Abstract:

Abstract:Transcription factors, the proteins with special structures, can bind to specific sites and regulate specific expression of target genes. NAC (NAM, ATAF1/2, CUC1/2) transcription factors, unique to plants, are composed of a conserved N-terminal domain and a highly variable C-terminal transcriptional activation domain. NAC transcription factors are involved in plant growth and development, responses to biotic and abiotic stresses and other processes, playing a regulatory role in flower development. In this paper, we reviewed the studies about NAC transcription factors in terms of discovery, structure, and regulatory roles in anther development, other floral organ development and flowering time. This review will provide a theoretical basis for deciphering the regulatory mechanism and improving the regulatory network of NAC transcription factors in flower development.

Abstract:GLKs (GOLDEN 2-LIKEs) are a group of plant-specific transcription factors regulating the chloroplast biogenesis, differentiation and function maintains by triggering the expression of the photosynthesis-associated nuclear genes (PhANGs). The GLKs also play important roles in nutrient's accumulation in fruits, leaf senescence, immunity and abiotic stress response. The expression of GLK genes were affected by multiple hormones or environmental factors. Therefore, GLKs were considered as the key nodes of regulatory network in plant cells, and potential candidates to improve the photosynthetic capacity of crops. Since numerous researches of GLKs have been reported in plants, the biological function, molecular mechanism of GLKs genes and its applications in breeding were summarized and a GLK-mediated signaling network model was developed. This review may facilitate future research and application of GLKs.

Abstract:Gene editing technology has been a hotspot in the field of biotechnology. CRISPR/Cas systems are efficient gene editing tools because of its specificity, simplicity and flexibility, these features enabled the rapid application of CRISPR/Cas systems in a variety of organisms. Moreover, the combination of transcriptional activator with dead Cas protein can achieve specific regulation of gene expression at the transcription level, which has made important contributions to the development of biotechnology in medical and agriculture. Overexpression of foreign genes is a common method to verify gene function and regulation. However, due to the limitation of vector capacity, it is difficult to achieve overexpression of multiple genes. CRISPR/Cas9 activation system can regulate the expression of multiple genes under the guidance of different guide RNAs to verify gene functions at the regulatory level. This review summarizes the composition of the CRISPR/Cas9 activation system and different activation strategies, and summarizes solutions for excessive activation. It may facilitate the application of CRISPR/Cas9 activation system in genetic improvement of cotton and herbicide resistance research.

Abstract:Iron (Fe) is an important trace element involved in many important plant physiological and metabolic processes such as photosynthesis, respiration and nitrogen metabolism. Plants maintain iron homeostasis through absorption, transporting, storage and redistribution of iron. Iron metabolism is strictly regulated in plants. Iron regulatory transcription factors and iron transporters constitute the regulatory network of plant iron absorption and transport in plants. Ferritin and iron transporter jointly regulate the response to excess iron in plants. In recent years, important progress has been made in understanding how abscisic acid (ABA) regulates iron metabolism in plants. ABA may be used as a signal to regulate the absorption, transportation and reuse of Fe, or to relieve the symptoms of iron stress by regulating the oxidative stress responses in plants. In order to gain deeper insights into the crosstalk of ABA and iron metabolism in plants, this review summarized the mechanisms of iron absorption and transport and metabolic regulatory network in plants, as well as the mechanisms of ABA in regulating iron metabolism. The relationship between ABA and FER-like iron deficiency-induced transcription factor (FIT), iron-regulated transporter 1 (IRT1), and oxidative stress of iron deficiency were highlighted, and future research directions were prospected.

Abstract:Xanthomonas albilineans (Ashby) Downson is a quarantine pest for importing plants to China that causes leaf scald bacterial disease on sugarcane. X. albilineans produces a potent phytotoxin/antibiotic called albicidin. As a pathogenic factor, albicidin causes typical white leaf stripes by inhibiting plastid DNA gyrase and disturbing chloroplast differentiation. Meanwhile, the antibacterial activity of albicidin gives X. albilineans a competitive advantage against rival bacteria during their colonization. Furthermore, albicidin has a rapid bactericidal activity against a variety of Gram-positive and Gram-negative pathogenic bacteria of human species at nanomolar concentrations, making it a potential antimicrobial drug for clinical application. This article reviews the advances of albicidin from the aspects of its molecular structure, traditional extraction methods, mechanism of action, biosynthetic genes and processes, chemical synthesis method and improvement, in order to provide insights into the prevention and treatment of the sugarcane leaf scald disease, and the development of new antibiotics.

