Interleukin-1 receptor-associated kinase-3 (IRAK-3) is a member of the IRAK family. It can regulate immune homeostasis and tolerance in a number of infectious and non-infectious diseases through its inhibition of pro-inflammatory cytokine production. Recently, few studies are focusing on the function of IRAK-3 in fish. Herein, an IRAK-3 transcript (PaIRAK-3) was obtained from the monocyte/macrophage transcriptome of ayu (Plecoglossus altivelis). It possessed an open reading frame of 1 722 bp, predictedly encoding 573 amino acids, and had a theoretical molecular weight of 63.2 kD. PaIRAK-3 exhibited a N-terminal death domain, a proline/serine/threonine-rich domain, a kinase and/or pseudokinase domain and a C-terminal domain of IRAK family. The phylogenetic tree showed that IRAK-3 was relatively conservative in fish, and PaIRAK-3 had the highest homology with rainbow trout (Oncorhynchus mykiss) (63.1%) and silver salmon (Oncorhynchus kisutch) (63.0%). Quantitative PCR analysis showed that PaIRAK-3 was mainly expressed in the kidney, gill, spleen, liver and intestine. After Vibrio anguillarum infection, PaIRAK-3 was significantly up-regulated at the mRNA level in the kidney, spleen and liver of ayu, reaching the maximum value at 8 h post infection. And the mRNA level of PaIRAK-3 increased only at 8 h post infection in the gill tissue. Recombinant PaIRAK-3 (rPaIRAK-3) was prokaryotically expressed using BL21 Rosetta strain of Escherichia coli, and the purified rPaIRAK-3 was used to immunize mice (Mus musculus) to prepare the polyclonal antibodies. Western blot analysis using the polyclonal antibodies against rPaIRAK-3 showed that PaIRAK-3 was significantly up-regulated against V. anguillarum infection at the protein level in the liver and spleen tissue and reached the maximum value at 12 h post infection. In conclusion, PaIRAK-3 is closely related to V. anguillarum infection, which provides a premise for further research on the antibacterial immunity of PaIRAK-3.

Long-chain acyl-CoA synthetase 1 (ACSL1) is a member of the acyl activating enzyme family, which plays an important role in the activation, transport, degradation and synthesis of fatty acids in vivo. In this study, the adipocytes of Qinchuan beef cattle (Bos taurus) were used as the research object. qRT-PCR and small interfering RNA (siRNA) interference were performed to explore the mechanism of ACSL1 gene on the synthesis of unsaturated fatty acid. Firstly, the temporal expression of ACSL1 gene and unsaturated fatty acid synthesis related genes, such as fatty acid synthase (FASN), stearoyl-CoA desaturease 1 (SCD1), peroxisome proliferator activated receptor gamma (PPARγ), fatty acid desaturase 1 (FADS1) and fatty acid binding protein 3 (FABP3) during adipocyte differentiation were detected, and it was found that the expression levels of ACSL1, FADS1, SCD1, FASN and PPARγ showed a trend of first increasing and then decreasing, and peaked at 4 d, while FABP3 had no significant changes during the differentiation of bovine adipocytes. Then, siRNA targeting the ACSL1 gene was transfected into bovine adipocytes, and found that the mRNA level of ACSL1 was down-regulated by more than 70% (P<0.01), and the protein level was also significantly down-regulated. After silencing the ACSL1 gene, the mRNA level of FABP3 and PPARγ were down-regulated, and FADS1 and FASN were up-regulated, while SCD1 showed a trend of increasing first and then decreasing. Furthermore, interfering ACSL1 gene expression significantly reduced the ratio of C18∶0 (P<0.05), so that the content of saturated fatty acid (SFA) in adipocytes was reduced. Meanwhile, the ratio of C16∶1, C18∶1, C18∶2, arachidonic acid (AA) and eicosapentaenoic acid (EPA) decreased significantly (P<0.01), so that the content of saturated fatty acid (SFA) and polyunsaturated fatty acids (PUFA) in adipocytes was decreased. In addition, Bodipy staining showed that silencing the ACSL1 gene could reduce the formation of lipid droplets in bovine adipocytes. In summary, the ACSL1 gene played an important role in regulating the expression of unsaturated fatty acid synthesis-related genes, fatty acid composition and lipid droplet formation, and provides a theoretical basis for further elucidating the molecular mechanism of unsaturated fatty acid formation in bovine adipocytes.

