Thioredoxin (TRX) is a kind of functionally diverse proteins with disulfide active sites, and plays an important role in many aspects including plant stress response. Nucleotoreoxin (NRX), a novel member of the TRX superfamily, was found to be associated with drought resistance in wheat (Triticum aestivum), but the mechanism of drought resistance is still unclear. Exploring the interaction protein of TaNRX1 will be helpful to further reveal the drought-resistant mechanism of TaNRX1. Based on previous studies of other researchers, the protein phosphatase 2A catalytic subunit (PP2Ac) was selected as candidate interaction protein. And according to bioinformatics analysis in STRING website (https://string-db.org/), other 2 proteins, protein disulfide isomerase (PDI) and TRX-h were also selected as TaNRX1-D candidate interaction proteins in this study. Then, the ORFs of TaPDI-A, TaPDI-B, TaPDI-D, TaTRX-h-A, TaPP2Ac-B, and TaPP2Ac-D were acquired by homologous cloning from drought-resistant cultivar 'Jinmai 47'. Furthermore, the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) were used to verify the interaction proteins. The correctly sequenced recombinant plasmids pGADT7-TaTRX-h, pGADT7-TaPDI, and pGADT7-TaPP2Ac were co-transformed into yeast (Saccharomyces cerevisiae) strain Y2H Gold with pGBKT7-TaNRX1, and then transferred to SD/-Trp-Leu-His-Ade/X-α-gal screening medium. Then on the medium, colonies were found to grow normally and turn blue, which indicated that all the 3 proteins could interact with TaNRX1-D. In order to eliminate false positives, plasmids pCAMBIA1302-CeYFP-TaPDI, pCAMBIA1302-CeYFP-TaPP2Ac, pCAMBIA1302-CeYFP-TaTRX-h and pCAMBIA1302-NeYFP-TaNRX1 were paired, respectively. Then the 3 paired plasmids were co-transformed into Agrobacterium tumefaciens GV3101 and injected into Nicotiana benthamiana leaves. Yellow fluorescent signal was found in the fluorescence confocal microscope, and the yellow signal was mainly located in the nucleus and cell membrane. To verify whether the 3 interacted proteins would respond to drought stress, mannitol was used to simulate drought stress. The recombinant plasmid pYES2-TaPDI, pYES2-TaPP2Ac and pYES2-TaTRX-h and the empty vector pYES2 were transfected into yeast strain BY4741, respectively and cultured in SD/-Ura medium containing 100 mmol/L mannitol, and the growth of the yeasts was observed. The results showed that the growth of yeasts which heterologous expressing TaPDI, TaPP2Ac and TaTRX-h was significantly better than that of empty vector colonies after drought stress, suggesting that TaPDI, TaPP2Ac and TaTRX-h all have positive regulation to mannitol stress. This study validated interaction of 3 candidate proteins with TaNRX1, and simulated drought stress analysis on yeast was conducted, which provide a reference for further elucidating the molecular mechanism of TaNRX1 drought resistance.

The c-type lysozyme is a key immune protein to resist bacterial infection for the fish with innate immune system. Common carp (Cyprinus carpio) c-type lysozyme has some characteristics that are common in fish c-type lysozyme: Its N-terminal is a signal peptide sequence, and its C-terminal region is a mature peptide sequence. Glu35 and Asp51 are 2 amino acid residues located in the mature peptide and form the active site of the c-type lysozyme. In addition, 8 conserved cysteine residues exist in the mature peptide and form 4 pairs of disulfide bonds in spatial structure which are related to the activity and stability of the c-type lysozyme. In the present study, the cDNA encoding common carp c-type lysozyme mature peptide was cloned by reverse transcription PCR (RT-PCR), and the 381 bp cDNA encoded the mature peptide consisting of 127 residues. PCR were performed to add XhoⅠ restriction site and 6×His-tag to its 5' end, and XbaⅠ site to its 3' end. This target gene was named CLYc (common carp c-type lysozyme), and ligated to pPICZαA vector and electronically transformed into competent Pichia pastoris X-33. The yeast transformants containing multi-copy gene insertions were screened using high-concentration zeocin. The target protein was induced for 120 h with 1.5% methanol at 29 ℃, 250 r/min. The recombinant protein with molecular weight of 15.4 kD was purified by immobilized metal ion affinity chromatography (IMAC); Western blot and MALDI-TOF/TOF mass spectrometry analysis proved that this recombinant protein was the expected recombinant CLYc, and its expression yield was about 40 mg/L. The antimicrobial activity of the culture medium supernatant containing recombinant CLYc was tested on Gram-positive (including Listeria monocytogenes and Staphylococcus aureus) and Gram-negative (including Escherichia coli and Vibrio parahemolyticus) bacteria using micro liquid-bacterial colony notation, demonstrating that the crude culture medium supernatant had notable antimicrobial activity against these bacteria, implying the potential use of cell-free P. pastoris medium in fish feed without the need for further peptide purification. Furthermore, the minimum inhibitory concentration (MIC) of purified recombinant CLYc was determined on these bacteria and Bacillus subtilis, indicating that the recombinant CLYc had the same MIC for L. monocytogenes, B. subtilis, E. coli and V. parahemolyticus. The results of this study provide a technical approach for the development and production of natural antibacterial agents from fish.

