Caragana intermedia is a pioneer species for wind-controlling and sand-fixing and distributes widely in arid and semi-arid desert area of north-west China. It has extremely strong resistance to stress and is a good feeding stock. A dehydration responsive element binding protein (DREB) transcription factor encoding gene has been isolated from the dought-treated transcriptome data set. In order to detect the drought and cold resistance of CiDREB1C, expression level of CiDREB1C gene under drought and cold in C. intermedia was detected by qRT-PCR. In order to identify the gene's function, the full length CiDREB1C (GenBank No. MG748598) were obtained through PCR, and was cloned into the expression vector pCanG-HA. Transgenic Arabidopsis thaliana had been obtained by Agrobacterium mediated genetic transformation, and the homozygous transgenic A. thaliana had been screened. The results showed that under mannitol treatment, the overexpression lines accumulated less malondialdehyde (P＜0.01), and had more chlorophyll compared with the wild type, showed obvious drought resistance phenotype. After cold treatment, the overexpression lines showed a significant increase in chlorophyll content compared with the wild type as well (P＜0.01). Under darkness treatment, the leaves of overexpression lines were greener than those of wild type, the cell death was less than that of wild type and the chlorophyll content was significantly higher than that of wild type (P＜0.01). These results suggested that overexpression of CiDREB1C could significantly increase tolerance of the transgenic A. thaliana to drought, cold and dark stress.The results of this research provide a theoretical basis for further analysis the function of CiDREB1C gene in stress resistance and the molecular mechanism of stress resistance of C. intermedia.

Radiation mutation breeding has the characteristics of high frequency of mutation, large variation range and short breeding period, which is one of the main methods of breeding. In order to speed up the work, the previous study used 60Co-γ rays to irradiate Sophora davidii germplasm with the best production performance and carry out radiation mutation breeding. In this study, the variation in twelve M2 mutant populations of S. davidii which were induced by 60Co-γ radiation was evaluated by morphological characters and inter-simple sequence repeat (ISSR) markers. The results showed that compared with the control without radiation, the mutants were characterized with the increase or decrease of plant height and crown diameter, the significant increase of stem diameter, leaf length, leaf width and leaf area, and the reduction of the branch site height. The coefficient of variation of 8 phenotypic traits were between 19.72% and 55.46%. The degree of variation of each index was branching site height (55.46%)＞ plant height (53.24%)＞ crown diameter (47.91%)＞ leaf area (38.30%)＞ number of branches (36.84%)＞ stem diameter (36.10%)＞ leaf width (23.60%)＞ leaf length (19.72%). A total of 141 amplified loci were detected by 20 ISSR primers, of which 75 were polymorphic, and the polymorphic rate (PPR) was up to 53.2%. The Nei's genetic diversity (He) and Shannon's information index (I) were 0.248 3 and 0.377 9, respectively. The genetic similarity index based on ISSR data between the mutants ranged from 0.627 7 to 0.839 4, with a mean of 0.733 6. All mutants were clustered into 4 groups based on the morphological characters or the ISSR molecular markers, but the clustering results between two levels were not fully consistent. This study provides a scientific foundation and germplasms for further radiation breeding of S. davidii.

Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine-threonine phosphatase mediating protein dephosphorylation to regulate the activities of many key pathways. PP2A is a heterotrimeric including catalytic subunit, structural subunit and regulatory subunit. To clone the coding sequence and analyze the character of expression and function of protein phosphatase 2A catalytic subunit α gene (PPP2CA), the specific primers of PPP2CA gene of Banna mini-pig (Sus scrofa) inbred line (BMI) was designed and the coding sequence was amplified using the PPP2CA mRNA sequences of pig and other species from GenBank as reference sequences. qRT-PCR was used to detect expression profiles of tissues mRNA. At the same time, the amino acid sequences were analysed by functional bioinformatics. A complete coding sequence of 930 bp of BMI PPP2CA was obtained, which encoded 309 amino acids. Comparing with other tissues, the PPP2CA gene expression in the urethral ball glands and seminal vesicle appeared extremely significance (P＜0.01). The expression in the testis was higher than other tissues. It was expressed moderately in the liver, colon, spleen, lung, duodenum, prostate, kidney, epididymis and brain, weakly in the stomach, heart and muscle. Functional prediction of PPP2CA protein indicated that the PPP2CA contained one conserved domain MPP_PP2A_PP4_PP6 and four kinds functional active sites, without transmembrane helix and signal peptide. The N-terminal and C-terminal were hydrophilic and located in cytoplasmic with 94.1%. It was predicted that the α helices content of the second structure were the highest which were the major structure of the N terminal, while the random coils were the major structure of the C terminal. The high similarity of PPP2CA amino acids alignment between BMI and other species indicated that this gene was highly conservative in evolution. This research can lay a further foundation for clarifying mechanism of the PPP2CA gene on sperm capacitation.

