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Article(id=1241357430345486613, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241357427292033288, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20230504, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1690819200000, receivedDateStr=2023-08-01, revisedDate=null, revisedDateStr=null, acceptedDate=1697472000000, acceptedDateStr=2023-10-17, onlineDate=1773892274699, onlineDateStr=2026-03-19, pubDate=1709481600000, pubDateStr=2024-03-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773892274699, onlineIssueDateStr=2026-03-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773892274699, creator=13701087609, updateTime=1773892274699, updator=13701087609, issue=Issue{id=1241357427292033288, tenantId=1146029695717560320, journalId=1192105938417971205, year='2024', volume='64', issue='3', pageStart='651', pageEnd='967', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1773892273972, creator=13701087609, updateTime=1773892616576, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241358864344478487, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241357427292033288, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241358864344478488, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1241357427292033288, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=767, endPage=779, ext={EN=ArticleExt(id=1241357430655865123, articleId=1241357430345486613, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Construction of a chromosome-plasmid balanced lethal system based onmurI in the attenuated live vaccine strain ofPseudomonas plecoglossicida, columnId=1241045257748533520, journalTitle=Acta Microbiologica Sinica, columnName=Research Articles, runingTitle=null, highlight=null, articleAbstract=

[Objective] To construct a chromosome-plasmid balanced lethal system based on the glutamate racemase (MurI) gene for the expression of exogenous antigens in the attenuated vaccine strain ofPseudomonas plecoglossicida (Pp ΔtssD-1), so as to provide new ideas and methods for the development of multi-component live vaccines. [Methods] We constructed amurI-deleted strain fromPp ΔtssD-1 by homologous recombination. First, we replaced the kanamycin resistance gene of the pBBR1MCS-2 plasmid withmurI to construct a balanced lethal plasmid. Subsequently, we inserted the green fluorescent protein gene into the multicloning site of the plasmid to examine the expression stability of the exogenous antigen. Finally, we characterized the recombinant strain in terms of the growth curve, plasmid stability, and expression of the exogenous antigen. [Results] ThemurI-deleted strain was unable to grow in the lysogeny broth medium without D-glutamate. The non-resistant complemented strain regained growth capability in the lysogeny broth medium without D-glutamate. However, its growth was slower than that of the starting strain. Exogenously introduced antigens were identified as stable in the absence of antibiotic selection, and distinct green fluorescence signals were observed under a fluorescence microscope. Additionally, the balanced lethal plasmid exhibited high genetic stability within the recombinant strain. [Conclusion] A novel chromosome-plasmid balanced lethal system targetingmurI was developed in this study. It enabled the expression of exogenous antigens inPp ΔtssD-1 without the need for antibiotic selection. This system provides a new method for the development of multi-component live vaccines, with no need of antibiotic resistance markers and high plasmid stability.

, correspAuthors=Zhen TAO, authorNote=null, correspAuthorsNote=
*TAO Zhen, E-mail:
, copyrightStatement=Copyright ©2024 Acta Microbiologica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Mingming ZHANG, Haoda YE, Chaozheng ZHANG, Pengcheng WANG, Kequan WANG, Xiaojun YAN, Zhen TAO), CN=ArticleExt(id=1241357434372018613, articleId=1241357430345486613, tenantId=1146029695717560320, journalId=1192105938417971205, language=CN, title=基于谷氨酸消旋酶基因(murI)构建杀香鱼假单胞菌减毒活疫苗株的染色体-质粒平衡致死表达系统, columnId=1192149544164012138, journalTitle=微生物学报, columnName=研究报告, runingTitle=null, highlight=null, articleAbstract=

【目的】构建一种基于谷氨酸消旋酶(MurI)基因的染色体-质粒平衡致死系统，用于杀香鱼假单胞菌减毒活疫苗株(Pseudomonas plecoglossicida ΔtssD-1,Pp ΔtssD-1)中表达外源抗原，为开发多联活疫苗提供新的思路和方法。【方法】利用同源重组技术，将亲本株Pp ΔtssD-1中的murI基因敲除，构建murI基因缺失突变株；将广宿主穿梭质粒pBBR1MCS-2的卡那霉素抗性基因替换为murI基因，构建平衡致死质粒(即无抗性回补质粒)；在平衡致死质粒的多克隆位点处插入绿色荧光蛋白以检测外源抗原是否稳定表达，对重组菌株进行生物学特性分析，包括生长曲线、质粒稳定性和外源抗原表达水平。【结果】murI基因缺失株在不含D-谷氨酸的LB培养基上无法生长；无抗性回补株在不含D-谷氨酸的LB培养基上恢复了生长能力，但生长速度低于亲本株；经鉴定外源抗原可在无抗性质粒中稳定表达，并可在荧光显微镜下观察到明显的绿色荧光信号；此外，平衡致死质粒在重组菌株中具有良好的遗传稳定性。【结论】本研究以murI为靶点构建了新型的染色体-质粒平衡致死系统，可在无抗性筛选条件下在Pp ΔtssD-1中表达外源抗原，为开发多联活疫苗提供了新的策略和方法。