Suspension culture of Eriobotrya japonica (loquat) can produce rich pentacyclic triterpenoids. Amyrine synthase (AS), a key enzyme in the triterpenoid synthesis pathway, determines the branching point of major metabolism and secondary metabolism. But the function of the AS in Eriobotrya japonica has not been reported before. In this study, using the AS overexpression plasmid pDONR207-AS as the template, the positive open reading frame of the AS gene was amplified by PCR, then ligated into the fusion expression vector pBWA(V)HS-GFP after digestion to construct the expression vector pBWA(V)HS-AS-GFP-Tnos. Next, using loquat suspension cells (homologous system with AS isolated) as receptor materials, transient expression was carried out under the mediation of Agrobacterium tumefaciens GV3101. Finally, the subcellular localization of AS protein were analyzed by imaging of co-transformed GFP under laser confocal microscopy, and the catalytic products of AS protein were analyzed by conjoint analysis of gas chromatography-mass spectrometry. Results were as follows: The subcellular localization of AS protein was in the endoplasmic reticulum in loquat. And the catalytic products of AS protein contained α-amyrin and β-amyrin, which indicated that AS of loquat belonged to the multifunctional enzyme. This study provides a reference for improvement of the synthesis of pentacyclic triterpenoid and for the further transgenic application in suspension cells of loquat.

Gibberellin receptor GID1 (gibberellin insentive dwarf 1), one of important elements in gibberellin (GA) signal transduction pathway, directly affects the effect of gibberellin on plant. A cDNA sequence encoding gibberellin receptor GID1 was cloned from epidermis of bulbils of 'Niuwei Yam' (Dioscorea opposita) with reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression of the cloned yam GID1 gene at different stages after bulbils were harvested and the response of the gene expression to paclobutrazole were tested by qRT-PCR. The results showed that the obtained gene had a full length of 1 123 bp with 996 bp opening reading fram encoding 331 amino acids, and named DoGID1A (GenBank No.MK268684). Bioinformation analysis showed that the molecular weight and theoretic isoelectric piont of the protein was 3.72×10⁴ D and 8.25, respectively. The DoGID1A protein was unstable protein with an abhydrolase domain (93~304 aa) but without transmembrance domain or signal peptide. Homologous alignment showed that DoGID1A protein shared 73.56% sequence similarity with 13 specieces including Musa acuminata, and shared conserved many domains including motifs linking hormone GA and protein DELLA. Phylogenetic tree showed all dicotyledons were cluster in a group, monocotyledonous plants except rice (Oryza sativa) and yam shared another group. Yam was clustered out alone, so was rice. qRT-PCR analysis showed the expression of DoGID1A gene increased during yam bulbil sprouting and was enhanced by paclobutrazol treatment. These results indicated that DoGID1A gene could be associated with yam bulbil sprouting. This study provides basic information for further research on expression pattern and function of this gene.

Pregnant Zizania latifolia is the result of stem organ expansion and development induced by Ustilago esculenta after infecting Zizania latifolia plants, but the regulatory mechanism of its expansion and development is still unclear. In this study, the full-length sequence of ZlGH3-8 (Zizania latifolia Gretchen Hagen 3-8) gene (GenBank No. MH355951) was cloned, the gene sequence was 1 916 bp in length and contained 2 introns. The cDNA was 1 174 bp in length, encoded 390 amino acids, which had a GH3 domain and a indoleacetic acid amide synthetase domain, and was highly similar to OsGH3-8 (GenBank No. XP_015647797.1) of Oryza sativa ssp. japonica. It was found that the expression change of ZlGH3-8 in plants was related to mycelial infection and proliferation of U. latifolia. The expression of ZlGH3-8 in stems was significantly higher than that in leaves (P<0.05). At the early stage of stem enlargement, the expression of ZlGH3-8 in gray Z. latifolia was significantly higher than that of white Z. latifolia, while the expression of male Z. latifolia was the lowest (P<0.05). Further analysis found that there were significant differences in the expression of ZlGH3-8 during the expansion and development of white Z. latifolia, indicating the ZlGH3-8 might be participated in the morphological development of stem organs of Z. latifolia, and it might be related to the down-regulation of immune defense response related to the infection of U. latifolia during pregnancy. This study preliminarily clarified the expression response of ZlGH3-8 in the development of Z. latifolia, which provides basic data for the study on the mechanism of expansion and development of Z. latifolia.

MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor family play an important role in regulating plant reproduction and growth, LaMYB transcription factors in Liliium Asiatic hybrids 'Little Kiss' were identified at the transcriptome level and applied bioinformation analysis which aim to screen out those genes regulating pollen abortion. Based on the transcriptome data of lily anther, the MYB protein sequences were identified by Pfam 31.0 and BLASTP, at the same time, conserved domain and phylogenetic tree analysis were constructed by using Web Logo 3.0 and MEGA 7.0 for those R2R3-MYB transcription factors with the largest proportion. The expression levels of meiosis stage and mononuclear stage of LaMYB were analyzed by Mev. The results showed that 69 MYB genes were identified in lily, 50 of them belonged to R2R3-MYB, 15 of them belonged to 1R-MYB and 4 of them belonged to 3R-MYB, in the mean time, all those LaMYB proteins were unstably hydrophilic proteins; subcellular localization prediction showed that LaMYB proteins were all localized in nucleus; Web logo3.0 analysis found that the protein domain of R2R3-MYB were relatively conserved, but the variability of its C-terminal transcriptional activation domain makes it functionally diverse. The phylogenetic of LaMYB proteins were divided into 22 subfamilies according to the classification method of Arabidopsis family. The homologous genes involved in the division of pollen abortion mainly belong to the S18、S23、S21 and S22 subfamilies. Meanwhile, the results of RNA-Seq analysis indicated that the expression level of 42 LaMYB genes were up-regulated in the key period of pollen abortion (mononuclear stage) compared with the normal developmental stage (meiosis stage)), the other 27 genes were down-regulated in all of LaMYB. Additionally, the result of qRT-PCR showed that the 6 candidate genes were significantly different between the normal developmental stage and abortion period of lily pollen. This study showed that LaMYB genes play an important regulatory role in pollen abortive process and providing basic information for further exploration of the function of LaMYB genes in pollen abortion.

The 'Yongjin' is a color-leafed variety of Cinnamomum camphora, and it has high ornamental and economic value. Exploring the physiological and biochemical mechanism of its special leaf color formation is of great significance to the new color-leafed variety breeding of C. camphora. In this study, the leaf color, chlorophyll, carotenoid and anthocyanin contents, and photosynthetic characteristics of 'Yongjin' and wild-type were compared in April, meanwhile expression differences of 15 chlorophyll synthesis genes in 2 types were analyzed using qRT-PCR. The results showed that there was significant difference for leaf color between 'Yongjin' and wild-type in April. 'Yongjin' leaf color values were significantly lower than that of wild-type during the determination period (P<0.05), and showed an increasing trend, with the minimum of 2.757 on April 10th and the maximum of 8.163 on April 21th. The chlorophyll a, chlorophyll b and total chlorophyll contents in current-year leaves of 'Yongjin' were obviously lower than that of wild-type on April 10th, the corresponding values were only 24.9%, 42.2% and 29.7% of the wild-type. Although chlorophyll contents in 'Yongjin' were extremely significant different from the wild-type at 3 time-points (P<0.01), there was no significant difference in carotenoid and anthocyanin content between 2 types. The photo response curves of 2 types was similar, and the maximum net photosynthetic rate and light saturation point in 'Yongjin' was separately 7.44 μmol/(m² ·s) and 396.7 μmol/(m² ·s), which were significantly lower than that of wild-type (P<0.05). The expression patterns of genes related to chlorophyll synthesis in 'Yongjin' were obviously different form the wild-type. The expression levels of 11 genes in 'Yongjin' were significantly lower than that of wild-type on April 10th, and the expression levels of genes encoding C.camphora glutamy l-tRNA reductase (CcHEMA) and C.camphora uroporphyrinogen Ⅲ synthetase (CcHEMD) were only 38.7% and 31.7% of the wild-type. Especially, the expression levels of CcHEMD and C.camphora coproporphyrinogen Ⅲ oxidase (CcHEMF) were significantly lower than that of the wild-type during the measurement period (P<0.05), which was consistent with the relatively low chlorophyll content. In summary, the relatively low chlorophyll content of 'Yongjin' in spring was the direct cause of its special leaf color change, which was associated with the relatively weak expressions coding genes of HEMA, magnesium chelatase H subumit (CHLH), CHLI, CHLD, HEMD and HEMF. The results lay an important foundation for further dissecting the special leaf color formation mechanism of this color-leafed variety, can also provide the theoretical basis for the breeding of new varieties in C. camphora.

