Studying the developmental process of gynoecium is very important for breeding. Proteomic technique is a powerful tool for system biology research, and proteomics in strawberry (Fragaria ananassa) gynoecium development has not been reported to date. In order to select a suitable method for large-scale proteomics analysis of strawberry gynoecium, DDA (data-dependent acquisition) and DIA (data-independent acquisition) techniques were applied for label-free quantitative proteomics analysis of strawberry gynoecium at 3 developmental stages of small bud stage, early blooming stage and full blooming stage. The two techniques were evaluated in terms of protein identification quantity, coefficient of variation (CV) of peptide ion peak intensity and the proportion of missing data. The result shows that the coverage, reproducibility, and accuracy of DIA data were higher than those of DDA. A total of 3 687 proteins were identified in DDA and 5 341 proteins were identified in DIA; The average CV value of peptide ion peak intensity for the 3 periods were 28.4%, 33.3% and 28.4% in the DDA data, respectively, while in DIA data the CV values were only 10.4%, 10.9% and 11.9%. Gene ontology (GO) function analysis was performed in the differentially expressed proteins (DEPs) screened by DIA. The DEPs between small bud stage and early blooming stage were mainly annotated to mitosis, methylation, and secondary metabolite synthesis, while the DEPs between early and full blooming stage were annotated to energy metabolism, photosynthesis, and primary metabolism. KEGG enrichment analysis showed that multiple peroxidases including peroxidase 4 (POD4) were involved in lignin synthesis, and their expression increased simultaneously with the flower developmental process. Further analysis of methylation-related proteins tapped into multiple proteins with important physiological functions, including flowering-regulated protein arginine methyltransferase 5 (PRMT5), transposon silencing related DNA (cytosine 5)-methyltransferase 3 (CMT3), and aromatic compounds synthesis related orcinol O-methyltransferase (OOMT). Moreover, some essential proteins such as PRMT5, CMT3 and POD4 were identified only in DIA data, which further demonstrated the superiority of DIA over DDA. This study established DIA as a superior method for large-scale proteomics analysis of strawberry gynoecium, and preliminarily explored the changes in gynoecium protein expression levels during 3 developmental periods of small bud stage, early blooming stage and full blooming stage, which could provide basic information for the research on the mechanism of strawberry gynoecium development process.

Arginine decarboxylase (ADC) is a key enzyme in the synthesis of polyamines, which plays an important role in the resistance of plants to stress. In the early stage of this study, by analyzing the transcriptome database of wheat (Triticum aestivum) and leaf rust fungus (Puccinia triticina) interaction, it was screend that the expression of TaADC in the compatible and incompatible combinations was signifigant difference. On this basis, qRT-PCR detection showed that the expression of TaADC was induced by Puccinia triticina infection in the incompatible combination, not induced in the compatible combination. Transient transformation of recombinant plasmid 35S::TaADC-GFP into tobacco (Nicotiana tabacum) revealed that TaADC localized in the cytoplasm. Virus-induced gene silencing (VIGS) and RNA interference (RNAi) techniques were used to silence TaADC gene. Compared with TaADC non-silent plants (control), silent plants showed increased number of haustorium mother cells (HMC) and area of host hypersentive reaction (HR) in single infection site. The above results proved that TaADC might play an important role in the occurrence of HR in wheat resistance to Puccinia triticina infection. This study might provide new evidence for elucidating the molecular mechanism of HR in the interaction between wheat and Puccinia triticina.

1,5 ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme of photosynthetic carbon assimilation in green plants, and its small structural subunits (Rubisco small subuni, RbcS) affect the function of Rubisco holoenzyme. In this study, the maize (Zea mays) inbred line B73 was material, DNA and RNA were firstly extracted from the leaves, the promoter sequence of ZmRbcS1 gene (GenBank No. 542212) was amplified and its structure was analyzed. Meanwhile, the ORF of the gene was amplified and the vector was constructed. Based on the analysis of the promoter by PlantCARE database, it was found that in addition to a large number of conservative light regulatory elements (such as G-box, GATA-motif, I-box, RbcS-CMA7c, etc.), the promoter sequence also contained a variety of conservative cis-acting elements (such as drought-induced response element (MBS) and gibberellin-responsive element (gibberellin-responsive element) in response to drought, hormones and other stress factors. P-box) and jasmonic acid response element (CGTCA-motif, TGACG-motif). According to the structure characteristics of ZmRbcS1 promoter and the distribution of regulating elements, three upstream primers and one downstream primer were designed for the amplification of promoter fragments of different lengths, with the lengths of 2 114, 1 021 and 566 bp, respectively. Subsequently, the 5' end of the promoter was deleted and the deletion expression vector of the reporter gene gap-glucuronidase (GUS) was constructed by fusing three segments of different lengths, and the promoter function of the instantaneous expression system of tobacco (Nicotiana benthamiana) leaves mediated by Agrobacterium tumefaciens was analyzed. Histochemical staining showed that the promoter activity increased first and then decreased with the reduction of promoter fragment. This study verified that the protein product encoded by ZmRbcS1 gene was located in the chloroplast, and the analysis and identification of its promoter clock-acting element provide the basis for further study on the function of ZmRbcS1 gene.

