Construction of Molecular Biology and Experiment Case Library for Graduate Students

Molecular Biology and Experiment is considered fundamental for graduate students specializing in aquaculture at Guangdong Ocean University.This discipline focuses on the examination of the structure and function of macromolecules,including proteins and nucleic acids.Moreover,it elucidates biological phenomena and principles at the molecular level,making it an essential foundational course for students pursuing various biology majors.As a foundational course for the basic application of aquaculture,Molecular Biology and Experiment requires guidance through numerous examples and cases.However,there are several challenges to address in developing the case library.Consequently,a case library has been established to meet the course requirements of Molecular Biology and Experiment for modern graduate students,with the central goal of reforming the educational model of higher education institutions and enhancing the effectiveness and quality of talent development.This strategy is designed to nurture highly skilled professionals who can address the current needs of the industry.

Reform and Exploration of Integrating"Service Learning"into the Teaching of Pathogenic Biology of Aquatic Animals to Help Rural Revitalization

This paper intends to combine the development trend of the subject,restructure and optimize the course content,and construct the service learning model of Pathogenic Biology of Aquatic Animals for postgraduates.At the same time,through service practice,it is needed to further consolidate curriculum knowledge and skills,stimulate the learning initiative and enthusiasm of postgraduates,expand professional knowledge,improve professional quality,and lay a solid foundation for serving the national rural revitalization strategy in the future.

In vitro Inhibitory Activity of Shisandra chinensis and Polygonatum sibiricum against Vibrio harveyi and Its Biofilms

[Objective] This study aimed to analyze the in vitro inhibitory activity of Shisandra chinensis and Polygonatum sibiricum against Vibrio harveyi and its biofilms. [Result] By agar diffusion test, in vitro inhibitory activity of 5. chinensis and P. sibiricum against V. harveyi was investigated. The minimal inhibitory concentration （ MIC） and minimal bactericidal concentration （MBC） of 5. chinensis and P. sibiricum against V. harveyi were determined by doubling dilution meth-od. The inhibitory activity of 5. chinensis and P. sibiricum on the formation of V. harveyi biofilms was evaluated by modified MTT assay. [ Result ] Both 5. chinen-sis and P. sibiricum had inhibitory activity against V. harveyi. The inhibition zone diameter of 5. chinensis against V. harveyi was 17. 95 mm; MIC and MBC of 5. chinensis were both 3.125 mg/ml. The inhibition zone diameter of P. sibiricum against V. harveyi was 12. 22 mm; MIC and MBC of P. sibiricum were 3.125 and 6.250 mg/ml, respectively. When the concentration was higher than 6. 25 mg/ml, 5. chinensis decoction had extremely significant inhibitory activity against V. harveyi （P 〈 0. 01） ; when the concentration was higher than 3. 125 mg/ml, P. sibiricum had extremely significant inhibitory activity against V. harveyi （P 〈0. 01）. [ Conclusion] 5. chinensis and P. sibiricum could significantly inhibit V. harveyi and its biofilms.

Inhibitory Activity of Prunus mume,Copitis chinensis and Crataegus pinnatifida on Vibrio harveyi and Its Biofilm

[Objective] This study was conducted to determine the in-vitro inhibitory effects of Prunus mume,Coptis chinensis and Crataegus pinnatifida on Vibrio harveyi and its biofilm. [Method]The inhibitory zone diameters of the three Chinese herbal medicines against V. harveyi were determined by agar diffusion method; the minimal inhibitory concentration( MIC) and minimal bactericidal concentration( MBC) values of the three Chinese herbal medicines against V. harveyi were determined by doubling dilution method; and the effects of the three Chinese herbal medicines on the formation of V. harveyi biofilm were determined by methyl thiazolyl tetrazolium( MTT) method. [Result]The three Chinese herbal medicines all inhibited V. harveyi to different degrees. C. chinensis and C. pinnatifida and P. mume exhibited the inhibitory zone diameters of( 17. 62 ± 0. 04),( 20. 16 ± 0. 08) and( 30. 76 ± 0. 26) mm against V. harveyi,respectively. P. mume and C. pinnatifida had strong inhibitory effects on V. harveyi. The MIC and MBC values of P. mume against V. harveyi were 7. 812 5 mg/ml; the MIC and MBC values of C. pinnatifida against V. harveyi were 31. 25 mg/ml; and the MIC and MBC values of C. chinensis against V. harveyi were 62. 5 mg/ml. P. mume had the strongest antibacterial and bactericidal ability. The MIC values of C. pinnatifida,C. chinensis and P. mume against V. harveyi were 7. 81,7. 81 and 1. 96 mg/ml,respectively,i. e.,P. mume exhibited the lowest MIC. [Conclusion] P. mume,C. pinnatifida and C. chinensis all have inhibitory effects on V. harveyi and its biofilm,and P. mume has the strongest bactericidal ability.

Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein Va1686 Gene of Vibrio alginolyticus

In this study, Va 1686 gene was cloned from Vibrio alginolyticus . The total length of the gene is 1 164 bp, and it could encode 387 amino acids. The physicochemical properties, protein structure, genetic evolutionary relationship and antigenic characteristics of the effect protein Va1686 of V. alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools. The results showed that Va1686 is a stable hydrophilic and acidic protein without a transmembrane region and a signal peptide, and secondary structure to α-helix. The evolutionary analysis showed that V. alginolyticus HY9901 and V. harveyi were clustered together, which indicated that the genetic relationship between the two species was the closest. Va1686 contains a Fic superfamily conserved domain associated with cell division. Bioinformatics analysis showed that the B-cell preponderant epitopes of Va1686 might be localized in the regions of 48-49, 82-85, 125-126, 150-153, 185-186, 236-237 and so on. The 3D structure model of Va1686 subunit was simulated by SWISS-MODEL software and it was found that the vopS of V. parahaemolyticus was similar and the similarity was 89.46%. In this study, the feasibility of Va1686 as a common antigen of Vibrio was verified from the perspective of bioinformatics, which laid the foundation for the next step in vaccine development.

Molecular Cloning and Bioinformatics Analysis of T3SS Effector HopPmaJ from Vibrio alginolyticus

In this paper, the hopPmaJ gene, which encodes type III effector HopPmaJ of Vibrio alginolyticus, was cloned, sequenced and bioinformatically predicted. The results showed that hopPmaJ （GenBank accession number： KX245315） is 345 bp in length and encodes 114 amino acids, with a theoretical molecular mass of 12. 774 kD, and a pI of 4.45. The protein has multiple functional domains and binding sites, but no signal peptide. The tertiary structure of the protein is a homodimer. Based on multiple parameters, the possible dominant B cell antigenic epitopes of HopPmaJ were predicted to be at positions 13 - 16, 29 -30, 40 -42, 45 -49 and 84 -91.

Construction of Case Database for Postgraduate Course Aquatic Animal Pathogen Biology

As an application instructional course for Aquatic Animal Medicine(AAM),Aquatic Animal Pathogen Biology needs to be guided by a large number of examples and cases,but the current case database construction faces many urgent problems.In view of this,this paper analyzes the characteristics of professional education of AAM postgraduate students.With the goal of cultivating applied personnel that meet the requirements of the times,and the guiding ideology of strengthening the reform of the education model in colleges and universities,and improving the quantity and quality of personnel training,it builds a case database for the course Aquatic Animal Pathogen Biology in accordance with modern postgraduate teaching needs.

Career Planning Education Paths for Students of Aquatic Animal Medicine Discipline in the Context of the Belt and Road Initiative: A Case Study of Construction Achievement of Guangdong Ocean University

In order to improve the professional competitiveness of students in the Aquatic Animal Medicine Discipline,combined with the case tracking and theoretical practice for aquatic animal medicine students for three years,with the aid of SPSS software,this paper elaborated the actual level and existing problems in career planning of aquatic animal medicine students in Guangdong Ocean University from five dimensions:career planning awareness,self-awareness,environmental awareness,goal planning,and implementation correction. Then,it discussed the opportunities and challenges of the Aquatic Animal Medicine Discipline in the context of the Belt and Road Initiative. Finally,it came up with solutions and reform recommendations in education,teaching,and counseling paths from the students,university,families,government,and society.

