Whirly family is a kind of plant-specific transcription factors, which have extensive and complex biological functions in both the nucleus and organelles. Because of the rotating appearance of its quaternary structure, it is called Whirly protein, and the corresponding gene is named Whirly gene. This family plays an important role in plant growth and stress response. In order to explore the function of wheat (Triticum aestivum) Whirly family, the basic characteristics of TaWHY gene family members were analyzed by bioinformatics method, and their responses to various stresses were verified by qPCR. The results showed that there were 6 members in TaWHY family, named TaWHY1-A, TaWHY1-A-1, TaWHY1-D, TaWHY2-A, TaWHY2-B and TaWHY2-D, respectively, which were distributed on 6 chromosomes of wheat, and all the member sequences contained introns. The results showed that TaWHYs protein instability index was 30~60, and the average coefficient of hydrophilicity was less than 0, which indicated that wheat Whirly protein was stabled in nature and belongs to hydrophilic protein. TaWHY protein was distributed in mitochondria, chloroplast, nucleus and cytoplasm. The secondary structure of TaWHY protein was mainly alpha helix and random curl, and there were 2 three-dimensional structure models. Transcriptional data analysis showed that TaWHY gene showed different tissue expression patterns and was regulated by different abiotic stresses. The results of qPCR showed that the relative expression of TaWHY1-A gene was significantly up-regulated under low temperature stress conditions(P<0.05), which could respond to low temperature stress. The results of this study can provide reference for further functional verification and mechanism analysis of TaWHY.

Glutathione peroxidase (GPX), as one of the most important antioxidant enzymes in plants, plays an important role in plant stress response. In the analysis of the transcriptome data of the wheat (Triticum aestivum) GPX gene family, it was found that the TaGPX8 (IWGSC accession No. TraesCS4D02G162000.1) had the characteristics of being induced by stress. In this study, TaGPX8 was cloned, its protein was analyzed by bioinformatics and subcellular localized, and the expression of TaGPX8 in roots and leaves under salt and drought stress were analyzed.The results of gene structure analysis showed that TaGPX8 had a full length of 14 754 bp, including 6 exons and 5 introns, and the full length of CDS was 564 bp, encoding 187 amino acid residues. The relative molecular mass of the encoded protein was 21.330 kD, its theoretical isoelectric point was 6.62, and its average hydrophilicity was -0.664. Promoter sequence analysis found that the promoter sequence of TaGPX8 contained some cis-acting elements involved in hormone response, light response, and stress response. qPCR results revealed that TaGPX8 gene responded to drought and salt stress. Under drought stress, the expression level of TaGPX8 in leaves at 12 h had no significant difference with the control, but was significantly induced at 24 h, its expression level was up regulated, which was 12 times that of the control, and the expression level returned to no significant difference with the control in 48 h. In addition, the expression in roots also increased at 48 h of drought stress treatment. Under salt stress, the expression of TaGPX8 in leaves was inhibited to a certain extent at 12 h of treatment, but the expression level increased at 24 h of treatment, and then recovered to no significant difference with the control at 48 h. However, the expression in the root was suppressed. The yeast transcription activation experiments verified that TaGPX8 did not have transcriptional activation activity. Subcellular localization showed that the TaGPX8 protein was localized in the nucleus and cell membrane. This study provides a reference basis for further research on the function of TaGPX8 gene.