Abstract:The aluminum stress in acidic soil areas of China is an important abiotic stress factor that hampers the normal growth and development of plants and seriously affects the agricultural yield. The forms of plant resistance to aluminum stress are complex and diverse, which include secretion of organic acids, increase of rhizosphere pH, secretion of mucus, cell wall fixation of Al³⁺, organic acid chelation of Al³⁺ in cell solute, and vacuolar area isolation. Most of studies focus on analyzing conventional physiological characteristics, but in-depth molecular biological analyses are lacking. This review summarizes the mechanisms how plants adapt to acidic aluminum stress. This includes the effect of acid aluminum stress on plant growth and physiological metabolism, the two main physiological mechanisms of plant adaptation to acid aluminum stress (aluminum exclusion mechanism, aluminum tolerance mechanism), and the aluminum resistance related genes. Finally, this paper puts forward some prospects for further revealing the mechanism of plant adaptation to acid aluminum stress and excavating high-quality crops suitable for cultivation in acidic soils.

Abstract:Extracellular vesicles (EVs) are membrane-bound particles actively released by cells. In prokaryotes and eukaryotes, EVs are effective bridges for communication between cells. EVs carry biological macromolecules, including proteins, lipids and nucleic acid, which affects different physiological functions of parent cells and recipient cells. Among them, the microRNA carried by EVs is the most reported and plays an important role in physiological function of organisms. During the development of follicles, only a few follicles can fully develop and ovulate, whereas most of them undergo atresia at different stages of development. In the whole process of follicular development, the changes at each stage and the regulation mechanism of follicular atresia are not completely understood. In this paper, we introduced the types, characteristics, isolation methods and uses of EVs, and emphasized how microRNA carried by EVs in follicular fluid regulated follicular atresia from the aspects of different cytokines and hormones. Additionally, the application prospect of microRNA carried by EVs in follicular fluid in reproductive regulation and reproductive disease diagnosis was discussed. This paper is significant for studying the regulation of follicular development and the effective utilization of oocytes.

Abstract:Recombinant proteins provide new means for disease treatment, while creating considerable economic benefits. Using commercial crops (mainly tobacco), cereal crops, legumes, and vegetable crops to produce recombinant proteins with medicinal value is a hot-spot for research in "molecular farming". Although many recombinant proteins have been expressed in plants, only a small number have been successfully put into use. To overcome the problems that greatly hamper the development of recombinant protein production in plants, researchers have improved expression systems to increase the yield of recombinant proteins. Starting from analyzing the problems of low yield and/or low biological activity of recombinant proteins produced by plants, the optimization strategies to solve these problems were reviewed, and future research directions for improving the yield of recombinant proteins produced by plants were proposed.

Abstract:Multicellular network analysis is a method for topological properties analysis of cells. The functions of organs are determined by their inner cells. The arrangement of cells within organs endows higher-order functionality through a structure-function relationship, though the organizational properties of these multicellular configurations remain poorly understood. Multicellular network analysis with multicellular models established by 3D scanning of plants, will further discover the plant development mechanism, and provide clues for synthesizing plant multicellular systems. In this paper, we review the development of multicellular models, summarize the process of multicellular network analysis, and describe the development and application of multicellular network analysis in plants. In addition, this review also provides perspective on future development of plant multicellular network analysis.

Abstract:Escherichia coli biofilm is a complex membrane aggregation produced by the adhesion and secretion of extracellular polymeric substances by E. coli cells aggregated on specific media. Pathogenic E. coli will evade the immune system and the impact of various harmful factors in the environment after the formation of biofilm, causing sustained and even fatal damage to the host. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger ubiquitous in bacteria and plays a crucial role in regulating biofilm formation. This paper reviewed the recent studies about the role of c-di-GMP in the movement, adhesion, and EPS production mechanism of E. coli during biofilm formation, aiming to provide a basis for inhibiting E. coli biofilm from the perspective of c-di-GMP.

Abstract:Probiotics can improve the microecological balance of the body and have special effects in promoting nutrient absorption, controlling intestinal infections, and regulating immune function. However, there are problems such as difficult colonization in the gastrointestinal environment and low oral bioavailability. Bacterial biofilms are organized bacterial cells that adhere to an abiotic or biotic surface and are enclosed in extracellular polymeric substances of exopolysaccharides (EPS), extracellular DNA (eDNA), proteins and lipids, with a three-dimensional spatial structure. Probiotics with the help of bacterial biofilms have obvious advantages over planktonic bacteria in stress resistance, combating pathogens and modulating the host's immune function, which provides a new research idea for the development of probiotics. This paper expounded on the advantages of probiotics with the help of bacterial biofilms, and focused on introducing substances that could promote the formation of probiotic biofilms and the mechanisms, and the safety of probiotic biofilms. Currently, research on probiotic biofilms is still in its infancy, and this paper is expected to provide references for future research in this field.