Practice Instruction of Innovative and Entrepreneurial Training Program for Aquatic Animal Medicine Students

As an important platform for the combination of theory and practice,college students' innovative and entrepreneurial training program has an important impact on improving the comprehensive ability of undergraduates. The major of aquatic animal medicine is a new sea-related major. The continuous development and transformation and upgrading of aquaculture in the new period have put forward newer and higher requirements for the innovative and practical ability and comprehensive quality of aquatic animal medicine graduates. Taking the innovation and entrepreneurship program of college students majoring in aquatic animal medicine as an example,this paper analyzes the significance of the innovation training program for college students,the cultivation of students' scientific research ability,and the problems existing in the application and operation of the program,in order to provide a reference for the innovative education and talent training for students majoring in aquatic animal medicine.

Construction and Biological Function Analysis of a dldh Deletion Mutant Strain of Vibrio alginolyticus

[ Objeetlve] This study aimed to investigate the role of dihydrolipoamide dehydrogenase （DLDH） in the pathopoiesis of Vibrio alginolyticua. [ Method] Using suicide plasmid pRE112 as the vector, d/dh deletion mutant strain ZJO3Adldh was constructed with insertion inactivation mutation method by Overlap PCR and homologous recombination technology to compare and analyze the differences in the growth, swimming ability, enzyme activity, biofilm formation and pathogenicity between wild-type strain ZJ03 and deletion mutant strain ZJO3△dldh. [ Result] dldh deletion mutant strain ZJ03 △d/dh exhibited prolonged lag phase, significantly reduced swimming ability （P 〈 0.05 ）, significantly reduced enzyme activity （P 〈 0.05 ）, significantly reduced biofilm formation ability （P 〈 0.05 ） and remarkably reduced pathogenicity to Epinephelus coioides （P 〈0.05） compared to wild-type strain ZJ03. [ Conclusion] This study provided theoretical basis for revealing the pathogenic mechanisms of Vibrio from the perspective of bacterial energy metabolism.

Inhibitory Effects of Prunus mume,Coptis chinensis,and Crataegus pinnatifida on Vibrio parahaemolyticus and Its Biofilm in Vitro

In order to explore the inhibitory effects of Prunus mume,Crataegus pinnatifida and Coptis chinensis on Vibrio parahaemolyticus and its biofilm in vitro,the agar diffusion method was applied. These three Chinese herbal medicines had different inhibitory effects on V. parahaemolyticus. The inhibition zone of C. pinnatifida to V. parahaemolyticus was( 15. 25 ± 0. 53) mm,and the minimum inhibitory concentration( MIC) and minimum bactericidal concentration( MBC) of C. pinnatifida on V. parahaemolyticus were both 31. 25 mg/m L; the inhibition zone of C. chinensis to V. parahaemolyticus was( 18. 08 ± 0. 10) mm,and the MIC and MBC of C. chinensis on V. parahaemolyticus were both15. 63 mg/m L; the inhibition zone of P. mume to V. parahaemolyticus was( 28. 99 ± 0. 47) mm,and the MIC and MBC of P. mume on V. parahaemolyticus were both 7. 81 mg/m L. The effects of three traditional Chinese medicines on the biofilm formation of V. parahaemolyticus were tested by MTT colorimetric method using methylthiazolyl tetrazolium( MTT). P. mume,C. pinnatifida and C. chinensis have significant inhibitory effects on V. parahaemolyticus biofilm and their MIC are 7. 81 mg/m L,3. 125 mg/m L,and 62. 5 mg/m L,respectively( P < 0. 01).The experimental results are expected to provide certain references for the development of new fishery drugs.