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unstructuredReference=WEIN T, DAGAN T.Plasmid evolution[J].Current Biology,2020,30(19):R1158-R1163., articleTitle=Plasmid evolution, refAbstract=null)], funds=[Fund(id=1241444400623046930, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, awardId=42376108, language=EN, fundingSource=National Natural Science Foundation of China(42376108), fundOrder=null, country=null), Fund(id=1241444400761458971, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, awardId=42376108, language=CN, fundingSource=国家自然科学基金(42376108), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1241444386853147581, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, xref=null, ext=[AuthorCompanyExt(id=1241444386865730494, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, companyId=1241444386853147581, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 School of Fishery, Zhejiang Ocean University, Zhoushan 316022, Zhejiang, China), AuthorCompanyExt(id=1241444386878313407, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, companyId=1241444386853147581, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 浙江海洋大学水产学院, 浙江 舟山 316022)]), AuthorCompany(id=1241444387037696971, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, xref=null, ext=[AuthorCompanyExt(id=1241444387046085581, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, companyId=1241444387037696971, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 School of Marine Sciences, Ningbo University, Ningbo 315211, Zhejiang, China), AuthorCompanyExt(id=1241444387058668496, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, companyId=1241444387037696971, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 宁波大学海洋学院, 浙江 宁波 315211)])], figs=[ArticleFig(id=1241444393152991399, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Figure 1, caption=Schematic diagram of the construction of the balanced-lethal plasmid pBBR1-murI., figureFileSmall=tU0BjTQ7NgJlEaaR9gZK0w==, figureFileBig=Cdp7vLXN87t3dAPNxk0J6A==, tableContent=null), ArticleFig(id=1241444393262043307, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=图1, caption=构建平衡致死质粒pBBR1-murI示意图, figureFileSmall=tU0BjTQ7NgJlEaaR9gZK0w==, figureFileBig=Cdp7vLXN87t3dAPNxk0J6A==, tableContent=null), ArticleFig(id=1241444393404649651, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Figure 2, caption=Alignments of MurI proteins ofPseudomonas plecoglossicida XSDHY-P andEscherichia coli K12. The residues are colored according to their conservation and similarity. Strictly conserved residues are shown in white text on the red background, and 75% conserved or similarly substituted residues are shown in red text on the white background. The UDP-N-acetyl-alpha-D-muramoyl-L-alanine and L-glutamine binding-site residues are indicated by hollow and solid triangles under the sequences, respectively. The active sites are indicated by asterisks under the sequences[20]., figureFileSmall=vUK3KBbME7SxvEZq3RO7rA==, figureFileBig=sCnSWCLR7BWSJFu0EF7dow==, tableContent=null), ArticleFig(id=1241444393526284472, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=图2, caption=MurI蛋白的序列比对图, figureFileSmall=vUK3KBbME7SxvEZq3RO7rA==, figureFileBig=sCnSWCLR7BWSJFu0EF7dow==, tableContent=null), ArticleFig(id=1241444393652113599, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Figure 3, caption=Construction of themurI gene knockout strain ofPseudomonas plecoglossicidaPp ΔtssD-1. A: PCR detection of the merged flanking arms using primers P7/P8. B: PCR detection ofmurI gene using primers P9/P10. C: PCR identification ofP.plecoglossicida using primers P5/P6. M: DL1000 marker; Lane 1:Pp ΔtssD-1; Lane 2−15: Putative positive colonies., figureFileSmall=zC7EWbqb0lHNZGCwx3Uw+g==, figureFileBig=27RbFx65Th3SCTomPHwmMw==, tableContent=null), ArticleFig(id=1241444393748582594, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=图3, caption=murI基因敲除株的鉴定, figureFileSmall=zC7EWbqb0lHNZGCwx3Uw+g==, figureFileBig=27RbFx65Th3SCTomPHwmMw==, tableContent=null), ArticleFig(id=1241444393845051592, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Figure 4, caption=Growth ofPp ΔtssD-1ΔmurI on LB agar without 10 mmol/L D-glutamate (A) and LB with 10 mmol/L D-glutamate (B)., figureFileSmall=ZTEFIN6JPFPV07tCvDUWfg==, figureFileBig=op5ViOPeuhj8VSbOtVupEQ==, tableContent=null), ArticleFig(id=1241444394058961100, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=图4, caption=Pp ΔtssD-1ΔmurI在无D-谷氨酸LB平板(A)和含D-谷氨酸LB平板(B)上的生长情况, figureFileSmall=ZTEFIN6JPFPV07tCvDUWfg==, figureFileBig=op5ViOPeuhj8VSbOtVupEQ==, tableContent=null), ArticleFig(id=1241444394172207312, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Figure 5, caption=PCR verification of the plasmid pBBR1-murI using primes P15/P16. M: DL2000 marker; Lane 1−6: PCR amplification of the putative colonies of recombinant strainPp ΔtssD-1ΔmurI (pBBR1-murI); Lane 7: Negative control., figureFileSmall=xHaDybM7ZxiGNLhBneiV4A==, figureFileBig=maoPNR/dbGnC5ARoeWbUlA==, tableContent=null), ArticleFig(id=1241444397569593554, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=图5, caption=pBBR1-murI质粒鉴定, figureFileSmall=xHaDybM7ZxiGNLhBneiV4A==, figureFileBig=maoPNR/dbGnC5ARoeWbUlA==, tableContent=null), ArticleFig(id=1241444397645091031, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Figure 6, caption=Confirmation of genetic stability ofPp ΔtssD-1ΔmurI (pBBR1-murI). M: DL2000 marker; Lane 1−6: PCR amplification of the 10th, 20th, 30th, 40th, 50th, and 60th generation ofPp ΔtssD-1ΔmurI (pBBR1-murI) using primers P9/P10; Lane 7: Negative