Transgene silencing is a widespread phenomenon in transgenic plants and animals, bringing new problems for the cultivation and application of genetically modified organisms. In this study, we analyzed the expression of exogenous Red Fluorescence Protein from Discosoma sp. (DsRed) gene in 2 healthy transgenic Cashmere goats (Capra hircus) and transgenic nuclear-donor cells were analyzed. The expression of an exogenous DsRed gene in fibroblasts obtained from the ear tip was significantly higher than that in the epidermis cells from transgenic Cashmere goats (P＜0.05). After bisulfite sequencing, the methylation status of CMV promoter in 2 healthy transgenic Cashmere goats fibroblast had been checked. The CMV promoter showed high methylation status in 2 samples: ZK0310-1: 89.09%, ZK110324-1: 86.36%. After treatment with the methylation inhibitor 5-Azacytidine (5-Az), the DsRed expression in transgenic goat cells was significantly increased(P＜0.05). After treatment with the histone deacetylase inhibitor Trichostatin A (TSA) in the concentration range of 0~1 μmol/L of TSA, DsRed mRNA expression quantity and concentration of the TSA showed dose-response relationship(R2=0.985, P＜0.01). The optimal drug concentration of 2 cell type isolated from 2 transgenic Cashmere goats were: ZK0311-1 fibroblast, 5-Az: 5 μmol/L、TSA: 1.0 μmol/L; ZK0311-1 epidermis cells, 5-Az: 20 μmol/L, TSA: 1.0 μmol/L; ZK110324-1 fibroblast, 5-Az: 10 μmol/L, TSA: 1.0 μmol/L; ZK110324-1 epidermis cells 5-Az: 5 μmol/L, TSA: 0.75 μmol/L. Time of 5-Az treatment was 7 d; time of TSA treatment was 24 h. After 5-Az and TSA treatment, the increased expression of cell lines DsRed mRNA also caused obvious improvement of red fluorescent protein expression(P＜0.05). After treatment with the methylation inhibitor 5-Az, and CMV promoter methylation in transgenic goat fibroblast was significantly decreased to ZK0311-1: 78.48%, ZK110324-1: 49.7%(P＜0.05). It was proved that 5-Az had obvious inhibition effect of exogenous gene promoter methylation. These results demonstrated that in adult-derived transgenic cells, TSA and 5-Az treatments are effective measures to improve the efficiency of exogenous gene expression. This study has important reference value on how to improve the exogenous gene silencing in transgenic animal production.

Polymorphisms in microRNAs (miRNAs) genes can potentially alter various biological processes by influencing the processing and/or target selection of miRNAs. To investigate if the single rs16057773 (A＞G) mutation, which we characterized resides in the terminal loop of pre-miR-1816, could affect its processing and had a biological function, association study of the polymorphisms with chicken (Gallus) carcass and growth traits using the Gushi-Anka F2 resource population showed that this SNP was significantly associated with birth weight and postnatal weight till 12 weeks as well as some body size indexes including shank girth, body slanting length, crista breadth (P＜0.05). Moreover, the SNP was significantly associated with carcass weight (P＜0.05). The result of association study suggested that the SNP in the precursor of miR-1816 played an important role in the growth of chicken. Mfold predicted that the SNP had no effect on miRNA secondary structure. Through constructing miR-1816 pcDNA3.1-EGFP expression vectors of 2 genotypes and transfecting DF1 cells subsequently, quantitative Real-time PCR (qRT-PCR) results showed that there was no significant difference between different genotype and mature miR-1816 expression level. The result indicated that this SNP in the precursor of miR-1816 could not alter the processing of miR-1816. This study revealed that the SNP in the miR-1816 was significantly associated with chicken economic performance. However, this SNP in the terminal loop of pre-miR-1816, which did not alter the stability of miR-1816 secondary structure, had no effect on miRNA processing. This study suggested that there was a different path that mediated the function of miRNA SNP, and will contribute to the study of miRNA processing.