Polyamines which play an important role in plant growth and development are aliphatic nitrogen-containing bases with low molecular weight and biological activity. Polyamine uptake transporters (PUT) mediate the intracellular and intercellular transport of amino acids, polyamines, paraquat and thiamine. Studies showed that Arabidopsis thaliana polyamine transporter 3 (AtPUT3) mutant atput3 has the ability to resist 0.1 μmol/L paraquat. In order to study the role of rice (Oryza sativa) polyamine transporters (OsPUT) in plant growth and development under paraquat stress, 6 polyamine transporters were identified in the genome of rice by bioinformatics methods. The results showed that OsPUT1 and AtPUT3 proteins which have similar molecular weights and structures were membrane proteins with 12 transmembrane structures and amino acid transport domains, and the N-terminal and C-terminal were on the cytoplasmic side. The overexpression vector pFGC1300-35S-OsPUT1 was constructed by double restriction endonuclease digestion and sequencing, and then transformed into Arabidopsis thaliana atput3 mutant. In the whole life cycle, under short-day condition, the phenotypic differences were not obvious among wild-type (WT), mutant atput3 and overexpression (OE) lines, and there was no significant difference between the length of bolting time and the number of rosette leaves during bolting. There was no significant difference among the 3 germination rate when seeds germinated on 1/2 MS medium (0 μmol/L PQ) for 48 h. However, the seed germination rate of WT and OE lines was significantly lower than that of atput 3 (P<0.05) when seeds germinated on 1/2 MS medium (0.1 μmol/L PQ) for 5 d. After 10 d of seedling growth on 1/2 MS medium (0.1 μmol/L PQ), the phenotype of OE lines returned to WT state. Compared with atput3, the length of taproot of WT and OE lines decreased significantly (P<0.05). It shows that the function of OsPUT1 gene restores the phenotype of atput3 to WT, which proved that rice OsPUT1 protein and Arabidopsis AtPUT3 protein had similar functions. This study provides a reference for further exploring the function of OsPUT1 gene, and provides some enlightenment for further exploring the resistance of rice to PQ.

APETALA2/ethylene responsive factor (AP2/ERF) family is one of the largest transcription factor families in plants. ERF subfamily is the main member of AP2/ERF family and plays an important role in plant response to biotic and abiotic stresses. In this experiment, the bioinformatics analysis, subcellular localization and gene expression analysis of potato (Solanum tuberosum) StERF5 gene were carried out to further clarify the function of the gene. The results showed that the coding region of the gene was encoding 243 amino acids and contained an AP2 domain. The subcellular localization showed that the protein was localized in the nucleus and cell membrane. Tissue specific expression analysis showed that StERF5 gene was expressed in roots, stems and leaves, with the highest expression level in roots. There were significant differences in the expression levels of StERF5 gene in stems of different potato varieties (P<0.01). Expression of StERF5 gene was up-regulated under drought and salt treatments. This study lays the foundation for the subsequent in-depth understanding of function of StERF5.

Dihydroflavonol-4-reductase (DFR) plays a key catalytic role in anthocyanin synthesis. Anthocyanin can protect tissues from oxidative damage induced by abiotic stress, and can also be used as chelating agents to promote plant iron absorption. In this study, GmDFR gene (GenBank No. MN547961) and its promoter pGmDFR fragment (GenBank No. MW455109) were cloned from soybean (Glycine max). The CDS length of GmDFR gene was 1 020 bp and encoded 339 amino acids. GmDFR had typical PLN02650 and WcaG superfamily domains, belongs to dihydroflavonol 4-reductase and nucleoside diphosphate sugar isomerase family, GmDFR protein was not a transmembrane protein. Phylogenetic analysis showed that soybean GmDFR was most closely related to wild soybean (G. soja) GsDFR, with a similarity of 99.12%. Semi-quantitative RT-PCR detection showed that GmDFR was expressed in soybean tissues and the highest expression level was in roots. The pGmDFR promoter fragment was obtained by cloning, which contained multiple hormone and stress response elements. Under iron deficiency, the anthocyanin content and chlorophyll content of GmDFR transgenic tobacco (Nicotiana tabacum) were significantly higher than that of WT (P<0.05); the contents of malondialdehyde, hydrogen peroxide and superoxide anion were significantly lower than that of WT (P<0.05); and the activities of antioxidant enzymes (SOD, POD and CAT) were significantly higher than that of WT (P<0.05). These results indicated that transgenic GmDFR tobacco had certain ability to resist iron deficiency. This study provides scientific basis for the application of GmDFR gene in soybean resistance to iron deficiency, and also provides new gene resources for soybean molecular breeding.