Abstract:Biofilm formation is closely related to pathogenicity and antibiotic resistance of bacteria, and plays important roles in a number of chronic and subchronic infections. Animal models are widely used in the research of bacterial biofilm-associated infections, and provide a powerful scientific tool for investigating its pathogenesis and control strategies. This review summarized the application of mammalian models (e.g. mouse, rabbit, and pig) and non-mammalian models (e.g. Drosophila melanogaster, Zebrafish, and Caenorhabditis elegans) in bacterial biofilm studies, and prospects the application of animal models in biofilm. This review may facilitate the selection of suitable animal models in the study of biofilm-associated infections, so as to prevent and control the potential adverse effects.

Abstract:With the development of vaccine research and development technologies, novel vaccines have been widely used in the prevention of various infectious diseases. Due to the excellent safety, novel vaccines have unique advantages in the application of vaccines against virulent pathogens. The major premise of developing novel vaccines is to screen protective antigens. With the development of various omics research, cutting-edge bioinformatics tools for eukaryotes have been well developed, while the much simpler structure of viruses compared with eukaryotic cells corresponds to relatively simple research methods. Strategies for screening protective antigens need to combine the advantages of both bioinformatics methods and traditional molecular biology methods. In this review, the strategies for screening virus protective antigens were discussed from the perspective of host and virus, and a series of bioinformatics tools developed based on eukaryotic cells that may be used for screening protective antigens were listed. This review also summarized the cases of using protective antigens to design novel vaccines, in order to better understand the strategies for screening virus protective antigens and facilitate the research and development of novel vaccines.

Abstract:Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which causes great economic losses. At the moment, no effective neutralizing antibody is available for scientific research and treatment. Therefore, developing a method for screening the neutralizing monoclonal antibodies is of great significance for the prevention and treatment of PRRSV and the screening of antigen sites. Monoclonal antibodies have been widely used in the treatment and diagnosis of many human and animal diseases. Therefore, screening effective neutralizing antibodies for different pathogens is an urgent task. Among the methods for monoclonal antibody screening, B cell immortalization is an effective method to obtain neutralizing monoclonal antibody. Specifically, in this study, the bcl-6 and bcl-xl genes were connected by f2a and then the yielded product was ligated to a vector for retrovirus packaging. The swine lymphocytes immunized with PRRSV were infected the yielded mature viruses and cultured in the complete medium containing CD40L and IL21 cytokines. Then, CD21 was used as the marker to screen B cells with the magnetic bead method. Finally, monoclonal B cells were obtained and the secretion of antibodies was tested. The results showed that the plasmid, either being transfected alone or with the packaged plasmids, could be expressed, and that the packaged retrovirus could infect the cells. Moreover, the infected lymphocytes secreted antibodies, so did the screened B cells. Therefore, the method for screening monoclonal antibody against PRRSV was successfully established.

Abstract:African swine fever virus (ASFV) infection leads to a mortality rate of up to 100%, causing devastating disasters to the pig industry. Understanding the ASFV infection and replication is therefore of great importance. ASFV has more than 150 open reading frames, among which the inner coat protein p17 encoded by the D117L gene is involved in the formation of the icosahedral structure of the virus. However, little is known about the mechanism how p17 regulates host cell function. In this study, the potential host proteins interacting with ASFV p17 were screened by immunoprecipitation technique combined with protein profiling analysis. The interactions of p17 with mitochondrial membrane protein TOMM70 and heat shock protein HSPA8 were confirmed by co-immunoprecipitation technique and laser confocal experiments. This study provides important information for further exploring the function of p17 during ASFV infection.

Abstract:In order to investigate the apoptosis triggered by porcine circovirus type 2 (PCV2) in lymphocytes and the underlying mechanism, the levels of apoptosis and the expression levels of miRNA were examined by flow cytometry, Western blotting and real-time PCR (qPCR). The mimics or inhibitors of miR-125a-5p, an apoptosis-related miRNA, were transfected into PK-15 cells, and the apoptosis rate was examined upon overexpression or inhibition of mir-125a-5p. The target gene of mir-125a-5p was predicted by bioinformatics method, and the regulation of mir-125a-5p on the target gene was analyzed by luciferase reporter assay. The expressions of Bcl-2, Bax, cytochrome C and caspase-3 were detected by Western blotting. The results showed that exosomes secreted by PK-15 cells infected with PCV2 significantly increased the lymphocyte apoptosis rate, which was dose-dependent in certain concentration range. The expression of miR-125a-5p was dramatically increased. The apoptosis rate was increased significantly in the cells transfected with miR-125a-5p. It was predicted that there were binding sites of miR-125a-5p at Bcl-2 3'UTR by TargetScan. The luciferase activity of wild-type pmir-Bcl-2 3'UTR was inhibited significantly by miR-125a-5p mimics, but that of mutant pmir-Bcl-2 3'UTR was not changed. By Western blotting, Bcl-2 was reduced significantly, while Bax, cytochrome C and caspase-3 increased significantly, and the ratio of Bcl-2/Bax was significantly decreased. These results showed that PCV2 up-regulated the expression of miR-125a-5p through exosomes, then inhibited the expression of Bcl-2 at both mRNA and protein level, activated mitochondrial apoptosis pathway and induced apoptosis in lymphocytes.