SET and MYND domain-containing protein 3 (SMYD3) is a histone H3 Lysine 4 trimethyltransferase, which overexpresses in many cancers and can promote cells proliferation, migration and invasion. In order to study the function of SMYD3 in bovine embryonic fibroblast cells (bEFs), SMYD3 CDS was obtained from bovine (Bos taurus) and inserted into the vector pIRES2-Zsgreen1 to construct overexpression vector pSMYD3-IRES2-Zsgreen1. pSMYD3-IRES2-Zsgreen1 was transfected into bEFs and qRT-PCR and Western blot were used to verify its expression. Also, the cell growth was recorded by detecting the absorbancy of 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide (MTT) every 24 h for 7 d. At last, the transgenic bEFs were infected with lentiviral vectors expressing the mouse (Mus musculus) octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2), avian myelocytomatosis viral oncogene homolog (c-Myc) and Kruppel-like factor 4 (Klf4) transcription factors, respectively, to obtain the bovine induced pluripotent stem cells (iPSCs). The enzyme digestion results showed that the vector pSMYD3-IRES2-Zsgreen1 was correctly constructed. The qRT-PCR and Western blot results indicated that SMYD3 overexpressed in pSMYD3-IRES2-Zsgreen1 transfected bEFs, which cells growth improved comparing with the control vector transgenic cells. After induction by the 4 transcription factors, the SMYD3 transgenic cells formed more stem cell-like clones, which displayed OCT4, NANOG homeobox (NANOG) and SOX2 immunofluorescence staining positively. These results established and firstly reported that SMYD3 could promote bEFs growth and induction efficiency for iPSCs, which provides basic data for the strategies of preparing bovine iPSCs and improves understanding of the molecular basis of iPSCs.

Glutathione reductase (GR) is one of the key enzymes in the plant antioxidant system. In this study, a full-length cDNA sequence designated as BnGR1 was cloned by RACE methods based on the relative unigene sequence in ramie (Boehmeria nivea (L.) Gaud.) transcriptome sequencing. The full-length cDNA sequence (GenBank accession: KF747758) of BnGR1 was 1 977, and the ORF was 1 494 bp, which encoded 497 amino acids with predicted pI of 5.68 and molecule weight of 53.7 kD, respectively. BnGR1 contained a pyridine nucleotide-disulphide oxidoreductases classⅠ active site, a nicotinamide adenine dinucleotide phosphate (NADP) binding site, a flavin adenine dinucletide (FAD) binding site, two L-glutathione oxidized (GSSG) binding sites and a special structure domain of cytosolic GR, respectively. Homology comparison analysis showed that the deduced BnGR1 amino acid sequence shared a 88% homology with GR gene (EOY05332) in cocoa (Theobroma cacao). Prokaryotic expression vector pGEX-4T-BnGR1 was established and transformed into Escherichia coli BL21 after isopropyl-β-d-thiogalactoside (IPTG) induction, which showed a successful gene expression. The expression pattern analysis carried out by quantitative Real-time PCR indicated that BnGR1 expressed in root, stem, stem apex and leaves, with the highest expression level in mature leaves and the lowest in stem. The BnGR1 gene was up-regulated by CdCl2, abscisic acid (ABA) and salicylic acid (SA) treatments. The results indicated that BnGR1 gene might play an important role in response to abiotic stresses of ramie， which provide the basic data for exploring the ramie resistance molecular mechanism of heavy metals Cd2+.

