is more than 150, through 2 intraperitoneal injection challenged with Edwardsiella tarda, and the paired sample t test showed that there no significant difference between two experiments (P＞0.05), besides, pearson correlation analysis demonstrated that there was a very significant linear correlation (P＜0.01) between the survival rate of the two experiments. Analysis of survival rate demonstrated that there were significant differences in different families on resistance to E. tarda infection. Among 46 families, 5 families were identified as strong disease resistance, the survival rate were over 70% (F1551, F1503, F1501, F1550, F1505), 15 families with a survival rate of 50%~70% were identified as disease-resistance families, 23 families with a survival rate of 20% ~50% were identified as normal disease resistance families, and 3 families with a survival rate of less than 20% were identified as susceptible families. According to the comparison of daily weight gain rate of 46 families, 4 fast-growth families were identified which daily weight gain rate was up to 0.225 0 g/d (F1533, F1551, F1514, F1502), 12 secondly fast-growth families were identified, 24 normal growth families were identified and 6 slow-growth families were identified. The heritability of disease-resistance was 0.177±0.020, which was low heritability and fit for selection and breeding in families. The breeding value of parents for disease-resistance range from (-1.962±0.646) to (2.110±0.546). The breeding value of F1551 and F1550 in female breeding value was higher than in the other families and the breeding value of F1551 in male breeding value was higher than in the other families. By the variance analysis, correlation analysis and analysis of breeding value. A F4 family (F1551) with excellent breeding performance has picked out, such as disease-resistance, fast-growing, and high breeding value. Our study finds that descendant enhance disease resistance inherited from their parents, indicating that the establishment of a disease-resistant strain based on these families is suitable for aquaculture.

With the development of high-throughput sequencing technique, the whole genome sequencing and transcriptome sequencing in maize (Zea mays) have been completed. The differentially expressed genes can be discovered by bioinformatic methods in different maize tissues, which will further reveal the molecular mechanism of the growth and development process. In this study, the transcriptome datasets of maize inbred lines Mo17 and B73, from the NCBI GEO database, were downloaded, and 5 genes (GRMZM2G311036, GRMZM2G336824, GRMZM2G059465, GRMZM2G124799, GRMZM2G147491), belonging to O-methyltransferase (OMT) gene family, which were expressed higher in roots than in shoots in both maize inbreds B73 and Mo17, were found, and additional 23 copies were identified from the maize B73 reference genome. The plant O-methyltransferases (OMTs) are dependent on S-Adenosyl-L-methionine (SAM), which can catalyze a variety of secondary metabolites such as flavonoids, alkaloids and phytoalexins that play an important role in various stages of plant growth and development, the resistance of the invasion of exotic pathogens. All 28 OMT genes were located on 8 chromosomes. Most of them contained 2 exons and one intron (except for GRMZM2G349791 and GRMZM2G104730, which contained one exon and 8 exons, respectively) with the length varied from 291 to 921 bp. The genes structures, conserved motifs and evolutionary analysis revealed that all the 27 OMTs belonged to a new type of OMT gene family that was different from the caffeic acid O-methyltransferase (COMT) and caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT). The new type of OMT genes contained more than 8 conservative motifs except GRMZM2G147503. They were the same protein family (O-methyltransferase COMT-type family) as COMT based on the prediction of protein family. Furthermore, the motifs 2, 4 and 9 were only presented in the new OMT gene family, while the motifs 5, 6 and 7 were shared by the new OMT and COMT, and the CCoAOMT (GRMZM2G127948) had no common conservative domain with them, which indicated that the new OMT and COMT were closely related to each other. This new OMT gene family could be divided into 5 clades, and 2 clades contained Bx7 and Bx10, respectively, which involved in the biosynthesis of benzoxazinoids, suggested that they play an important role in insect resistance in maize. This OMT gene family were expanded mainly by tandem duplication and segment duplication based on the locations and phylogeny. Expression analysis of the parents of maize NAM population showed that OMT genes had differential and complicated expression patterns. Furthermore, most of genes were expressed higher in vegetative growth stage than in reproductive period. In vegetative growth period, genes were expressed higher in roots than in other tissues, while genes were highly expressed in seeds of the reproductive growth stage. These results provide a certain theoretical basis and reference for further understanding the biological function and its application of OMT gene family, and provide an important guiding significance for studying the catalytic mechanism and expression regulation mode of the gene family.