The members of the WRKY transcription factor (TF) family play important roles in mediating plant tolerance to salt stress. Previously, TaWRKY46, a member of WRKY gene family, was revealed to obviously respond to salt stress. Thus transgenic tobacco (Nicotiana tabacum) lines overexpressing this wheat gene were generated. The experiment indicated that TaWRKY46 targeted onto nucleus at subcellular level. Using the culture methods of vermiculite-based and Murashige & Skoog (MS) hydroponic solution, the phenotype of wild type (WT) and overexpression lines (OE) under salt stress treatment was investigated. OE1 and OE5, two OE lines overexpression TaWRKY46, displayed increased growth vigor and leaf area of plants, together with enhanced plant fresh weight and contents of soluble sugar and proteins upon salt stress with respect to WT. Assays on the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), the enzymes worked as cellular protector, showed the higher activities in OE lines than those in WT plants. Reverse transcription PCR (RT-PCR) analysis indicated the expression levels of some protection enzymes mentioned above from roots were higher in OE1 and OE5 than those in WT under salt stress treatment. Further histochemical staining study via nitroblue tetrazolium (DAB) and diaminobenzidine (NBT) dyeing methods revealed that the accumulative amounts of O₂^-· and H₂O₂ were lower in the OE plants than that of WT plants. The expression analysis revealed that the pyrabactin resistance-like abscisic acid receptor gene (PYL8) and a suite of sucrose non-fermenting-1-related protein kinase 2 (SnRK2) family genes were upregulated in expression in the OE plants. Observation on stomata movement showed that the OE plants possessed enlarged stomata aperture and increased closure rate upon salt stress compared with the WT plants. Using yeast-two hybridization assay, TaWRKY46 was identified to be interacted with TaSAP1-1, a stress-related protein in T. aestivum. Therefore, TaWRKY46 played an important role in salt tolerance through enhancing osmotic regulation and protective enzyme system, crossing with ABA signaling pathway, as well as initiating protein interaction. The results enrich the knowledge as to wheat plants coping with salt stress and provide theoretical guidance for breeding stress tolerant cultivars of crops.

The PIN-FORMED (PIN) gene family of trans-membrane proteins as auxin efflux carriers plays a crucial role in polar auxin transport and then influences the growth and development of plants. The objective of this study is to understand the distribution, structure and evolution of PIN protein genes in peach (Prunus persica) genome, to study the expression characteristics of PIN family members in different tree architectures of peach. In this study, PpPIN family was identified from the whole peach genome according to the reported 8 PINs of Arabidopsis thaliana and Mem_trans (PF03547) conserved domains of AtPIN proteins. Several bioinformatics softwares, including TBtools v0.6733, GSDS 2.0, MEME 4.9, Protparam V12.1, MEGA 7.0 and PlantCARE were used to identify chromosome location, gene structure, physical and chemical properties of protein, motif analysis, phylogeny analysis and cis-elements of promoters on PpPIN protein family, respectively. Meanwhile, the expression characteristics of PpPIN genes in the shoot tips of different tree architecture of peach were analyzed based on the results of transcriptome sequencing. And the expression characteristics of PpPIN genes were also analyzed by real-time quantitative RT-PCR (qRT-PCR) in different tissues (organs) of different types of peach. Fifteen PpPIN genes were identified and unevenly distributed on 6 chromosomes of peach. Except for PpPIN3 and PpPIN5, all other PpPIN genes contained introns, with the number of introns ranging from 1 to 10. The number of amino acids and the isoelectric point of PpPIN proteins which were hydrophilic proteins were 357~678 and 4.97~9.40, respectively. Phylogenetic analysis results indicated the PIN proteins of 8 species, including peach, apple (Malus×domestica), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), Populus (Populus trichocarpa), potato (Solanum tuberosum), maize (Zea mays) and soybean (Glycine max), were divided into 3 groups (canonical type, noncanonical type 1 and noncanonical type 2). According to the results of evolutionary analysis, 15 PpPIN proteins were divided into 3 groups. Canonical type, Noncanonical type1 and Noncanonical type 2 included 6, 2 and 7 PIN proteins, respectively. The number of conserved motifs of 8 PpPIN proteins of Canonical type and Noncanonical type 1 was 10, and the order of 10 motifs in these 2 types was all the same. While the number of conserved motifs of 7 PpPIN proteins of noncanonical type 2 was 8~11, and the arrangement of conserved motifs was not the same. The cis-element analysis of 15 PpPIN gene promoters showed that the members of PpPIN gene family were affected by light, temperature, drought stress and other environmental conditions, as well as abscisic acid, gibberellin and other hormones. Fifteen PpPIN genes were differentially expressed in different tree architecture of peach. According to the transcriptome analyses, the expression level of PpPIN12 (the homologous gene of AtPIN5) in the shoot tip of 'Fenhuashouxingtao' (dwarf type) was significantly higher than that of 'Qiumihong' (standard type), while the expression level of PpPIN12 in the shoot tip of 'Sahonglongzhu' (pillar type) was significantly lower than that of 'Okuba' (standard type). The expression level of PpPIN2 (the homologous gene of OsPIN2) in the upper phloem of the branch junctions of 'Okubo' (standard type) was obviously higher than that of 'Sahonglongzhu' (pillar type), while the expression level of PpPIN13 gene (the homologous gene of OsPIN13) was opposite to PpPIN2. The 3 candidate genes, including PpPIN12, PpPIN2 and PpPIN13, may play an important role in regulating the formation of plant height and branch angle of peach trees.