Fatty acid desaturation 2 (FADS2), as a rate-limiting enzyme in the synthesis of polyunsaturated fatty acids (PUFAs）, such as arachidonic acid (AA) and docosahexaenoic acid (DHA), can dehydrogenate its substrate to form a double bond at the 6~7 position. In this study, FADS2 gene (GenBank No. MK292654), with 1 335 bp length of the CDS and encoding 444 amino acids, was cloned and had high homology with Ovis aries (XM_012146317.2) and Bos taurus (NM_001083444.1). Using siRNA technique in goat mammary epithelial cells (GMECs), the mRNA and protein levels of FADS2 decreased 60%~70% (P<0.01) and 15%~18% (P<0.05), respectively. Interfering FADS2 expression significantly up-regulated the expression of genes related to the synthesis of PUFAs, such as elongase of very long chain fatty acids 2 gene (ELOVL2) (P<0.01), ELOVL6 (P<0.01), fatty acid desaturation 1 gene (FADS1) (P<0.05); It also promoted the expression of sterol regulatory element-binding transcription protein gene (SREBP1a) (P<0.01), fatty acid synthase gene (FASN) (P<0.05), acetyl-CoA carboxylase gene (ACACA) (P<0.05), and stearoyl-CoA desaturase1 gene (SCD1) expression (P<0.05), respectively. In addition, interfering FADS2 expression could significantly decreased the proportion of AA and DHA (P<0.05) and restrained PUFAs synthesis (P<0.05). Furthermore, interfering FADS2 gene expression could significantly inhibit diacylglycerol acyltransferase1 (DGAT1) (P<0.01) and DGAT2 (P<0.05) gene expression to decrease triacylglyceride (TAG) content in GMECs (P<0.05). In conclusion, FADS2 gene plays an important role in regulating PUFAs synthesis and triglyceride metabolism in dairy goats, which provides experimental basis for the study of PUFAs metabolism in goat milk.

Bone morphogenetic protein 2 (BMP2) is named for its ability to initiate, promote and maintain the regeneration of cartilage and bone. In recent years, studies have found that BMP2 played an important role in promoting fat formation and was associated with the formation of sheep (Ovis aries) tail type. Therefore, BMP2 was chosen to be a novel candidate gene for the sheep tail traits in order to find new molecular markers for breeding. In the 533 sheep experiment group, 30 DNA samples of Hu sheep or Tibetan sheep with a fixed concentration were selected to construct the DNA pool, respectively, primers were designed to extend exons and 1 000 bp of upstream and downstream regulation regions. PCR products were tested and the objective strap-was sequenced. Sequenom Mass-array technology was used for genotype of detecting SNP. Haploview 4.1 was used to construct haplotypes and analyze linkage disequilibrium of the polymorphic loci. Association analysis between the SNPs in BMP2 gene and the tail traits were performed using SPSS 19.0 software. 7 SNPs were detected. rs402970343, rs594116558, rs160577302 and rs429072090 were related to tail length, tail width and tail perimeter. rs417201901 and rs402226298 were involved in tail width and tail perimeter. rs413373794 was only related to tail width. Meanwhile, the combined genotype of rs417201901-rs402970343 and rs413373794-rs402226298-rs429072090 correlation analysis revealed that: Individuals with CCGG and CCCCGG combined genotypes were significantly better than the other individuals on tail length, tail width and tail perimeter (P<0.05 or P<0.01). The results showed that the different SNPs and combined genotypes in BMP2 gene had effect on sheep tail traits with the SNPs being potentially valuable as genetic markers for breeding.