The current study aimed at analyzing gene expression of tyrosine kinase A(TrkA) gene and SNPs as well as their association with litter size in sheep (Ovis aries), and thus to offer a reference data for the candidate gene research of reproduction trait in sheep. Therefore, the qRT-PCR was conducted to investigate the expression patterns of TrkA gene in heart, liver, spleen, lung, kidney, rumen, duodenum, longissimus dorsi muscle, fat, hypothalamus, pituitary and ovarian tissues of Hu sheep, and DNA pooling sequencing and restriction fragment length polymorphism (RFLP) were carried out to screen SNPs and their association with litter size in sheep population of Hu sheep (n=526) and Small tail han sheep (n=437). The results revealed that TrkA gene showed the highest expression in ovary, lung and liver which was significantly higher (P＜0.05) than that in spleen, kidney, rumen, duodenum, longissimus dorsi muscle, fat, hypothalamus, pituitary tissues of Hu sheep, and the expression in heart was in the middle expression level. The lowest expression level was observed in spleen, kidney, rumen, duodenum, longissimus dorsi muscle, fat, hypothalamus, pituitary. According to DNA pooling sequencing and RFLP technologies, 2 polymorphisms, NC_019458.2:g.105281586C＞T and NC_019458.2:g.105284248G＞C were identified in intron 9 and intron 14 of TrkA gene, respectively. In NC_019458.2: g.105281586C＞T locus, 3 genotypes, CC, CT and TT were found in both Hu sheep and Small tail han sheep. CT was the dominant genotype, and C was the dominant allele. In addition, the homozygosity (Ho), heterozygosity (He), effective allele number (Ne), polymorphic information content (PIC) for Hu sheep were 0.50, 0.50, 1.99 and 0.37, respectively, and were 0.49, 0.51, 1.95 and 0.37 for Small tail han sheep, respectively. The χ2 test indicated that Small tail han sheep population deviated Hardy-Weinberg equilibrium, while Hu sheep was in Hardy-Weinberg equilibrium. Same as NC_019458.2:g.105281586C＞T locus, 3 genotypes, GG, GC and CC were identified in NC_019458.2:g.105284248G＞C locus in both Hu sheep and Small tail han sheep. Of which, GC was the dominant genotype, and C was the dominant allele. The Ho, He, Ne, PIC for Hu sheep were 0.50, 0.50, 2.00 and 0.37, respectively, and were 0.49, 0.51, 1.96 and 0.37 for Small tail han sheep, respectively. In contrast, the χ2 test indicated that Hu sheep population deviated Hardy-Weinberg equilibrium, while Small tail Han sheep population was in Hardy-Weinberg equilibrium. The association analysis of polymorphisms in TrkA gene with litter size revealed that NC_019458.2: g.105281586C＞T was significantly correlated with litter size in Hu and Small tail Han sheep (P＜0.05), and NC_019458.2:g.105284248G＞C was significantly associated with litter size in Hu sheep (P＜0.05). In summary, this study indicated that expression of TrkA gene in ovary and lung was higher than remaining tissues in addition to liver. SNPs of TrkA gene associated with litter size in sheep could be used as a candidate gene for litter size selection of sheep at early stage of growth. This study provides a scientific basis for searching molecular markers associated with reproductive traits, and setting breeding strategies, as well as improving the production performance of sheep.