4-coumaric acid coenzyme A ligase (4CL) is a key enzyme in the flavonoid synthesis pathway, catalyzing the hydroxycoumaric acid to the formation of coumaroyl-CoA, which is a key enzyme in plant flavonoid biosynthesis at the starting substrate. In this research, the welsh onion (Allium fistulosum) was used as the material to clone the Af4CL gene, which encoding the welsh onion 4-coumaric acid-CoA ligase, and the bioinformatics analysis were also carried out. At the same time, the localization and prokaryotic expression system of Af4CL gene were analyzed. The results showed that the full-length coding region of Af4CL was 1 644 bp and it encoded 547 amino acids. The bioinformatics analysis showed that there were 2 conserved structural regions: BoxⅠ and BoxⅡ; Evolutionary analysis results indicated that Af4CL was most closely related to As4CL of garlic (Allium sativum) . The 35S:Af4CL-GFP vector was constructed, and the localization of Af4CL was analyzed by Agrobacterium-mediated transient expression system in tobacco (Nicotiana tabacum) leaves. The results showed that Af4CL was localized in the cell membrane; and the target protein was successfully induced and purified by the prokaryotic expression system. In conclusion, Af4CL gene was successfully cloned and the bioinformation analysis were carried out. At the same time, the target protein was successfully induced. These studies provide reference for the study of flavonoid metabolism and in vitro synthesis of flavonoids in A. fistulosum.

Lysin motif (LysM) receptor kinases is an important class of receptor-like kinases found in plants and play a key role in plant defense against stress and diseases. The purpose of this study is to explore the members of LysM receptor kinases gene family in Vitis spp. and their functions. Based on the genomic data of Pinot Noir (Vitis vinefera cv. Pinot Noir) from the Eurasian species grapevine and Shanputao (Vitis amurensis) from East Asian species grapevine, VvLysM and VaLysM gene family members were identified using bioinformatic methods, and the protein physicochemical properties, secondary structure, chromosome distribution, conserved motifs, promoter cis-acting elements and other characteristics of these gene family members were compared and analyzed. The expression of VvLysMs under different tissues and abiotic stress were verified by gene chip data and qPCR. The results showed that VvLysM and VaLysM gene family each contained 12 members, and distributed in 9 chromosomes of Pinot Noir and 7 chromosomes of Shanputao, respectively, and the expansion of this family was based on tandem duplication and fragment duplication. The analysis of the physical and chemical properties of the proteins revealed that the amino acid sequence, molecular weight, isoelectric point, fat coefficient and other characteristics of the different members of the two grapevines were different. Most of LysM receptor kinase proteins had poor structural stability, low protein fat solubility, and were hydrophobic; the subcellular location indicated that this gene family was mainly distributed on the plasma membrane, and the secondary structure was dominated by α helix and random coils. According to evolutionary analysis, 24 gene members were divided into 3 subgroups. The quality and distribution characteristics of exons and introns, and the number and distribution of conservative motifs were highly similar among the different genes under the same subgroup. The quantity variation of introns arranged from 0 to 12 presented diversity. The analysis of cis-elements showed that there was a large number of light response elements, hormone response elements and stress response elements in the promoters of these genes members. The expression of VvLysMs in various tissues of grapevine showed obvious tissue expression specificity, especially the expression levels of them were higher in the late development of fruit and flower organs. The results of qPCR showed that different members of this gene family presented different expression patterns due to the types of stress after treatment with exogenous abscisic acid, salicylic acid, methyl jasmonate, low temperature and chitosan especially when VvLysM8 and VvLysM9 were treated with chitosan for 12 h, and VvLysM9 was treated with abscisic acid for 3 h, their relative expression levels were the highest and significantly higher than those of other members under all treatments. VvLysM8 and VvLysM9 could play an important role in abscisic acid signal transduction and chitin induced immune response in plants. This study may provide a reference for further research on the function of grapevine LysM receptor kinase.

