WRKY transcription factors constitute a class of proteins involved in growth, development, stress and defense responses in plants. In this study, the cDNA sequence of WRKY69 was cloned through reverse transcription PCR (RT-PCR) from oilseed rape (Brassica napus) (GenBank No. KC246583). Sequence analysis indicated the encoded protein had 279 amino acids with a single WRKY domain and a C2H2-type zinc finger motif, which belonged to GroupⅡ. Yeast transcriptional activity analysis showed that BnaWRKY69 might have transcriptional activation activity, and its activity was mainly determined by the carboxyl terminus. GFP-based subcellular localization showed that BnaWRKY69 was located not only in the nucleus but also at the cell membrane. A prediction of BnaWRKY69 protein sequence suggested it might undergo 2 types of post-translational modifications, myristoylation and farnesylation, which possibly determined its special membrane localization. Under hydroxylamine (NH₂OH) treatment, the BnaWRKY69 signals at the plasma membrane decreased gradually and even disappeared. The transcriptional level of BnaWRKY69 under various abiotic stresses, hormone and Sclerotinia sclerotiorum treatments was analyzed by quantitative real-time PCR (qRT-PCR). It was found that BnaWRKY69 was specifically induced by jasmonic acid (JA) and S. sclerotiorum. Further detection by qRT-PCR showed that transient overexpression of BnaWRKY69 gene in tobacco (Nicotiana benthamiana) leaves caused significant up-regulation of Pathogenesis-related 1a (PR1a), PR2 and Non-expressor of PR1 (NPR1) and down-regulation of Plant defense 1.2 (PDF1.2). In conclusion, this study preliminarily analyzed the basic features of BnaWRKY69 in different hormones and abiotic stress treatments and molecular mechanism responding to JA and S. sclerotiorum, which could provide a reference to breed oilseed rape varieties with improved disease resistance.

Transcription factors play important roles in the response of plants to various stresses. The transcription factor family of bZIP (basic domain leucine zipper) is involved in the defense response against pathogen infection. In this study, the expression pattern of TabZIP3 (Genbank No. BAD97365.1), a gene from the bZIP transcription factor family was obtained by screening the transcriptome database of the wheat (Triticum aestivum) infected by leaf rust fungus (Puccinia triticina). The expression patterns were visualized using heatmap, and confirmed by qRT-PCR. The expression of TabZIP3 was up-regulated after 8, 12 and 16 h post inoculation in the incompatible combination, and was obviously higher than that of the compatible combination (P<0.05). TabZIP3 was predicted to contain salicylic acid (SA) response elements, and the expression could be induced by spraying exogenous SA on the leaf surface. By using Barley stripe mosaic virus (BSMV) as a vector, the virus-induced gene silencing (VIGS) technique was used to silence TabZIP3, followed by Puccinia triticina inoculation. Hypersensitive response (HR) area and the number of haustorial mother cell (HMC) of single infection sites was much higher than that of control at 48 and 96 h (P<0.05). The research results showed that the SA inducible TabZIP3 might participated in the induction of basic defense response against Puccinia triticina infection in wheat, and might played a positive regulation role in HR response induced by Puccinia triticina. The results lay a foundational data for further study on the mechanism of the transcription factor TabZIP3 in wheat resistance to Puccinia triticina infection, and also provide an excellent candidate gene for breeding new wheat varieties resistant to Puccinia triticina.