Plant WRKY transcription factors play important regulatory roles in plant response to low temperature. However, there are few researches about genome-wide identification and characterization of melon (Cucumis melo) WRKY genes and their expression patterns under low-temperature stress. Thus, in this study, the genome-wide identification of WRKY genes and bioinformatics analysis were carried out in melon, and the expression patterns under low-temperature stress were detected by qRT-PCR. The results showed that there were 59 CmWRKYs identified in melon, which unevenly distributed on the chromosomes. Based on phylogenetic analysis, the CmWRKY proteins were classified into 3 groups (Ⅰ~Ⅲ) and the second group was further divided into 5 subgroups (Ⅱa~e). Synteny analysis showed that there were 28 collinear gene pairs in melon and Arabidopsis thaliana WRKY gene family and 10 pairs of CmWRKYs were identified as segmental duplication. Cis-regulatory elements related to stress and hormone responses were found in the promoters of CmWRKYs. Gene Ontology (GO) analysis displayed that CmWRKYs widely participated in physiological processes such as biological process, cellular component and molecular function. The qRT-PCR detection showed that the expression of 11 CmWRKYs was significantly up-regulated under low temperature stress. The results of this study can provide reference for further cloning of CmWRKYs and analysis of low temperature tolerance function.

2C type protein phosphatases (PP2C) widely exist in organisms and play important regulatory roles. Many studies have indicated that PP2C play important roles in plant stress resistance and response to hormone regulation. However, the PP2C in moso bamboo (Phyllostachys edulis) have not been investigated. In this study, moso bamboo was used as experimental material and bioinformatics method was used to identify 125 genes with complete PP2C conserved domain from the whole genome of moso bamboo. The length of the proteins encoded by PP2C ranged from 110 to 1 086 aa with molecular weights from 12.07 to 121.02 kD. According to phylogenetic analysis result, 125 PP2Cs were clustered into 9 subgroups, and 4 genes (PH02Gene36278.t1, PH02Gene48541.t1, PH02Gene37946.t1, PH02Gene29918.t1) were not divided into any subgroups and formed a branch independently; According to expression profile analysis of transcriptome, 121 PP2Cs exhibited different levels in different tissues and growth stages. Most PP2Cs were highly expressed in different stages of bamboo shoot development and some PP2Cs had high expression in roots and leaves; qRT-PCR results showed that 9 PP2Cs were up-regulated and 1 PP2C was down-regulated after abscisic acid treatment; whereas 6 PP2Cs were up-regulated and 5 PP2Cs were down-regulated after salt stress and methyl jasmonate treatment, which indicated that PP2Cs might play different roles in the regulation of stress and hormone responses. The present study could provide some reference for further functional study of PP2C gene family in Phyllostachys edulis.