Abstract:In order to evaluate the immune effect of the genotype Ⅰ Japanese encephalitis virus prM-E DNA vaccine and the prM-EⅢ fusion protein subunit vaccine on mice using DNA prime-protein boost strategy, the prM-E gene was inserted into the pVAX1 eukaryotic expression vector. The recombinant expression vector prM-E-pVAX1 was constructed as a DNA vaccine for initial immunity, and the recombinant prM-EⅢ fusion protein was obtained using a prokaryotic expression system as a subunit vaccine for enhanced immunity. Thirty two female BALB/c mice aged 4-6 weeks were randomly divided into four groups, and a prM-E-pVAX1 DNA vaccine group, a DNA prime-protein boost immune group, a prM-EⅢ subunit vaccine group, and a pVAX1 vector control group were set up. The specific antibody level in serum was monitored by ELISA, the neutralizing antibody titer was detected by plaque reduction neutralization, and the cellular immune responses induced by different vaccine immune groups were analyzed by cytokine expression abundance and lymphocyte proliferation experiments. The results showed that the neutralizing antibody titers induced by mice immunized with the DNA prime-protein boost strategy were close to that of the group immunized with the single prM-EⅢ subunit vaccine, but significantly higher than that of the group immunized with the single prM-E-pVAX1 DNA vaccine. DNA prime-protein boost strategies induced effective Th1/Th2 immune responses in mouse models, in particular the Th1 cell-mediated immune responses. This study provides a new immune strategy that may facilitate the prevention of Japanese encephalitis.

Abstract:Very long chain polyunsaturated fatty acids (VLC-PUFAs) are unique fatty acids in tissues of mammals such as retina and testis, and the key enzyme of its biosynthesis is very long chain fatty acid elongase 4 (Elovl4). Development of an animal model of tissue-specific knockout of Elovl4 gene is conducive to the in-depth study of the biological function of VLC-PUFAs. Therefore, we constructed Stra8-Cre mice and Elovl4 floxed mice based on Cre/loxP system, and obtained the (Elovl4[flox/+], Stra8-Cre) heterozygous knockout mice by hybridization. Subsequently, female mice were selected to cross with male mice with homozygous Elovl4[flox/flox] to gain homozygous mice (Elovl4[flox/flox], Stra8-Cre) through genotype identification and screening. RT-PCR, qRT-PCR, Western blotting, immunohistochemistry and immunofluorescence techniques were used to detect the knock-out efficiency of Elovl4 in testis. The expression of Elovl4 in testis of both heterozygous and homozygous knockout mice were significantly down-regulated at mRNA and protein levels, but were not affected in other tissues. In summary, we constructed a mouse model with specific knockout of Elovl4 gene in testis, which provides a reliable animal model for studying the effect of VLC-PUFAs on the reproductive function of male mice and the underpinning molecular mechanisms.

Abstract:In this study, we cloned the complete coding sequence (CDS) of chicken foxp3 (chfoxp3) gene, analyzed its structure, and investigated its expression profile in different chicken tissues. To be specific, chfoxp3 was cloned from the splenic tissue of 50-day-old specific-pathogen-free chickens, and analyzed by using online bioinformatics tools or software. The expression profiles of the chfoxp3 gene in different chicken tissues were detected by quantitative real-time PCR (qRT-PCR). The results indicated that the chfoxp3 gene contains an 882-bp open reading frame, encoding 293 amino acids hydrophilic protein with a molecular weight of 33.44 kDa. The chFoxp3 protein has a forkhead domain and carries a nuclear localization signal, which is typical in the Fox transcription factor family. The secondary structure of chFoxp3 consists of α-helix (29.35%), extended chain (10.92%), β-turn (5.12%) and random coil (54.61%). The expression of chfoxp3 varied in different tissues. The expression levels of chfoxp3 in chicken heart and pancreas were higher than in spleen, bursa of Fabricius, thymus, and other immune organs (P<0.01), which was quite different from that of mammals. Phylogenetic tree analysis showed that chFoxp3 belonged to the same clade as other wild birds did, but was far different from that of mammals. These results may facilitate further research on the role of chFoxp3 in immune regulation.