Drought stress is a major limiting factor affecting high and stable yield of crop and the transcription factors (TFs) play crucial roles in plant response to drought stress. In this study, 20% PEG6000 was used to simulate drought stress in the seedling stage, and the changes in the expression of expressed transcription genes in maize (Zea mays) leaves under drought stress for 60, 96 h and re-watering after 3 d were analyzed by RNA-sequence (RNA-seq) technology. The results showed that, a total of 56 transcription factor families were detected in transcriptome sequencing, with a total of 2 270 transcription factor genes, and the number of differentially expressed transcription factor genes was 556, accounting for 24.49% of the total. Compared with the control, the number of differentially expressed transcription factor genes was the lowest under drought stress at 96 h. After re-watering 3 d, the number of differentially expressed transcription factor genes was the highest and it was mainly down-regulated. Among the differentially expressed TF genes, members of the bHLH (basic helix-loop-helix), C2H2, ERF (ethylene responsive factor), MYB (v-avian myeloblastosis viral oncogene homolog), NAC (NAM, ATAF1/2, CUC1/2) and WRKY families were more abundant. According to the Venn plot analysis of differentially expressed transcription factor genes, a total of 37 transcription factor genes were differentially expressed at 3 treatment points under drought stress and rewatering, distributed among 15 transcription factor families, of which the number of WRKY family was the most enriched. The results would provide theoretical basis for further exploring the molecular response mechanism of maize transcription factor family to drought stress.

Plant glycosyltransferases (GTs) are a large class of enzymes that modify the secondary metabolites for glycosylation. GTs can convert photosynthesis products into disaccharides, oligosaccharides and polysaccharides, and can also catalyze some important products including cell wall polysaccharides, glycoproteins, and many different types of small molecular species. GTs can promote the glycosylation of certain hormones and signaling molecules, and thus participate in signal regulation.In order to study the regulation pattern of the Eucommia ulmoides glycosyltransferase gene (EuCGT1), this study obtained a length of 1 671 bp upstream of the 5' end of EuCGT1 gene of E. ulmoides. The Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) predictive analysis revealed that the fragment contained the eukaryotic promoter core elements TATA-box and CAAT-box. At the same time, the fragment also contained a plurality of induction-related cis-acting elements such as a methyl jasmonate response element TGACG-motif, a drought-related MBS element, a gibberellin response element GARE-motif, and an auxin response element TGA-element. The promoter fragment of different lengths was used to regulate the expression of GUS gene to construct plant expression vector, and the activity and function of EuCGT1 promoter in transgenic tobacco (Nicotiana tabacum 'Xanthi') were explored. It was found that the core region of the EuCGT1 promoter was mainly located in the -268~-1 bp upstream of the gene. The GUS histochemical staining of roots, stems and leaves of transgenic tobacco and the expression analysis of EuCGT1 gene in E. ulmoides showed that the promoter of this gene was more likely to be expressed in leaves. After hormone induction and environmental stress in E. ulmoides, the expression of EuCGT1 was regulated by methyl jasmonate, auxin, gibberellin and high salt. The results of this study may provide a reference for further revealing the function of the EuCGT1 promoter of E. ulmoides and the promoter-mediated regulation of gene expression.

PRKAA1 (protein kinase, AMP activated α1) plays a very important regulatory role for the processes of fat metabolism, cell proliferation, fat formation, etc. In this study, Guizhou fatty pig breed Congjiang Xiang pig (Sus scrofa) and commercial pig breed Yorkshire pigs (S. scrofa) were taken as research objects. Based on the 3'UTR sequence of PRKAA1 (GenBank No.XM_021076521), NCBI and NONCODE (http://www.noncode.org/) were used to screen the LncRNAs related to PRKAA1. The screened LncRNAs were cloned, and the secondary structure, ORF, target site of PRKAA1 and regulated miRNAs were predicted. qRT-PCR was used to detect the screened LncRNAs expression in different tissues of Congjiang Xiang pigs and Yorkshire pigs. Two candidate LncRNAs, NONSUST030689.1 (named ALNC1) and NONSUST004358.1 (named ALNC2), were selected for next experiments. The sequenced results revealed that ALNC2 had 46 bp inserted compared with original sequence. ALNC1 had 4 ORFs, and ALNC2 had 5 ORFs. In addition, ALNC1 and ALNC2 each had 1 target site for PRKAA1. Predicted miRNAs which could bind with LncRNAs including miR-1226-5p, miR-487b-5p, miR-7156-5p, etc. The results of qRT-PCR showed that the 2 LncRNAs had universal expression in all tissues of Congjiang Xiang pig and Yorkshire pig and were significantly different among different tissues. In Congjiang Xiang pig, ALNC1 and ALNC2 were the highest expressed in the liver and spleen and the lowest in the longissimus dorsi muscle. But ALNC1 and ALNC2 were the highest expressed in the large intestine and lung and the lowest in the longissimus dorsi muscle in Yorkshire pig. ALNC1 had significant higher expression in liver, spleen, small intestine, fat, and longissimus dorsi muscle of Congjiang Xiang pigs than that of Yorkshire pigs (P<0.01), but lower expression in heart and large intestine than that of Yorkshire pigs (P<0.01), and no significant difference in kidney and lung. ALNC2 had significant higher expression in liver, kidney and longissimus dorsi muscle of Congjiang Xiang pigs than that of Yorkshire pigs (P<0.01), but lower expression in heart, lung, small intestine, and large intestine (P<0.01). In fat tissues, ALNC2 expression levels of Congjiang Xiang pig were significantly lower than that of Yorkshire pigs (P<0.05). The ALNC2 expression levels in spleen had no significant difference between Congjiang Xiang pigs and Yorkshire pigs. The present results could provide basic data for further studies on the regulatory role of PRKAA1 and LncRNAs in fat deposition process of pigs.