In mammals, genomic imprinting is an epigenetic phenomenon by which certain genes are expressed from one allele, in a parent-of-origin-specific manner. ANO1 (Anoctamin 1) protein is a subunit of calcium-activated chloride channel. Overexpression of ANO1 gene is associated with a variety of human (Homo sapiens) cancers. In human, ANO1 gene exhibits biallelic expression in embryos and adult tissues, but shows maternal allele expression in placenta. At present, the imprinting status and regulation mechanism of ANO1 gene in bovine (Bos taurus) have not been reported. In this study, in order to analyze the imprinting status of ANO1 gene in cattle, SNP was first found on ANO1 gene by direct sequencing of PCR products, and then reverse transcription-PCR (RT-PCR) was performed on RNA derived from tissues and placentas of heterozygous cattle. Direct sequencing showed that the imprinting status of ANO1 gene in cattle was placenta-specific imprinting and maternal allele-specific expression. The specific methylation status of the promoter region of ANO1 gene in bovine placenta, bovine sperm and adult bovine tissues was further analyzed by sulfite sequencing. It was found that the promoter methylation of ANO1 gene did not participate in the gene imprinting expression. The results showed that bovine ANO1 gene was a placenta-specific paternal imprinting gene, which provides a reference for further study of the relationship between ANO1 gene expression and related diseases.

Puerarin has been proved to have antipyretic effects in animals. However, how puerarin works on the cellular or molecular levels is still poorly understood. The aim of the present study is to determine if puerarin has protective effect on the pig (Sus scrofa) kidney proximal tubular cells (LLC-PK1) subjected to heat stress and its possible underlying mechanisms in vitro. The LLC-PK1 cells were divided into control (C) group, heat stress (HS, 42 ℃) group, puerarin (Pue, 10 µmol/L) group, and heat stress + puerarin (HS+Pue) group. Cell viability was measured by CCK-8; cells apoptosis rate was detected by flow cytometry; mRNA and protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cytochrome c (Cyto c) and apoptosis inducing factor (AIF) were detected by qRT-PCR and Western blot, respectively. Cysteinyl aspartate specific proteinase-9 (Caspase-9) and Caspase-3 activities and total protein content of heat shock protein 72 (HSP72) and HSP90 were detected with the corresponding kits. The results showed that puerarin suppressed Cyto c and AIF mRNA expressions and their proteins released from mitochondria, and decreased both Caspase-9 and Caspase-3 activities of the LLC-PK1 cells under HS. Moreover, puerarin further increased Bcl-2/Bax ratio and HSP72 expressions, and decreased HSP90 expressions of the cells under HS. The results indicated that puerarin attenuates HS-induced apoptosis of the LLC-PK1 cells through inhibiting mitochondrial apoptotic pathway activation, and up-regulating HSP72 expression. This study reveals a part of the mechanism by which puerarin inhibits heat stress-induced damage in LLC-PK1 cells, providing basic materials for the use of puerarin as an effective drug against heat stress in practice.

Mammal's reproductive function is the most vulnerable to heat stress in all physiological functions. The main objectives of this research were to explore the influence of baicalin on oxidative damage of uterus tissue in mice (Mus musculus), and understand the anti-oxidation principle of baicalin. Female mice were divided into control (C) group, baicalin (Bai) group, heat stress (H) group and baicalin plus heat stress (H+Bai) group. TUNEL, spectrophotometry, Western blot and immunohistochemistry were used to detect uterine tissue structure, apoptosis and signal transduction. The results showed that, compared with the H group, the uterine tissue injury and apoptosis in mice treated with heat stress were reduced by baicalin, the content of MDA (malondialdehyde) was extremely significantly decreased (P<0.01), the activity of T-SOD (total superoxide dismutase dismutase) was extremely significantly increased (P<0.01), the activity of CAT(catalase) and GSH-Px (glutathione peroxidase) was significantly increased (P<0.05) in group H+Bai. The expression levels of Keap1 (kelch-like ECH-associated protein-1) and Nrf2 (NF-E2-related factor 2) proteins were remarkably decreased by baicalin (P<0.05), and the expressions of Bcl-2 (B-cell lymphoma-2), Bax (Bcl-2 associated X protein), Apaf-1 (apoptotic protease activating factor-1), Caspase-9 (cysteinyl aspartate specific proteinase-9) and Caspase-3 proteins in endometrial epithelial cells and adenocarcinoma epithelial cells were decreased in group H+Bai. Overall, baicalin might attenuate oxidative damage and apoptosis in endometrial epithelial cells of heat-stressed mice by activating Keap1/Nrf2 signaling pathway and then regulating the activity of antioxidant enzymes. The results of this research provide a theoretical approach to addressing animal heat stress, and provide basic information for screening anti-heat stress drugs and solving the decline of fecundity in summer dairy cattle (Bos taurus).