Abstract:The aim of this study was to investigate the effect of activating transcription factor 3 (ATF3) on the differentiation of intramuscular preadipocytes in goat, and to elucidate its possible action pathway at the molecular level. In this study, the recombinant plasmid of goat pEGFP-N1-ATF3 was constructed, and the intramuscular preadipocytes were transfected with liposomes. The relative expression levels of adipocyte differentiation marker genes were detected by quantitative real-time PCR (qRT-PCR). After transfection of goat intramuscular preadipocytes with the goat pEGFP-N1-ATF3 overexpression vector, it was found that the accumulation of lipid droplets was inhibited, and the adipocyte differentiation markers PPARγ, C/EBPα and SREBP1 were extremely significantly down-regulated (P<0.01), while C/EBPβ and AP2 were significantly down-regulated (P<0.05). The ATF3 binding sites were predicted to exist in the promoter regions of PPARγ, C/EBPα and AP2 by the ALGGEN PROMO program. The overexpression of goat ATF3 inhibits the accumulation of lipid droplets in intramuscular preadipocytes, and this effect may be achieved by down-regulating PPARγ, C/EBPα and AP2. These results may facilitate elucidation of the regulatory mechanism of ATF3 in regulating the differentiation of goat intramuscular preadipocytes.

Abstract:This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×10⁶ (±4.34%) Da and 2.40×10⁶ (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×10⁶ (±2.94%) Da and 2.88×10⁶(±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×10⁶ (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all <1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R² of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.

Abstract:In this study, we cloned the complete sequence coding for aminoacids in protein (CDS) of goat ST13 gene, analyzed the bioinformation of it, and explored the expression pattern in different goat tissues and goat subcutaneous preadipocytes at different differentiation stages. To be specific, ST13 gene was cloned by reverse transcription PCR (RT-PCR), and the bioinformation was analyzed by online tools or software. The expression in various goat tissues and subcutaneous preadipocytes at different differentiation stages was detected by quantitative reverse transcription PCR (qRT-PCR). The results showed that the cloned goat ST13 gene was 1 380 bp, with CDS of 1 101 bp, encoding 366 amino acids. Protein prediction results showed that ST13 had 26 phosphorylation sites and that some sequences were highly hydrophilic and unstable. Moreover, ST13 was a non-transmembrane and non-secretory protein. Subcellular localization demonstrated that ST13 was mostly distributed in the nucleus (69.6%). Phylogeny analysis suggested that goat ST13 had the highest identity to sheep ST13. Tissue expression pattern showed that ST13 gene expressed in all of the collected 13 tissues of goat, including heart, liver, spleen, lung and kidney, especially in triceps brachii and subcutaneous fat (P<0.01) and that the expression among heart, liver, spleen, lung, kidney, large intestine, small intestine and pancreas was insignificantly different (P>0.05). In addition, according to the temporal expression pattern in adipocytes, the expression of ST13 was up-regulated in differentiated adipocytes, and the expression was the highest at the 108th hour of induction, significantly higher than that at other time points (P<0.01). In conclusion, this gene expresses in various tissues of goat and regulates the differentiation of goat subcutaneous adipocytes.

Abstract:TCP (teosinte branched1/cincinnata/proliferating cell factor) is a group of plant-specific transcription factors that play important roles in plant growth and development. To date, there are no report about TCP transcription factors in eggplant (Solanum melongena L). In this study, twenty-nine eggplant TCP (SmTCP) family genes distributed on 11 chromosomes were identified from the genome database of eggplant using bioinformatics methods. The results showed that all members of the family contained sequences encoding TCP conserved domains with length of amino acids ranging from 201 to 538 and exon numbers of 1 or 2. Subcellular localization revealed that three SmTCP proteins (SmTCP02/03/21) were located in the cytoplasm and the other SmTCP proteins were located in the nucleus. The 29 TCP transcription factors were divided into ClassⅠ (PCF) and ClassⅡ (CIN and CYC/TB1) by phylogenetic tree and sequence analysis. Collinearity analysis showed that 17 pairs (21) of SmTCP genes had collinearity, and these collinearity genes belonged to segmental duplication. Analysis of gene expression patterns showed that all 29 members of SmTCP gene family were expressed in 15 tissues or organs, but the expression patterns were different. Among them, four gene (SmTCP18/19/20/25) of CIN subfamily were highly expressed in leaves at different growth stages. Analysis of cis-acting elements in the promoter region of SmTCP showed that there were four types of cis-acting elements, which were light response related cis-acting elements, growth and development related cis-acting elements, hormone response related cis-acting elements and stress related cis-acting elements. In summary, the molecular basis of SmTCP genes in eggplant and the influence of TCP gene on the growth and development of eggplant provided a theoretical basis for molecular breeding of eggplant.