The CMV promoter is often used as a transcriptional regulatory sequence in eukaryotic expression vectors, and considered as a universal element capable of transcriptional activity in a wide rang of cells in vitro. However, reports documenting the transcriptional activity of this control element in different tissues of transgenic animals are relatively less. In this study, we produced the CMV-EGFP integrated transgenic pigs (Sus scrofa) through somatic cell nuclear transfer technique and CMV-EGFP integrated transgenic mice (Mus musculus) through pronuclear microinjection. Transgenic positive animals were identified through PCR screen. Then the expression level of reporter gene EGFP was investigated in liver, heart and muscle of the transgenic pigs through qPCR assay. The results showed that EGFP is most abundant in heart, and modest in muscle, and relatively less abundant in liver. We further examined the expression pattern of EGFP in a variety of tissues in both transgenic pigs and mice through detecting the fluorescence signal from cryosections. The results showed that the CMV promoter can drive EGFP expression in all the examined tissues of transgenic pigs and mice, but on closer inspection, we found that the fluorescence intensity of EGFP was obviously different in different tissues, where the signal was most strong in lung, small intestine and pancreas, modest in muscle and heart, and weak in spleen, kidney and liver of transgenic pigs. Similarly, expression level of EGFP was highest in lung, modest in brain, liver and muscle, and lower in spleen and kidney of transgenic mice. These experiment results suggested that the CMV promoter could drive the expression of EGFP in a wide range of tissues, however, its activity varies notably across different tissue, where it is most active in lung, modest active in muscle, and less active in kidney and spleen in both the transgenic pigs and transgenic mice. This study indicates that the abundance of transcription factors capable of activating the CMV promoter varies significantly in different tissues of animals. Therefore, whether to use the CMV promoter for establishing a transgenic animal model, should be base on the targeted expression site of the extragenous gene in the animal.

Akirin, a conserved nuclear factor, is associating with immune functions in animal via regulating cytokine transcription and immune cell chemotaxis. Here, an akirin1 cDNA sequence (Paakirin1) from ayu (Plecoglossus altivelis) was cloned. The Paakirin1 was 1817 bp in length encoded a 182 aa polypeptide with a molecular weight of 20.68 kD(GenBank accession No. KP278589). Sequence comparisons for similarity showed that Paakirin1 was highly similar to akirin1 in fish, such as, rainbow trout (Oncorhynchus mykiss) akirin1 (86%), followed by Arctic char (Salvelinus alpinus) and Atlantic salmon (Salmo salar) akirin1 (85%). Multiple sequence alignment analysis showed that Paakirin1 possessed nuclear localization signal. Phylogenetic analysis showed that fish akirin1 genes grouped tightly into one big cluster. Paakirin1 clustered together with the akirin1s of salmonidae family. Semi-quantitative PCR data showed that Paakirin1 was mainly expressed in head-kidney, spleen, intestine, and monocytes macrophages. After Vibrio anguillarum infection, Paakirin1 mRNA was up-regulated in head-kidney and spleen, while down-regulated in intestine. Furthermore, Paakirin1 mRNA was also up-regulated in monocytes macrophages after lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (PolyI:C) treatment. After Paakirin1 siRNA treatment in monocytes macrophages, the mRNA expression of interleukin-1β (IL-1β) and tumor necrosis factor (TNF) induced by V. anguillarum was unaltered, while its chemotaxis toward to recombinant CXC chemokine ligand 8 (CXCL8) protein was down-regulated. In summary, Paakirin1 alters its expression after infection and mediates chemotaxis of ayu monocytes macrophages. The research provides a theoretical basis and reference for the further investigation of function and regulation of monocytes macrophages in fish.

Choline monooxygenase (CMO) is an important rate-limiting enzyme in the biosynthesis of betaine. To investigate the application that transform cmo gene in Salicornia bigelovii to the Nicotiana tabacum, basic plasmid pCAMBIA1300UR was used to construct the expression vector pCAMBIA1300UR-cmo. Then the reconstructed expression vector was mediated into agrobacterium EHA105 by electric shock. So that the recombinant vector were introduced into the tobacco genome by Agrobacterium tumefaciens mediated transformation. Antibiotics (kanamycin) screening, PCR and qRT-PCR were all used to confirm the success of transformation. After treated with different concentrative NaCl for 10 d, the transgenic tobacco was searched for the evidence of salt tolerance. The results showed that the transformative efficiency of cmo were estimated up to 80%. When treated by the condition of 200 mmol/L of NaCl, the activity of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and proline (PRO) of transgenic tobacco were stronger obviously than that in the non-transgenic plants except the content of MDA and H2O2. So the cmo gene could improve the salt tolerance in the tobacco plants. The study laid the foundation of finding and exploitation of resistant genes from stress-tolerant plants.