Indigenous chicken (Gallus gallus) breeds are important part of China's poultry genetic resources. Zhejiang province has a long history of breeding chicken and has various chicken genetic resources. In order to study the genetic diversity and genetic structure of main chicken breeds in Zhejiang province and promote the protection and rational development and utilization of different chicken breeds resource, the present study used short tandem repeat (STR) typing technique to detect the genetic diversity of 6 chicken breeds Queshan chicken, Xianju chicken, Xiaoshan chicken, Silky chicken, Longyou Ma chicken, and Baier Huang chicken (total of 358) at 26 microsatellite loci. The results showed that 153 effective alleles were detected at 26 sites in 6 populations, and the average values of effective number of alleles (Ne), expected heterozygousity (He), gene flow (Nm) and Shannon information index I were 5.906 9, 0.795 9, 2.007 and 1.925 1, respectively, and the polymorphism information content (PIC) was 0.24 to 0.87. Nei genetic distance was 0.103 5~0.380 9, among which the genetic distance between Queshan chicken and Xiaoshan chicken was the farthest (0.3809), followed by Queshan chicken and Baier Huang chicken (0.1072), and the genetic distance of Longyou Ma chicken and Xianju chicken was the smallest (0.1035). The results of cluster analysis showed that Queshan chicken and Baier Huang chicken were a single cluster and Xianju chicken and Longyou Ma chicken were a group. All the 6 groups had high genetic diversity, among which the Baier Huang chicken had the highest genetic diversity, the Silky chicken had the lowest genetic diversity, and the similarity order of Queshan chicken with other chicken breeds was Baier Huang chicken, Silky chicken, Longyou Ma chicken, Xianju chicken and Xiaoshan chicken consecutively. In this study, the genetic diversity of 6 chicken breeds in Zhejiang province was analyzed, and the genetic structure of the main chicken breeds in Zhejiang province was further improved, which might provide basic data for the development and utilization of chicken genetic resources.

Inositol triphosphate receptor type Ⅲ(IP3R3) is mediator of second messenger-induced intracellular calcium release. It can combine with inositol triphosphate (IP3) to form calcium channel and regulate intracellular calcium release. In order to explore the genetic effects of IP3R3 gene on duck (Anas platyrhyncha domestica) eggshell quality and enrich the molecular genetic basis of important economic traits related to laying ducks. In this study, qRT-PCR was used to detect the tissue expression of IP3R3 gene in Guizhou Sansui ducks. According to the sequence of IP3R3 gene in GenBank, PCR amplification primers were designed, and the amplified products were recovered, purified and sequenced directly.. The SNP loci of 120 Sansui ducks were screened and identified by DNA Star software MegAlign program and sequencing peak map, and SPSS18.0 general linear model (GLM) was used to analyze the relationship between SNPs. The results showed that IP3R3 gene expression was detected in 11 tissues of ducks. The results showed that the mRNA of IP3R3 gene was detected in 11 tissues of ducks, among which the expression abundance of eggshell gland was the highest, and the relative expression quantity of lung, chest muscle and muscle stomach was the lowest. The expression level of IP3R3 gene in different tissues of duck was as follow: Eggshell glands>heart>spleen>glandular stomach>pancreas>kidney>liver>small intestine>lung>pectorals>muscular stomach. SNPs identification results showed that 4 SNPs were found in the IP3R3 gene, including g.21663 C>T and g.21699 G>A in exon 20, g.21646 G>C in intron 19, and g.21825 C>A in intron 20. Further analysis showed that exons g.21663 C>T and g.21699 G>A were synonymous mutation sites, which did not cause changes in tyrosine (TAC) and lysine (AAG). The results of genetic analysis of 4 SNP loci showed that all four SNP loci were moderately polymorphic and deviated from Hardy-Weinberg equilibrium (P<0.05) and results of association analysis showed that the influence of g.21646 on the egg shape index reached a significant level (P<0.05), in which the GG egg shape index was significantly higher than the CC genotype, and the effect of g.21663 on eggshell strength reached a significant level (P<0.05), among which the CT genotype eggshell strength was significantly higher than the TT and CC genotypes, and the other 2 SNP sites did not reach a significant level. Comprehensive correlation analysis and expression results showed that IP3R3 gene expression in the uterus of laying ducks was higher than that in other tissues. It was preliminarily speculated that the high expression of IP3R3 in the eggshell glands might be related to the regulation of eggshell quality, and the mutations of g.21663 and g.21646 loci had significant effects on duck eggshell quality, which could be used as candidate markers assisted by molecular markers for duck eggshell quality traits and provide scientific data for improving duck eggshell quality and poultry breeding.