Abstract:To study the molecular mechanism of salt stress response of peanut small GTP binding protein gene AhRabG3f, a 1 914 bp promoter fragment upstream of the start codon of AhRabG3f gene (3f-P) from peanut was cloned. Subsequently, five truncated fragments (3f-P1-3f-P5) with lengths of 1 729, 1 379, 666, 510 and 179 bp were obtained through deletion at the 5' end, respectively. Plant expression vectors where these six promoter fragments were fused with the gus gene were constructed and transformed into tobacco by Agrobacterium-mediated method, respectively. GUS expression in transgenic tobacco and activity analysis were conducted. The gus gene expression can be detected in the transgenic tobacco harboring each promoter segment, among which the driving activity of the full-length promoter 3f-P was the weakest, while the driving activity of the promoter segment 3f-P3 was the strongest. Upon exposure of the transgenic tobacco to salt stress, the GUS activity driven by 3f-P, 3f-P1, 3f-P2 and 3f-P3 was 3.3, 1.2, 1.9 and 1.2 times compared to that of the transgenic plants without salt treatment. This suggests that the AhRabG3f promoter was salt-inducible and there might be positive regulatory elements between 3f-P and 3f-P3 in response to salt stress. The results of GUS activity driven by promoter fragments after salt treatment showed that elements included MYB and GT1 between 1 930 bp and 1 745 bp. Moreover, a TC-rich repeat between 682 bp and 526 bp might be positive cis-elements responsible for salt stress, and an MYC element between 1 395 bp and 682 bp might be a negative cis-element responsible for salt stress. This study may facilitate using the induced promoter to regulate the salt resistance of peanut.

Abstract:In order to characterize the chloroplast genome and phylogenetic relationships of Isopyrum anemonoides, we performed Illumina Hiseq high-throughput sequencing to sequence the complete chloroplast genome of this plant and constructed a whole-genome map based on contig assembly and annotation. The chloroplast genome of I. anemonoides is 161 034 bp in length and has a typical tetrad structure, comprising 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The genome also contains a total of 44 dispersed repeat sequences and 47 simple sequence repeats. Among the genome's 53 678 codons, the largest proportion are leucine-encoding codons (5 251), whereas the smallest proportion encode tryptophan (712). Colinear analysis revealed an absence of inversions and rearrangements between I. anemonoides and related species at the chloroplast genome level. Whereas phylogenetic analysis indicated that I. anemonoides did not cluster in a clade with I. manshuricum, it did show a very close phylogenetic relationship with Paraquilegia microphylla. The findings of this study provide basic data that will contribute to further species identification and phylogenetic study of the genus Isopyrum.

Abstract:Perilla (Perilla frutescens L.) is an important edible-medicinal oil crop, with its seed containing 46%-58% oil. Of perilla seed oil, α-linolenic acid (C18:3) accounts for more than 60%. Lysophosphatidic acid acyltransferase (LPAT) is one of the key enzymes responsible for triacylglycerol assembly in plant seeds, controlling the metabolic flow from lysophosphatidic acid to phosphatidic acid. In this study, the LPAT2 gene from the developing seeds of perilla was cloned and designated as PfLPAT2. The expression profile of PfLPAT2 gene was examined in various tissues and different seed development stages of perilla (10, 20, 30, and 40 days after flowering, DAF) by quantitative real-time PCR (qRT-PCR). In order to detect the subcellular localization of PfLPAT2 protein, a fusion expression vector containing PfLPAT2 and GFP was constructed and transformed into Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration. In order to explore the enzymatic activity and biological function of PfLPAT2 protein, an E. coli expression vector, a yeast expression vector and a constitutive plant overexpression vector were constructed and transformed into an E. coli mutant SM2-1, a wild-type Saccharomyces cerevisiae strain INVSc1, and a common tobacco (Nicotiana tabacum, variety:Sumsun NN, SNN), respectively. The results showed that the PfLPAT2 open reading frame (ORF) sequence was 1 155 bp in length, encoding 384 amino acid residues. Functional structure domain prediction showed that PfLPAT2 protein has a typical conserved domain of lysophosphatidic acid acyltransferase. qRT-PCR analysis indicated that PfLPAT2 gene was expressed in all tissues tested, with the peak level in seed of 20 DAF of perilla. Subcellular localization prediction showed that PfLPAT2 protein is localized in cytoplasm. Functional complementation assay of PfLPAT2 in E. coli LPAAT mutant (SM2-1) showed that PfLPAT2 could restore the lipid biosynthesis of SM2-1 cell membrane and possess LPAT enzyme activity. The total oil content in the PfLPAT2 transgenic yeast was significantly increased, and the content of each fatty acid component changed compared with that of the non-transgenic control strain. Particularly, oleic acid (C18:1) in the transgenic yeast significantly increased, indicating that PfLPAT2 has a higher substrate preference for C18:1. Importantly, total fatty acid content in the transgenic tobacco leaves increased by about 0.42 times compared to that of the controls, with the C18:1 content doubled. The increased total oil content and the altered fatty acid composition in transgenic tobacco lines demonstrated that the heterologous expression of PfLPAT2 could promote host oil biosynthesis and the accumulation of health-promoting fatty acids (C18:1 and C18:3). This study will provide a theoretical basis and genetic elements for in-depth analysis of the molecular regulation mechanism of perilla oil, especially the synthesis of unsaturated fatty acids, which is beneficial to the genetic improvement of oil quality of oil crops.