The new germplasm WB29 with an average height of 57 cm was a dwarf line of wheat (Triticum aestivum L.) which was selected from the derived lines after crossing and backcrossing multi-generational between common wheat 7182 and two-rowed barley (Hordeum vulgare ssp. distichon Hsü.). Cytology observation, plants height investigation, gibberellic acid reaction and molecular markers technology were used to identify this material and its heredity of dwarf traits. Cytology identification showed that the chromosome number of root-tip cell was 2n=42, no obvious hybridization signal was observed in genomic in situ hybridization (GISH) with two-rowed barley genomic DNA acted as a probe. Specific sequence-tagged site (STS) markers of barley ABG459 (2HS) could amplify specific bands of barley from WB29. Ten pairs of specific molecular markers for wheat dwarfing genes (reduced height gene, Rht) were applied to identify two-rowed barley, 7182, WB29 and Chinese spring and the result showed that 7182 and WB29 had Rht-D1b while none of the 10 Rht were detected in barley and Chinese spring. BC1F1 and F2 population were derived from crossing between WB29 and Chinese, and the statistical analysis of these two derived population indicated the dwarf traits of WB29 was controlled by one pair of incomplete dominance gene. Gibberellic acid reaction pointed that WB29 was a gibberellic-insensitive type of wheat. STS of barley ABG459 (2HS) was used to evaluate F2 population and 343 plants were found to have the barley 2HS chromosome. Wheat dwarfing gene Rht-D1b in F2 population was also detected and the result showed that 322 plants contained Rht-D1b. Linkage analysis showed that STS marker ABG459 was closely linked to the main dwarfing gene of WB29 at a genetic distance of 7.0 cM, while Rht-D1b was not linked to this main dwarfing gene. The result above had defined that WB29 did contain the barley 2HS chromosome and the dwarfing gene of WB29 which had played an important role in reducing the plant height may be located right in the barley 2HS chromosome sequence or near its insertion site. The conclusion of this research had fixed that WB29 was a wheat-barley hybrids which not only enriched the germplasm resources of wheat-barley hybrids, but also enriched the dwarf germplasm and had a certain significance in expending the application field of dwarfing genes since the main dwarfing gene of WB29 may be derived from barley and it may be a new dwarf resource.

The cellular retinol-binding proteins (CRBPs) play important roles in intestinal vitamin A absorption and cellular retinol transport due to the extreme hydrophobicity of retinol, and also regulate the metabolism and homeostasis of retinoids through interaction with metabolic enzymes. Ovocalyxin-32 (OCX-32) is a component of the avian uterine milieu present during formation of the eggshell. OCX-32 has attracted much attention for its antibacterial function and potential termination of calcification in egg shell. The purpose of this study was to find the association of　single nucleotide polymorphisms of CRBP4 and OCX-32 genes with egg production traits and some important molecular markers for such traits of Lingkun chicken (Gallus gallus). The SNPs in the exon 2, 5 of CRBP4 gene and exon 5 of OXC-32 gene were screened by DNA direct sequencing in 276 individuals of Lingkun chicken. The results showed that there were 3 SNPs (A337C, G2727A and A2847G) in CRBP4 gene, the mutation site of A337C was significantly associated with the total egg number of 300 d (P＜0.05), the mutation sites of G2727A and A2847G were significantly associated with egg shape index and albumen height (P＜0.05). In CRBP4 gene, genotype AA was the advantage genotype. Through inheritance and selection of the AA dominant genotype, there was a potential to further improve Lingkun chicken egg shape index and albumen height. There were 3 SNPs (G6566A, A6612G and A6760T) in the intron 4 of OCX-32 gene, the mutation site of G6566A was significantly associated with the albumen height (P＜0.05), the mutation sites of A6612G and A6760T were significantly associated with yolk weight (P＜0.05). In OCX-32 genotype BB was the advantage genotype. Through inheritance and selection of the BB dominant genotype, there was a potential to further improve Lingkun chicken albumen height and yolk weight. The 6 SNPs were in the Hardy-Weinberg state of equilibrium. The results showed that egg production might be closely related to the expression of CRBP4 and OCX-32 genes, and can be used as a molecular marker for egg production traits in marker assisted selection (MAS) breeding of Lingkun chicken, which provide a theoretical basis for Lingkun chicken breeding.