In the cattle industry, the appendages such as horns have become undesirable because they are dangerous to the breeders and animals. The polled (hornless) condition in cattle has existed since domestication, and it has been selected because of its economic importance and ease of management. A single dominant mutation is considered to be the cause of polled phenotype, called POLLED locus. But in realistic production, there are few cattle with this site. In order to produce polled phenotype more efficiently, the present study used CRISPR/Cas9 gene editing technique to mediate knock-in of P_202ID genotype in POLLED locus of horned Mongolian cattle (Bos taurus). First, four pairs of single guide RNA (sgRNA) targeting to bovine POLLED site, named as sgRNA1~4, were designed, and inserted into the pCas-Guide-EF1α-GFP plasmid. The results of surveyor assay showed that the mutant efficiency for 4 vectors amounted to 24%, 26%, 55%, and 17%, respectively. pCas-Guide-EF1α-GFP-sgRNA3 vector had the highest activity in the vectors. The high active vector and targeting carrier containing P_202ID sequence were co-transfected into Mongolian cattle fetal fibroblasts by electrotransfection. The positive single cells were separated by flow cytometry and further cultured into monoclonal cells. By PCR amplification and sequencing, 4 strains of heterologous recombinant monoclonal cell lines were obtained, which could be used as a donor cell for subsequent somatic cell nuclear transfer (SCNT). This study could provide experimental materials and technical support for cultivation of POLLED cattle with SCNT, and contribute important materials for the understanding of the mechanism of horn origin and development.

Light is one of the crucial environmental factors affecting the reproductive performance of female rabbits (Oryctolagus cuniculus). This study aimed to reveal the influence of artificial light on the reproductive system development of prepubertal female rabbits as well as the underlying mechanisms. A total of sixty 48-day-old female Tianfu black rabbits were equally divided into the light and control groups. After 10 d of acclimation, the light group was treated with artificial light for consecutive 12 d (16 h per day), while the control group was fed under natural light. After treatment, 6 rabbits randomly selected from each group were slaughtered. Thereafter, the relative weight of reproductive organs was recorded, the concentrations of plasma melatonin (MLT), estradiol (E₂), prostaglandin E₂ (PGE₂), and progesterone (P₄) were measured using ELISA, histological changes in the ovary, oviduct, and uterus were observed using hematoxylin & eosin (HE) staining, and qRT-PCR was used to detect the relative expression of the MLT receptor (MTNR1A) gene, the P₄ receptor (PR) gene, the E₂ receptor (ERA and ERB) genes, and the PGE₂ receptor (EP1~4) genes in the reproductive system. The results showed that compared to the control group, artificial light significantly increased the weight of bilateral ovaries, oviducts, and uteruses as well as the relative weight of bilateral oviducts and uteruses (P<0.05). Histological results showed that both tertiary and mature follicles were present in the ovaries treated with artificial light but were absent in the control group. Meantime, artificial light increased the thickness of the endometrium, the muscular layer, and the serosa of either the oviduct or uterus. In addition, compared to the control group, artificial light decreased plasma levels of MLT, increased those of PGE₂ and P₄, and significantly decreased those of E₂ (P<0.05). Finally, the mRNA levels of MTNR1A and ERB in both the ovary and uterine body, PR in all reproductive organs, EP1~3 in the ovary, EP1 and EP3~4 in the uterine horn, and EP2~4 in the oviduct were significantly or extremely significantly induced by artificial light (P<0.05 or P<0.01). These results indicated that artificial light could influence plasma levels of reproduction-related hormones through both its indirect effects on the hypothalamic-pituitary axis and direct actions on the reproductive system, which promoted the development of the ovary, oviduct, and uterus of prepubertal female rabbit by further activating respective signaling transduction pathways. This study provides the theoretical basis and fundamental information for elucidating the mechanisms initiating puberty in female rabbits.