MYB transcription factor is an important regulatory factor in plant growth and secondary metabolism, and plays key roles in anthocyanin biosynthesis. In this research, 9 VdMYB genes were identified and cloned from Vitis davidii callus based on previous transcriptome data. The MYB gene structure and function were analyzed and predicated by bioinformatic softwares, and their expression patterns were analyzed under different light quality. The results showed that most MYB genes included 3 exons and 2 introns; the number of amino acid residues of VdMYBs were 216~335, the relative molecular mass were 24.09~36.17 kD, and the isoelectric point were 5.42~9.58. The phylogenetic tree analysis indicated that the 9 VdMYB proteins were divided into 6 groups, and were predicted to be located in cytoplasm, mitochondria and nucleus, respectively. Analysis of promoter cis-acting elements showed that a large number of light-response and bio/abio-response elements were predicted in the 9 VdMYB promoters. The qRT-PCR analysis showed that light quality significantly affected the expression of VdMYBs, their expression pattern corresponded to the structural characteristics under different light quality. Among 9 VdMYB genes, VdMYB31/VdMYBB1 and VdMYB4A might play as positive and negative regulators involved in anthocyanin biosynthesis.This study provides theoretical reference for further elucidating the light-response molecular mechanism of MYB family genes involved in the regulatory pathways of anthocyanins in Vitis davidii.

Tea plants (Camellia sinensis), as one of the important woody economic plants, are vulnerable to low temperature and freezing injury, which seriously affecting the yield and quality of tea. In order to further clarify the molecular mechanism on cold response of tea plants, in this study, based on the cold treatment transcriptome library of 2 Guizhou cultivars, Qiancha 1 (QC1) and Qianmei 601 (QM601), a cold-upregulated differentially expressed mitogen-activated protein kinase (MAPK) gene (transcriptome library number: CSS0027011) was isolated and cloned, and named as CsMAPK15 (GenBank No. ON39909). The open reading frame of CsMAPK15 contains 1 701 nucleotides encoding a protein of 566 amino acids. Motif analysis revealed that CsMAPK15 had 3 conserved motifs. Phylogenetic analysis showed that CsMAPK15 shared high sequence similarity with homologs from other species. It shared 79.17% identity with AtMAPK16 (AT5G19010) from Arabidopsis and 80.51 % similarity with OsMAPK15 (LOC_Os07g47490) from rice (Oryza sativa). The tissue pattern analysis revealed that expression level of CsMAPK15 was highest in roots. Promoter analysis showed that the CsMAPK15 promoter had several cis-acting elements, including stress responsive element (STRE) and salicylic acid (SA) responsive element (TCA element). qPCR confirmed that CsMAPK15 could response to cold signal and SA. Furthermore, the transgenic rice plants which overexpressed CsMAPK15 were successfully obtained by callus transformation. The preliminary results showed that there was no significant change in plant growth and development between transgenic plants and wild-type plants. This study provides research materials and theoretical basis for further clarifying the role of CsMAPK15 in plant cold resistance regulation．

G protein subunit alpha Q (GNAQ) regulate G protein-coupled receptors and mediate downstream signaling pathways through encoding Gαq protein, which involves many physiological processes such as nutrient metabolism, reproduction regulation and cell signal transduction. This study aimed to explore the expression of GNAQ in reproductive axons of female yak (Bos grunniens). The samples of hypothalamus, pituitary and ovarian of healthy female yaks (adult) during follicular phase were collected. The CDS of GNAQ gene was cloned with yak ovarian cDNA as template by reverse transcription-PCR (RT-PCR) and it was analyzed by bioinformatics software. The hypothalamic-pituitary-ovarian reproductive axis (HPOA) expression characteristics of yak GNAQ gene and protein levels were detected by qRT-PCR, Western blot and immunohistochemistry (IHC) .The results showed that the CDS of yak GNAQ gene (GenBank No. OP811271) was 1 080 bp in length and encoded 359 amino acids. GNAQ was close to amino acid homology of ordinary cattle (B. taurus), and distant to mouse (Mus musculus), and it was highly conserved in the evolution of the species. It was found that GNAQ gene and protein levels were expressed in hypothalamus, pituitary and ovary of yak by qRT-PCR and Western blot, and the highest expression level was found in pituitary, the expression level in hypothalamus was significantly higher than in ovary (P<0.05). Immunohistochemistry results showed that GNAQ protein was strongly positive in ovarian granulosa cells and was mainly expressed in cytoplasm, which was consistent with the prediction of subcellular localization. This study provides theoretical basis for further exploration of the role of GNAQ gene in reproductive physiology regulation of yak, which is helpful to study the regulation and mechanism of seasonal estrus in yak.