Molecular Cloning,Bioinformatics Analysis and Expression Analysis of Type III Secretion System(T3SS)Injectisome Gene vscX from Vibrio alginolyticus

In order to enrich the research about type III secretion system （T3SS） injectisome of Vibrio alginolyticus, vscX gene was cloned from E algianlyticus strain HY9901 for bioinformatics analysis and expression analysis. Specific primers were designed according to the full-length geanme sequence of V. alginolyticus in GenBank. vscX gene （C, enBank accession number： FR780679） contained a 378 bp open reading frame （ORF）, encoding a putative protein of 125 amino acids. The theoretical molecular weight was 14.209 2 kD and theoretical pI was 5.75. By using Signal 4.1 Server and TMHMM Server 2.0, it was predicted that VscX protein had no transmembrane domain or signal peptide. The results of prediction using SoftBerry-Psite software showed that VscX protein contained three casein ki- nase lI phosphorylation sites, one N-myristoylation site and three C-terminal targeting signal sites. Subcellular localization revealed that VscX might be located in cytoplasm with the possibility of 56.5%. According to SMART prediction, VscX had one Pfam （ 1 - 125 aa） domain. Phylogenetic analysis revealed that VscX from V. alginolyticus and VscX from E parahemolyticus were clustered into the same group. Network interaction analysis showed that vscX was adjacent to vseY, vopB and sycN. By real-time fluorescent quantitative PCR technique and 2-△△△ method, the differences in expression levels of VscX mRNA in V. alginolyticus strain HY9901, T3SS deletion strain AvscO and complementary strain C-vscO at different growth stages were analyzed. The results showed that the expression levels of VscX mRNA in V. algianlyticus strain HY9901 and C-vscO were significantly up-regulated at stable growth stage （ P 〈0.01 ） ; the expression levels of VscX mRNA in deletion strain △vscO were significantly up-regulated at late growth stage （ P 〈0.01 ）. This study provided the basis for revealing the transport mechanism of T3SS injectisome of E alginolyticus.

Molecular Cloning and Bioinformatics Analysis of kdpD Gene from Vibrio alginolyticus

In this study, histidine kinase gene kdpD in the two-component regulatory system of Vibrio alginolyticus strain HY9901 was cloned for bioinformatics analysis. Sequence analysis revealed that kdpD gene （GenBank accession number： KJ544668） was 1 374 bp in length, encoding a putative protein of 457 amino acids. The predicted molecular weight （MW） of KdpE was 51.60 kD with a theoretical isoelectric point （pl） of 6.02. Using SignalP 4.0, TMHMM Server 2.0 and SoftBerry-Psite software, it was predicted that KdpE protein was equally located in Golgi apparatus, plasma membrane and endoplasmie reticulum （33.3%） , which did not contain a signal peptide but contained three transmembrane domains. KdpE protein had five casein kinase 1I phosphorylation sites, five protein kinase C phosphorylation sites and one cAMP- and cGMP-dependent protein kinase phosphorylation site. A phylogenetic tree was constructed by MEGA 5.0 software, which revealed that KdpD from V. alginolyticus had close genetic relationship with corresponding proteins from V. campbellii and V. parahaemolyticus. Using SWISS- MODEL Workspace, the three-dimensional structure of HATPase_c conserved domain in KdpD protein was constructed. These results may provide the basis for fur- ther studies on two-comoonent regulatory system KdpD/KdpE of V. alginolyticus.

Inhibitory Effects of Prunus mume,Coptis chinensis,and Crataegus pinnatifida on Vibrio alginolyticus and Its Biofilm In Vitro