Plant aquaporin is involved in rapid transmembrane transport of water and regulates various physiological processes such as nutrient transport, seed germination and lateral root growth in plants. In this study, three plasma membrane intrinsic proteins, RcPIP1;3 (Ricinus communis plasma membrane intrinsic proteins genes 1; 3)(GenBank No. MT068547), RcPIP2;1 (GenBank No. MT068546) and RcPIP2;2 (GenBank No. MT068545), were isolated from leaves of Tongbi 5 genotype by RT-PCR. Bioinformatic analysis showed that these 3 genes all contained 4 exons and 3 introns, 6 transmembrane helics and 2 specific NPA (Asn-Pro-Ala) motifs of major intrinsic proteins (MIPs) superfamily. Multiple sequence alignment indicated the homology of RcPIP1;3 with PtPIP1;3 reached 92.36%, and the homology of RcPIP2;1 and RcPIP2;2 with PtPIP2;1 and PtPIP2;2 were as high as 87.80% and 89.58%. Phylogenetic analysis confirmed RcPIP1;3 was the member of PIP1s family, RcPIP2;1 and RcPIP2;2 were the members of the PIP2s family. Sequence analysis of the promoter region showed that the 3 genes had cis-acting elements in response to hormone induction and environmental stress, which would suggested that they might have potential functions of regulating growth and development and adapting to environmental stress. qRT-PCR analysis found that RcPIP1;3 and RcPIP2;2 were up-regulated by cold stress, and RcPIPP2;1 was down-regulated by cold stress, which indicated that 3 genes may have antagonistic regulation modes in response to cold stress. This research provides an important theoretical basis for exploring the cold adaptation regulation process mediated by RcPIPs.

Pollination and fertilization are key reproductive processes that affects the seed setting of plants. MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor is one of the important transcription factor families involved in the regulation of these processes. An unigene sequence sharing high homology with MYB family gene was obtained from gynoecium transcriptome database of previously constructed tree peony (Paeonia ostii 'Feng Dan Bai') and a complete coding region gene named as PoMYB1 (GenBank No. MT360920) was cloned with the expressed sequence tag and rapid amplification of cDNA ends (RACE) methods. PoMYB1 contained a 864 bp long ORF, indicating that it could encode 267 amino acids residues with 2 typical MYB domains. Bioinformatics analysis showed that the molecular formula of PoMYB1 protein was C₁₄₃₄H₂₂₃₅N₄₁₅O₄₄₁S₁₂, the relative molecular weight was 32.73 kD and the isoelectric point was 7.01, the fat coefficient was 68.99, and the instability coefficient was 48.18. These characteristics showed that it was an unstable protein. Amino acids alignment revealed that PoMYB1 was composed of highly conserved and incompletely duplicated R2R3-MYB domains. PoMYB1 had 3 characteristic conserved motifs. Phylogenic analysis showed that the PoMYB1 shared the highest similarity with that of PqMYB, followed by MrMYB1, which were obtained from P. qiui and Morella rubra, both belonged to R2R3-MYB transcription factor family of subgroup 6. The expression characteristics of PoMYB1 gene were analyzed by qRT-PCR. The results indicated that the highest in sepal and followed by leaf and petal, which showed that it played a role in the development of flowers and leaves. The highest expressed increment appeared in 24 h after fertilization, and the highest abundance in 48 h after fertilization was observed, which speculated its putative function in double fertilization. Besides, construction of yeast (Saccharomyces cerevisiae) recombinant vectors containing 3 different missing fragments of PoMYB1 and introduced into yeast cells and the transactivation activation activity of PoMYB1 was also verified by yeast one hybrid. The results indicated that PoMYB1 was a transcriptional activator, and the transcriptional regulatory regions in the C-terminus. The study provides theoretical basis for exploring the molecular mechanisms underlying double fertilization in P. ostii and establishes a foundation for further research transcriptional regulation mechanism of PoMYB1.