control., figureFileSmall=5/PbXvDbWUMVj99Y3pxNfA==, figureFileBig=UmE+LZJOhjP2dMXQPB4csA==, tableContent=null), ArticleFig(id=1241444397783503068, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=图6, caption=pBBR1-murI质粒稳定性验证, figureFileSmall=5/PbXvDbWUMVj99Y3pxNfA==, figureFileBig=UmE+LZJOhjP2dMXQPB4csA==, tableContent=null), ArticleFig(id=1241444397955469536, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Figure 7, caption=The growth curves for different strains in lysogeny broth medium. The optical density (OD600) values are plotted as the average±standard deviation (n=3) at each sampling time., figureFileSmall=J0zcnKEE77YmsMellMuwCA==, figureFileBig=5Sgf9r4MsQ2zB0O6O95Leg==, tableContent=null), ArticleFig(id=1241444398064521444, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=图7, caption=不同菌株在LB液体培养基中的生长曲线, figureFileSmall=J0zcnKEE77YmsMellMuwCA==, figureFileBig=5Sgf9r4MsQ2zB0O6O95Leg==, tableContent=null), ArticleFig(id=1241444398160990439, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Figure 8, caption=Construction of the plasmid of pBBR1-murI-gfpuv. M: DL5000 marker; Lane 1−5: PCR amplification of the putative colonies of recombinant strainPp ΔtssD-1ΔmurI (pBBR1-murI-gfpuv)., figureFileSmall=D5FxpmCc1br1IoXcoo7Dww==, figureFileBig=dItVTMeyCvrqk045nJMOaQ==, tableContent=null), ArticleFig(id=1241444398261653740, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=图8, caption=质粒pBBR1-murI-gfpuv验证, figureFileSmall=D5FxpmCc1br1IoXcoo7Dww==, figureFileBig=dItVTMeyCvrqk045nJMOaQ==, tableContent=null), ArticleFig(id=1241444398362317038, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Figure 9, caption=Expression ofgfpuv in the recombinant strains (400×). A:Pp ΔtssD-1ΔmurI (pBBR1-murI-gfpuv). B:Pp ΔtssD-1ΔmurI (pBBR1-murI)., figureFileSmall=dLQN0mGMsaOuJnNyH8Gpbg==, figureFileBig=ehfOCjuLeOGNqgVYe7iQEQ==, tableContent=null), ArticleFig(id=1241444398475563254, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=图9, caption=gfpuv基因在重组菌株中的表达(400×), figureFileSmall=dLQN0mGMsaOuJnNyH8Gpbg==, figureFileBig=ehfOCjuLeOGNqgVYe7iQEQ==, tableContent=null), ArticleFig(id=1241444398601392377, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Table 1, caption=

Bacterial strains and plasmids used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Strains and plasmidsDescriptionSource
Cam: Chloramphenicol; Kan: Kanamycin.
Strains
   Pseudomonas plecoglossicida
     Pp ΔtssD-1Wilde type knockouted thetssD-1, gene site is DVB73_06795; Camr[14]
   Escherichia coli
     E.coli DH5αClone strainVazyme
     E.coli S17-1λpirHost for π requiring plasmids, conjugal donor[14]
Plasmids
   pK18mobsacBsacB-based gene replacement vector; Kanr[14]
   pBBR1MCS-2Mobilizable shuttle and expression vector. Replicates in many Gram-negative bacteria; Kanr[15]
   pDSK-gfpuvBroad host green fluorescent expression; Kanr[16]
   pK18-murIsacB-based gene replacement vector andmurI homologous arms upstream and downstream of themurI gene; KanrThis study
   pBBR1-murIpBBR1MCS-2 expressing vector, containing gene encodingmurIThis study
   pBBR1-murI-gfpuvpBBR1MCS-2 expressing vector, containing gene encoding green fluorescent protein andmurIThis study
), ArticleFig(id=1241444398718832896, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=表1, caption=

本研究所用菌株及质粒

, figureFileSmall=null, figureFileBig=null, tableContent=
Strains and plasmidsDescriptionSource
Cam: Chloramphenicol; Kan: Kanamycin.
Strains
   Pseudomonas plecoglossicida
     Pp ΔtssD-1Wilde type knockouted thetssD-1, gene site is DVB73_06795; Camr[14]
   Escherichia coli
     E.coli DH5αClone strainVazyme
     E.coli S17-1λpirHost for π requiring plasmids, conjugal donor[14]
Plasmids
   pK18mobsacBsacB-based gene replacement vector; Kanr[14]
   pBBR1MCS-2Mobilizable shuttle and expression vector. Replicates in many Gram-negative bacteria; Kanr[15]
   pDSK-gfpuvBroad host green fluorescent expression; Kanr[16]
   pK18-murIsacB-based gene replacement vector andmurI homologous arms upstream and downstream of themurI gene; KanrThis study
   pBBR1-murIpBBR1MCS-2 expressing vector, containing gene encodingmurIThis study
   pBBR1-murI-gfpuvpBBR1MCS-2 expressing vector, containing gene encoding green fluorescent protein andmurIThis study
), ArticleFig(id=1241444400216199427, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=EN, label=Table 2, caption=

Primers used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
PrimersPrimer sequences (5′→3′)Product size (bp)
Restriction sites are underlined.
P1AATGAATTCGCGTGGGCGAACTGC600
P2ACGTTTTTTTTAAGCCATCGCGCTTGCCGCATA
P3AGCGCGATGGCTTAAAAAAAACGTGAAAAACCGCTGAACTATCAAG600
P4AATGGATCCCAGGGTATAGGTTTCAACCCCATCG
P5TGCTGAAGGACGAGCGTTCG520
P6ATCATCTTGCCGACAACAGC
P7TGGACTGCTCCGACAACTTC1 472/674
P8CCGTCCCAATAGCCAGTCAG
P9ATGGCTGAGCGTTCGGCGCC798
P10AGGCAGCGTGCAGCCTTTTCAGTTGTAA
P11CAGGATGAGGATCGTTTCGCATGGCTGAGCGTTCGGCGC798
P12TTCGAACCCCAGAGTCCCGCTTACAACTGAAAAGGCTGCACGCTGCCT
P13TGCAGCCTTTTCAGTTGTAAGCGGGACTCTGGGGTTCGAAAT4 352
P14GGCGCCGAACGCTCAGCCATGCGAAACGATCCTCATCCTGT
P15CAGGTAGCTTGCAGTGGGCTTAC1 094
P16CCAACCTTTCATAGAAGGCGGC
P17AATGGATCCTGTAAAACGACGGCCAGTGAATTC995
P18AATCCGCGGTTATTTGTAGAGCTCATCCATGCCATGTG
), ArticleFig(id=1241444400371388680, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1241357430345486613, language=CN, label=表2, caption=

  本研究所用引物

, figureFileSmall=null, figureFileBig=null, tableContent=
PrimersPrimer sequences (5′→3′)Product size (bp)
Restriction sites are underlined.