[Objectives]To explore the inhibitory effects of Prunus mume,Coptis chinensis,and Crataegus pinnatifida on Vibrio alginolyticus and its biofilm in vitro. [Methods] The agar diffusion method was applied to determine the inhibition zone diameter of three Chinese herbal medicines on V. alginolyticus; double dilution method was used to determine the minimum inhibitory concentration( MIC) and minimum bactericidal concentration( MBC) of three Chinese herbal medicines on V. alginolyticus; methylthiazolyl tetrazolium( MTT) method was applied to evaluate the effects of three Chinese herbal medicines on the biofilm formation of V. alginolyticus. [Results]The three Chinese herbal medicines had different inhibitory effects on V. alginolyticus. The inhibition zones of P. mume,C. chinensis,and C. pinnatifida on V. alginolyticus were( 18. 31 ± 0. 29) mm,( 20. 31 ± 0. 30) mm,and( 30. 24 ± 0. 14) mm respectively,and P. mume had strong bacteriostatic effects on V. alginolyticus. The double dilution method was used to determine the bacteriostatic effects of three Chinese herbal medicines on V. alginolyticus. Results showed that P. mume has the highest antibacterial and bactericidal ability. The MIC and MBC of P. mume,C. pinnatifida,and C. chinensis on V. alginolyticus was 3. 91,31. 25,and 125 mg/m L,respectively. Finally,MTT method was applied to evaluate the effects of C. pinnatifida,C. chinensis,and P. mume on the biofilm formation of V. alginolyticus,and results showed that the MIC on the biofilm formation of V. alginolyticus was 15. 63,7. 81,and 3. 91 mg/m L,respectively; the MIC of P. mume was the lowest. [Conclusions]This experiment proves that P. mume,C. chinensis,and C. pinnatifida have inhibitory effects on V. alginolyticus and its biofilm formation. It is expected to provide references for prevention and control of pathogenic vibrio induced diseases of aquatic animals.

Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein HY 322 Gene of Vibrio alginolyticus

In this study,Hy322 gene was cloned from Vibrio alginolyticus.The total length of its gene was 969 bp,and it could encode 322 amino acids.The physicochemical properties,protein structure,genetic evolutionary relationship and antigenic characteristics of the effector protein Hy322 of V.alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools.The results showed that Hy322 is an unstable hydrophilic and acidic protein without a transmembrane region and a signal peptide,and secondary structure to α-helix.The evolutionary analysis showed that V.alginolyticus HY9901 and V.harveyi were clustered together,which indicated that the genetic relationship between the two species was closest.HY322 contains a FliN super family conserved domain associated with Flagellar motor switch.Bioinformatics analysis showed that the B-cell preponderant epitopes of Hy322 might be localized in the regions of 32-33,100-102,138-140,215-216,235-238 and 246-249.The 3D structure model of Hy322 subunit was simulated by SWISS-MODEL software and itwas found that the yscQ of Yersinia were similar and the similarity was 42.25%.In this study,the feasibility of Hy322 as a common antigen of Vibrio was verified from the perspective of bioinformatics,which laid the foundation for the next step in vaccine development.

Analysis of Biological Activity and Immunogenicity of Extracellular Products from Streptococcus iniae

[Objectives] This study aimed to analyze the biological activity and immunogenicity of extracellular products from Streptococcus iniae .[Methods] S. iniae was incubated with brain heart infusion agar medium （BHIA ＋ 4% calf serum） for 60 h. The bacterial liquid was rinsed with PBS, centrifuged, and filtered through microporous filtering film to collect extracellular products （ECPs）.[Results] Extracellular proteinase （ECPase） of S. iniae exhibited amylase, protease, lecitinase, gelatinase, lipase activities and hemolytic activity but had no urease activity. EDTA, DTT and PMSF could reduce ECPase activity to 72.4%, 77.6% and 72.4%, respectively. Cu^2＋ , Ca 2＋ , K^＋ and Mg^2＋ exhibited an inhibitory effect on ECPase activity, whereas Fe^3＋ , Co^2＋ and Mn^2＋ could activate ECPase activity. ECPs had good heat stability and exhibited relatively high activities under alkaline conditions. The optimal temperature for ECPs was 55 ℃. Two-dimensional electrophoresis was performed to analyze the main protein of ECPs. The results indicated that there are 12 main bands of ECPs, and the molecular weights mainly ranged between 28-68 kDa. About 120 protein spots were detected, and the molecular weights mainly ranged between 26-95 kDa. The mouse anti- S. iniae was used for Western-blot analysis of ECPs, and the results showed that there were four proteins, with molecular weights of 26, 37, 95, and 97 kDa, respectively. The pathogenicity assay indicated that ECPs of S. iniae were highly pathogenic to tilapia. The mortality rate of tilapia was enhanced as the concentration of ECPs increased.[Conclusions] This study provided a certain theoretical basis for revealing the pathogenic mechanism of Streptococcus iniae .