Haptoglobin (Hp), mainly synthesized by the liver, plays an important role in the acute phase response (APR) induced by infection, trauma, and inflammation. In this study, the full-length cDNA sequence of the Hp gene (LjHp) (GenBank No. MK820673) of the Japanese sea bass (Lateolabrax japonicus) was obtained from the transcriptome sequencing data of Japanese sea bass. The cDNA sequence of LjHp consisted of 2 285 nucleotides, which contained a large open reading frame predicted to encode a precursor protein comprising of 309 amino acids with a molecular weight of approximately 33.82 kD. A predicted signal peptide of 22 amino acids was present in the N-terminal of LjHp. Sequence analysis showed that LjHp had the typical characters of fish Hp, and shared the highest amino acid sequence identity (76.8%) with that of European seabream (Dicentrarchus labrax). Phylogenetic tree analysis confirmed that LjHp fell into the fish Hp cluster, and grouped tightly with swamp eel (Monopterus albus) Hp. LjHp mRNA was mainly expressed in the liver of healthy Japanese sea bass, and its expression increased significantly in liver, spleen and head kidney after infection with Vibrio harveyi (P<0.05). Western blot analysis showed that LjHp was modified by N-glycosylation in the serum of the Japanese sea bass. Moreover, the serum content of LjHp up-regulated significantly after infection with V. harveyi (P<0.05). The above results showed that the mRNA and protein expression of LjHp was closely related to V. harveyi infection, revealing that it might be a positive acute phase protein in fish against bacterial infection. The present study provides a theoretical basis for studying the functions of fish Hp and its molecular mechanism in APR induced by pathogens.

As a key factor in the initiation of T cell receptor (TCR) signal transduction, tyrosine protein kinase ZAP-70 (70 kD zeta-associated-protein) is critical to the proliferation and activation of T cells, contributing to the fight against bacterial infection. However, current studies on ZAP-70 in fish are quite limited. In this study, a series of experiments were conducted to uncover the potential role of ZAP-70 in hybrid snakehead (Channa maculata ♀ ×Channa argus ♂)(CmaZAP-70), a feature economic species in China. The complete ORF of CmaZAP-70 gene (GenBank No. MK134563) was cloned and its response to Aeromonas schubertii and Nocardia seriolae infection was analyzed. The structure, homology and phylogenetic tree of CmaZAP-70 sequence were also analyzed. Moreover, the plasmid pEGFP-N1-ZAP70 was constructed to detect the effect of overexpression on the activity of nuclear factor kappa-B (NF-κB) and activating protein 1 (AP-1). Sequence analysis showed that the complete ORF of CmaZAP-70 gene was 1 818 bp, which encoded 605 aa with relative molecular weight and theoretical point of 68.2 kD and 8.06, respectively. SignalP 4.1 Server analysis showed that CmaZAP-70 was a non-secretory protein without signal peptide, which contained 2 tandem SH2 domains and 1 SH1 kinase domain. In addition, CmaZAP-70 was highly conserved with some phosphorylation sites and catalytic motifs of mammal ZAP-70. Phylogenetic analysis showed that CmaZAP-70 was clustered into 1 group with other teleost and it was clustered closely with Dicentrarchus labrax, which indicated that 2 species had high homology. Moreover, the qRT-PCR results showed that CmaZAP-70 was widely expressed in 10 tissues of healthy hybrid snakehead, with the highest expression in spleen (P<0.05) and relatively highly in the thymus, head kidney and blood, whereas it was expressed at lowest levels in the skin (P<0.05). The expression changes of CmaZAP-70 were similar and mainly up-regulated in the liver, spleen, and head kidney at different time points after infection with the pathogens A. schubertii and N. seriolae, respectively. After infection with A. schubertii, CmaZAP-70 showed the highest expression in the liver, spleen and head kidney at 1 d, 1 d and 2 d, respectively (P<0.05), while it all displayed the highest expression in the liver, spleen, and head kidney at 3 d after infection with N. seriolae (P<0.05). Furthermore, the dual-luciferase reporter assay showed that overexpression of pEGFP-N1-ZAP70 significantly enhanced the activity of NF-κB and AP-1, which were 4.3 and 3.5 times higher than that of control, respectively (P<0.05). The results conclude that CmaZAP-70 might play a crucial role in the defense against pathogen infection, and participates in the regulation of signal transduction. This study would lay a solid foundation for further revealing the anti-infection mechanism of TCR signaling pathway in fish.