Chinese tongue sole (Cynoglossus semilaevis) is an economical sea-water species which cultured in coastal areas of China. The development of the Chinese tongue sole industry was impeded due to the high frequency of disease outbreak. In this study, 1 999 and 1 715 individuals from 22 families established in 2016 were selected for challenge test with Vibrio harveyi intraperitoneal injection, respectively. The challenge test lasted 288 h, and ended at 300 h, the survival rate of two tests after injection ranged from 0 to 95.45% and 0 to 87.30%, respectively. The average survival rate of two experiments at end of test were 31.17% and 26.88%. respectively. There were no significant differences between survival rate of two experiments based on results of paired-t test (P=0.05). Two types of records, 0/1 (0 for death, and 1 for survival) and the survival time of individual until death, were used as phenotype to estimate genetic parameters for resistance against V. harveyi in C. semilaevis using three models (binary linear model (BLM), linear model with 0/1 as phenotype; binary threshold model (BTM), threshold (logit) model with 0/1 as phenotype; test-days linear model (DLM) linear model with survival time as phenotype). Besides, correlation analysis and paired-t test were conducted to test differences of predictive ability between three models based on family estimated breeding value (equals averaging of estimated breeding values (EBVs) of individuals of each family). In the present study, resistance against V. harveyi in C. semilaevis belonged to moderate-to-low heritability trait, and the heritability ranged from 0.17±0.05 to 0.36±0.20. The highest heritability was estimated by model DLM (0.36), the lowest was estimated by model BTM (0.17), the result by BLM (0.28) was between them. The Pearson correlation coefficients between family EBVs estimated by three models were 0.94 to 0.97, it revealed strong positive correlation. The three models had same ranking based on family EBVs and the ranking had no significant differences according to results of paired-t test (P=0.05). In conclusion, the resistance against V. harveyi in C. semilaevis belonged to moderate-to-low heritability trait, and there were abundant genetic variations among the selected group. Family selection was a suitable method to select promising Chinese tongue sole strains with excellent ability of V. harveyi. Three models had same ability to estimate EBVs for V. harveyi resistance in C. semilaevis. Besides, this research supplemented relevant studies on estimating genetic parameters of V. harveyi resistance in C. semilaevis, and provided reference for Chinese tongue sole disease-resistance breeding.

Yak (Bos grunniens) has a typical cotyledon placenta which undergoes changes from formation, growth, and maturation during pregnancy, and it is the link between maternal and fetal during pregnancy. It is play an important role in the exchange of nutrients and metabolic waste, hormone secretion. Studying on the relationship between the phenomenon of apoptosis and the main physiological function in the placentome is very improtant for analyzing the mechanism of dynamic development of yak placentome in the angle of apoptosis. Placenta tissues during pregnancy, pre and post parturation of 4 ~ 8-year-old yaks were collected. They were paraffin-embedded and HE dyeinged to observe the changes of placental structure in all stages of pregnancy. The apoptosis of yak placental tissue was observed and analyzed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and 4,6-diamidino-2-phenylindole (DAPI) dyeing technology. The structure of yaks placentomes during pregnancy was constantly changing, so the different stage of gestation could present different morphological characteristics. Yaks had formed a typical structure of the epithelial chorionic placenta in 45 days of pregnancy. With the progress of pregnancy, fetal villi gradually branched and deeply into the maternal caruncula crypts. The number of binucleate cell was increased in placentomes gradually. Caruncula crypt epithelium in 6 months of pregnancy began to appear obviously degenerative changes, such as cell vacuolization. The entire crypt epithelium was not found at pre-parturition. Yak placentomes existed in obvious apoptosis phenomenon. Fetal villi epithelium and maternal crypt epithelium was the main distribution of TUNEL positive cells during gestation and uterine meat Fu matrix had a very small amount of apoptotic cells in early stage of gestation. With the grown of the fetus, TUNEL-positive cells in the two layer epithelial tissue generally showed an upward trend, and the maternal crypt epithelium was significantly lower than the fetal villus epithelium. Fetal villi epithelium was significantly higher in the pre-delivery and achieve maximum after delivery. The experimental study found that yak placentomes processed in a series of changes about the morphological structure from the formation, growth and maturation of physiological. Its apoptosis plays an important role in the normal renewal of cells and structural changes of placental tissue, and the result provides a theoretical data for solving the problems such as abortion appearing in the yak production process.