Epigenetic mechanisms preside over genetic information to enable development from the fertilized, totipotent oocyte to the adult body. Methylation of histones at specific residues is one of the most important epigenetic modifications and has an essential role in both transcriptional repression and activation during embryonic development, including lineage specification and cell differentiation. The UTX (ubiquitously transcribed tetratricopeptide repeat X chromosome) and JMJD3 (jumonji domain containing 3) are identified as a histone H3 lysine 27 trimethylation (H3K27me3) demethylase, which promote muscle development, regulates metabolism and other important physiological functions. The principle of in vitro transcription system is based on DNA template by RNA polymerase in vitro synthesis of RNA. In this study, 3 pairs of primer were designed based on the mRNA sequence of JMJD3 and UTX in mouse (Musculus mus), and the in vitro transcription vectors pMD19T-T7UTX and pMD19T-T7JMJD3 were constructed, respectively. The in vitro transcription of mRNA was injected into the mouse oocytes, and the H3K27me3 was detected by immunofluorescence assay. The results showed that the in vitro transcription of UTX and JMJD3 was detected by electrophoresis, and the bands were clear and consistent with the expected size. The electrophoresis of the correct size of UTX and JMJD3 in vitro transcription products were gel recovery and purified. The use of microinjection technology taking UTX and JMJD3 gel product was injected into mouse oocytes. H3K27me3 expression was detected by laser confocal immunofluorescence technology. Result showed that the expression of H3K27me3 after injection of oocytes was significantly decreased. In this study, in vitro transcription of UTX and JMJD3 vectors were successfully constructed, respectively, using the piecewise PCR method, and proved to had very good enzyme activity at the cell level. This study successfully constructed pMD19T-T7UTX and pMD19T- T7JMJD3 vectors. At the cellular level, pMD19T-T7UTX and pMD19T- T7JMJD3 vectors could be transcribed and translated into active proteins. These vectors can be used to study the role of UTX and JMJD3 in the development and related diseases.

The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. At present, the most effective method for the production of transgenic mice is the pronuclear injection technology. Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic (Tg) mice (Mus musculus). However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. The main disadvantage of this technique is that the efficiency is not high. In this paper, the injection needle diameter, concentration of exogenous gene, single-/double-pronuclear microinjection, embryo transfer and other major steps were optimized. The results showed that different concentrations of exogenous gene had a great impact on the positive rate of transgenic mice. When the injection of exogenous gene concentration in 1.00, 2.50, 3.00 and 4.00 μg/mL, the positive rates of transgenic mice were 0.67%, 14.9%, 30.6% and 21.4%, respectively. Among them, the exogenous gene concentration at 3.00 μg/mL was significantly higher than that of other groups (P＜0.01). In the single-/double-pronuclear microinjection test, the concentration of exogenous gene was 3.00 μg/mL. The positive rate of the double nucleus injection group was significantly higher than that of the single injection group (24.3% vs. 18.3%, P＜0.01). After micro-injection, the 2 cell stage embryo transplantation of 0.5 d, 1.5 and 2.5 d, to the pseudopregnant females through the fallopian tube. The birth rates were 68.6%, 67.6% and 40.1%, respectively, and the positive rate were 17.4%, 22.5% and 10.1% respectively. In comparison, the birth rate had no difference in 0.5 d and 1.5 d surrogate mothers group, but the transgenic positive rate (22.5%) in the 1.5 d group was significantly higher than that in the other groups. The results indicated that the fine as much as possible of the injection needle; 2~4 μg/mL exogenous gene concentration; double-pronuclear microinjection; 1.5 days pseudopregnant were the key to obtaining high efficiency of transgenic mice. This study may provide some useful reference for the research of transgenic animal technology.