Water fern Azolla is a striking plant-cyanobacteria mutualistic association. N2-fixing cyanobacteria and numerous bacteria have been found within the leaf cavities of Azolla using traditional techniques. The aim of the present study is to explore the genetic diversity of microbial community, particularly fungi and archaea, employing scanning electron microscopy (SEM), fluorescence in situ hybridization (FISH) and high throughout sequencing technique. 250 microbial samples isolated from 120 leaf cavities of fresh and healthy Azolla microphylla fronds were examined with both fluorescence microscopy and electron microscopy. Fungi-like structures, including hyphae, ascus, ascospore, conidium and basidium, were found in 230 samples. Eighty percent of fungi-like structures were positioned in leaf 7th to leaf 15th, indicating the older of leaf age, the more number of fungi detected. Archaea was investigated positively in 86% of the samples, and there was no correlation between the abundance and the leaf age. Through high throughout sequencing both fungi and archaea were identified qualitatively and quantitatively. The results showed that there were Ascomycota(67.39%), Phragmoplastophyta (31.72%), Cordycipitaceae (0.56%), Entomophthoromycota (0.33%), totally 4 fungal phyla, and 2 archaeal phyla with dominance of Euryarchaeota (99.68%) co-existing in Azolla. The results of this study implied that the microecosystem within Azolla was much more complex than we originally thought. It also indicates that Azolla is a valuable model plant for the study of plant-microbe interaction.

Liver injury has a high incidence in pet clinical cases, which can be caused by many factors. In recent years, with the development of cell therapy research, mesenchymal stem cell (MSCs) transplantation has gradually become a hot spot and main directions in the treatment of liver injury. At present, drug-induced animal models are the main research methods in the treatment of liver injury. It is very important to select a suitable and effective method to induce MSCs differentiate into hepatocytes. Some studies have shown that the treatment of MSCs with melatonin (Mel) can improve their viability in vitro and enhance the effect of transplantation therapy. It is also used in the treatment of diseases. In this review, adipose-derived mesenchymal stem cells (ADSCs) combined with melatonin transplantation for the treatment of liver injury are systematically described, which will provide a theoretical basis and guidance for further study.

As a host cell chaperone protein, P58^IPK (58 kD inhibitor of PKR) plays an important role in regulating the body's antiviral and immune response. In this study, CDS region of porcine (Sus scrofa) P58^IPK gene was cloned and its prokaryotic expression vector was constructed. Purified P58^IPK protein was used as an antigen to produce polyclonal antibody, which would provide basic materials for the further study on the interplay between Porcine circovirus type 2 (PCV2) and its host. A specific pair of primers with the usage of amplifying the partial P58^IPK CDS sequence (containing no signal peptide sequence) by reverse transcription PCR (RT-PCR) was designed according to the complete genome sequence of porcine P58^IPK (GenBank No. HQ287801.1). After double digestion by NdeⅠ/XbaⅠ restriction enzymes, the products were cloned into the prokaryotic expression vector pCzn1. After sequencing, the generated recombinant expression plasmid pCzn1-P58^IPK was transformed into bacterium Arctic Express^TM Escherichia coli. The expressed His-P58^IPK recombinant proteins were denaturated, renaturated and purified by using His-Band Ni⁺ affinity chromatography, then were used as target antigens to immunize rabbits (Oryctolagus?cuniculus) to prepare polyclonal antibody (pAb). After the pAb was purified, its titer and specificity were detected by indirect ELISA and Western blot, respectively. The results showed that the length of the cloned P58^IPK gene fragment was 1 425 bp, the expressed His-P58^IPK recombinant protein with a molecular weight of about 54.44 kD mainly existed in a form of inclusion body. The titer of the pAb prepared from this recombinant protein was above 1∶512 000, and the specificity of this pAb was verified very well as it could not only recognize the His-P58^IPK recombinant protein but also porcine P58^IPK protein. These data indicated that the partial CDS region of porcine P58^IPK gene has been successfully cloned, expressed, purified and the prepared polyclonal antibody could be used for the detection of porcine P58^IPK protein. The obtained polyclonal antibody could be used as basic material for further studying of the function of porcine P58^IPK protein and interaction mechanism between PCV2 virus and the host.