The vertebrate central nervous system is highly sensitive to hypoxia. As a species with long-term adaptability to the hypoxic environment, yak's (Bos grunniens) central nervous system, especially the brain, must have its own special adaptation mechanism. Therefore, it is necessary to study how the yak brain adapts to this hypoxic environment. Several hypoxic neuroprotective factors have been discovered, of which neuroglobin (NGB) and hypoxia-inducible factor-1α (HIF-1α) are mainly expressed in the vertebrate nervous system. But currently, the expression and distribution characteristics of NGB and HIF-1α in yak brain, especially in diencephalon, still remains unclear. This study was aimed to discover the expression and distribution differences or similarities of NGB and HIF-1α in yak's diencephalon and explore the reasons,and qRT-PCR, Western blot and immunohistochemical techniques were used to detect the expression and localizations of NGB and HIF-1α. The results showed that the expression levels of NGB and HIF-1α mRNA were the highest in the pituitary and pineal of yak diencephalon (P<0.05). The expression characteristics of NGB in yak diencephalon were coincident with HIF-1α, and The order from high to low was pituitary, pineal, thalamus, epithalamus, optic nerve and hypothalamus. There was no difference in the expression of NGB and HIF-1α among thalamus, epithalamus, optic nerve and hypothalamus. The expression levels of NGB protein had the highest expression in the pituitary and hypothalamus, while HIF-1α protein had the highest expression in the pituitary (P<0.05). There was no difference in the expression of NGB protein among thalamus, pineal, epithalamus, and optic nerve. There were significant differences in the expression of HIF-1α protein in the yak pituitary, thalamus, epithalamus, and optic nerve. The expression characteristics of NGB in yak diencephalon, including pituitary, thalamus, pineal, epithalamus, optic nerve, were coincident with HIF-1α while the expression characteristics of NGB and HIF-1α was opposite in the hypothalamus. The positive products of NGB and HIF-1α were mainly distributed in the cytoplasm of neurons, and the distribution characteristics of HIF-1α in yak diencephalon were coincident with NGB. The intensity of the immunopositive response of HIF-1α protein was higher than that of NGB protein. NGB and HIF-1α proteins were mainly expressed in the cytoplasm of polymorphic cells of the thalamus, epithalamus, hypothalamus; There were mainly expressed in the cytoplasm of acidophages in the pituitary; in the pineal gland were mainly expressed in the cytoplasm of the pineal cells; There were mainly expressed in the cytoplasm of visual nerve cells and nerve glial cells in the optic nerve, and the 2 proteins were not found in all negative controls. The above results suggested that the pituitary in the yak's diencephalon may have a strong susceptibility to hypoxia. There may be a synergy between NGB and HIF-1α in improving the ability of the yak's diencephalon to adapt the hypoxia environment, however, some regions may have certain regional selectivity depending on their different functions. The coordination and selection mechanisms remain to be determined in further research. This study provides a valuable theoretical basis for further research on the mechanism of yak brain tissues adaptation to hypoxia, and also provides reference data support for in-depth exploration of the possible physiological functions of mammalian brain tissue NGB and HIF-1α.

Large yellow croaker (Larimichthys crocea) is a kind of warm-temperature fish. Low temperature in winter can cause large death of cultured L. crocea, so it is necessary to carry out researches on how to deal with low temperature stress. In order to study the sequence characteristics of stearoyl coenzyme A desaturase 1 (SCD-1) gene of Donghai No.1 large yellow croaker and their responses to cold stress, the full length of SCD-1a (GenBank No. MT325796) and SCD-1b (GenBank No. MT325797) cDNA were cloned by rapid amplification of cDNA ends (RACE) technique, and expression level under chronic and acute cold stress were analysed by qRT-PCR. The full length of SCD-1a cDNA was 3 095 bp which contained a 1 014 bp ORF and encoded 337 amino acid. The full length of SCD-1b cDNA was 2 636 bp which contained a 1 008 bp ORF and encoded 335 amino acid. SCD-1a and SCD-1b both contained 3 conserved histidine rich regions, and the sequence similarity was 72.2%. The predicted tertiary protein structure of SCD-1a and SCD-1b were highly similar and both contained 4 transmembrane helical structure. In the phylogenetic tree based on amino acids, the SCD of teleost divided into 2 clusters, the SCD-1a and SCD-1b of L. crocea located in 2 clusters separately, indicating that they were different subtypes. Under normal growth temperature (18 ℃), the expression levels of SCD-1a and SCD-1b were both high in liver and lower in other tissues. During chronic-cold stress (decreasing slowly from 12 ℃ to 6 ℃ by the method of cooling at the rate of 0.5 ℃/12 h and continuing to cool down after maintaining for 12 h), the expression of SCD-1a significantly increased in gill, skill, muscle, intestine and brain, and the expression of SCD-1b significantly increased in skill, muscle, intestine, brain and heart, nevertheless SCD-1a expression decreased significantly in liver. During acute cold stress (from 12 to 8 ℃ immediately), the expression level of SCD-1b significantly increased in all tissues, and SCD-1a expression significantly increased in all tissues except skill. The results of this study have certain reference significance for understanding the mechanism of response and adaptation of L. crocea to low temperature stress.