P1AATGAATTCGCGTGGGCGAACTGC600
P2ACGTTTTTTTTAAGCCATCGCGCTTGCCGCATA
P3AGCGCGATGGCTTAAAAAAAACGTGAAAAACCGCTGAACTATCAAG600
P4AATGGATCCCAGGGTATAGGTTTCAACCCCATCG
P5TGCTGAAGGACGAGCGTTCG520
P6ATCATCTTGCCGACAACAGC
P7TGGACTGCTCCGACAACTTC1 472/674
P8CCGTCCCAATAGCCAGTCAG
P9ATGGCTGAGCGTTCGGCGCC798
P10AGGCAGCGTGCAGCCTTTTCAGTTGTAA
P11CAGGATGAGGATCGTTTCGCATGGCTGAGCGTTCGGCGC798
P12TTCGAACCCCAGAGTCCCGCTTACAACTGAAAAGGCTGCACGCTGCCT
P13TGCAGCCTTTTCAGTTGTAAGCGGGACTCTGGGGTTCGAAAT4 352
P14GGCGCCGAACGCTCAGCCATGCGAAACGATCCTCATCCTGT
P15CAGGTAGCTTGCAGTGGGCTTAC1 094
P16CCAACCTTTCATAGAAGGCGGC
P17AATGGATCCTGTAAAACGACGGCCAGTGAATTC995
P18AATCCGCGGTTATTTGTAGAGCTCATCCATGCCATGTG
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基于谷氨酸消旋酶基因(murI)构建杀香鱼假单胞菌减毒活疫苗株的染色体-质粒平衡致死表达系统
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张明明 1 , 叶浩达 2 , 张朝政 1 , 王鹏程 1 , 王克诠 1 , 严小军 1 , 陶震 1, *
微生物学报 | 研究报告 2024,64(3): 767-779
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微生物学报 | 研究报告 2024, 64(3): 767-779
基于谷氨酸消旋酶基因(murI)构建杀香鱼假单胞菌减毒活疫苗株的染色体-质粒平衡致死表达系统
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张明明1, 叶浩达2, 张朝政1, 王鹏程1, 王克诠1, 严小军1, 陶震1, *
作者信息
  • 1 浙江海洋大学水产学院, 浙江 舟山 316022
  • 2 宁波大学海洋学院, 浙江 宁波 315211
Construction of a chromosome-plasmid balanced lethal system based onmurI in the attenuated live vaccine strain ofPseudomonas plecoglossicida
Mingming ZHANG1, Haoda YE2, Chaozheng ZHANG1, Pengcheng WANG1, Kequan WANG1, Xiaojun YAN1, Zhen TAO1, *
Affiliations
  • 1 School of Fishery, Zhejiang Ocean University, Zhoushan 316022, Zhejiang, China
  • 2 School of Marine Sciences, Ningbo University, Ningbo 315211, Zhejiang, China
出版时间: 2024-03-04 doi: 10.13343/j.cnki.wsxb.20230504
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摘要
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【目的】构建一种基于谷氨酸消旋酶(MurI)基因的染色体-质粒平衡致死系统，用于杀香鱼假单胞菌减毒活疫苗株(Pseudomonas plecoglossicida ΔtssD-1,Pp ΔtssD-1)中表达外源抗原，为开发多联活疫苗提供新的思路和方法。【方法】利用同源重组技术，将亲本株Pp ΔtssD-1中的murI基因敲除，构建murI基因缺失突变株；将广宿主穿梭质粒pBBR1MCS-2的卡那霉素抗性基因替换为murI基因，构建平衡致死质粒(即无抗性回补质粒)；在平衡致死质粒的多克隆位点处插入绿色荧光蛋白以检测外源抗原是否稳定表达，对重组菌株进行生物学特性分析，包括生长曲线、质粒稳定性和外源抗原表达水平。【结果】murI基因缺失株在不含D-谷氨酸的LB培养基上无法生长；无抗性回补株在不含D-谷氨酸的LB培养基上恢复了生长能力，但生长速度低于亲本株；经鉴定外源抗原可在无抗性质粒中稳定表达，并可在荧光显微镜下观察到明显的绿色荧光信号；此外，平衡致死质粒在重组菌株中具有良好的遗传稳定性。【结论】本研究以murI为靶点构建了新型的染色体-质粒平衡致死系统，可在无抗性筛选条件下在Pp ΔtssD-1中表达外源抗原，为开发多联活疫苗提供了新的策略和方法。

杀香鱼假单胞菌ΔtssD-1株  /  营养缺陷株  /  平衡致死系统  /  D-谷氨酸

[Objective] To construct a chromosome-plasmid balanced lethal system based on the glutamate racemase (MurI) gene for the expression of exogenous antigens in the attenuated vaccine strain ofPseudomonas plecoglossicida (Pp ΔtssD-1), so as to provide new ideas and methods for the development of multi-component live vaccines. [Methods] We constructed amurI-deleted strain fromPp ΔtssD-1 by homologous recombination. First, we replaced the kanamycin resistance gene of the pBBR1MCS-2 plasmid withmurI to construct a balanced lethal plasmid. Subsequently, we inserted the green fluorescent protein gene into the multicloning site of the plasmid to examine the expression stability of the exogenous antigen. Finally, we characterized the recombinant strain in terms of the growth curve, plasmid stability, and expression of the exogenous antigen. [Results] ThemurI-deleted strain was unable to grow in the lysogeny broth medium without D-glutamate. The non-resistant complemented strain regained growth capability in the lysogeny broth medium without D-glutamate. However, its growth was slower than that of the starting strain. Exogenously introduced antigens were identified as stable in the absence of antibiotic selection, and distinct green fluorescence signals were observed under a fluorescence microscope. Additionally, the balanced lethal plasmid exhibited high genetic stability within the recombinant strain. [Conclusion] A novel chromosome-plasmid balanced lethal system targetingmurI was developed in this study. It enabled the expression of exogenous antigens inPp ΔtssD-1 without the need for antibiotic selection. This system provides a new method for the development of multi-component live vaccines, with no need of antibiotic resistance markers and high plasmid stability.