Soybean (Glycine max) seeds provide an ideal host for the production of foreign recombinant proteins because of their robust capability for biosynthesis and accumulation of the native proteins. To further enhance the expression of foreign proteins in soybean seeds, a polypeptide fusion strategy was utilized to investigate the influence of the maize (Zea mays) γ-Zein fusion tag on accumulation of the foreign recombinant protein. The GFP (green fluorescent protein) and Zein-GFP driven by the soybean seed-specific promoter BCSP were individually introduced into the cultivated soybean genotype by Agrobacterium tumefaciens-mediated transformation, respectively. Totally 41 Zein-GFP and 46 GFP transgenic plants were obtained in this study. Reverse transcription PCR (RT-PCR) and Western blot analysis confirmed transcript and translation of the foreign genes in the transgenic soybean seeds. The transgenic lines with similar expression of Zein-GFP and GFP at mRNA levels in seeds were selected and accumulation of the recombinant proteins was quantified by enzyme linked immunosorbent assay (ELISA). Average accumulation level of the fusion protein Zein-GFP reached to 1.51% TSP (total soluble protein) ranging from 1.37% to 1.60% TSP in the transgenic seeds, increased by 10.78 fold compared with 0.14% TSP of the unfused GFP (0.13%~0.15% TSP). Subcellular targeting analysis by laser scanning confocal microscopy showed that the γ-Zein fused GFP were present in the protein bodies (PBs), while the unfused GFP was mainly distributed in the cytoplasm. Taken together, the results showed that the γ-Zein polypeptide fusion induced the formation of PBs and significantly enhanced accumulation of the foreign recombinant protein in the transgenic soybean seeds, and thus provide the basis for application of the soybean-based bio-reactor.