Potato (Solanum tuberosum) is one of the most important crops in the world. Effective identification of potato varieties (lines) and evaluation of genetic diversity will help to obtain high yield and nice quality potato varieties with high tolerance to natural environments. In this study, 65 potato varieties were provided by Zhangjiakou Academy of Agricultural Sciences. The genetic diversity of 65 potato germplasm and the construction of DNA fingerprinting were accomplished by SSR molecular markers and phenotypic traits. 29 pairs of SSR primers with high polymorphism and clear bands were selected. These primers were used for amplification of all varieties (lines), and a total of 145 bands were amplified, including 100 polymorphism bands. Each pair of SSR primers amplified bands of 3~11, and the average percentage of polymorphic bands (PPB) of primers was 65.77%. According to the results of electrophoresis, SSR fingerprints of 65 germplasm resources were constructed with 5 pairs of primers, which were M03, M10, M12, S182 and STI039. Through polymorphism analysis based on SSR analysis of 65 potato varieties (lines), Nei's genetic diversity index (H)(0.35) and Shannon index (I) (0.52) indicated that the varieties (lines) had medium genetic diversity. The result of the population structure analysis showed that the corresponding Delta K value was maximum at K=3. Therefore, 65 potato varieties (lines) could be divided into 3 populations (Pop). Clustering analysis found that the genetic similarity coefficient of 65 varieties (lines) was between 0.62 and 0.91, and the varieties (lines) could be divided into 3 groups at the genetic similarity coefficient of 0.67. The results of the two clustering methods were basically consistent by SSR. The analysis of 32 phenotypic traits showed that the coefficient of variation (CV) of 23 phenotypic traits was higher above 36%. The average I and H of the population were 0.94 and 0.55, respectively. Significant correlation was found among phenotypic traits, including the color-related traits. It was significantly positive correlated between tuber skin color and stem color (r=0.48), between tuber skin color and flesh color (r=0.35) and between flesh color and eye depth (r=0.38). Using principal component analysis (PCA) to analyze phenotypic traits, 12 principal components (PC1~PC12) explained the total variation of 74.55% in 65 varieties (lines), and PC1~PC3 accounted for 30.08%. The two-dimensional figure showed the genetic distance among the phenotypes of 65 varieties (lines) reflected by PC1 and PC2 could be divided into 4 groups. Based on the clustering analysis of 32 phenotypic traits, the genetic similarity coefficient of 65 varieties (lines) was between 0.29 and 0.75, and all of the varieties (lines) could be divided into 3 groups at the similarity coefficient of 0.38. In summary, 5 SSR primers were used to construct the 65 potato varieties (lines) fingerprint. SSR markers clearly identified the population genetic structure among germplasm materials. In addition, the results of the investigation of molecular markers and phenotypic traits were encoded with QR codes, which could efficiently and conveniently view and identify the genetic diversity of 65 varieties (lines). This study will provide a theoretical basis for potato evaluation and breeding in the future. And the construction of fingerprints of 65 varieties (lines) will provide a basis for variety identification and intellectual property protection.

With the rapid development of genetically modified technology, the environmental biosafety of transgene introgression into wild relatives has attracted much attention. To evaluate whether the transgene endowing glyphosate-resistance to Brassica napus could successfully introgress to the different populations of wild Brassica juncea, the fitness of the glyphosate-resistant second backcross generations BC2mF₁ and BC2pF₁ (m and p represent wild B. juncea as maternal plants and paternal plants, respectively) between 4 populations of wild B. juncea (Jurong, Nantong, Xining, Xi'an) and glyphosate-resistant oilseed rape was studied in the greenhouse and field. Vegetative growth indicators and reproductive growth indicators of these 4 second backcross generations were measured and used to calculate their composite fitness. Furthermore, the differences of the composite fitness between the second backcross generations and wild B. juncea were analyzed. The results showed that the composite fitness of all BC2mF₁ and BC2pF₁ were similar with that of their respective wild B. juncea under greenhouse conditions. Similarly, in the field, the BC2mF₁ of Jurong, Xining, and Xi'an and BC2pF₁ of Nantong and Xi'an were equivalent in composite fitness to their respective wild B. juncea, However, composite fitness of BC2pF₁ of Jurong, Xining and BC2mF₁ of Nantong surpassed that of their respective wild B. juncea progenitor. In conclusion, the glyphosate-resistant BC2mF₁ and BC2pF₁ of transgenic oilseed rape and the 4 wild B. juncea had a strong survival ability and potential possibility of establishing populations in the field. It was necessary to prevent initial gene flow and introgression to backcrosses between the herbicide-resistant transgenic oilseed rape and wild B. juncea upon commercial release of the transgenic crop. The results will provide data on the possible ecological consequences of the introgress of transgene from glyphosate-resistant transgenic oilseed rape into different populations of wild B. juncea.