Previous study had shown that Wnt family member 5A (Wnt5a) mediated Wnt/β-catenin signal was significantly enriched in the formation of chicken (Gallus gallus) male germ cells by high-throughput sequencing. To investigate the role of Wnt5a-mediated Wnt/β-catenin signaling pathway in the differentiation of chicken embryonic stem cells (ESCs) into male germ cells, a chicken ESCs line stably interfering with Wnt5a gene was established by Lentivirus mediated RNA interference technique. Three interference target sequences and one negative control sequence were designed for Wnt5a gene and recombined with the pGMLV-SC5 lentivirus vector. Chicken DF-1 cells were infected with lentivirus recombinant vector, and the expression of Wnt5a gene was detected by qRT-PCR to determine the interference efficiency of each interference vector, and the recombinant vector with the best interference effect was wrapped with lentivirus. Chicken ESCs were infected with lentivirus wrapped with interference vector, and the expression levels of Wnt5a and Wnt signaling pathways key genes: Axis inhibition protein 2 (Axin2), Beta Catenin (β-catenin), phospholipase C (PLC) and ubiquitin specific peptidase 54 (USP54) were detected by qRT-PCR and Western blot. At the same time, a Wnt5a overexpression vector (pCDNA3.0-Wnt5a) was constructed to carry out the rescue experiment of Wnt5a gene. The recombinant plasmid of Wnt5a-shRNA1, Wnt5a-shRNA2 and Wnt5a-shRNA3 was successfully constructed, and the inhibition effect of the Wnt5a-shRNA2 was the best, and the interference efficiency was up to 80% (80%±0.02). The results of qRT-PCR showed the Wnt5a-shRNA2 recombinant plasmid was packaged into a retrovirus with a virus titer of 5×10⁸ TU/mL. After transfecting Wnt5a-shRNA2 lentivirus into chicken ESCs, The results of qRT-PCR and Western blot showed that Wnt5a mRNA and protein expression levels in the interference group were significantly lower than those in the control group (0.16±0.03 vs 1.03±0.02)(P<0.05).The expression levels of key genes Axin2 and β-catenin downstream of the Wnt signaling pathway were significantly down-regulated (0.41±0.08 vs 1.03±0.05; 0.25±0.04 vs 1.03±0.05)(P<0.05). At the same time, the Wnt5a overexpression vector (PCDNA3.0-Wnt5a) was transfected into the interference group, and the expression level of Wnt5a in the interference group was recovered (1.19±0.07 vs 1.03±0.02). In this study, a lentiviral vector expressing shRNA targeting chicken Wnt5a was successfully constructed, which could effectively silence the expression of Wnt5a gene in chicken DF-1 cells and ESCs. Moreover, ESCs lines stably expressing Wnt5a-shRNA2 could interfere with Wnt signaling transduction. These results will provide a theoretical basis for further research on the function of Wnt5a gene and its mediated Wnt signaling pathway at the cellular level.