Pseudomonas plecoglossicida ΔtssD-1  /  auxotroph  /  balanced lethal system  /  D-glutamate
张明明, 叶浩达, 张朝政, 王鹏程, 王克诠, 严小军, 陶震. 基于谷氨酸消旋酶基因(murI)构建杀香鱼假单胞菌减毒活疫苗株的染色体-质粒平衡致死表达系统. 微生物学报, 2024 , 64 (3) : 767 -779 . DOI: 10.13343/j.cnki.wsxb.20230504
Mingming ZHANG, Haoda YE, Chaozheng ZHANG, Pengcheng WANG, Kequan WANG, Xiaojun YAN, Zhen TAO. Construction of a chromosome-plasmid balanced lethal system based onmurI in the attenuated live vaccine strain ofPseudomonas plecoglossicida[J]. Acta Microbiologica Sinica, 2024 , 64 (3) : 767 -779 . DOI: 10.13343/j.cnki.wsxb.20230504
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大黄鱼是我国重要的海水经济养殖鱼类之一，其养殖过程中面临多种疾病威胁，尤其是大黄鱼内脏白点病(visceral granulomas disease)，该病由杀香鱼假单胞菌(Pseudomonas plecoglossicida)引起，具有流行范围广和发病死亡率高等特点，给大黄鱼养殖业造成了严重的经济损失[1-2]。为了能安全有效地防治大黄鱼内脏白点病，研究人员已经通过基因工程方法，敲除了杀香鱼假单胞菌的一个毒力基因tssD-1 (type Ⅵ secretion system tube protein Hcp，基因位点DVB73_06795)，构建了一株杀香鱼假单胞菌减毒活疫苗候选株(Pp ΔtssD-1)，并证实Pp ΔtssD-1在大黄鱼中的高免疫效价和安全性[3]。然而，Pp ΔtssD-1只能预防由杀香鱼假单胞菌引起的大黄鱼内脏白点病，不能预防其他常见的细菌性疾病，如弧菌病、诺卡氏菌病等[4-5]。因此，研发广谱多联疫苗成为新方向。
将多种外源抗原导入到安全的活细菌载体中进行表达是一种构建多联疫苗的技术方法。然而，传统的含抗性基因的质粒系统存在一些缺陷，如安全性风险和质粒丢失导致抗原无法表达等[6]。为解决这一问题，研究人员提出了染色体-质粒平衡致死系统(chromosome-plasmid balanced-lethal system)这一新型抗原表达递送策略[7-8]。该系统通过敲除染色体上对生长必需的基因并由质粒补偿实现，非抗性筛选压力下即可稳定存在和表达外源抗原。目前，平衡致死系统多基于营养合成相关基因，如asd、thyA和glnA等[9-11]。谷氨酸消旋酶(glutamate racemase, MurI) (EC5.1.1.3)催化L-谷氨酸向D-谷氨酸的转化，是肽聚糖合成途径中的关键一步[12]。因此，murI可能是构建新型平衡致死系统的理想靶点。为验证这一假设，本研究以Pp ΔtssD-1为亲本株，基于细胞壁合成关键基因murI，构建一种新型平衡致死系统。该系统能够在无抗生素选择压力下稳定存在并表达外源抗原，确保外源抗原的稳定表达和高效递送。由于murI基因在细菌细胞壁合成中至关重要且高度保守[13]，所以该方法可能具有较广泛的适用性和可移植性。
1 材料与方法
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1.1 菌株、质粒及培养条件
本研究所用菌株和质粒见表1。大肠杆菌菌株在37 ℃条件下培养，杀香鱼假单胞菌菌株在30 ℃条件下培养。所有菌株均用LB固体或液体培养基进行培养，必要时添加合适浓度抗生素或D-谷氨酸。液体培养物在摇床中振荡培养时，使用的转速为150−200 r/min。
1.2 主要试剂
实验中所需抗生素如氨苄青霉素(ampicillin, Amp)、卡那霉素(kanamycin, Kan)及氯霉素(chloramphenicol, Chl)等购自生工生物工程(上海)股份有限公司；细菌培养基购自青岛海博生物有限公司；2×ESTaq Master Mix、DNA提取试剂盒购自康为世纪生物科技有限公司；限制性内切酶、T4连接酶购自纽英伦生物技术(北京)有限公司；高保真PCR预混液2×Phanta Max Master Mix、FastPure Plasmid Mini Kit及ClonExpress® Ultra One Step Cloning Kit (吉普森克隆)试剂盒购自诺维赞生物科技有限公司；PCR产物回收试剂盒购自Omega公司；D-谷氨酸购自北京索莱宝科技有限公司。
1.3 引物设计及合成
参照GenBank中公布的杀香鱼假单胞菌XSDHY-P基因组(登录号：NZ_CP031146)、pK18mobsacB质粒(登录号：FJ437239)序列，以及Addgene网站(www.addgene.org)公布的pBBR1MCS-2质粒及pDSK-gfpuv质粒序列设计引物(除P5/P6外)。参照Izumi等的方法合成验证杀香鱼假单胞菌特异性引物(P5/P6)[17]。引物序列见表2。引物均由生工生物工程(上海)股份有限公司合成。