Phosphorus Solubilizing bacteria (PSB) can improve utilization of phosphorus in soil, increase soil fertility, promote plant growth, and alleviate continuous cropping obstacle. In this study, four PSB strains were screened from the rhizosphere soil, and all of them had phosphorus solubilizing ability. By the analysis of comparisons, one of them was screened and classified as Sphingomonas sp., named strain CL01. Further, the efficiency of phosphorus-solubilizing and the secreting level of indole acetic acid (IAA) were determined for the strain CL01, meanwhile, the effects on the root growth of watermelon (Citrullus lanatus) seedling were studied. The results showed that the strain CL01 was the strongest one on the capacity of phosphorus solubilizing than the other strains. Its maximum amount of phosphorus solubilizing was 642.60 mg/L, and the secretion level of IAA was up to 22.87 mg/L, which could promote the development of roots and growth of plants. The watermelon seedlings were treated with the bacterial suspension (in LB medium, OD600=0.5), for 30 d, the underground dry weight, root length, root volume, the number of root tips and the proportion of fibrous roots of watermelon seedlings were increased significantly compared with the controls (CK1, sterilized continuous cropping soil; CK2, unsterilized continuous cropping soil). The underground dry weights of the seedlings were increased by 27.04% and 103.90% compared with CK1 and CK2, respectively, The root length of the seedlings were increased by 83.60% and 63.75% compared with CK1 and CK2, respectively. The root volume of the seedlings were increased by 14.29% and 100% compared with CK1 and CK2, respectively. The number of root tips were increased by 15.16% and 85.45% compared with CK1 and CK2, respectively. The proportion of fibrous roots were increased by 47.81% and 26.80% compared with CK1 and CK2, respectively. Also the average diameter of roots was decreased by 47.89% and 66% compared with CK1 and CK2, respectively. Noticeably, if the concentration of the bacterial suspension was doubled (OD600=1.0), the similar results were obtained, but they were not as good as the lower ones. Based on the results mentioned above, we proposed that strain CL01 had the capacities to dissolve organic phosphorus efficiently and secrete IAA. While the watermelon seedling continuous cropping with the bacterial suspension (OD600=0.5), the growth of root was promoted, root morphology was optimized and the composition of root was improved. The strain CL01 is an excellent candidate for reducing continuous cropping obstacle.

Chitinase (Chi) which is an important pathogenesis-related protein can degrade cell wall components of fungal pathogens, and be induced by salicylic acid (SA), ethylene, plant pathogenic fungi, bacteria and virus. To explore the function of chitinaseⅢ (ChiⅢ) gene in Pyrus bretschneideri Yali, the CDS of ChiⅢ was cloned from Yali which was named PbChiⅢ (GenBank accession No. KJ872675), and the CDS length of PbChiⅢ was 897 bp. Homology analysis showed that the amino acid sequence of PbChiⅢ was highly homologous with Pyrus Huangguan (ACM45715.1), and the nucleotide acid sequence had the closest relationship with Huangguan (FJ589785.1). Quantitative Real-time PCR (qRT-PCR) analysis revealed that PbChiⅢ highly expressed in root and fruit, and its expression had extremely significant difference compared with stem and leave (P＜0.01). Expression of PbChiⅢ was regulated by SA, and enhanced up to the peak at 12 h after treatment with SA during 96 h, and had extremely significant difference compared with control (P＜0.01). PbChiⅢ protein successfully expressed by prokaryotic expression system and a specific protein band of 31 kD was successfully induced. The CDS sequence of PbChiⅢ gene was firstly obtained from Yali and researched the expression of PbChiⅢ in different tissues and the expression characteristic after treatment with SA. This study provided informative references for further study of the function of PbChiⅢ.