Fatty acid desaturases (FADS) play an important role in the biosynthesis of polyunsaturated fatty acids, and their expressions are affected by many factors. Bioinformatics softwares were used to systematically analyze the collinearity, phylogenetic relationship, gene structure, conserved motifs and functional domains of chicken (Gallus gallus) FADS gene family members. Real-time quantitative PCR (qRT-PCR) was adopted to analyze the spatio-temporal expression of FADS gene family. The effects of estrogen and trophic factor on the expressions of the FADS gene family members in vivo were also investigated. The research results showed that FADS3 gene might not exist or annotated in poultry, while FADS1L1 and FADS1L2 were only found in poultry. The FADS6 located chromosome segment region was conserved among species. Phylogenetic analysis showed that the FADS6 genes of different species were clustered into one branch, and other members were clustered into another branch. The gene structure analysis and conserved motif analysis showed that the FADS6 gene was greatly different from other members of the family in exon number and the composition of conserved motif among species. The protein domain prediction results indicated that except FADS6, FADS1, FADS2, FADS1L1 and FADS1L2 genes owned the same functional structural domain. The tissues expression analysis showed FADS gene family members were widely expressed in different tissues. FADS1, FADS2 and FADS6 were relative highly expressed in the liver, FADS1L1 in the pectoral muscle. and FADS2L2 in the pancreas. The expression of FADS gene family in the liver of hens at the 30-week-old was significantly higher than that at 20-week-old (P<0.05). In vivo assays displayed that 17β-estradiol could significantly up-regulate the expressions of FADS1, FADS2 and FADS1L1 in chicken liver (P<0.05); and the addition of 8% soybean oil in the basal diet could significantly increase the expression of FADS2 in the chicken liver (P<0.05). In summary, this study comprehensively and systematically analyzed the biological characteristics of FADS gene family, and the effects of 17β-estradiol and soybean oil on their expressions which laid a foundation for further studying on the mechanism of FADS gene family.

Jinding duck (Anas platyrhynchos domestica) is a good breed of laying duck in China. It has many advantages, such as more eggs, strong resistance to stress and so on. It is excellent materials for breeding of new breed (matching line) of laying duck. In this study, 487 Jinding ducks were selected as the research objects, and the ovoinhibitor gene (OIH) and gonadotropin inhibitor gene (GnIH) were detected by DNA direct sequencing for SNP and genetic analysis, and the correlation with the age at first egg, egg numbers at 300 d and 500 d old was analyzed. The results showed that there were 7 polymorphic loci in the OIH gene exon and 4 polymorphic loci in the GnIH gene exon of Jinding duck, and all the loci produced 3 genotypes, 7 of which were missense mutations. The results of genetic analysis showed that all the 11 loci were in Hardy-Weinberg equilibrium (P>0.05), 8 of them were moderate polymorphism (0.50>PIC>0.25), 3 of them were low polymorphism (PIC<0.25). The results of linkage disequilibrium analysis showed that G2344A and T2363C, C2385T and C5749T were complete linkage disequilibrium, G1946A, T1949C and T2218C were complete linkage disequilibrium between any two of them. The results of correlation analysis showed that the C469T locus of OIH gene was significantly correlated with the age at first egg and egg numbers at 300 d old (P<0.05), and AA was the dominant genotype; A517G, C2385T, C5749T and G5801A loci of OIH gene had significant influence on the age at first egg (P<0.05); C2089T locus of GnIH gene was significantly correlated with the egg numbers at 500 d old (P<0.05). In this study, SNPs with genetic effect on laying performance of Jinding duck were screened, which could provide reference for the establishment of candidate genes of laying performance and molecular marker assisted selection.