Ambient temperature is significantly affected flower bud development and flower opening of sweet osmanthus (Osmanthus fragrans). To further understand SQUAMOSA promoter-binding protein-like (SPL) genes and miR156 (microRNA156) on how to respond to ambient temperature in regulating flower bud development of sweet osmanthus, the variety of Osmanthus fragrans 'Yanhonggui' were selected, and 10 OfSPLs (OfSPL1A: MT756841; OfSPL1B: MT756842; OfSPL5: MT756843; OfSPL6: MT756844; OfSPL7: MT756845; OfSPL8: MT756849; OfSPL10: MT756846; OfSPL11: MT756847; OfSPL12: MT756848; OfSPL13: MT756850) were cloned by PCR. Meanwhile, the expression of miR156 and OfSPLs were identified by real-time quantitative PCR (qRT-PCR) to further analyze their temporal and spatial expression patterns. The results of OfSPL sequences analysis revealed that the cloned OfSPLs cDNA length between 1 004~3 824 bp, the sizes of OfSPL proteins ranged from 232 amino acid (aa) to 1 006 aa, and all of OfSPLs included a conserved SBP domain. Furthermore, all of OfSPL genes showed high homology with OlSPL genes from the species of Olea europaea by amino acid sequence alignment and evolutionary analysis. To investigate the intracellular localization of the proteins coded by the OfSPL genes, the bioinformatics method was used for predicting. The predicted results showed that all of the OfSPL proteins located in the nucleus. miR156 plays its role through regulating SPL gene expression by translational repression or degradation of mRNA in a sequence-specific manner. Therefore, the binding site of SPL genes was further analyzed. The expression of OfPL6, OfPL10, OfPL11, and OfSPL12 might be affected by miR156 through targeting their binding sites. Under different temperature treatments, the expression of OfSPL1A, OfSPL5, OfSPL6, OfSPL10, and OfSPL13 significantly increased at 19 ℃ comparing with the control 25 ℃. In contrast, the expression of miR156-1 and miR156-2 under 19 ℃ treatment were lower than the control 25 ℃. The results indicated that the expression of miR156-1 and miR156-2 may be inhibited by low ambient temperature, and resulted in the genes of OfSPL6 and OfSPL10 upregulation and then promoted the flower bud differentiation. Additionally, OfSPL1A, OfSPL5, and OfSPL13 may also involve in the flower bud development of O. fragrans at low ambient temperature, but they were not directly regulated by miR156-1 and miR156-2. This study could provide a reference for further research on the regulation of flower bud differentiation and flowering time by ambient temperature in O. fragrans.

Thioredoxin interacting protein (Txnip), a redox regulatory protein, binds to thioredoxin (Trx) and inhibits its activity, regulating the redox state in various physiological processes of cells, but its role in differentiation of porcine fat cells regulated by nutrient is unclear. In this study, the cDNA sequence of Sus scrofa Txnip gene was synthesized and the recombinant lentivirus (LV) overexpressing Txnip (LV5-Txnip) was constructed. The isolated porcine preadipocytes were cultured and transfected with LV5-Txnip and the transfection efficiency was around 90%. The Txnip mRNA level was raised by about 80 folds. The porcine preadipocytes transfected with LV5-Txnip were induced by adipogenic differentiation medium and treated with 5 or 15 mmol/L concentration of glucose, respectively. The differentiation and mRNA expressions of adipogenic-related genes for the cells were detected every other day using oil red O staining extraction assay and qRT-PCR, respectively. The results showed that differentiation of the cells transfected with LV5-Txnip was significantly inhibited (P<0.05), compared to the cells transfected with negative control virus and to the non-transfected cells, the expression level of peroxisome proliferator-activated receptor γ (PPARγ) and fatty acid synthase (FAS) was also reduced significantly (P<0.05). The differentiation and mRNA expression of PPARγ, carbohydrate response element binding protein (ChREBP) and glucose transporter 4 (Glut4) of control preadipocytes were induced significantly by high concentration of glucose, but this promotion was reduced by Txnip-overexpression (P<0.05). Txnip-overexpression suppressed differentiation of porcine preadipocytes through down-regulating PPARγ expression, which indicating Txnip could be an inhibitor of porcine adipocyte differentiation. Txnip impaired the promotion of glucose on the cell differentiation via decreased Glut4 expression leading to lower glucose transport and expression of ChREBP and PPARγ. The present study provides a reference for the investigation of the adipogenesis regulated by nutrient.