Xenotransplantation is an effective solution for resolving the problem of inadequate supply of donor organs. Pigs (Sus scrofa) are considered the ideal donor for transplantation to human (Homo sapiens) recipients. However, xenograft would be cleared up by the host macrophages after entering into the receptor. The reason is that the singal regulatory protein α (SIRPα) receptors on macrophages can't specifically recognize the surface molecule integrin associated protein (CD47) on xenograft cells. This study was to generate α-1, 3-galactosyltransferase gene (GTKO) knockout pigs expressing hCD47 which could avoid hyperacute rejection and weaken the phagocytosis by host macrophage. Firstly pCAGGS-hCD47-Neo vector to expressing the hCD47 gene under the cytomegalovirus early enhancer/chicken β actin promoter (CAG) was constructed. The ear fibroblasts of GTKO Bama miniature pig were transfected. Then the transgenic cloned piglets were generated by somatic cell cloning techniques. The transgenic cloned piglets were identified by PCR. The hCD47 gene expression in transgenic pig's heart, liver, spleen, lung, kidney and pancreas was analyzed by qRT-PCR, Western blot and immunohistochemistry. The haemal physiological parameters were detected by drawing blood from the survival piglets to monitor health condition. The results showed that 13 transgenic cloning piglets were all hCD47 gene positive expression. The hCD47 gene had the highest expression level in pancreas, followed by liver, lung, spleen, kidney and the lowest expression level in heart. Western blot and immunohistochemistry results also showed that expression of the hCD47 gene was strong in the pancreas and liver, consistent with the results of qRT-PCR. The haemal physiological parameters showed that survival piglets were in good health. All above, this study had successfully generated the GTKO Bama miniature pig which expressed hCD47 genes. Expression model of hCD47 in porcine organs was identified, which would be helpful for studying the role of hCD47 gene in weakening the phagocytosis of host macrophages for various xenograft organs.

Lethal thrombocytopenia that accompanies liver xenotransplantation is one of the barrier to clinical application. Asialoglycoprotein receptor 1 (ASGR1) in pig (Sus scrofa) sinusoidal endothelial cells can bind and phagocytose human (Homo sapiens) platelets, causing thrombocytopenia. ASGR1 knockout pigs were generated by clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) in α-1,3-galactosyltransferase (GGTA1) gene knockout pigs, which could avoid hyperacute rejection and diminish platelet destruction in pig-to-human xenotransplantation. Single guide RNA1 (sgRNA1) was assembled and transferred to ?broblasts with enhanced green fluorescent protein (EGFP) plasmid. Forty-one colonies were finally selected, and the targeting efficiency reached 90% (37/41), more remarkably 73% (30/41) of colonies were biallelic knockout, among which 4 colonies with different genotypes were selected as nuclear donors for somatic cell nuclear transfer (SCNT). Cloned embryos were co-transferred into four recipient gilts. Pregnancy was established in 2 of 4 transfers. Two pregnancies were maintained to term, resulting in 6 live-born piglets with biallelic mutations of the ASGR1 gene. Western blot results showed that the ASGR1 gene expression was abrogate in the liver. Thirteen potential off-target sites were investigated, but no off-target event was detected in the live piglets. Taken together, the use of the CRISPR/Cas9 system of fibroblasts followed by SCNT enabled the production of ASGR1 biallelic knockout pigs. Thus, deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.

Platelet endothelial cell adhesion molecule-1 (CD31) is an important endothelial cell surface membrane molecules, which has been used to identify endothelial cells. However, whether CD31 can be used as specific marker of purified pig (Sus scrofa) sinusoidal endothelial cells (porcine liver sinusoidal endothelial cell, pLSEC) should be verified. Therefore, in this study, whether purified pLSEC had iconic aperture structure were observed by flow sorting separation pLSEC with CD31 marker. α-1,3-galactosidase transferase gene knockout (GTKO) of Wuzhishan miniature pigs were used as experiment material. These methods including liver perfusion in situ collagenase digestion in vitro, percoll selective gradient centrifugation and adherent culture, fluorescein isothiocyanate-CD31(FITC-CD31) marker pLSEC streaming sorting were adopted. The results of observations of endothelial cells of isolated pLSEC by inverted microscope showed that typical cell morphology, cobblestone neatly arranged, TEM cells ultrastructure fenestrae. Cells could be observed to have fenestrations through environmental scanning electron microscope,cell surface structure of pLSEC and porcine aortic endothelial cells were obvious different from each other which could be observed through transmission electron microscope, pLSEC had a special aperture structure. qRT-PCR was used to detect CD31, cell adhesion molecules (CD146), hemophilia factor Ⅷ gene (Ⅷ-F) and von willebrand factor gene (vWF) of pLSEC, pAEC, porcine ear fibroblast cell (pEFC). The results of expression showed that pLSEC of CD31, CD146, Ⅷ factor and vWF expression levels have were higher than pAEC and pEFC significantly. Then phagocytosis was detected using immunofluorescence pLSEC which had an innered cell membrane fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate acetylated low density lipoprotein (DiI-Ac-LDL) uptake. In a word, CD31 can be used as a special marker to purify pLSEC, which provides an effective way for the separation of liver sinusoidal endothelial cells.