Study on Case Library Construction of Inspection and Quarantine of Aquatic Animal

By making use of the technical advantages of Guangzhou Huibiao Testing Technology Center in the field of testing, the project analyzes the characteristics of undergraduate professional education in aquatic animal medicine, and intends to build a case library of Inspection and Quarantine of Aquatic Animal suitable for modern undergraduate teaching needs, with training applied talents to meet the requirements of the times as the goal, and strengthening the reform of college education mode and improving the quantity and quality of talent training as the guiding ideology.

Construction of Bilingual Teaching Mode of Aquatic Animal Bacteriology in the Context of the Belt and Road Initiative

Aquatic Animal Bacteriology is a basic course of Aquatic Animal Medicine discipline.Bacteriology is not only a frontier subject in the field of life sciences,but also one of the most rapidly developing scientific fields today.The Belt and Road Initiative has brought opportunities and challenges to the upgrading and development of this discipline,so this discipline faces many problems that need to be solved.In view of this,this study combined the development trend of the discipline,reorganized and optimized the course content,and prepared sophisticated multimedia courseware,to stimulate students'learning initiative and enthusiasm,expand the field of professional knowledge,improve their English translation and writing skills,so as to lay a solid foundation for their future service to the Belt and Road Initiative.

High-quality Development Path of Graduate Education in Aquaculture Discipline:A Case Study of Guangdong Ocean University

Aquaculture is a discipline system that focuses on exploring the growth,development,reproduction,aquaculture,and resources of aquatic animals and plants,and their complex relationships.Under the tide of the"high-quality development"strategy,the aquaculture discipline is also facing new opportunities and challenges for transformation,upgrading,and deepening development.Therefore,exploring and practicing an effective path for the high-quality development of graduate education in aquaculture is not only the key to promoting the transformation of graduate education in aquaculture from scale expansion to quality improvement,but also has immeasurable value for implementing the strategy on developing a quality workforce and the strategy of scientific and technological powerhouse.The high-quality development of graduate education in aquaculture can be promoted from the following aspects:optimizing and improving the construction of the graduate education system,focusing on enhancing the quality of high-level talent cultivation,strengthening the overall strength of the graduate supervisor team,actively promoting the adjustment and upgrading of the disciplinary and professional structure,strengthening the construction of resource platforms and deepening the implementation of collaborative education mechanisms,and continuously expanding and deepening the new pattern of international exchange and cooperation.Through the comprehensive promotion of the above paths,the aim is to fully build a model for the improvement and governance of graduate education quality.

GCRV 096 VP6 protein and its impacts on GCRV replication with different genotypes in CIK cells

Grass carp reovirus(GCRV)is the causative agent of grass carp hemorrhagic disease,which has detrimental effects on the grass carp aquaculture industry.There are four known genotypes of GCRV and strains of different GCRV genotypes differ greatly.In this study,the diversity of the protein VP6 from different GCRV stains and the effect of genotype GCRV 096 on replication was investigated in CIK cells.Our results showed that the VP6 protein of GCRV 096(genotypeІ)exhibited limited homology to that of GCRV GD108(genotypeШ),with few residues conserved in predicted protein-protein interaction domains.GCRV 096 VP6 protein was expressed and purified and an antiserum against it was characterized.Addition of purified VP6 protein or antiserum to culture media of CIK cells inhibited the replication of GCRV 096 in these cells.In contrast,replication of GCRV GD108(genotypeШ)was not affected in CIK cells under the same condition.Overall,our results indicated that the protein VP6 and VP6 antiserum did not provide cross-protection against GCRV strains and this can be attributed to differences among GCRV genotypes.It will be important to consider multiple GCRV genotypes in the development of effective GCRV vaccines and other therapies against grass carp hemorrhagic disease.In addition,bioinformatics analysis also suggested that the protein VP6 may have a role in the process of GCRV infection.This study lays the foundation for the prevention of grass carp hemorrhagic disease and further detailed studies on the pathogenesis of GCRV.