Haemonchus contortus is one of the most important parasites of ruminants such as Ovis aries and Capra hircus, and can bring huge economic losses to the breeding industry. In preliminary transcriptome study of present research group, the resistant gene ATP-binding cassette (ABC) transporter gene (GenBank No. MT478136) was identified from the resistant strain of Haemonchus contortus. The purpose of this study was to explore the bioinformatics character of the ABC transporter gene, to establish a system of soluble expression and purification for ABC transporter, and to lay the foundation for further exploration of ABC transporter-involved drug resistance mechanism. The online software was used to predict the protein structure and antigen epitope. The results showed that the molecular formula of ABC was C₉₂₈H₁₅₀₂N₂₇₀O₂₆₅S₇, which was a hydrophilic protein without transmembrane region and signal peptide. The secondary structure was mainly extended chains. The prediction of antigenic epitopes indicated that ABC transporter had many antigenic epitopes with high antigenicity. The ABC transporter gene was amplified by reverse transcription PCR (RT-PCR), and the recombinant plasmid pET30a(+)-ABC was constructed by restriction digestion. The plasmid was transformed into Escherichia coli BL21 to optimize inducing conditions. It was found that 1 mg/mL recombinant ABC transporter could be expressed with 0.6 mmol/L isopropyl β-D-thiogalactoside (IPTG) at 37 ℃ for 7 h. In this study, recombinant ABC transporter was successfully expressed and purified in Haemonchus contortus, which could provide a reference for further mechanism study on potential drug resistance and immunogenicity.

Pleurotus pulmonarius has the characteristics of changing temperature and fruiting. Low temperature stimulation can induce the formation of fruiting body. Using Illumina sequencing technology, the complete transcriptome including four different stages of growth and development: Mycelial culture at normal temperature stage, low temperature induction stage, recovery to normal temperature stage after low temperature induction and primordium formation stage of Pleurotus pulmonarius were sequenced. There were 41 544 496, 45 722 382, 46 642 306 and 49 980 456 clean reads obtained at four different growth stages respectively. The clean reads of four different stages samples transcriptome sequencing were aligned to the reference sequences which were spliced by Trinity software. The alignmengt rate of the four sequencing samples was 83.22%, 82.85%, 83.10% and 82.59%, respectively. Analysis of differentially expressed genes showed that there were 1 216 genes up-regulated while 1 046 of them were down-regulated expressed between two libraries with normal and low-temperature induction. Meanwhile, 1 331 of genes were up-regulated while 1 784 of them were down-regulated in the primordium library compared with library of mycelium induced by low-temperature. Functional annotation with Blast-Nr database indicated that these up-regulated genes in mycelium induced by low-temperature were involved in transferring alkyl, aspartic-type endopeptidase activity, oxidation-reduction process, cell wall integrity and intracellular protein transport. And in primordium, these up-regulated genes were involved in regulation of transcription, ribosome synthetize, methelation and ion channel. Gene Ontology functional enrichment analysis showed that the differentially expressed genes involved in catalytic activity, single organism metabolic process and oxidoreductase activity were significantly enriched at two stages of normal temperature treatment and low temperature induction, while at the stages of recovery to normal temperature stage after low temperature induction and primordium formation catalytic activity, single tissue process and single tissue metabolism process were significantly enriched. KEGG metabolic pathway analysis showed that there were significant differences in gene enrichment in two metabolic pathways: TCA cycle and endoplasmic reticulum protein processing. Through the comparative study of transcriptome in different stages of growth and development, 2 262 and 3 115 differentially expressed genes were screened out. qRT-PCR analysis showed that the expression changes of the selected genes at different stages were consistent with the results of transcriptome sequencing. This study lays the foundation for further excavation of the key genes of primordium formation and fruiting body growth of Pleurotus pulmonarius, and also provides guidance for the application of genetic engineering to cultivate new high-quality and high-yield Pleurotus pulmonarius varieties.