In the development of nearly 20 years, genetically modified organisms (GMOs) planting area has been growing and the number of GMOs has also been increasing. With the global trade liberalization and commercialization of GMOs, the requirements of analytical approaches also enhances unceasingly. Due to the diversity of genetic modifications in GMOs and low level presence of genetically modified products, the analytical approaches with high sensitivity and accuracy are required to meet the needs of GMO detection. Currently, qPCR is widely used in quantification of GMOs. However, this method relies on the standard curve and reference material, which leads it to a relative quantification method. Digital PCR (dPCR) makes the accurate and absolute quantification of DNA into reality compared with the qRT-PCR. QuantStudioTM 3D digital PCR (3D-dPCR) is a novel absolute quantification method for nucleic acids analysis which based on a hydrophilic and hydrophobic chip with ultra-high density to realize partition. 3D-dPCR has a huge potential applicability in accurate quantification of GMOs. Herein, we developed the simplex and duplex 3D-dPCR assays to quantify the GM maize (Zea mays) MON863 event, and the results was also compared with those from qRT-PCR. The GM content tested by 3D-dPCR was about half of that tested by qRT-PCR. This because the genotype of GM maize grain was heterozygous. When analyzing the qRT-PCR data we regarded the samples and reference materials were homozygous since the genotype of the samples and reference materials were unknown. However, digital PCR was an absolute quantification method which did not depende on any other reference standard, and realized absolute quantification through limit dilution, Poisson distribution and endpoint PCR to ensure the accuracy and reliability of the quantitative results. What′s more, simplex and duplex 3D-dPCR assays showed better consistency than qRT-PCR. The maximum difference between simplex and duplex 3D-dPCR was 13.79%, while the maximum difference between simplex and duplex qRT-PCR was 54.75%. It meaned duplex 3D-dPCR assay was more stable than duplex qRT-PCR assay. Concluded from the results, 3D-dPCR was well developed as one accurate quantification method for GMO products, especially when lacked of the information of sample purity and genotype. Digital PCR could eliminate the influence of these factors and obtain the accurate value. Moreover, digital PCR could also be used as a new method to verify the genotype of GMOs, providing reference for other methods. In addition, compared with qRT-PCR, 3D-dPCR was more suitable for quantitative detection of GMOs by duplex assays. The results, based on 3D-dPCR simplex and duplex quantification methods, provide a good prospect in GMO quantification detection.

Targeted mutagenesis is becoming an important way to modify genes in various cells and organisms, and has been widely used for functional study of genes and for genetic modification in plants and animals. Currently, three technologies, including zinc-finger nucleases (ZFN), transcription activator-like effector nuclease (TALEN)， and clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (CRISPR/Cas9), have been developed for targeted mutagenesis. Among them, TALEN technology has been successful in targeted gene modification in many agricultural plants, and has become an effective tool for molecular breeding in crops. In this study, we employed TALEN to generate site-specific modification in CYP81A6, which confers bentazone resistance in rice (Oryza sativa), to obtain bentazone-sensitive mutant applicable in rice hybrid breeding. TALEN-targeted site was selected near the 5' end of CYP81A6 coding region to ensure complete knockout of gene function. A total of 110 T0 transgenic plants were generated by Agrobacterium-mediated transformation of rice callus (Japonica, Wuyunjing 7). High resolution melting (HRM) analysis indicated that 3 T0 transgenic lines carried targeted mutation (i.e. mutagenesis frequency of 2.73%). These 3 lines were sensitive to bentazone in the field test. Sequence analysis revealed multiple mutations in CYP81A6 in the three lines, indicating the transgenic lines as chimera. The 3 T0 transgenic lines were self-pollinated, and the T1 population were subjected to bentazone-sensitivity test. We found many T1 transgenic seedlings still sensitive to bentazone, but none was homozygous mutant. T1 non-transgenic seedlings were all resistant to bentazone-treatment. These results indicated that the TALEN employed in this study was not efficient in generating mutations in germline cells, although it was active in somatic cells. The wide presence of chimeric mutants in T0 and T1 plants indicated that mutations generated by TALEN need to be reconfirmed in the next generation, preferably in the plants that does not contain the transgene. Although an inheritable mutant was not obtained, the study generated valuable information that can improve future experiments in targeted mutagenesis and screening of mutant plants.