Feruloyl transferase is one of the key enzymes responsible for transferring ferulic acid from ferulic acid CoA to arabinolxylan molecules, which plays a key role in the connection between arabinoxylan and lignin molecules. Therefore, ferulolyl transferase is closely related to the formation of anti-degradation barrier in plant cell wall. In this study, the cDNA sequence of Bra1 (PlantGDB No.: 5g14720) gene in Brachypodium distachyon was cloned by qRT-PCR technique, and the length of which was up to 1 369 base pairs. The open reading frame (ORF) of Bra1 gene encoded a deduced protein with 443 amino acid residues, with a theoretical molecular weight of 48.45 kD. The prokaryotic expression and mass spectrometry analyses further confirmed that the gene can encode the protein correctly, and the molecular weight was consistent with the theoretical value. Bioinformatics analysis indicated that the amino acid sequence of Bra1 protein contained a HXXXD domain and a DFGWG conserved domain that were unique to the BAHD acyltransferase family, as indicated that Bra1 protein was a member of BAHD acyltransferase family. The phylogenetic analysis of 89 BAHD acyltransferases in Brachypodium distachyon suggested that these acyltransferases could roughly be divided into four subfamilies, each of them containing several small groups, and the Bra1 protein (subfamily I) did not belong to the same subfamily as the other previously characterized BAHD acyltransferases (subfamily Ⅲ) whose functions were mainly related to the metabolism of p-coumalic acid (p-CA), and thus, Bra1 protein may be different in function from those BAHD acyltransferases. In addition, the expression profiles of 10 BAHD acyltransferase genes in the subfamily Ⅲ and the subfamily I which was far related to the subfamily Ⅲ were analyzed by the qRT-PCR, and the results showed that the Bra1 gene was with a higher and more stable expression compared with other genes, and furthermore the expression of Bra1 gene in the mature stem, leaf and spike were almost twice as high as that in young tissues, and its expression character was consistent with the accumulation of ferulic acid (FA) in Brachypodium distachyon suggesting that the Bra1 gene may be involved in regulating the cross-linked reaction mediated by ferulic acid in plant cell walls. This study provides important data for the identification of biological function of Bra1 gene as well as for the development and utilization of grass energy crops and the recycle utilization of crop straw biomass.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis, but the role of GAPC (cytosolic glyceraldehyde-3-phosphate dehydrogenase), which is a cytosolic GAPDH isoform and catalyzes the conversion of 3-phosphoglyceraldehyde to 1,3-diphosphoglyceric acid, against abiotic stresses is largely unknown. In this study, the TaGAPC1 gene encoding 337 amino acids and the TaGAPC1 promoter named P973 were both cloned. We fused the TaGAPC1 to green fluorescent protein gene (GFP) and transformed it into onion (Allium cepa) epidermal cells, the result showed that TaGAPC1 protein localized on the cytomembrane. Quantitative real-time PCR was used to detect TaGAPC1 expression in leaf, root and stem or under PEG8000, NaCl, ABA, and 4 ℃ stresses, the results indicated that the expression levels of TaGAPC1 gene in leaf, root, and stem gradually declined, and TaGAPC1 expression was induced by PEG8000, NaCl, and ABA treatments, but it did not response to 4 ℃ treatment. The PLACE (http://www.dna.affrc.go.jp/PLACE/) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database indicated that some cis-acting elements responsive to abiotic stress were present in P973 promoter, such as Drought-responsive element (DRE), ABA-responsive element (ABRE), MYB-binding site (MBS) and WUN-motif. Based on the position of cis-acting elements in TaGAPC1 promoter, 5 deletions named P844, P738, P605, P475, and P256 were respectively amplified. After the 6 promoters were fused to β-glucuronidase gene (GUS) and transformation into tabacco (Nicotiana batacum) was performed, GUS activity driven by the 6 promoters under PEG8000, NaCl, ABA, and 4 ℃ stresses were determined. The result suggested that TaGAPC1 promoter region from -973 to -605 was essential for the response to PEG8000 and NaCl, the region from -973 to -475 was essential for the response to ABA. The study illustrated the relationship of TaGAPC1 gene with abiotic stress response at molecular level, and lay foundation for further exploring TaGAPC1's molecular mechanism under stress.

Grain weight is an important factor determing the yield of wheat (Triticum aestivum) production, and is one of the main objectives in high-yield wheat breeding. In order to develop molecular markers of potential grain weight related genes in wheat, the TaCYP78A16 (Triticum aestivum cytochrome P450 78A 16, GenBank No.: MH572527) gene was isolated from the whole wheat genome, then polymorphism analysis was implemented in its genome sequence. The results showed that high expression of TaCYP78A16 was detected in young panicles immature seeds by real time PCR analysis expression pattern of TaCYP78A16 and it indicated that TaCYP78A16 might play an important role in grain development and have an impact on grain weight. Polymorphism analysis showed that there were 7 polymorphic loci in 16Ap, which formed 3 haplotypes (16Ap-Hap1, 16Ap-Hap2 and 16Ap-Hap3). A CAPS (cleaved amplified polymorphic sequences) marker, 16Ap-Hap, was developed based on SNP to distinguish the promoter of TaCYP78A16-A alleles. A total of 3 genotypes were detected among the 30 wheat accessions and the 323 nature populations based on the CAPS marker. The functional marker, CAPS-16Ap, can be used in marker-assisted selection breeding in wheat.