Abstract:Brassica juncea is a yearly or biennial vegetable in Brassica of Cruciferae. The yield and quality of its product organs are affected by flowering time. WRKY proteins family can respond to biological and abiotic stresses, developmental regulation and signal transduction. WRKY75 is an important member of WRKY family which can regulate flowering, but the flowering regulation mechanism in B. juncea has not been reported. In this study, a gene BjuWRKY75 in B. juncea was cloned, and the encoded-protein belonged to the group Ⅱ of WRKY protein with highly conserved domain. BjuWRKY75 had the highest homology with BriWRKY75 of Brassica nigra. The relative expression level of BjuWRKY75 in flowers was significantly higher than that in leaves and stems, and it was expressed stably in leaves. BjuWRKY75 protein was localized in the nucleus and interacted with the promoter of the flowering integrator BjuFT, which contained the W-box response element for the interaction between protein and DNA. Thus, it could transcriptionally activate the expression of the downstream genes. The overexpression of BjuWRKY75 in Arabidopsis led to earlier flowering significantly. In conclusion, BjuWRKY75 could directly target the promoter of BjuFT and accelerate flowering. These results may facilitate further study on the regulation of flowering molecules of BjuWRKY75.

Abstract:Influenza C virus is an important respiratory pathogen not only infecting people, but also pigs, dogs, and other animals. Polymerase is central to the replication of influenza C virus and is an important target for studying the mechanism of viral replication. However, there is no commercial monoclonal antibody (MAb) targeting influenza C virus polymerase, which hampers the development of relevant research to some extent. In order to prepare MAb targeting the polymerase basic protein 2 (PB2) of influenza C virus, influenza C virus RNA-dependent RNA polymerase (RdRp, consists of PB1, PB2 and P3) was co-immunoprecipitated with Flag-tagged human acidic nuclear phosphoprotein 32A (huANP32A-Flag) from 293T cells based on the interaction between huANP32A and influenza virus RdRp. The purified RdRp was used as antigen to immunize BALB/c mice. Six positive hybridoma cell lines (7B11-5, 8A4-5, 13D9-6, 8D4-1, 8D4-3, 9F9-4) that stably secrete and recognize PB2 MAb were screened by indirect ELISA and Western blotting. The subtypes of MAb 7B11-5, 8A4-5, 8D4-1 and 8D4-3 antibody were identified as IgG1, the subtypes of MAb 13D9-6 and 9F9-4 were IgG2a and IgG3, respectively. All the light chains of the MAbs were κ chain. A hybridoma cell line 8D4-1 with high titer was further selected to prepare ascites. The titer of mouse ascites antibody was determined to be 1:64 000. Western blotting results showed that the MAb 8D4-1 had a specific immune response with ICV PB2; laser confocal assay showed that the prepared MAb 8D4-1 accurately detected the subcellular localization of PB2 subunits. Moreover, ICV RdRp was highly enriched by ANP32A. The high specific of the prepared PB2 MAb 8D4-1 may facilitate the polymerase detection, structural analysis and mechanism study of influenza C virus.