Nescient helix-loop-helix 2 (NHLH2), as nervous system-specific transcription factor, can not only regulate the number of nerve fibers in adipose tissue, but also play an important role in maintaining the energy balance of animals. Searching for DNA markers related to growth traits can provide scientific basis for the protection, development and utilization of beef cattle (Bos taurus) genetic resources. In this study, the genetic variations in 5'UTR of NHLH2 gene and their association with growth traits in Jiaxian red cattle (B. taurus) were explored by the techniques of PCR-single strand conformation polymorphism (PCR-SSCP), DNA sequence analysis and bioinformatics. The 5'UTR of NHLH2 gene was amplified by PCR using the genomic DNA of 374 different individuals of Jiaxian red cattle as templates, and about 400 bp amplified products were obtained. It was found that the 5'UTR of NHLH2 gene was polymorphic by SSCP analysis. Three SSCP genotypes were identified and denominated as genotype AA, AB and BB, respectively. After sequencing the polymorphic products, it was found that there were 2 SNPs (25G>C and 179G>A) in the 5'UTR of NHLH2 gene, which constructed 2 alleles (A and B). A total of 133 amino acids were encoded in bovine NHLH2 gene. The result of homology analysis showed the high similarity of amino acid sequences among different species, which confirmed that the gene was very conservative in the evolution process. The linkage disequilibrium analysis of 2 SNPs showed that the linkage disequilibrium parameter r² was 1, which indicated that the 2 SNPs were completely linked without cross or recombination. The result showed that there were 2 alleles A and B in the locus with corresponding frequencies of 0.239 and 0.761. And the frequencies of genotype AA, AB and BB were 0.139, 0.201 and 0.660, respectively. The frequency of allele B was higher than that of allele A. Allele B was the dominant allele, and genotype BB was the dominant genotype. The χ²-test value was 0.904, which suggested that the 5'UTR locus of NHLH2 gene was in Hardy-Weinberg equilibrium in this experimental population of Jiaxian red cattle. The polymorphism information content (PIC) value was 0.298, which indicated that the polymorphic locus belonged to moderate polymorphism. Using SPSS 19.0 software and the least square method, the correlation between different genotypes of polymorphic locus and growth traits in Jiaxian red cattle was analyzed. The polymorphisms for 5'UTR locus of NHLH2 gene had significant association (P<0.05) with chest circumference, body length and body weight, while had no significant association with other growth traits. The growth trait indexes of genotype BB individuals were significantly higher than those of genotype AA individuals (P<0.05). The above results confirmed that the correlation was detected between 5'UTR mutation of NHLH2 gene and the growth traits of Jiaxian red cattle, and the polymorphic locus could be used as a molecular marker for the growth traits, which could be used for improvement and breeding of Jiaxian red cattle.

Debao pony (Equus caballus) is an excellent horse breed in Guangxi of China. It has significant differences in body height compared with other breeds of horses in China. However, the regulation mechanism of body height growth of Debao pony is still poorly understood. This study was carried out from sampling and transcriptome sequencing of Mongolian horse and Debao pony childhood and adulthood pituitary and long bone epiphysis. There were 847 long-chain non coding RNAs (lncRNAs) differentially expressed at different stages of development, including 512 up-regulated and 335 down-regulated; and 1 633 lncRNAs were differentially expressed in different varieties, including 882 up-regulated and 751 down-regulated. In addition, there were 41 lncRNAs differentially expressed in pituitary tissue and 5 lncRNAs in long bone tissue during adult and juvenile development period of Debao pony; 41 lncRNAs were differentially expressed in pituitary tissue and 7 lncRNAs in long bone during the two development period of Mongolian horse. There were 59 lncRNAs differentially expressed in pituitary tissues and 34 lncRNAs in long bone tissues during juvenile period of Mongolian horse and Debao pony; 32 lncRNAs were differentially expressed in pituitary tissues and 35 lncRNAs in long bone tissue during adult period of the two horse breeds. KEGG and Gene Ontology (GO) analyses were performed on the corresponding target genes of these differentially expressed lncRNAs, and the results showed that they were mainly involved in the development of nervous system and endocrine system. According to the function enrichment, pathway enrichment and previous studies of key candidate genes, 20 protein coding genes were screened out, and 13 differentially expressed lncRNAs targeted growth hormone (GH) and insulin-like growth factor-1 (IGF-1) genes by trans action. The results of this study could provide a reference for the study on the mechanism of Debao pony body size development and the breeding of domestic animals targeting dwarfism.