Inhibin (INH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have important effects on the development of follicles. This study aimed to detect the expression of INHβB, FSH receptor (FSHR) and LH receptor (LHR) genes and proteins in yak (Bos grunniens) antral follicles with different diameters by qPCR and Western blot, and immunohistochemistry (IHC) was used to detect their distribution in yak antral follicles. The results showed that INHβB gene and protein expression in antral follicles increased first and then decreased with the increase of follicle diameter, and the highest expression level was found in large follicles (5~8 mm in diameter) (P<0.05). The FSHR gene and protein expression in large follicles (5~8 mm in diameter) were significantly higher than that in other follicles(P<0.05), while the expression of FSHR in other follicles with different diameters were not significantly different. LHR gene and protein expression increased with the increase of follicles diameter, and the highest expression level was found in the extra large follicles (diameter>8 mm)(P<0.05). IHC results showed that INHβB, FSHR and LHR mainly distributed in granulosa cells, cumulus cells and follicle membrane. The above results indicates that INHβB might has an important effect on follicle atresia or ovulation in yaks, and FSHR and LHR might enhance the effect of FSH and LH on follicle growth and development. This study provides a reference for improving the reproductive efficiency of yaks.

Glucagon-like peptide-2 receptor (GLP2R) is one of the key mediators of regulating body energy balance, which may have important effects on organ development and growth performance of sheep (Ovis aries). This study was conducted to investigate the association between GLP2R single nucleotide polymorphism and slaughter performance and weight of internal organs such as heart, liver, spleen, lung and kidney in Hu sheep. Firstly, the expression of GLP2R gene in different tissues was analyzed by qPCR. Furthermore, the polymorphism of GLP2R gene locus was detected by kompetitive allele specific PCR (KASP) typing technique in 1 351 male Hu lamb with complete pedigree, and the correlation analysis was conducted with the slaughter traits and weight of internal organs of lambs. The results showed that GLP2R gene was widely expressed in various tissues of Hu sheep, and the relative expression level was significantly higher in kidney and rumen tissues (P<0.05). There were 3 genotypes GG, GA and AA at g.34409249 G>A locus of GLP2R gene exon, which showed moderate polymorphism (0.25<PIC<0.50). This locus was not in the Hardy Weinberg equilibrium state in the population (P<0.05). The single nucleotide polymorphism of GLP2R gene was closely related to the heart, lung and kidney weight of Hu sheep, and the heart, lung and kidney weight of GG genotype group was significantly higher than that of AA genotype group (P<0.05). In conclusion, the mutation site of GLP2R gene exon in G.34409249 G>A can be used as a candidate molecular marker site for heart, lung and kidney organ development, which provides a theoretical basis for the breeding of Hu sheep.