Myelocytomatosis protein2 (MYC2) is the core transcription factor of jasmonic acid signaling pathway and involves in plant defense response, secondary metabolism regulation, growth and development. Screening interaction proteins of Salvia miltiorrhiza transcription factor SmMYC2 is helpful to further study the biological function of SmMYC2. The MYC2 gene in S. miltiorrhiza was cloned and constructed into pGBKT7-GW (BD) to obtain the yeast (Saccharomyces cerevisiae) bait expression vector of BD-SmMYC2. The cytotoxicity and self-activation of BD-SmMYC2 were detected. The results showed that SmMYC2 has strong self-activation activity. In order to analyze the self-activation region, the N-terminal region (including the bHLH-MYC-N superfamily domain) and the C-terminal region (including the HLH domain) of SmMYC2 were cloned and constructed into the yeast expression vector BD. The transcription activity domain of the SmMYC2 was analyzed through detecting the transcription activity of the N-terminus and C-terminus of the SmMYC2. The SmMYC2-N-BD yeast turned blue on SD/-Trp/X-α-gal medium and could grow on SD-Trp-His and SD-Trp-Ade mediums, However, the SmMYC2-C-BD yeast was colorless on SD/-Trp/X-α-gal medium and no growth on SD-Trp-His and SD-Trp-Ade medium. These results indicated that the N-terminus of SmMYC2 has obvious self-activation activity and the C-terminus of SmMYC2 had no self-activation activity. The interactive proteins were screened from cDNA library of S. miltiorrhiza using yeast two-hybrid system through the bait vector of SmMYC2-C-BD. Function annotation showed that these candidate interaction proteins included WRKY protein containing the WRKYGQK domain, the peroxiredoxin reductase of Prxs protein family and succinate semialdehyde dehydrogenase. The experimental results provide basic date for further studying on the interaction proteins of MYC2.

Neurotrimin (NTM) plays an important role in the nerve development. The NTM is an imprinted gene in human (Homo sapiens), but the imprinting state in cattle (Bos taurus) has not been revealed. The purpose of present study is to analyze the sequences of cattle NTM gene, the structure of its coding protein, and to identify the imprinting status of NTM gene in adult tissues and placenta in cattle. A new splice NTMxd (GenBank No.: MH037574) of cattle NTM gene was obtained by the technology of rapid amplification of cDNA ends (RACE), and a more complete transmembrane signal was found in the N-terminal of amino acid sequence encoded by the splice. In order to analyze the imprinting status of NTM gene in cattle, a locus of single nucleotide polymorphism (SNP) was found in cattle NTM gene by direct sequencing of PCR products, the accession number of the SNP locus in NCBI website (www.ncbi.nlm.nih.gov) is rs42185569. The total RNA was extracted from the tissue samples and placentas of heterozygous individuals, and performed expression analysis by reverse transcription-PCR (RT-PCR). Direct sequencing of the RT-PCR amplified products revealed that NTM gene exhibited monoallelic expression in cattle placenta and adult tissues, indicating that NTM gene is an imprinted gene in cattle. The results of present study provides basic data for further studying of NTM gene function and related neurodevelopmental diseases.

The water sources in the livestock areas in the central and western parts of northern China are mostly high-salt water sources. Ordinary livestock cannot adapt to such water sources, thus affecting the development of animal husbandry in the northern regions. Bactrian camel (Camelidae bactrianus) has a species capable of adapting to high salt water source in the extreme environment of northern desert, but its adaptation mechanism is still unclear. In this study, bactrian camel's salt adaptability was concerned and bactrian camel colon tissue under salt stress conditions and normal conditions was sequenced to understand the mechanism of adaptability of camel's colon tissues to a salt stress environment. The results showed that 52 882 246 reads were measured in the test group and 51 533 346 in the control group; the total mapped rate of reads in the test group was 86.41%, the uniquely mapped rate was 84.61%, the total mapped rate of reads in the control group was 92.48%, the uniquely mapped rate was 91.62 %; 2 450 differentially expressed genes were obtained, among which 1 136 genes were up-regulated and 1 314 were down-regulated. Gene Ontology (GO) analysis results showed that there were 17 terms that had significantly down-regulated expression of genes and 28 terms that had significantly up-regulated expression of genes. There was no statistically significant enrichment term in the up-regulated expression of genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the down-regulated expression genes were significantly enriched to include spliceosome, RNA transport, basal transcription factors, ribosome biogenesis in eukaryotes, RNA degradation，oxidative phosphorylation and protein export; up-regulation of expression of genes were not found in enriched pathways. The above results indicated that genes with down-regulated expression in colon were more than up-regulated expressed genes in salt stress. Statistically significant biological function analysis showed that down-regulated expression genes were involved in RNA processes and protein synthesis pathways. These genes were favorable for reducing RNA synthesis and reducing the metabolic rate of colon tissue. This study analyzes the salt adaptation mechanism of regional characteristic livestock breeds, and provides salt adaptation molecular theory for breeding, thereby improving regional resource utilization.