Burbot (Lota lota) is a cold-water fish with economic value, which is the only freshwater species of Gadiformes. Therefore, it is an important material for studying the evolutionary mechanism of temperature and salinity adaptation. The transcriptome of brain, kidney and liver tissues from L. lota were sequenced based on Illumina Hiseq-2500 high throughput sequencing platform. A total of 20.78 Gb clean data were obtained after removing raw reads by quality control. The high quality clean reads of different tissues were 22 254 637 (brain), 22 843 364 (kidney) and 24 168 330 (liver), respectively. 106 084 unigenes with the average length of 706 bp were assembled by Trinity software. The unigenes were subjected to annotation analysis by matching sequences against related databases. The results showed that 32 745 (30.87%) of these unigenes were significantly matched. In addition, all annotated unigenes were screened against the Gene Ontology (GO) database, in which all the unigenes were divided into 3 categories with 56 branches. The unigenes were divided into 25 categories according to Cluster of Orthologous Groups of Proteins (COG) function classification, and were grouped into 6 categories with 195 classes based on KEGG database. The transcriptome sequencing data and functional annotation in this study will be expected to provide reference materials for the further exploration of the functional genetic resources for L. lota in the future.

Agrilus zanthoxylumi is an important trunk-boring pest on the Zanthoxylum bungeanum, which lays a foundation for understanding the mechanism of olfactory perception and studying the role of chemosensory proteins (CSPs) in its chemical sensory system. Based on the transcriptome sequencing results of the team, cloning of the cDNA of AzanCSP3; and the signal peptide, isoelectric point, molecular mass, and three-dimensional structure of the signal peptide were predicted by using online tools (Expasy; Signal P4.1; Swiss model); the homology was compared by using BLAST, the phylogenetic tree was constructed using a neighbor-joining method with MEGA 10.0 software, and the affinity between AzanCSP3 and CSPs from other 11 Coleoptera species was analyzed. The correct recombinant plasmid of prokaryotic expression vector pET28a(+) was ligated and cloned into Escherichia coli BL21 competent cells and expressed by isopropyl β-D-thiogalactoside (IPTG), the expression of the target protein was analyzed by SDS-PAGE, and using Western blot to detect whether the expressed protein was the target fusion protein. The results showed that the cDNA sequence of a chemosensory protein of A. zanthoxylumi was cloned and named as AzanCSP3. Consisted with the results of the transcriptome data, the full-length cDNA was 846 bp, contained a complete open reading frame of 396 bp, encoding 132 amino acids, the molecular weight of the mature protein was 15.038 kD, the isoelectric point was 9.16, and the signal peptide with 28 amino acids at the N-terminus was predicted. The three-dimensional structure of AzanCSP3 had 6 conserved α-helices and formed a hydrophobic binding cavity with typical characteristics of the chemosensory protein family. The amino acid sequence alignment indicated that AzanCSP3 had higher homology with other Coleoptera CSP sequences, and had the highest amino acid sequence homology with AmalCSP4, which was 87.79%, phylogenetic results indicated that AzanCSP3, AmalCSP4 and AplaCSP3 were clustered with 100% confidence. Western blot showed that AzanCSP3 was successfully expressed in E.coli. The structural characteristics of chemosensory protein AzanCSP3 in A. zanthoxylumi were defined, which would lay a theoretical foundation for the in-depth study of chemosensory protein of A. zanthoxylumi and the rule of the relationship between A. zanthoxylumi and environmental chemical information.