Rabbit hemorrhagic disease (RHD) is a highly lethal infectious disease of rabbits (Oryctolagus cuniculus) caused by Rabbit hemorrhagic disease virus (RHDV). Annexin A2 (ANXA2) is closely associated with the infection process of many viruses. In order to investigate the role of rabbit ANXA2 in the process of RHDV infection of host cells, in this study, rabbit ANXA2 gene fragments were amplified by PCR, and the eukaryotic expression vector pCD513B-ANXA2 was constructed and transfected into 293T cells with packaging plasmids pLP1, pLP2, and pLP/VSVG, then packaged lentiviral particles containing ANXA2 gene, infected rabbit kidney cells (RK13), and screened the cell line RK13-513B-ANXA2 for stable overexpression of ANXA2 protein using puromycin, detected the expression of ANXA2 gene and its protein by qPCR and Western blot. siRNA was synthesized and transfected into RK13 to analyze the gene expression. After 2 h, the relative change of RHDV adsorption was detected by qPCR, immunological fluorescence assay (IFA) and Western blot. The results showed that viral particles with high titers of 5.0×10⁷~1.0×10⁸ TU/mL were obtained using lentiviral vector packaging. The expression of ANXA2 gene and its protein in RK13-513B-ANXA2 cells was significantly up-regulated (P<0.01). The adsorption of RHDV by RK13 overexpressing ANXA2 protein was significantly up-regulated (P<0.01), and interference with ANXA2 expression protein significantly down-regulated the adsorption of RHDV to RK13 (P<0.05). The results suggest that ANXA2 may be involved in the infection of host cells by RHDV, which provides a firm basis for further investigation of the mechanism of RHDV infection in host cells.

Valgus-varus Deformity (VVD) is one of the most common leg diseases in broiler (Gallus gallus) breeding industry. To explore the relationship between the methylation level of the promoter region of the VVD related gene ST3 β-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) and the activity and gene expression of alkaline phosphatase (ALP), the differential expression of ST3GAL4 in different tissues was detected by qPCR, and the differential expression tissues were screened. Further, bisulfite-sequencing PCR (BSP) were carried out to detect the methylation level of ST3GAL4 in blood and differentially expressed tissues. The results showed that the ALP activity of broilers in VVD group was significantly lower than that in normal group (P<0.05); The expression level of ST3GAL4 gene in leg muscle of broilers in VVD group was significantly higher than that in normal group (P<0.05). The results of methylation level showed that the overall methylation level of blood ST3GAL4 promoter region in VVD group was higher than that in normal group (P=0.051), and the methylation level of CpG25 was significantly different (P<0.05). In leg muscle tissue, the overall methylation level of ST3GAL4 promoter region of broilers in VVD group was significantly lower than that in normal group (P<0.01), and the methylation levels of CpG11, CpG14 and CpG15 were significantly different (P<0.05). The prediction results of transcription factors in ST3GAL4 promoter region showed that the key site CpG15 might bind to the transcription factors cAMP response element binding protein 1 (CRE-BP1), cAMP response element binding protein (CREB) and cytoplasmic polyadenylation element binding protein (CPE bind), and the key site CpG25 might bind to the transcription factors specific protein 1 (Sp1) and activator protein-2α (AP-2α). Correlation analysis showed that the methylation level of CpG25 in the ST3GAL4 promoter region in blood was negatively correlated with serum ALP activity, and the methylation level of CpG15 in the ST3GAL4 promoter region in leg muscle was also negatively correlated with gene expression. This study reveals that the methylation level of the promoter region of ST3GAL4 gene in VVD broilers is correlated with gene expression level and serum ALP activity, which provides reference data for disease resistance breeding of broilers.

As a serious global invasive species with strong adaptability to the environment, the golden apple snail (Pomacea canalicata) had spread to 16 provinces cities and regions in the southern China. Heat shock proteins (HSPs) are an important type of molecular chaperone proteins produced by organisms in response to environmental stress and environmental factor stimulation can induce its expression to cope with adverse effects of organism; the heat shock factor (HSF) is a key factor of transcription regulating of heat shock protein genes. In order to explore the role of HSF and HSP genes in the regulation of temperature tolerance of snails, in this study, only 1 HSF1 gene were identified by bioinformatics methods from the P. canaliculata as well as other 3 apple snails (P. maculata, Lanistes nyassanus, Marisa cornuarietis) which were in the same genus or family, and also 63, 66, 67, 64 HSP genes were identified, respectively. The amino acid length and gene structure of HSF1 gene in 4 snails were similar and highly conservative, which showed low gene diversity during the evolution of species. The HSP genes in the 4 snails had little difference, but the subfamily composition of the gene family was quite different. The HSP90 gene was relatively conserved, while HSP20 gene changed greatly. The HSP40 gene had the most members in the gene family among the 4 apple snails. Based on the gene expression of RNA-seq data, the results showed that the expression of HSF1 gene from high to low were gill, foot muscle, liver and ovary, respectively. Under high temperature stress, the HSF1 gene expression in 4 tissues were highly expressed, and the expression pattern was consistent in foot muscle and liver tissue. According to the HSP gene expression heat map, 17 HSP genes were specifically highly expressed under high temperature stress, results of 12 genes were consistent with the qPCR. The present study provides a reference basis for exploring the mechanism of HSF and HSP gene family in temperature tolerance.