β-glucosidase plays a vital role as a rate-limiting enzyme in the field of bioenergy and food industries. In this study, different promoters and anchor proteins were screened in the surface display system of Saccharomyces cerevisiae to explore the effects of different promoters and anchor proteins on the BGL activity from Aspergillus niger. pYES2/CT/α-factor was used as the starting plasmid, green fluorescent protein (GFP) as the reporter protein and mating agglutinin protein (Sag1p), suppression of exponential defect protein (Sed1p) and cell wall protein 2 (Cwp2p) as anchors protein, to construct 3 different surface display plasmids of anchor protein GAL1-eGFP-Sag1/Sed1/Cwp2 and yeast surface display system. The original promoter GAL1 of plasmid pYES2/CT/α-factor was replaced with constitutive promoters promoter of glycerol 3-phos‐phate dehydrogenase (GPD) and promoter of suppression of exponential defect protein (SED1) by In-Fusion technology. Plasmids pYES2-GPD and pYES2-SED were constructed and digested , then they were separately ligated with the digested GAL1-eGFP-Sag1/Sed1/Cwp2 constructed above to construct 6 recombinant plasmids GAP/SED1-eGFP-Sag1/Sed1/Cwp2. Finally, the eGFP on the plasmids GPD/SED1-eGFP-Sag1/Sed1/Cwp2 were replaced with the target protein gene bgl1 derived from A. niger and 6 recombinant yeasts, PBy-GBSa, PBy-GBSe, PBy-GBCw, PBy-SBSa, PBy-SBSe, PBy-SBCw, were successfully constructed to explore the effects of different promoters (SED1 and GPD) and anchoring proteins (Sag1p, Sed1p and Cwp2p) on BGL activity. Green fluorescent protein was observed by laser confocal microscope and was found to be located on the cell surface of S. cerevisiae, revealing that the surface display platform of S. cerevisiae was successfully established. The activities of BGL displayed on the surface of 6 recombinant yeasts were compared with each other, which showed that the GPD promoter performed better than the SED1 promoter; no matter what kind of promoter, Sag1p showed the highest enzyme activity among the 3 anchor proteins (Sag1p, Sed1p, Cwp2p); When GPD was used as the promoter, the differences in enzyme activity between different anchor proteins were more obvious. The BGL recombinant strain PBy-GBSa was able to display BGL more efficiently, and the enzyme activity reached a maximum of (18.29±1.05) U/g when cultured for 24 h. The types of promoters and anchoring proteins had important effects on the surface display of BGL in S. cerevisiae, which would provide a theoretical basis for the more efficient and stable surface display of BGL in S. cerevisiae and the industrial application of BGL whole-cell catalysts.

Foot-and-mouth disease serotypes Southern African Territories 2 (SAT2-FMD) is an acute, heat, and highly contactive infectious disease caused by Foot-and-mouth disease virus serotypes Southern African Territories 2 (SAT2-FMDV). At present, global trade and mutual visits are frequent, and the risk of SAT2-FMD entering China is extremely high, which will seriously threaten the sustainable and healthy development of Chinese breading industry. This experiment was aimed to prokaryotic soluble expression and analysis the immunogenicity of VP1 gene of SAT2-FMDV (GenBank No. JX014256). The gene was amplified from the recombined plasmid pUC57-His-SUMO-SAT2-VP1 by PCR, with a product of approximately 993 bp. The identified recombined plasmid pET32a-HisSUMO-VP1 was expressed by Escherichia coli BL21 (DE3) and was induced by 1 mmol/L isopropy-β-D-thiogalactoside (IPTG). The HisSUMO-VP1 protein was purified by Ni-NTA, and the purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDA-PAGE) and Western blot. The HisSUMO-VP1 protein was used as an immunogen for immunization of Balb/C mice (Mus musculus), and the serum antibody, spleen lymphocyte proliferation and cytokine levels were detected. The results of SDA-PAGE and Western blot showed that the HisSUMO-VP1 protein with a relative molecular weight of 63 kD was highly soluble and had good reactivity. The immune experiment found that the serum antibody levels in the immunized mice vaccinated with the HisSUMO-VP1 fusion protein group was significantly higher than those in the HisSUMO protein vaccinated group and the mock group (P<0.001), and the mice spleen lymphocytes had a significant lymphocyte proliferation response (P<0.001). The levels of IFN-γ and IL-2 were significantly higher than those in the PBS group (P<0.05). The HisSUMO-VP1 protein with excellent immunogenicity is solublely expressed in E. coli, providing data support for the diagnosis and vaccine development of SAT2-FMDV.