Enzootic nasal adenocarcinoma (ENA) caused by Enzootic nasal tumour virus of goats (ENTV-2) has been reported in many provinces in China in recent years. In order to clarify the virology and genome-wide characteristics of ENTV-2FJ strain, In this study, the nasal fluid of the diseased sheep was collected, and the virus of ENTV-2FJ strain was purified by differential centrifugation combined with sucrose density gradient centrifugation, and then subjected to electron microscopic observation and SDS-PAGE; Seven cDNA fragments of ENTV-2FJ isolate were amplified by reverse transcription PCR (RT-PCR), the amplified products were cloned into pMD19-T plasmid vector and sequenced, the whole genome sequence of the ENTV-2FJ strain was obtained by splicing with DNAstar 7.1 software and the sequence was analyzed. After purification of the virus of ENTV-2FJ strain, pure virions can be observed under electron microscope. The virions were spherical, 90~110 nm in diameter, with envelope and spikes; By SDS-PAGE analysis, the structural proteins of the ENTV-2FJ strain showed 9 bands with molecular weights of 118, 79, 60, 51, 28, 20, 16, 13 and 11 ku, the genome of the ENTV-2FJ strain was 7 469 bp in length, exhibiting 95.1%, 95.4%, 95.1%, 94.9% and 95.4% homology with ENTV-2SC, ENTV-2Shaanxi, ENTV-2Shaanxi2, ENTV-2Shaanxi3, ENTV-2Shaanxi4, respectively. Phylogenetic analysis based on the nucleotide sequence of ENTV-2 whole genomes, gag and env genes revealed that the ENTV-2FJ strain was in the same evolutionary branch as ENTV-2SC, ENTV-2Shaanxi, ENTV-2Shaanxi2, ENTV-2Shaanxi3, and ENTV-2Shaanxi4. These results lay the foundation for the establishment of the ENTV-2 assay and genetic variation analysis.

Fat tissue accumulation is closely related to growth efficiency and meat quality in pigs (Sus scrofa). The development of adipose tissue depends on the increasing of adipocytic numbers and size, which leads to the adipogenesis through coordination and interaction. Adipocyte differentiation is controlled by a precisely regulated transcriptional cascade that activates or inhibits each other's expression in a continuous manner. The process of porcine adipocyte differentiation, the research model of adipocytes, the method of preadipocytes differentiation, the marker factors of adipocyte differentiation, and the research progress of several important transcription factors related to porcine adipocyte differentiation were reviewed in this paper, which aims to provide the reference for studying the mechanism of differentiation of pig adipocytes and lipid metabolism.

Laccase is a polyphenol oxidase containing copper ions, which occur widely in fungi, plants, bacteria and insects. In fungi, laccases are generally in the form of gene families encoding isozymes which are involved in many important biological processes including fungal morphogenesis, pigment synthesis, infection and pathogenesis. Laccase catalyze the oxidation of a wide variety of substrates such as polyphenols, aromatic amines and lignin. However, the research about laccases mainly focuses on the degradation and decolorization of artificial compounds such as toxic compounds. There are few studies on the identification and analysis of natural catalytic substrates and their metabolic pathways in fungi. In this paper, the biological functions of fungal laccase and its natural catalytic substrate are reviewed to clarify the mechanism of laccase biological function.

China is one of the main consumer of genetically modified soybean (Glycine max) and the safety of genetically modified soybeans received wider attention. It is imperative to study fast and accurate detection methods for genetically modified ingredients in soybeans. In this study, based on TaqMan probe technology, a duplex real-time PCR method was established to detect MON87701 and MON87708 transgenic soybean strains simultaneously, and its specificity, sensitivity and applicability were analyzed. The results showed that the method could accurately detect target genes from 18 transgenic samples and 5 non-transgenic samples, indicating good specificity. The sensitivity test showed that the sensitivity of this method was 0.01%. The applicability of this method had been tested by 10 real samples and the results were as same as the EU standard. These results suggested that the duplex real-time PCR method was an effective tool for detection of soybean MON87701 and soybean MON8770 in the exit and entry inspection laboratory. This study provides a reference for the regulation of genetically modified products.