Jiaobai as an important aquatic vegetable in China, nearly 20% of the grey Jiaobai in the field has a serious impact on its yield. Previous research showed that the formation of 2 phenotypes of normal Jiaobai and grey Jiaobai in the field was closely related to the differentiation of MT and T type strains of Ustilago esculenta. Grey Jiaobai is caused by the infection of Zizania latifolia by T type strain of U. esculenta. Therefore, 129 494 SNP loci with difference between MT and T-type strains was obtained in this study, by resequencing, SNP calling and SNP filtration. After that, 647 specific SNP loci with specificity of T type of strain were screened by Hardy-Weinberg equilibrium test strategy and short fragment sieving. Finally, based on the 48 loci selected by randomization principle, 8 loci with high accuracy were screened by allele specific PCR (AS-PCR) with designed specific primers for field test. The results showed that the occurrence ratio of grey Jiaobai could be reduced 37.59% after eliminating the seedlings with positive identification results using the combination of the selected 8 specific loci to identify the T-type strain. This research provides technical support for further improving the purity of seedlings in improved seed breeding and subsequent seedling quality detection.

Fat mass and distribution are important factors determining the economic traits of pigs (Sus scrofa). Subcutaneous fat influences carcass quality, and intramuscular fat (IMF) content is strongly correlated with meat quality traits such as tenderness, juiciness and flavor. There is a genetic correlation between fat deposition in meat and subcutaneous adipose tissue, so it has been widely concerned improving IMF content and fatty acid composition through nutrition, thereby increasing the sensory quality and nutritional value of pork. This paper aimes to review on progresses in effect of dietary energy and protein levels, amino acids, polyunsaturated fatty acid (PUFA) and other nutrients on the fat deposition and fatty acid composition in meat and subcutaneous adipose tissue of pigs and their potential molecular mechanism, and provides reference for taking nutritional measures to improve pork quality.

The microbiota-gut-brain axis (MGBA) is one of the most important physiological metabolic regulation mechanisms in animals. The gut microbiota plays a key role in the bidirectional response system mediated by the gut and the brain through neural and endocrine substances. Currently, there have been many reports on MGBA in the field of nutritional metabolism of livestock and poultry, while there are few studies on production and application and functional diseases of livestock and poultry. Based on the relationship between microorganisms and their metabolites and the host intestines, neuroendocrine system, and immune system, this article summarizes the research progress of livestock and poultry MGBA regulation, with a view to providing new ideas for the research and application of livestock and poultry MGBA, to achieving the purpose of scientific and efficient breeding, thereby improving the economic benefits and animal welfare of livestock and poultry and promoting the efficient and healthy development of the livestock and poultry breeding industry.

Insulin-like growth factor 1 receptor (IGF-1R) plays an important role in the growth, development and metabolism of mammals. In this study, the small guide RNA (sgRNA) was inserted into lentiCRISPR v2 plasmid by Gibson assembly and gene splicing by overlap extension PCR (SOE PCR), and pSMART LCKan vector was used to construct IGF-1R gene knockout vector. The vector was verified by bacterial liquid PCR, double enzyme digestion and gene sequencing. After that, the vector was verified by bacterial liquid PCR, double enzyme digestion. The expression of IGF-1R protein in knockout group and control group was compared by Western blot. The results of PCR, double enzyme digestion and gene sequencing showed that the vector was successfully constructed. Western blot showed that the expression of IGF-1R protein decreased extremely significantly in the knockout group (P<0.01), indicating the successful knockout of IGF-1R gene in mouse (Mus musculus) embryonic fibroblast (MEF) cells. This study improved the construction process of expression vector of CRISPR/Cas9 knockout system, and provides a powerful tool for further studying the role of IGF-1R gene at the cellular level.