During the development of cashmere goat (Capra hircus), different gene expression regulation plays an important role in hair follicle metabolism. In this study, ALX4 (aristaless-like homeobox 4) was selected as the candidate gene to verify the correlation between ALX4 gene and cashmere goat traits, and to provide a basis for molecular marker-assisted selection of cashmere goats. The blood DNA of 20 cashmere goats with maximum and minimum was first extracted, and the 3'-UTR, 5'-UTR, exon, and promoter sequences of the ALX4 gene were amplified and sequenced by PCR amplification technology. According to the results of Blast comparison, SNP loci with mutations in ALX4 gene were counted, and 315 cashmere goat individuals were detected using KASP (kompetitive allele-specific PCR)genotyping technology. The correlation analysis between SNPs loci and villi traits was performed using a general linear model. The results showed that SNP1 (T-1700C) exists in the promoter region of ALX4 gene, SNP2 (T-14621C), SNP3 (T-15433C), SNP4 (G-16377C), SNP5 (A-17780G) and SNP6 (A-18044G) in the 3'-UTR. Among them, the frequency of CG genotype of SNP4 locus of ALX4 gene is the highest (0.4459), which is the dominant genotype, and the G allele is the dominant allele; the frequency of GG genotype of SNP6 locus is the highest (0.5641), which is the dominant genotype, and the G allele is the dominant allele. All SNP loci were in Hardy-Weinberg equilibrium in cashmere goat population (P>0.05), and the homozygosity was higher and the heterozygosity was lower in the population. The effective allele of SNP4 (1.8947) was the highest among all SNPs, and that of SNP1 was the highest. The number of effective alleles (1.2534) was the lowest than other SNPs; except for SNP1 in low polymorphism (PIC<0.25), all other SNPs loci were in moderately high (0.5>PIC>0.25). Correlation analysis showed that the SNP4 locus of ALX4 gene was significantly associated with wool length traits (P<0.05), the wool length of individuals with GG genotype was significantly higher than those of CC and CG individuals (P<0.05); the SNP6 was significantly associated with cashmere fineness traits (P<0.05) the cashmere fineness of AA individuals was significantly higher than that of GG and AG individuals (P<0.05); A total of 12 haplotypes were constructed using PHASE software, with 4 haplotypes having a frequency>5%, which were: H1 (TCCAA, 20.384%), H4 (TCCGG, 15.029%), H7 (TCGGG, 38.498%), and H12 (CTGGG, 22.248%). In the haplotype combination of 5 SNPs of the ALX4 gene, there were 9 haplotype combinations with a frequency>5%. Correlation analysis of these 9 haplotype combinations with cashmere production traits indicated that different haplotype combinations of 5 SNPs in the 3'-UTR region of ALX4 gene were significantly associated with wool length correlation (P<0.05), the wool length of H1H1 individuals was significantly higher than that of HIH7, H1H12, H4H7 and H7H7 (P<0.05). The above results indicate that the ALX4 gene can be used as a key gene for molecular marker-assisted breeding in cashmere goats. In the breeding work of cashmere goats, individuals with AG genotypes at the SNP6 locus can be selected for ultrafine cashmere goat breeding.

Long noncoding RNA (LncRNA) was widely transcribed in the mammalian genome. LncRNA can play biological functions through the corresponding micRNA and target genes. By combining bioinformatics prediction and experimental verification, the LncRNA target genes can be rapidly and efficiently obtained and screened. To further explore the expression characteristics and predict its interaction molecules in melanoma pathogenesis of LncRNA XLOC_15448, the tissue expression profile LncRNA XLOC_15448 was detected using qRT-PCR method in different tissues of Youzhou dark goat (Capra hircus) and Youzhou blank goat (Capra hircus), as well as the LncRNA XLOC_15448 expression characteristics were detected in B16 cells at different developmental stages. The target genes were also predicted using miRBase, TargetScan7.2, miRTarBase, RNA22 version 2.0, and RNAhybrid2.2, and finally the lncRNA-miRNA-mRNA regulatory network was constructed in Capra hircus. The results demonstrated that LncRNA XLOC_15448 was widely expressed in different tissues of the goat, especially with the highest expression in the heart and lowest level in spleen, and it was also widely expressed in muscle, brain, skin, liver, kidney, lung and spleen. Comparison analysis illustrated that the skin LncRNA XLOC_15448 was expressed significantly higher in Youzhou dark goat than that of Youzhou blank white goat, and that the expression pattern of the lncRNA was increasing during the B16 cell proliferation. Bio-information analysis indicated that LncRNA XLOC_15448 might regulate gene expression of MITF (microphthalmia-associated transcription factor) by targeting to miR-182-5p. It was speculated that there may be a lncRNA-miRNA-mRNA network which modulate the dermal melanogenesis and melanin deposition in Youzhou dark goat, which would provide the basic data for further research on the regulatory mechanisms of lncRNAs related to goat skin pigmentation.