As is known to all, rice (Oryza sativa) is one of the most important food crops to human beings, but a variety of adverse environmental stress severely affect the growth and development and limits the production of rice. Further study of the structure, function and expression pattern of plant promoter have great significance for further exploring the adaptation mechanism of plants to its growing environment and creating stress-resistance improved transgenic rice. Calcium-dependent and calmodulin-independent protein kinases (CDPKs) are a Ser/Thr protein kinase family which exist only in the plant and parts of protista, and play a very important role in mediating the signal transductions of growth and development, and the abiotic stresses. The research mainly analyzed the expression pattern and cis regulatory elements of OsCDPK12 gene promoter which named 12P. Firstly, 12P with 2 109 bp in length of upstream regions was cloned by PCR from the genomic DNA of rice Nipponbare (Oryza sativa ssp. japonica) and transformed into rice zhonghua11 (Oryza sativa ssp. japonica) after fused with the report gene β-glucuronidas (GUS). The histochemical staining results showed that GUS gene driven by the promoter of 12P mainly express in root-stem transition zone, stem node, anther and seed. Subsequently, eight different length of 12P 5' deletions were fused to GUS gene and transformed into rice zhonghua11, respectively. The result of deletion analysis indicated that GUS gene driven by the deletions of 12P1975, 12P1828, 12P1570, 12P1351, 12P1194, 12P966 and 12P472 expressed in root-stem transition zone, tender panicle, branch, stem node and mature seed, respectively, but not in leaf, sheath and root. 12P687 drived GUS gene expressed in all the tissues above, and showed a constitutive expression pattern. The histochemical staining results of different deleting promoters transgenic plants showed that there are several important negatively regulatory elements locate at the fragments from -966 bp to -687 bp, several positive regulatory elements locate at the fragments from -687 bp to -472 bp. Quantitative analysis of GUS activity showed that GUS activity of the 12P687 promoter in the tissue of leaf and leaf sheath was about half of the 35S promoter, and 12P687 GUS activity in the root was about 1/3 of the 35S promoter. Furthermore, the different deleting promoters transgenic plants undertook low temperature, low nitrogen and high salt stress treatment, respectively, the result showed that GUS gene in12P1828 transgenic rice plants was up-regulated expressed in the leaf after low temperature treatment, while, in 12P687 transgenic rice plants it's down-regulated in the root after salt stress treatment. These results identified the expression characteristics of 12P which can drive downstream target genes expressed and some potentially important regulatory element that existing, and also verified the expression of OsCDPK12 gene is regulated by low temperature and high salt stress preliminarily, so it will be conducive to provide reference for further work of cultivating stress resistant transgenic rice and researching the function of gene.

Translocation line is an individual formed by one-way or reciprocal translocation between chromosomes of two different species. It is significantly important for the application of excellent exogenous gene to study the genetic stability of exogenous chromosome in translocation lines. In order to obtain Chinese cabbage (Brassica oleracea var. capitata)-cabbage (Brassica compestris ssp. pekinensis) translocation lines, Chinese cabbage-cabbage alien addition line 7＃(AC7) and its parents Chinese cabbage 85-1 and cabbage 11-1 were used as experimental population in present study. Using rays 45 Gy 60Co-γ radiating pollens of AC7, M1 plants were obtained by backcrossing irradiated pollens with Chinese cabbage 85-1, and then M2 plants were obtained by selfing M1. Using 31 specific InDel (Insertion-Deletion) markers (compared with Chinese cabbage) distributed in linkage group No.1 of cabbage, and combining with cytological observation, ten translocation lines were identified from selfing progenies of M2 plants. Two translocation lines AT7-1 and AT7-2 with different chromosome fragments of cabbage were selected and studied, for which isolated microspore culture, selfing and backcrossing were further performed. Using specific InDel markers, progeny plants from selfing, backcrossing and microspore culture of two translocation lines were identified. We also had carried on investigation of phenotypic traits in selfing progenies of AT7-1 and AT7-2. The results showed that genetic stability of two translocation lines AT7-1 and AT7-2 with different chromosome fragments of cabbage was different. The genetic stability of selfing progenies, backcross progenies and microspore culture plants in the same translocation line was also different. In AT7-1, the ratio of keeping exogenous fragment in selfing progenies, backcross progenies and microspore culture plants was 73%, 28% and 50%, respectively. In AT7-2, the ratio of keeping exogenous fragment in selfing progenies, backcross progenies and microspore culture plants was 84%, 62% and 25%, respectively. Head formation related traits in selfing progenies of AT7-1 were segregated, but without segregation in selfing progenies of AT7-2. Studying genetic stability of exogenous chromosomes in Chinese cabbage background could be useful in using new germplasm for Chinese cabbage breeding.