In mammalian ovary, folliculogenesis, development and follicle maturation toward ovulation is a complex biological process. This process requires the precise coordination of oocyte and its companion somatic cells. During this process, oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factors 9 (GDF9) regulate granulosa cells growth, differentiation, promote folliculogenesis from the primordial stage, determine ovulatory follicle survival and ultimately affect ovulation by paracrine/autocrine mechanisms. It has been found that mutations of the BMP15 and GDF9 gene cause the distinct alteration in ovarian function in different species. In mice (Mus musculus) , homozygous null mutation of BMP15 reduce the fertility of female mice, but GDF9-nulll female mice are sterility. In sheep (Ovis aries), heterozygous mutant of these two genes decrease the BMP signaling and lead to the hyper prolificacy, whereas the homozygous sheep exhibit the inhibition of ovarian follicular development and infertility. In this review, we described the changes of ovarian function and ovulation in BMP15/GDF9 knockout and overexpressing mice and sheep with natural mutations. We summarized that BMP15, GDF9 and their dimmers orchestrate follicular development, participant in selection of dominant follicular and regulate ovulation by the dynamic balance of their bioactivity. The recent findings relating the functions of BMP15 and GDF9 opened up new perspectives for regulation of sheep ovulation and breeding new prolific sheep.

With the improvement of people's living standards, the demand for high-quality lamb is increasing. Meat quality traits such as drip loss, flesh color, marble texture, water power, intramuscular fat content, and muscle fatty acid composition and pH also directly or indirectly affect people's consumption tendency. Because most of the current methods for determining meat quality are carried out after slaughter, it is difficult for conventional breeding methods to quickly and effectively improve the quality of mutton at a low cost. In recent years, breeders have begun to pay attention to the use of molecular biology methods to find candidate genes and molecular markers related to mutton quality, which has opened a new path for accurate and rapid improvement of meat quality. This article reviews the indicators of mutton quality evaluation and important candidate genes related to some indicators, with a view to helping the future genetic improvement and breeding of mutton quality.

Tomato black ring virus (TBRV) is a kind of harmful organisms that can infect a wide range of economically important herbaceous and woody species. TBRV now is a port quarantine pest in many countries including China. Strengthening the inspection and quarantine of TBRV at ports is the key to prevent TBRV from invading China. Serological assays are well known to be quick, simple, low-cost and high-throughput techniques for plant viral detection. The aim of present study was to develop serological techniques for TBRV detection using the prepared specific monoclonal antibody (MAb) against TBRV, which could serve TBRV detection, inspection and quarantine at ports. The purified TBRV virions were used as the immune antigen to immunize BALB/c female mice (Mus musculus). Four hybridoma cell lines (10A8, 21A8, 21B8 and 8D3) secreting anti-TBRV MAbs were successfully prepared by fusing mouse myeloma cells (Sp2/0) with splenocytes from the immunized BALB/c mouse, screening hybridomas and antibodies, and cloning cells. The hybridomas were intraperitoneally injected into pristine-primed BALB/c mice, and about 20 mL ascetic fluids contained MAbs for each hybridoma cell line was obtained. The titers of all 4 MAbs in ascitic fluids determined by an indirect enzyme-linked immunosorbent assay (indirect-ELISA) were up to 10^-7. Isotypes and subclasses of all these 4 MAbs were determined to be IgG1, κ light chain using a commercial MAb isotyped kit. The IgG yields of 4 MAbs in ascetic fluids ranged from 5.44 to 12.93 mg/mL. Western blot assay of MAb specificity showed that all 4 MAbs had a specific immune reaction with the approximately 43 kD subunit of TBRV capsid protein in infected plant tissues, but had a negative immune reaction with healthy plant tissues. Dot enzyme-linked immunosorbent assay (Dot-ELISA) and Tissue print enzyme-linked immunosorbent assay (Tissue print-ELISA) serological approaches for TBRV detection were developed using the created MAbs as primary antibodies. Both developed Dot-ELISA and Tissue print-ELISA could specifically detect TBRV in infected plants, while all the detection with healthy plants and the plants infected by Tobacco ringspot virus (TRSV), Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV) and Tomato yellow leaf curl virus (TYLCV) respectively showed negative reactions. Furthermore, established Dot-ELISA serological approaches based on MAbs 21B8, 8D3, 10A8 and 21A8 could reliably and effectively detect TBRV in infected tomato leaf crude extracts diluted up to 1∶10 240, 1∶10 240, 1∶5 120 and 1∶5 120, respectively. The above results indicated that the 4 created MAbs and the newly established Dot-ELISA and Tissue print-ELISA serological approaches had very high specificity and sensitivity for TBRV detection in plants, and could be accurately and effectively used for the inspection and quarantine of TBRV at ports and the detection and diagnosis of TBRV in field plants.