Prox1 (prospero-related homeobox protein 1) protein is a transcription factor that plays a key regulatory role in embryonic development. In order to investigate the subcellular localization of chicken (Gallus gallus) Prox1 and its gene expression pattern in the growth and development of different chicken tissues, the recombinant eukaryotic expression vector pEGFP-C1-Prox1 was constructed based on the pCR2.1-Prox1 cloning vector. The expression and subcellular localization of the recombinant protein were identified by Western blot and fluorescence observation in plasmid-transfected human (Homo sapien) embryonic kidney cells (HEK-293T). In addition, the expression of Prox1 gene in different chicken tissues was detected by qRT-PCR at embryo 14 d (E14 d), 0-day-old (0 d), 7-day-old (7 d), and 14-day-old (14 d). The results showed that pEGFP-C1-Prox1 was successfully constructed and the recombinant protein EGFP-Prox1 was correctly expressed and localized mainly in the nucleus. The qRT-PCR results showed that Prox1 gene expression was highest in the stomach at E14 d and 7 d, but highest in the lung at 0 d and 14 d. The expression pattern showed that Prox1 expression was stable in the eyeball and cerebrum from E14 d to 14 d, but increased in the heart until 7 d. Prox1 expression was on the rise in liver and lung, which showed the lowest expression at E14 d and the highest expression at 14 d. In stomach and leg muscle, Prox1 expression was highest at E14 d, lowest at 0 d, and higher at 7 d, which showed a trend of W pattern. The expression of Prox1 in the chest muscle gradually increased from E14 d to 7 d, and then decreased from 7 d to 14 d. This study provided a reference for further studying the regulation mechanism of Prox1 gene in the growth and development of different chicken tissues.

In the summer of 2018, a large-scale persistent outbreak of Liza haematocheila occurred in a freshwater fishing farm in Ningbo, Zhejiang province, with the cumulative mortality rate more than 60%. The moribund fish swimming alone or spinning on the water surface with unresponsive, anorexic, swollen abdomen and exophthalmos, thickened opaque eyes. The dissected mullet showed ascites, enlarged liver, spleen, petechiae and local bleeding and other symptoms, reddish macular pseudomembrane was observed in the connection of the swim bladder and kidney in the abdomen with the inflated compartment. To study the pathogen and main pathogenesis of cultured L. haematocheila and other related species for further diseases scientific control, eight bacterial strains (named Lh-1~Lh-8) were isolated from the brain, liver, spleen and kidney of 3 diseased fish with typical symptoms by using nutrient agar (NA) and brain heart infusion agar (BHIA) medium. Round, smooth and greyish white colonies sized 1~4 mm were found on BHIA and confirmed as Gram-positive coccus with short chain. Lh-3, one of the isolates, was identified physiologically and biochemically as Lactococcus garvieae with 99% similarity by using VITEK-2-Compact. The 16S rDNA and gyrase B (gyrB) were used for molecular identification. Sequence homology alignment and construction of a phylogenetic tree showed that the similarity to Lactococcus garvieae reached 100% and 93.5%, respectively, and both showed Lactococcus garvieae. The symptoms similar to natural disease occurred in L. haematocheila 2 d after was injected intraperitoneally with Lh-3, with the LD₅₀ of 5.86×10⁴ CFU/ind. Lh-3 showed typical alpha hemolysis on blood agar plates, and no transparent circles appeared on skimmed milk plates. Institute of Cancer Research (ICR) mice (Mus musculus) were infected by intraperitoneal injection of Lh-3. The mice showed bristled, trembling and dispirited with reddish and swollen eyes after 12 h of challenging. Pathological changes such as congestion of liver, lung and abdominal wall were observed in infected mice, and the mortality within 24 h was 100% (1×10⁹ CFU/ind). It could be concluded that the isolated Lactococcus garvieae was a causative pathogen to Lactococcus haematocheila with strong virulence, and potential zoonosis pathogen to mammals with certain pathogenicity. This study provides a reference for disease control of L. haematocheila and other aquaculture.