Major histocompatibility complex (MHC) and interleukin (IL) are important immune molecules, in which MHC classⅠ molecules present endogenous antigen and induce initial immune response, and ILs are critical immune regulators. Little information about relationship between chicken (Gallus gallus) MHC classⅠ(also named as B-Fα) and IL genes is reported. The present study focused on effects of downgraded expression of B-Fα on IL genes using the method of gene silencing. First, according to the sequence of B-Fα gene and the structure require of shRNA (short hairpin RNA) gene sequence, 3 shRNA fragments with potential gene silencing were selected and synthesized, and inserted into lentiviruse expression plasmids pLL3.7 with restriction enzyme, respectively. The 3 recombinant plasmids were then transfected into 293T cells, which could express label protein in the cells and also be packed into lentiviruses. The recombinant viruses could infect HD-11 cells with efficiency of over 95%, and the gene transcription levels in the HD-11 cells detected by qRT-PCR were downgraded to 39%, 97% and 61% respectively. Finally the effects of B-Fα gene silencing on the transcription levels of cytokine genes IL-2, IL-4, and IL-6 were detected with recombinant lentiviruses (containing pLL-shB-Fα-257), which had the strongest silencing (downgraded to 39%). The results showed that in the cells with down regulated B-Fα gene, the transcription levels of IL were influenced: IL-2 was down-regulated (P＜0.01), IL-4 was up-regulated (P＜0.05), and IL-6 had no change (P＞0.05). All above results indicated that B-Fα gene transcription could be interfered by the gene silencing method, while down regulation of B-Fα gene expression influenced IL transcription level. These suggest that there might be critical association between B-Fα genes, which inducing cellular immune responses, and cytokine genes which are the downstream regulatory factors. The present results provide basic data for further study about regulation mechanism of immune responses.

Jiangsu Province possesses a large number of excellent indigenous chicken (Gallus gallus domesticus) resources. These breeds have been selected for an array of traits including better meat quality, better resistance to disease, and better adaptability to intensive management systems making them good candidates for increased crossbred production. The objective of this study was to determine the genetic diversities and population structures of chickens that are traditionally raised in Jiangsu province. To address this question, the complete mitochondrial DNA D-loop sequence of 149 chickens from 5 native breeds (Black langshan, White langshan, Luyuan, Liyang, and Taihu) of Jiangsu province were analyzed . Sequences read lengths of the native breeds were 1 231 to 1 232 bp, with a single-base deletion from the 859 bp site in the 1 231 bp haplotype. A total of 33 variable sites that defined 19 haplotypes were identified. The average haplotype diversity and nucleotide diversity were 0.862±0.017 and 0.005 91±0.001 35. The median network showed that genetic structure of the mtDNA haplotypes of Jiangsu chickens are distributed across 5 clades (haplogroups): Clades A, B, C, D, and E. However, most of the individuals characterized in this study belonged to clades A and B. The analysis of molecular variance (AMOVA) showed that genetic differentiation indexes F-statistics (Fst) was 0.334 97(P＜0.01), genetic variation within and between populations accounted for 66.5% and 33.5% of the total genetic variation respectively. The results of this study indicated that Jiangsu chicken populations have relatively low nucleotide and haplotype diversity and likely share 5 common maternal lineages, but there were no obvious differentiation between them, and that some chicken populations may have been mixed with exotic lineage chickens. The results of the current study can be used as baseline genetic information for genetic conservation program and in breeding of Jiangsu chickens.