Cell lines provide an important in vitro system for investigations in virology and functional genomics in fish. A new heart cell line, epinephelus moara heart cell line (EMH), was established from Kelp Bass (Epinephelus moara). The cell line was maintained in Leibovitz's L15 medium supplemented with HEPES, antibiotics, 2-Mercaptoethanol (2-ME), fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF). The cultured EMH cells, fibroblastic and epithelial in morphology, could be subcultured once every 4 or 5 days and had been subcultured for more than 20 passages. The suitable temperature for the cell growth was 18~30 ℃ with the optimum growth at 30 ℃ and an unfavorable growth at 36 ℃. The suitable FBS concentration for the cell growth was 10%~20% with the optimum concentration at 15%. Cytogenetic analysis revealed that 62% of the cells maintained a normal diploid chromosome number (2n=48) in the EMH cells at Passage 20. The fluorescent signals were observed in EMH cells after the cells were transfected with enhanced green fluorescent protein (EGFP) reporter plasmid. The EMH established here can be used as a useful tool for transgenic and genetic manipulation studies and may be potentially used for fish virus isolation, propagation and vaccine development.

Histone variant H3.3 is an essential maternal factor for oocyte reprogramming. After SCNT(somatic cell nuclear transfer), the H3.3 protein in oocyte will replace its counterpart in donor nucleus, opening chromatin structure and facilitating reprogramming. However, the H3.3 protein, no matter it is in oocyte or in donor cell, have same amino acid sequence. Besides, there is no specific antibody for H3.3. In order to study the replace process, we used a method of adding a tag. H3f3b is the coding gene of H3.3. Firstly, we got the CDS of h3f3b from bovine oocyte cDNA, By primer designing, we added a Flag or HA coding sequence to its 5' end. Then the sequence was connected to the eukaryotic expression vector pcDNA3.1(+).The constructed vectors of pcDNA3.1(+)-HA-h3f3b and pcDNA3.1(+)-Flag-h3f3b were identified by restriction enzyme digestion and nucleotide sequencing. After transfection of 293T cell, we confirmed the expression and location of the HA and Flag tagged H3.3 protein. Reverse transcription-PCR (RT-PCR) and Western blot showed that only the group transfected with reconstructed plasmid could transcribe reconstructed H3.3 mRNA and expressed the fusion protein. Immunofluorescent staining manifested that the protein successfully locates to nucleus. In this study, we constructed two epitope-tagged h3f3b expression vector and confirmed its expression and location, our work can be useful for the research of H3.3 exchange. Furthermore, this method can be used to study other maternal factors which have the feature of replace its somatic counterpart.

Bacterial pustule is a serious seed-borne disease, which dramatically reduces soybean(Glycine max) yield. The disease becomes more and more serious around the country these years. This research selected house-keeping gene of DNA replication and repair protein (recF) as target gene, and developed a bacterial pustule detection system based on padlock probe combined with dot-blot hybridization. The results indicated that the Padlock probe could detect the bacterial pustule pathogen specifically, and its sensitivity could be up to 100 fg/μL. The system also facilitated the detection of Xanthomonas axonopodis pv. glycines in soybean seedlots with 0.1% artificially infested seed. On the other hand, 45 samples of commercial soybean seeds were detected by using this method, and detected 4 samples positive with the bacterial pustule pathogen. The results indicated that the detecting system based on padlock probe combined with dot-blot hybridization could detect Xanthomonas axonopodis pv. glycines accurately, and provide useful references for prevention and control of bacterial pustule.