As one of the common expression elements in nature, the bidirectional promoters (BDPs) can provide new tools for genetic circuit design and metabolic pathway assembly and optimization. However, there is a lack of high-throughput screening and characterization methods for BDPs. In order to obtain the efficient BDPs for multi-gene regulation of metabolic pathways based on genome-level high-throughput screening, a BDPs probe vector p19BDP based on fluorescence generated by the interaction of the green fluorescent sensor for transiently expressed proteins 1 (gSTEP1) and the sensor for transiently expressed proteins tag (STEPtag) was constructed. The gSTEP1 and STEPtag gene were amplified by PCR, and ligated to the p19-0 vector in a "head-to-head" manner. After inserting the BDPs sequence between the two genes, the BDPs activity was assessed by detecting the fluorescence intensity. The p19BDP was used to characterize the activity of 8 BDPs screened by transcriptome data analysis in Corynebacterium glutamicum. The results showed that the fluorescence intensity of the BDP_cat and BDP_icl were 15.9 and 6.2 times higher than those before induction, respectively, the fluorescence intensity was positively correlated with the concentration of inducer in a certain range. For the other 6 endogenous BDPs (BDP₁~BDP₆), the fluorescence intensity of BDP₆ was 6.8 times higher than that of the control promoter BDP_cat (uninduced). The probe vecotr did not affect the growth of recombinant strain, and its fluorescence value remained stable after the formation of gSTEP1/STEPtag complex. The above results showed that the probe vector p19BDP could characterize sensitively and effectively the strength of BDPs in C. glutamicum. This study provides a powerful tool for high-throughput mining of natural BDPs in C. glutamicum and a new idea for characterizing BDPs in prokaryote.

Alternaria tenuissima is one of the pathogenic fungi that causes black spot disease of Fritillaria thunbergii. In order to solve the problem of rapid detection of F. thunbergii black spot disease in the field, this study prepared the monoclonal antibodies (McAbs) that against A. tenuissima and the colloidal carbon immunochromatographic test strip. The cell strains secreting specific McAbs against A. tenuissima were obtained by hybridoma technique using the mixture of hypha and spores of A. tenuissima as immunogen. The titer, sensitivities and specificities of the McAbs were determined by indirect ELISA. The McAbs binding protein was detected by Western blot. The colloidal carbon immunochromatographic test strip was prepared by double antibody sandwich method. Finally, five cell strains were obtained and named as AtA1, AtA8, AtD2, AtG7 and AtH9, respectively. The McAbs did not cross-react with the common pathogenic fungi of F. thunbergii, such as A. alternata, Fusarium solani, F. incarnatum, F. equiseti, F. oxysporum, Botrytis cinerea, Phomopsis oblonga, Phoma sp. and so on. The titer of the McAbs were all higher than 10⁵ times. The relative molecular weight of McAbs binding protein were 30, 30, 43, 148 and 50 kD, respectively. The types of antibodies are IgG2a, IgG3, IgM, IgG2a and IgM, among which the detection sensitivities of AtA1, AtA8 and AtG7 were 12.21 ng/mL, and the detection sensitivities of AtD2 and AtH9 were 24.41 ng/mL. The protein of F. thunbergii had no effect on the detection sensitivity of the 5 McAbs. The test strips that based on AtA1 and AtH9 were prepared for the detection of A. tenuissima and the black spot disease of F. thunbergii infected by A. tenuissima. The strip has the advantages of high sensitivity, strong specificity, simple operation, fast and economical, suitable for large-scale test and so on, which provides technical support for early detection and prevention of black spot disease of F. thunbergii.