The warm temperature acclimation related 65 kD protein-2 (Wap65-2) is a glycoprotein and so far only found in teleost fish. The expression pattern of Wap65-2 discloses its function in response to the stresses of temperature, infection and heavy metal contamination, and especially the function in anti-bacterial immunology attracts broad attention in recent years. In this study, the recombinant ayu (Plecoglossus altivelis) Wap65-2 mature peptide (rPaWap65-2m) was expressed in an insect baculovirus vector system (BEVS), by which we sought to explore a new way to produce biologically active rPaWap65-2m. The recombinant Bacmid-PaWap65-2 was obtained by cloning, and then used to transfect Spodoptera frugiperda cells (Sf9) for the viral stock. Sf9 cells in exponential growth phase were infected with high-titer P3 viral stock and then detected the expression of rPaWap65-2m by Western blot. Sf9 cells were cultured in optimized large scale serum-free system for rPaWap65-2m expression. rPaWap65-2m was purified by anion-exchange chromatography and nickel chelate affinity chromatography from the collected culture supernatant. The interaction between rPaWap65-2m and Complement 3 (C3) was identified by His-tag pulldown method. Results showed that after P3 viral stock infected Sf9 cells, rPaWap65-2m was expressed and secreted into the culture supernatant as a non-glycoprotein detected by Western blot. The maximum expression was obtained when Sf9 cells were infected with the multiplicity of infection (MOI) of 5 and collected at 72 h after infection. The purity of rPaWap65-2m was greater than 93%. rPaWap65-2m was verified to interact with full-length nature C3 by pulldown. In summary, the biologically active rPaWap65-2m was obtained for further study on its immunological function.

Celery (Apium graveolens) is one of the most consumed leaf vegetables worldwide. With the rapid expansion of the cultivation area of greenhouse vegetables in recent years, the year-round production, continuous cropping, etc. lead to the deterioration of the environment which may cause the emergence of new features of plant disease. In this study, a strain QC02 was be isolated from the celery rotted samples of Changping district in 2015. Based on the analysis of morphology, physiology and biochemistry, the 16S rDNA sequence as well as the average nucleotide identity (ANI) of the genome sequence, a comprehensive identification for QC02 was conducted. The results showed that the QC02 colonies on KB medium were milky white, circular, smooth in surface with even edges, and produced fluorescent pigment under the UV light, and produced pits on CVP (cavity formation on crystal violet pectate) medium. By artificial inoculation, the strain could infect celery and cause stalk rot symptoms, which were similar to those in the fields. The bacterium could also infect potato (Solanum tuberosum), carrot (Daucus carota), garlic (Allium sativum), and onion (Allium cepa). The PCR amplification with Pseudomonas-specific primers Ps-F/Ps-R produced the targeted fragment from QC02 and the Pseudomonas spp. The LOPAT characteristics of this bacterium were in accordance with those of the previously reported Pseudomonas marginalis. Biolog test also identified QC02 as P. marginalis. Among the 13 Pseudomonas type strains whose entire 16S rDNA gene sequence had the over 99% similarity with that of QC02 (GenBank No. MG765472), the characteristic of average nucleotide identity (ANI) based on the whole genome sequence from these strains showed that only the ANI values between QC02 and P. marginalis ICMP 3553T were as high as 98.46%, which was higher than the threshold value (95%) between intra- and inter-species. These results revealed that the strain QC02 causing bacterial rot disease on celery is P. marginalis. This study is the first report about the occurrence of Pseudomonas marginalis on celery in China, and provides a foundation for the disease control measures as well as celery breeding for disease resistance.