Plant growth-promoting rhizobacteria (PGPR) are microorganisms that live in the rhizosphere and root surface of plants, and can directly or indirectly promote plant growth and development. The application of microbial fertilizers containing plant growth-promoting rhizobacteria can significantly improve the nutritional status and growth of plants. The demand of modern ecological agriculture for non-chemical fertilizers and the improvement of people's awareness of environmental protection make it great potential for the development and research of microbial fertilizers. The collection and preservation of bacterial resources is fundamental to the development of microbial fertilizers, and thus it is extremely important to screen for excellent strains. Therefore, this study collected rhizosphere soil of wheat (Triticum aestivum), maize (Zea mays), tomato (Solanum lycopersicum) and cucumber (Cucumis sativus) grown in Beijing. A total of 500 bacterial strains were isolated and screened with spread plate method, and their plant growth-promoting properties were tested. Through pot experiment in the greenhouse, 15 bacterial strains with excellent growth-promoting effects on tomato seedlings were identified. The 16S rRNA gene sequence analysis revealed that 11 strains 4-L, 14-L, z4-3, z7-2, z13-5, S6, 1-18, Z20-2, Z23-3, Z28-4, Z30-2 were Paenibacillus sp., 2 strains z13-2 and z13-3 were Pseudomonas azotoformans, strain z14-1 was Alcaligenes faecalis, and strain 56 was Bacillus subtilis. On this basis, Paenibacillus sp. S6, 1-18 and B. subtilis 56 with good plant growth-promoting ability were inoculated onto wheat for field plot experiment. In this experiment, 5 kinds of treatment were set, including control 1 (without strain inoculation and the amount of urea was reduced by 10%); control 2 (without strain inoculation and the amount of urea was not reduced), treatment 3 (Paenibacillus sp. S6 was inoculated and the amount of urea was reduced by 10%), treatment 4 (B. subtilis 56 was inoculated and the amount of urea was reduced by 10%), and treatment 5 (Paenibacillus sp. 1-18 was inoculated and the amount of urea was reduced by 10%). At the returning green stage of wheat, the effects of different treatments on the growth of wheat were evaluated. The results showed that the plant height of wheat in treatments 3, 4, 5 was significantly higher than that in control 1 (P<0.05), but had no significant difference from control 2. Similarly, the fresh weight of wheat in treatments 3, 4, 5 was significantly higher than that in control 1 (P<0.05), but had no significant difference from control 2. In addition, wheat yield was counted at maturity. The results showed that under the condition of reducing 10% urea, the yield of wheat inoculated with Paenibacillus sp. S6, Paenibacillus sp. 1-18 and B. subtilis 56 increased by 6.9%, 8.8%, and 10.4% (P<0.05), respectively, compared with the control 1; while there was no significant difference compared with the control 2. The above results indicated that it was feasible to replace 10% of chemical fertilizers with microbial fertilizers containing above 3 strains. Among them, B. subtilis 56 performed best in reducing fertilizer use and increasing yield, and thus it could be demonstrated and promoted on a large scale. This study could provide excellent strain resources for the development of microbial fertilizers, and provide reference for spread and application of the technique using microbial fertilizers replacing some chemical fertilizers.

Ustilago esculenta is a dimorpic fungi with the ability to infect Zizania latifolia and causes stem enlargement. The fungal dimorphism is vital for the infection ability and pathogenicity of U. esculenta.The mitogen-activated protein kinase (MAPK) cascades and cyclic adenosine monophosphate-protein kinase A(cAMP-PKA) pathway are the core of signaling pathways in regulation of the fungal dimorphism. In this study, the phenomenon of multi-budding growth of cells after the addition of cAMP was found, and the hyphal growth was affected during the mating process. Furthermore, a gene encoding PKA catalytic submit (GenBank No. KR870334.1) was cloned. Sequence analysis results showed that the gene had a total length of 1 346 bp, with 1 intron and 2 exons forming 1 233 bp open reading frame, encoding 410 amino acids. This amino acid sequence was homologous to the amino acid sequence of PKA catalytic subunit in other species, such as U. hordei and U. maydis. The expression pattern analysis showed that UePkaC was up-regulated in the process of mating. The phenotype analysis of the UePkaC deletion mutants showed that they were longer and had no mating ability and pathogenicity. Furthermore, the ability of conjunction tube was lost in UePkaC mutant during mating and the expression of pheromone synthesis gene was significantly inhibited, indicating that UePkaC affects the conjugation tube formation and mating process by regulating the expression of a genes.The above results showed that UePkaC gene played a key role in the haploid growth and dimorphism of U. esculenta, which provided basic data for studying the pathogenic mechanism of U. esculenta.