Abstract:Cytosine methylation is one of the major types of DNA epigenetic modifications and plays an important role in maintaining normal cell function and regulating gene expression. Bisulfite sequencing PCR (BSP) based cloning and sequencing is a general method for detecting DNA methylation at specific sites, which can clarify the methylation status of each CpG site in the target fragment. However, this method requires large amounts of single-clonal sequencing, which is complicated to operate, time consuming and expensive. Therefore, the development of an accurate, efficient and convenient DNA methylation detection technology is of great significance to improve the efficiency of epigenetic research. Based on the high-throughput mutation detection platform Hi-TOM (high-throughput tracking of mutations) developed by our group, we further established a site-specific DNA methylation high-throughput detection platform Hi-Meth (High-throughput Detection of DNA Methylation). After bisulfite treatment of DNA samples, the specific site-specific DNA methylation analysis results could be obtained through the Hi-Meth platform by performing only one round of PCR amplification. Using the Hi-Meth platform, the DNA methylation status of two promoter regions of rice were detected. The DNA methylation results from Hi-Meth were consistent with the results from BSP-based method. Thus, site-specific DNA methylation analysis results could be obtained accurately and conveniently through the Hi-Meth platform. In conclusion, Hi-Meth provides an important methylation detection platform for specific DNA regions, which has important significance for epigenetic research.

Abstract:A mutant strain ΔVcrV was constructed by using homologous recombination method for investigating the function of the VcrV gene in Vibrio alginolyticus type Ⅲ secretion system. The genetic stability of ΔVcrV was detected by PCR, and the biological characteristics between the mutant and the wild type strains were compared. ΔVcrV muntat had no significant changes in growth rate and autoagglutination compared with the wild type strain, but the ability to form biofilms was reduced, and the LD₅₀ was increased by 16.5 times. The swimming and swarming motility of the mutant strain ΔVcrV were significantly enhanced, while cell adhesion was significantly reduced than the wild strain (P<0.01). The tolerance of ΔVcrV mutant to H₂O₂ and NaCl was decreased. Compared with that of the wild type strain, the sensitivity of ΔVcrV mutant to cefuroxime, medimycin and clindamycin was increased, but to amikacin and polymxin B was decreased. The reactive oxygen species (ROS) content of ΔVcrV mutant was significantly decreased (P<0.01), and the indexes of proline, peptidoglycan, β-lactamase, catalase, superoxide dismutase and glutathione peroxidase of ΔVcrV mutant were significantly increased than that of the wild type strain (P<0.01). The biological characteristics of ΔVcrV mutant indicated that VcrV gene was involved in pathogenicity and various biological functions of V. alginolyticus type Ⅲ secretion system.

Abstract:Myostatin (Mstn) is known as growth/differentiation factor-8 (GDF-8). Knockout or knockdown of Mstn gene promotes muscle development and reduces fat content. Here we prepared Mstn knockdown mice by RNA interference, then the morphology of the skeletal muscle, the content of triglyceride (TG), the content and composition of fatty acids in the skeletal muscle were detected. The expression of Mstn reduced in muscle of Mstn knockdown mice compared to the controls. The cross sectional areas of the skeletal muscle myofibers were significantly larger while the content of TG was less than that of the controls, and the ratios of n-3/n-6 and unsat/sat in the knockdown mice increased significantly. Subsequently, we detected the expression of genes associated with fatty acid metabolism. The expression of the genes associated with lipolysis and fatty acid transportation were up-regulated, while the genes associated with fatty acid synthesis were down-regulated. Of these genes, the up-regulation of a gene associated with β oxidation, Cpt1b, was up-regulated remarkably. We further detected the enzyme activity of CPT1 in skeletal muscle and obtained the same results with gene expression. Moreover, chromatin immunoprecipitation assay was performed and we found that SMAD3, a transcription factor downstream of Mstn, directly binds to the promoter of Cpt1b gene. These results showed that knockdown of Mstn up-regulated the expression of Cpt1b through the binding of SMAD3 to the promoter of Cpt1b, then promoted the β oxidation metabolism of intramuscular fatty acids.

Abstract:After the outbreak of COVID-19, the widespread application of online teaching has brought challenges and opportunities for higher education. Developing an effective teaching system is the focus of curriculum teaching reform in the post pandemic era. According to the characteristics of Human and Animal Physiology, the course teachers has developed a new teaching system by updating the teaching concept, reconstructing the contents of the course, changing the teaching modes, strengthening the integration of moral and intellectual education, and improving the assessment approaches. This teaching system is aimed at meeting the need of personalized learning for students and adapting to a new teaching environment. This article introduces the exploration and practice of the curriculum reform.

Abstract:Microbiology is a key basic professional course for all the students specializing in biology, biotechnology and related majors. To date, microbiology is mainly taught in Chinese within colleges and universities in China. Development of a microbiology course that is taught in English may satisfy the diversified learning needs of the students and promote the "Double First-Class" initiative. We started to teach the microbiology course in English at the East China University of Science and Technology since 2016. This practice was associated with reform and innovation in the teaching methods and contents. The microbiology course taught in English greatly attracted the interest of the attending students and helped improve their professional English learning as well as scientific research. This course provided important support for fostering innovative professional first-class undergraduates under the context of the "Double First-Class" initiative.