Diarrhea is common in piglets and causes serious economic losses. In this study, an oligonucleotide microarray was developed for simultaneous the detection of Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine rotavirus group A (PRoV A) and Porcine delta-coronavirus (PDCoV). Specific primers were designed according to the genes of PEDV spike gene (S), membrane gene(M), TGEV spike gene (S) nucleocapsid gene (N), PoRV A outer capsid protein 7 gene (VP7), non-structural protein 4 gene (NSP4) and PDCoV membrane gene (M), nucleocapsid gene (N), and the corresponding oligonucleotide probes were designed according to the conservative region of each target gene fragment. The 5' end of downstream primers was labelled Cy3 fluorophores directly for multiple PCR. The microarray reaction parameters of each reaction step were optimized, which were as follows: The dilution ratio of 100 μmol/L original probe and sample buffer was 1∶2, the optimal hybridization condition was 46 ℃ for 120 min. Signal-to-noise ratio (SNR)≥2 or signal value >1 300 was defined as positive. The sensitivity of chip was 10⁵ copies/μL. No cross reaction was detected with other non-target pathogens. The shelf life test showed that the microarray can be stored at 4 ℃ for at least 120 d. The detection of the 209 clinical samples by oligonucleotide microarray showed the positive rates of PEDV, TGEV, PDCoV and PoRV A were 68.42%, 4.30%, 0.95% and 2.87%, respectively, which was consistent with the results by Reverse transcription PCR. In conclusion, the constructed oligonucleotide microarray provides a new alternative method for high-through screening of the 4 porcine enteric diarrheic viruses.

As powerful tool, fish cell lines have been widely used in virology, environmental toxicology, cell biology, genomics and genetics. Spotted knifejaw (Oplegnathus punctatus) is an excellent marine fish species. In this study, the tissue block method was used to establish the O. punctatus liver cell line (OPL) from spotted knifejaw liver. The culture medium used for the cell line was L-15 which was supplemented with mycillin, fetal bovine serum (FBS), antibiotics, basic fibroblast growth factor (bFGF) , and 2-mercaptoethanol (2-ME) . The cultured OPL cells, fibroblastic in morphology, had been subcultured to more than 30 passages in a good proliferating state. By comparing the effects of different medium of L-15 (Leibovitz-15), DMEM/F12 (Dulbecco's modified Eagle medium/Ham's F-12), DMEM and MEM on cell growth, the result showed that the optimum medium was L-15. The effects of temperature, FBS and bFGF on the growth of OPL cells were examined. The cells grew well in the temperature of 18~30 ℃, and the optimum growth temperature was 24 ℃. The results showed that the significant growth rate of OPL cells were accompanied with the increase of serum concentration range of 0%~20%. In addition, bFGF was benefit for growth of OPL cells. Chromosome analysis indicated that the passage 20 cells exhibited a normal diploid karyotype (2n=2sm+46t), but the ratio was only 68%. And the significant fluorescent signals were detected in OPL cells after transfection with pEGFP-N3 plasmid. The cytochrome oxidase subunitⅠ (COⅠ) gene was used to identified that OPL cell line originated from O. punctatus. Moreover, the establishment of the OPL cell line could provide basic experimental materials for studying the mechanisms of infective viruses and the function analysis of cells and genes.

Small ubiquitin-like modifiers (SUMOs) are widely used as labels for soluble expression of recombinant protein. Ubiquitin-like-specific protease 1 (Ulp1) is a major member of SUMOs proteases; However, its poor stability hampers its applications. Moreover, studies indicated that Ulp1 fragment from residues 304 to 621 displays full proteolytic activity in cleavage reactions. Here, a cyclized recombinant truncated Ulp1 (Gly304~Lys621) was constructed with SpyTag/SpyCatcher technology, it was observed that cyclization was efficiently and spontaneously completed under a variety of environmental conditions, resulted in strong stability and maintained enzyme catalytic activity and function. To objectively verify the effect of SpyTag/SpyCatcher technology on recombinant truncated Ulp1 stability, 3 proteases were designed in this study, including recombinant truncated Ulp1-His6 (rtUlp1), SpyTag-rtUlp1-SpyCatcher-His6 (crtUlp1) and Spy-TagΔDA-rtUlp1-SpyCatcherΔEQ-His6 (lrtUlp1). The results showed that crtUlp1 exhibited higher levels of thermostability and alkali-tolerance than the other 2 variants. Additionally, the optimal temperature of the circular variant was 5 ℃ higher than that of rtUlp1 and 10 ℃ higher than that of the linear variant. These results indicated that SpyTag/SpyCatcher-mediated cyclization increased the thermodynamic and pH stability of rtUlp1 and suggested that this technology represent a promising and effective strategy for enhancing rtUlp1 stability.