1.4 杀香鱼假单胞菌murI基因的鉴定
为鉴定杀香鱼假单胞菌XSDHY-P基因组中的murI基因，以Escherichia coli K12菌株MurI蛋白的氨基酸序列(GenBank登录号：AAC76949.2)作为查询序列，利用假单胞菌数据库(www.pseudomonas.com)提供的在线工具BLASTp，在杀香鱼假单胞菌XSDHY-P基因组编码的蛋白数据库中寻找同源蛋白，BLASTp比对使用系统默认参数。使用NCBI提供的在线工具Conserved Domain Search (https://www.ncbi.nlm.nih.gov/cdd/)对目标蛋白的保守结构域进行鉴定。蛋白的氨基酸序列使用Clustal Omega进行了多序列比对[18]，使用ESPript工具对序列比对结果进行可视化[19]。
1.5murI基因缺失突变株(ΔmurI)的构建及鉴定
本研究基于质粒pK18mobsacB同源重组双交换的方法，实现Pp ΔtssD-1基因组中murI基因的无痕敲除，实验过程参考Tao等的方法[14]。在Pp ΔtssD-1基因组中murI (基因位点DVB73_RS19840)基因处，分别用引物对P1/P2和P3/P4扩增murI基因上下游600 bp的同源臂片段，将2个片段连接到已双酶切的pK18mobsacB质粒(酶切位点：EcoR I、BamH I)中，得到murI基因敲除质粒pK18-murI。再将连接产物转化至Escherichia coli DH5α感受态细胞，筛选阳性克隆菌落，进行PCR检测，并通过测序进行验证。选择验证后的阳性克隆菌落，扩大培养后提取质粒，将其转化至E.coli S17-1λpir中。将含有重组质粒的E.coli S17-1λpir与受体菌株Pp ΔtssD-1按1:10比例结合培养，在含卡那霉素(50 μg/mL)和氯霉素(35 μg/mL)的LB平板上筛选阳性克隆菌落。用引物对P5/P6和P7/P8进行PCR验证，选择P5/P6扩增阳性且P7/P8扩增得到双条带(674 bp和1 472 bp)的重组菌株，在含有10%蔗糖、10 mmol/L D-谷氨酸和氯霉素(35 μg/mL)的LB平板上进行筛选培养，用引物对P5/P6、P7/P8及murI检测引物P9/P10进行PCR检测(以亲本菌株Pp ΔtssD-1作为对照)，筛选Pp ΔtssD-1的murI基因缺失株，即Pp ΔtssD-1ΔmurI。
1.6Pp ΔtssD-1ΔmurI的表型及遗传稳定性检测
将Pp ΔtssD-1ΔmurI的菌液分别接种于普通LB平板和含10 mmol/L D-谷氨酸的LB平板上，培养24 h观察生长情况，确认D-谷氨酸营养缺陷表型。此外，将Pp ΔtssD-1ΔmurI转接至含10 mmol/L D-谷氨酸的LB液体培养基中，连续传代培养40代(每12 h重新接种1次，计为一代)，每10代取样1次，提取基因组DNA，利用引物对P7/P8进行PCR鉴定，以确定缺失基因在传代过程中的稳定性。
1.7 平衡致死质粒的构建及鉴定
以广宿主穿梭载体pBBR1MCS-2质粒为载体，使用Gibson assembly克隆方法，将质粒上的卡那霉素抗性基因nptII替换为Pp ΔtssD-1的murI基因，构建平衡致死质粒(无抗性回补质粒) (图1)。以Pp ΔtssD-1的DNA为模板，用引物对P11/P12进行PCR扩增，获得murI基因片段；同时以pBBR1MCS-2质粒为模板，使用引物P13/P14进行PCR扩增，获得不含nptII基因的线性化质粒片段。将2个片段连接后转化至Pp ΔtssD-1ΔmurI，30 ℃培养48 h，挑取单克隆菌落进行培养，用引物P15/P16进行PCR鉴定，以Pp ΔtssD-1ΔmurI为阴性对照，同时进行测序验证，将重组质粒命名为pBBR1-murI，携带有该质粒的重组菌株命名为Pp ΔtssD-1ΔmurI (pBBR1-murI)。
1.8 重组菌株的生物学特性分析
1.8.1 pBBR1-murI质粒在重组菌株中的稳定性检测
将重组菌株Pp ΔtssD-1ΔmurI (pBBR1-murI)接种至LB液体培养基，并进行连续传代培养，每次按1:50的比例接种至新鲜的LB培养基中，连续转接60次。每隔10次取部分培养物，用引物P9/P10进行PCR扩增检测，验证pBBR1-murI质粒在重组菌株中的遗传稳定性。
1.8.2 重组菌株的生长特性鉴定
分别将Pp ΔtssD-1ΔmurI (pBBR1-murI)、Pp ΔtssD-1ΔmurI和Pp ΔtssD-1菌株接种于LB液体培养基(按需添加D-谷氨酸)中培养过夜，按1:100稀释在新鲜培养基中，振荡培养至OD600在0.5−0.8，4 000×g离心10 min，弃上清后加入不含D-谷氨酸的LB液体培养基，并分别将浓度调整至OD600值为0.1。然后，将上述重悬菌液按200 μL/孔转至96孔板中，30 ℃、150 r/min振荡培养，每隔2 h在酶标仪上测定OD600值，连续测定12 h，绘制生长曲线。
1.9 外源基因在重组菌株中的表达情况鉴定