Peste des petits ruminants virus (PPRV) is the causative agent of Peste des petits ruminants (PPR). Nectin-4 is the receptor of PPRV-infected host epithelial cells. In this study, Nectin-4 gene was synthesized to construct the recombinant plasmid pLOV-eGFP-Nectin-4. To obtain retrovirus-like particles,the recombinant plasmid was co-transfected into 293T cells with the packaging plasmid pSPAX2 and the outer membrane plasmid pMD2.G. To construct the MDBK (Madin-Darby bovine kidney) cell line stably expressing Nectin-4 receptor, the MDBK cells were firstly infected with the supernatant of cell culture which containing the retrovirus-like particles, and then screened and purified using puromycin. The MDBK cell line stably expressing Nectin-4 protein was obtained, which was named as MDBK-Nectin-4. The fluorescence of 293T cells co-transfected with pLOV-eGFP-Nectin-4, pSPAX2 and pMD2.G, respectively. Plasmids showed that the lentivirus was successfully packaged. The results of the minimal lethal concentrations of puromycin for MDBK cells showed that the best screening concentration of puromycin was 1 μg/mL. RT-PCR analysis showed that the Nectin-4 gene could be transcribed into mRNA in MDBK-Nectin-4 cells. The results of Western blot assay showed that Nectin-4 protein could be expressed stably in the MDBK-Nectin-4 cells. The results of indirect immunofluorescence assay (IFA) demonstrated that the expressed Nectin-4 protein was mainly distributed on the cell membrane. After continuous passaged to the 20th generation, the MDBK-Nectin-4 cell line still stably expressed the Nectin-4 protein, which indicated a good genetic stability of this cell line. MDBK-Nectin-4 cell line infected with PPRV was more rapidly cytopathic than the MDBK cells. The established cell line provides experimental materials for further investigation of the interaction mechanism between PPRV and receptor Nectin-4, as well as clinical rapid isolation of PPRV.

The genotyping system based on short tandem repeats (STR) and sex locus can not only provide important molecular genetic marker information, but also effectively solve the problem of individual identification and genetic relationship in animals. To establish a multiplex detection system for STR and CHD (chromobox-helicase-DNA binding gene) of pigeon (Columba livia domestica) and evaluate its forensic application value, in present study, a 6-dye fluorescence-labelling multiplex PCR amplification system for 19 STR (short tandem repeats) loci and 1 CHD sex locus was established to evaluate its forensic application and population research. In total, CliμD11, PG4, PG1, PG2, CliμT02, CliμD17, CliμD35, CliμT17, CliμD16, PIGN04, CliμD32, PIGN57, PIGN26, PG5, PG6, CliμD19, PIGN12, PIGN15, PIGN10, and gender-identification gene CHD were picked up. The results showed that the multiplex amplification system was reliable and easy for identification via feather examples from 241 pigeons. It had been proved that the sensitivity could be low to 0.0625 ng DNA and the species identification was rather specific. The statistic analysis of all alleles of 19 STR loci in the pigeon population showed that all 19 STR loci could be qualified to be used in individual identification and paternity test. Finally it was proved that the pigeon STR loci multiplex amplification system established in present research was rather stable, sensitive and species-specific, which was the most effective pigeon detection system and the first report in China as well. The present study research provides molecular basis for individual identification and paternity testing in pigeons.

Foxtail millet (Setaria italica) isan important cereal crop in China, its nutritional qualities have been gradually attracted by people. With the development of biotechnology, foxtail millet has gradually become a new hot-spot in genetic improvement and functional genomic research, due to its hereditary features and small genome. In this paper, the breeding process and its application of the sterile line in millet are reviewed. Meanwhile, the formation of male sterility and related genes in millet are explained from different levels. Finally, the states of heterosis utilization in millet are summarized, and the future researches on the heterosis in millet are prospected consulting the results of other crops of Gramineae. The review can lay a theoretical foundation for study on utilization and mechanism of heterosis in foxtail millet.

The testes are the most important reproductive organs in male mammals as they are the site of testosterone production and spermatogenesis. Normal testicular structure and physiology are the basis for reproductive activity. Proteomics is one of main methods to study proteome at post-genomics era, which may reflect the dynamic nature of proteins in whole tissue or cell. Thus it is of great significance to explore the protein compositions of testis by proteomics techniques. In this review, we mainly focus on the technological means of quantitative proteomics, as well as its application in the studies of testicular development, and responses to diseases conditions, which provides insight into the further research on genes and regulatory networks that control testicular development and size.