Diversified crops are needed to optimize crop single production, which can increase biodiversity at yellow river pumping irrigation area in the central arid zone of Ningxia. In order to investigate the influence of different crops on the bacterial community structure and diversity of farmland soil, this study took millet (Setaria italica), black beans (Glycine max), quinoa (Chenopodium quinoa) and recreational soil as the objects, and measured the soil pH, total nitrogen, organic matter, available potassium, available phosphorus and alkali nitrogen content. Furthermore, the bacterial community structures were compared using Illumina MiSeq high-throughput sequencing technology. The results showed that the contents of soil organic matter, alkali nitrogen, available potassium and available phosphorus increased after planting quinoa, which were 2.88%, 19.12%, 112.04% and 39.90% higher than those in fallow land, respectively, but the pH value decreased, only 0.01 unit. A total of 908 120 effective sequences were obtained from the 5 groups of soil samples, and 4 623 to 5 394 bacterial OTU were obtained from different soil samples, covering 31 phyla, 92 classes, 130 orders, 217 families, 363 genera of bacteria. The flora classification found that Protrobacteria, Chloroflexi and Actinobacteria were the main bacterial groups with relative abundance accounting for 55%~75% of the community. The dominant genus in rhizosphere soil of different crops were also different. Compared with fallow land, planting crops could improve soil bacterial richness index, Shannon index and dominance index, among which quinoa treatment was the most obvious, increasing by 19.59%, 22.20%, 7.84%, 0.36%, respectively. Through multivariate analysis and correlation analysis, it was found that soil bacterial community structure was greatly affected by soil available potassium. The results showed that planting different crops had great influence on the change of soil fertility, and could change the composition of soil bacterial communities, providing theoretical support for a deeper understanding of the relationship between soil bacterial communities and crop species.

Soil salinization has caused serious harm to the security of cultivated land and hinders the sustainable development of agriculture. Bioremediation has great potential because of its lasting effect and high ecological benefit. This study adopted field test methods, six treatments were set up: Conventional fertilizer (CK), DF-3 bacterial fertilizer (T1), DF-7 bacterial fertilizer (T2), LT bacterial fertilizer (T3); LP bacterial fertilizer (T4) and commercial bacterial fertilizer (T5), to study their effects on the chemical properties and the enzyme activities of of saline soils of Lycium barbarum, and to analyze the changes of bacterial community structure and diversity by using high-throughput sequencing technology, and the best microbial fertilizer for improving saline-alkali soils was found. The results showed that compared with the conventional fertilizer (CK), the pH and total salt content of different bacterial fertilizer treatments significantly decreased in that the pH values decreased 0.93%~3.50% (P<0.05), total salt content decreased 1.30%~9.42% (P<0.05), and the inhibition effects of T1 and T4 were very obvious; The contents of available K and P increased most after the application of T1, which were 14.57% and 129.69% (P<0.05) and higher than those of CK, respectively. The contents of alkali hydrolyzed N increased most by using T5, which was 80.70% (P<0.05) and higher than that of CK, and the content of organic matter increased most when T2 was used, which was 47.61% (P<0.05) and higher than that of CK. The activities of urease and sucrase in the soil treated with T1 were the highest, which were 2.31 and 19.44 (P<0.05) times of CK, respectively. The activities of alkaline phosphatase and protease in the soil treated with T4 were the highest, which were 1.72 and 0.97 (P<0.05) times of CK, respectively. In addition to T1, the combined application of bacterial fertilizers increased the bacterial operational taxonomic units number and Chao1 index, and T4 and T5 increased the bacterial Shannon index. Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes were the dominant groups, and their relative richness together accounted for the total bacteria 82.72%~87.24% of the total. According to redundancy analysis, 69.88% of the change in the bacterial communities was due to physical and chemical factors and enzyme activities of the soils. The dominant phyla were Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi in all treatments. At the genus levels, the relative abundance of Pseudomonas and Bacillus were higher than in other treatments by using T1. There was a positive correlation between Proteobacteria and Firmicutes. Soil protease, urease, sucrase, and catalase activities were positively correlated with Bacteroidetes. Combined application of microbial fertilizer could improve soil fertility and soil enzyme activities, change the composition and structure of bacterial communities, and was beneficial to the improvement of saline-alkali soils, T1 and T4 were better in eliminating soil salinity and recovering beneficial microorganisms in salinized Lycium barbarum farmland, which would provide theoretical basis and technical reference for scientific improvement of saline-alkali soils.

Nitrogen is the main component of proteins, nucleic acids and enzymes, and plays an important role in plant growth and development. NH₄⁺ uptake and assimilation consumes low energy compared to NO₃- assimilation, NH₄⁺ absorption and utilization efficiency directly affects plant growth and development. Plants can activate complex regulatory networks to optimize NH₄⁺ uptake and utilization due to external fluctuations in NH₄⁺ supply. This review summarized the ways in which ammonium transporter participates in NH₄⁺ perception and the regulatory mechanism at the transcription and protein levels. In addition, it was summarized that the stress responses of AMTs and NH₄⁺ assimilation-related genes under abiotic stresses such as low nitrogen, drought, and salt stress. This paper provides a theoretical basis for high nitrogen use efficiency of plant under abiotic stress.