使用引物P17/P18扩增pDSK-gfpuv中的编码绿色荧光蛋白的gfpuv基因及其启动区域(995 bp)，将该片段插入pBBR1-murI质粒的Sac I和BamH I酶切位点之间，构建pBBR1-murI-gfpuv质粒。然后将该质粒转化至Pp ΔtssD-1ΔmurI菌株，并在无D-谷氨酸的LB平板上进行筛选。挑取单菌落培养，使用引物P17/P18进行PCR鉴定，并通过测序进行验证，经过验证的重组菌株命名为Pp ΔtssD-1ΔmurI (pBBR1-murI-gfpuv)。然后，在LB液体培养基中培养至对数生长期，取样进行荧光显微镜观察，检测GFPuv蛋白的表达情况，Pp ΔtssD-1ΔmurI (pBBR1-murI)作为阴性对照。
1.10 数据处理
生长曲线数据采用Graphpad Prism 8软件进行绘制和分析。
2 结果与分析
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2.1 杀香鱼假单胞菌XSDHY-P基因组中murI基因的鉴定
使用E.coli K12菌株MurI蛋白的氨基酸序列作为查询序列，在杀香鱼假单胞菌XSDHY-P的蛋白数据库中进行BLASTp比对。结果显示，只有一个蛋白与查询序列具有显著的同源性，其编码基因位于XSDHY-P基因组(GenBank登录号：NZ_CP031146.1)的DVB73_ RS19840位点，长度为798 bp。该基因编码的蛋白与E.coli K12菌株的MurI蛋白之间的氨基酸序列一致性为37.8%，并且在一些关键位点上具有保守性(图2)。此外，利用Conserved Domain Search工具对该蛋白进行了保守结构域的分析，结果表明该蛋白是MurI超家族(COG0796)成员，参与细胞壁合成的关键步骤。综上所述，将DVB73_RS19840位点基因鉴定为murI基因。
2.2Pp ΔtssD-1ΔmurI的构建及鉴定
利用基因敲除质粒pK18mobsacB的两步同源重组方法，在Pp ΔtssD-1菌株中敲除murI基因。第1步同源重组时，重组染色体带有卡那霉素抗性基因，作为筛选标记；第2步同源重组时，利用sacB蔗糖敏感性，作为负筛选标记，在含有10%蔗糖、10 mmol/L D-谷氨酸和35 μg/mL氯霉素的LB平板上培养，选择能够生长的菌落作为可能成功构建的murI缺失株。通过以下3对引物对murI基因缺失株进行PCR验证：P7/P8用于检测目标染色体上下游同源臂内侧融合片段(图3A)，P9/P10用于检测murI基因是否完全缺失(图3B)，P5/P6用于检测杀香鱼假单胞菌特异性序列(图3C)。只有当P7/P8扩增出单一条带(674 bp)，P9/P10扩增阴性，以及P5/P6扩增阳性(520 bp)时，才能确认为murI缺失株。图3显示了PCR产物电泳胶图的结果，其中泳道3、7、9、13符合上述标准，为murI基因敲除株Pp ΔtssD-1ΔmurI。
2.3Pp ΔtssD-1ΔmurI的表型及遗传稳定性检测
体外培养实验结果显示，Pp ΔtssD-1ΔmurI丧失了在普通LB平板上生长的能力(图4A)，但在补充10 mmol/L D-谷氨酸后恢复了生长(图4B)，表明该菌株为D-谷氨酸营养缺陷型。此外，将Pp ΔtssD-1ΔmurI在含D-谷氨酸的LB液体培养基中连续培养40代，取10、20、30、40代的菌种，用P9/P10引物检测murI基因。结果显示，所有代次的菌种检测结果均为阴性(均无扩增条带，结果未显示)，表明该营养缺陷型可以稳定遗传。
2.4 平衡致死质粒pBBR1-murI的构建及鉴定
本研究通过Gibson assembly克隆的方法构建了平衡致死质粒pBBR1-murI，并将其转化至Pp ΔtssD-1ΔmurI，在无D-谷氨酸添加的LB平板上进行筛选。挑取阳性克隆，进行PCR验证。结果如图5所示，使用引物对P15/P16进行PCR扩增检测，携带有pBBR1-murI质粒的菌株在1 094 bp处有明显条带；阴性对照组(Pp ΔtssD-1ΔmurI)则无条带。结合PCR验证和测序结果，表明平衡致死质粒pBBR1-murI构建成功。
2.5 重组菌株的生物学特性分析
2.5.1 pBBR1-murI质粒在Pp ΔtssD-1ΔmurI中的稳定性检测
随机挑取一株重组菌株Pp ΔtssD-1ΔmurI (pBBR1-murI)，在不含D-谷氨酸的LB液体培养基中连续传代60次，取第10、20、30、40、50、60次的菌种，用P9/P10引物进行PCR扩增检测，均可获得798 bp的目标条带(图6)，测序结果比对正确，表明pBBR1-murI质粒可以在无抗生素选择压力下稳定存在于Pp ΔtssD-1 ΔmurI中。
2.5.2 重组菌株的生长特性鉴定
在LB培养基中无外源D-谷氨酸补充条件下，如果重组菌株可以在此培养基中生长，则说明重组菌株在回补了质粒pBBR1-murI后可以自身合成D-谷氨酸。如图7所示，亲本株Pp ΔtssD-1生长良好，说明该菌株具有合成D-谷氨酸的能力。与亲本株相比Pp ΔtssD-1ΔmurI几乎未生长，Pp ΔtssD-1ΔmurI (pBBR1-murI)恢复了生长，但生长速度低于亲本株。
2.6 外源抗原在重组菌株中的表达情况鉴定
将表达绿色荧光蛋白的gfpuv基因克隆至pBBR1-murI中构建质粒pBBR1-murI-gfpuv，并电转至Pp ΔtssD-1ΔmurI菌株，用引物P17/P18进行PCR扩增，在995 bp处有明显条带(图8)。对所得产物进行测序分析，综合PCR验证和测序结果，证明Pp ΔtssD-1ΔmurI (pBBR1-murI-gfpuv)构建成功。将Pp ΔtssD-1ΔmurI (pBBR1-murI-gfpuv)与对照菌株Pp ΔtssD-1ΔmurI (pBBR1-murI)的菌液取样进行荧光显微观察，在Pp ΔtssD-1ΔmurI (pBBR1-murI-gfpuv)中观察到明显的绿色荧光(图9A)，而对照菌株Pp ΔtssD-1ΔmurI (pBBR1-murI)未观察到荧光信号(图9B)，表明以载体pBBR1-murI为基础构建含gfpuv基因的重组质粒可以在Pp ΔtssD-1ΔmurI中稳定表达外源基因。