Gene chip standardization is the basic guarantee for the specific sensitive detection of pathogens, In this study, the optimal hybridization conditions of gold label silver stain visual DNA microarray such as the use of spotting buffer, spotting frequency, hybridization temperature, hybridization time, nanogold concentration and silver enhancement time were screened respectively to analysis the influence on the visual DNA microarray. Primers were designed based on the sequences of S and M gene of Porcine epidemic diarrhea virus (PEDV); S and N gene of Transmissible gastroenteritis virus (TGEV); VP7 and NSP4 gene of Group A porcine rotavirus (GAR). The 60-mer oligonucleotides probes with 15 T bases on their 5' end were designed according targets DNA sequences and sprayed on the aldehyde-modified glass slides with spotting buffer in ratio of 1∶1 and nonmiscible in once, twice and trice respectively. The positive target DNAs which were extracted from three porcine diarrhea viruses were modified biotinylated by asymmetrical PCR and hybridized in 30, 60, 90 and 120 min respectively with prepared microarray at 40, 45, 50 and 55 ℃. The nanogold particles of 10, 20, 30, 40, 50 and 60 times dilution concentration were introduced by nanogold-streptavidin particles according the specific links of biotin and streptavidin. The results could be observed by naked eyes after different minutes of silver enhancement system. To evaluated the visual DNA microarray, a total of 173 clinical samples, which collected from piglet diarrheic cases were detected using this visual DNA microarray and RT-PCR respectively. The results of 10 times of repeated tests showed that the optimal conditions of visual DNA microarray were that targets DNA hybridized with prepared microarray which is spotted once with the mixture of probes and spotting buffer in the ratio of 1∶1 at 50 ℃ in 90 min, and then enhanced by silver buffer in 12~14 min after combined with 40 μg/mL nanogold-streptavidin particles. The clinical samples results detected by microarray were consistent with that by RT-PCR. In this study, the optimal reaction conditions of viral diarrhea visual inspection were determined, which laid a foundation for the clinical application standardization research of visual microarray technology.

DELLA protein is a subfamily of GRAS protein family, and plays negative role in the gibberellin (GA) signal pathway. DELLA is not only involved in the GA signaling, but also involved in a variety of signal transduction in plant hormones, such as auxin, abscisic acid (ABA), ethylene, jasmonic acid (JA) etc. It plays an important role in the process of plant growth and development. The article summarizes the molecular cloning and analysis of DELLA protein-coding genes, and mainly about the expression of timal and spacial expression and regulation of DELLA gene expression under abiotic stress, and explains the DELLA protein interactions with the signal transduction of hormone and other factors. The article reveals the signal network system and its mechanism of DELLA.

With the rapid development of molecular biology techniques, animal breeding has entered the era of molecular breeding guided by quantitative genetics, molecular genetics and other disciplines. Animal biology breeding exhibits significant application value and industrial prospects, and will be helpful to promote the localization process of livestock and poultry breeding in China. This review summarized the development history, research progress and applications of animal biology breeding around the world, including genomic selection and genome editing, etc. It also analyzed the development status of the existing problems in China's animal biology breeding in the perspective of technological innovations and technological industrialization, and also put forth the countermeasures and proposals for promoting animal breeding industry, including strengthening independent innovation research.