3 讨论
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本研究采用以murI为靶点构建染色体-质粒平衡致死系统的方法，实现了在杀香鱼假单胞菌减毒活疫苗株中稳定地表达外源蛋白的目的。该系统通过敲除Pp ΔtssD-1菌株中的murI基因，使其成为D-谷氨酸营养缺陷株，并利用广宿主穿梭载体pBBR1MCS-2同时表达murI基因和外源抗原基因，从而实现了在无抗生素选择压力下的稳定表达。该系统具备以下几个特征：首先，与传统方法相比，该系统无须使用抗生素抗性基因作为筛选标记，避免了潜在的生物安全问题和耐药性传播的风险。质粒在宿主中的稳定维持和表达不再依赖外源抗生素，而是通过细菌生存所必需的基因来实现。其次，D-谷氨酸是一种特异于原核生物的氨基酸，在哺乳动物和鱼类体内不存在或仅以微量存在[21-22]。通过破坏Pp ΔtssD-1菌株的D-谷氨酸合成途径，使其无法在鱼体内获得足够的D-谷氨酸，从而导致细菌失去生存能力。这种基于营养缺陷的策略确保了质粒的稳定存在和高效表达。
染色体-质粒平衡致死系统是一种利用非抗生素标记的载体表达系统，其宿主菌是染色体上某些关键基因的缺陷型突变体，这些基因对细菌的生存和代谢是必需的[7]。当这些缺陷型突变体导入带有相应互补基因的质粒后，就形成了染色体-质粒平衡致死系统，质粒对宿主菌起到了功能性互补的作用。如果质粒丢失，细菌就会因为缺乏必需物质而死亡。在先前的研究中，已经选择了其他营养代谢途径中的必需基因，如asd、thyA和glnA等作为靶点[23-27]，在不同的细菌中构建了平衡致死系统，并实现了稳定的抗原表达。
本研究选择杀香鱼假单胞菌基因组中单一拷贝的murI基因作为靶点，该基因广泛存在于细菌中。经过敲除murI基因，成功构建了D-谷氨酸营养缺陷株Pp ΔtssD-1ΔmurI。实验结果表明，Pp ΔtssD-1ΔmurI在普通LB培养基中失去生长能力，但在补充外源D-谷氨酸后恢复生长，验证了其为D-谷氨酸营养缺陷株。通过转化含有murI基因的pBBR1-murI质粒，Pp ΔtssD-1ΔmurI (pBBR1-murI)在LB平板上恢复了生长能力，证明了互补质粒上murI基因在体外成功表达。同时，传代实验结果显示，该系统在体外的遗传稳定性良好。
然而，本研究观察到Pp ΔtssD-1ΔmurI (pBBR1-murI)的生长速度似乎低于亲本株Pp ΔtssD-1。这可能是由于宿主细胞内存在多个拷贝的质粒，可能导致MurI蛋白的过量表达，从而改变了细胞的活性，进而影响了细胞的分裂[28]。此外，质粒的复制和维持也可能会对细胞的代谢造成负担，消耗了细胞的资源和能量，从而降低了细胞的生长速率[29]。为了提高重组菌株的生长效率，未来的研究需要进一步优化该表达系统。值得注意的是，本研究通过转入含有gfpuv基因的pBBR1-murI-gfpuv重组质粒，在Pp ΔtssD-1ΔmurI菌株中成功表达了GFPuv蛋白。这为将质粒中的gfpuv基因替换为特定的外源抗原基因，实现对外源抗原的高效表达提供了可能性。
4 结论
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本研究构建了一种基于murI基因的染色体-质粒平衡致死表达系统，用于在杀香鱼假单胞菌减毒活疫苗株中高效表达外源抗原，为多联活疫苗的开发提供了一种创新的策略。
基金
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  • 国家自然科学基金(42376108)
文献
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2024年第64卷第3期
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doi: 10.13343/j.cnki.wsxb.20230504
  • 接收时间：2023-08-01
  • 首发时间：2026-03-19
  • 出版时间：2024-03-04
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  • 收稿日期：2023-08-01
  • 录用日期：2023-10-17
基金
National Natural Science Foundation of China(42376108)
国家自然科学基金(42376108)
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    1 浙江海洋大学水产学院, 浙江 舟山 316022
    2 宁波大学海洋学院, 浙江 宁波 315211

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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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