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Article(id=1226598461756322661, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226598456190484999, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20240719, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1731427200000, receivedDateStr=2024-11-13, revisedDate=null, revisedDateStr=null, acceptedDate=1737648000000, acceptedDateStr=2025-01-24, onlineDate=1770373462379, onlineDateStr=2026-02-06, pubDate=1743696000000, pubDateStr=2025-04-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1770373462379, onlineIssueDateStr=2026-02-06, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1770373462379, creator=13701087609, updateTime=1770373462379, updator=13701087609, issue=Issue{id=1226598456190484999, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='4', pageStart='1', pageEnd='1823', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1770373461053, creator=13701087609, updateTime=1770542963395, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1227309400608653689, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226598456190484999, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1227309400608653690, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226598456190484999, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1774, endPage=1787, ext={EN=ArticleExt(id=1226598462536463254, articleId=1226598461756322661, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Functions of five chitin lyases from Pseudoalteromonas arabiensis N1230-9, columnId=1226598457239061007, journalTitle=Acta Microbiologica Sinica, columnName=Development and application of microbial resources, runingTitle=null, highlight=null, articleAbstract=

[Objective] Chitin lyases are the key enzymes for bacteria to degrade chitin. Pseudoalteromonas arabiensis N1230-9 possesses five chitin lyase-encoding genes (woc28159, woc28160, woc28161, woc27404, and woc27232). Functional identification of these five genes is crucial for determining the ability of strain to degrade chitin. [Methods] The distribution patterns of five chitin lyases from strain N1230-9 in 44 model strains of Pseudomonas were analyzed by bioinformatics methods. The transcription levels of five chitin lyase-encoding genes of strain N1230-9 cultured in the medium with chitin as the sole carbon source were analyzed by RT-qPCR. The mutants with deletion of chitin lyase-encoding genes were constructed by homologous recombination, and their abilities to degrade chitin were evaluated. [Results] Thirty-three Pseudoalteromonas strains possessed chitin lyase homologous proteins of strain N1230-9. Chitin significantly upregulated the transcription levels of woc28159, woc28160, woc28161, and woc27404, and the deletion of each of the four genes weakened the chitin-degradation ability. The transcription level of woc27232 was not affected by chitin, and the deletion of this gene did not affect the chitin-degradation ability of the strain. [Conclusion] WOC28159 and WOC28161 are necessary for the degradation of chitin by strain N1230-9. WOC28160 and WOC27404 endow strain N1230-9 with efficient chitin-degrading ability, while WOC27232 does not participate in the chitin degradation process.

, correspAuthors=Jigang CHEN, authorNote=null, correspAuthorsNote=
*E-mail:
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【目的】几丁质裂解酶是细菌有效降解几丁质的关键酶。阿拉伯海假交替单胞菌(Pseudoalteromonas arabiensis) N1230-9拥有5个几丁质裂解酶编码基因(woc28159、woc28160、woc28161、woc27404和woc27232)，对这5个基因的功能进行鉴定是解析该菌株具有高效几丁质降解能力的关键。【方法】通过生物信息学方法分析菌株N1230-9中的5个几丁质裂解酶在假交替单胞菌44个模式菌株中的分布规律；采用实时荧光定量PCR技术(RT-qPCR)检测菌株N1230-9中5个几丁质裂解酶编码基因在以几丁质为唯一碳源培养基中的转录水平；利用同源重组策略构建菌株N1230-9中5个几丁质裂解酶编码基因的单基因缺失突变体，并分析突变体的几丁质降解能力。【结果】有33株假交替单胞菌模式菌株拥有与菌株N1230-9几丁质裂解酶同源的蛋白；几丁质可以强烈诱导woc28159、woc28160、woc28161和woc27404的转录水平上调，且这4个基因的单基因缺失均在不同程度上削弱了菌株的几丁质降解能力。然而woc27232的转录水平不受几丁质的诱导，且该基因的缺失对菌株的几丁质降解能力无影响。【结论】几丁质酶WOC28159和WOC28161是菌株N1230-9降解几丁质所必需的酶，WOC28160和WOC27404增强了菌株的几丁质降解效率，而WOC27232并不参与菌株N1230-9的几丁质降解过程。

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作者贡献声明

兰肖敏：实验设计与实施，分析数据、撰写稿件；周敏婕：协助实验操作和数据处理；王景：协助实验操作；朱四东：实验技术支持；杨季芳：论文框架指导；陈吉刚：组织实施、论文修改和润色。

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Extremophiles, 2008, 12(4): 541-552., articleTitle=Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium Moritella marina and biochemical characterization of the recombinant enzyme, refAbstract=null), Reference(id=1227303269094044599, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, doi=null, pmid=null, pmcid=null, year=2015, volume=93, issue=null, pageStart=84, pageEnd=93, url=null, language=null, rfNumber=[25], rfOrder=26, authorNames=GARCÍA-FRAGA B, da SILVA AF, LÓPEZ-SEIJAS J, SIEIRO C, journalName=Biochemical Engineering Journal, refType=null, unstructuredReference=GARCÍA-FRAGA B, da SILVA AF, LÓPEZ-SEIJAS J, SIEIRO C. A novel family 19 chitinase from the marine-derived Pseudoalteromonas tunicata CCUG 44952T: heterologous expression, characterization and antifungal activity[J]. Biochemical Engineering Journal, 2015, 93: 84-93., articleTitle=A novel family 19 chitinase from the marine-derived Pseudoalteromonas tunicata CCUG 44952T: heterologous expression, characterization and antifungal activity, refAbstract=null)], funds=[Fund(id=1227303263834387174, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, awardId=GASI-04-HYDZ-02, language=EN, fundingSource=National Program on Global Change and Air-sea Interaction (Phase Ⅱ)(GASI-04-HYDZ-02), fundOrder=null, country=null), Fund(id=1227303264023130860, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, awardId=GASI-04-HYDZ-02, language=CN, fundingSource=全球变化与海气相互作用专项(二期)(GASI-04-HYDZ-02), fundOrder=null, country=null), Fund(id=1227303264119599855, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, awardId=2023J301, language=EN, fundingSource=Ningbo Natural Science Foundation(2023J301), fundOrder=null, country=null), Fund(id=1227303264203485939, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, awardId=2023J301, language=CN, fundingSource=宁波市自然科学基金(2023J301), fundOrder=null, country=null), Fund(id=1227303264312537847, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, awardId=CX2024017, language=EN, fundingSource=The First-class Discipline of Biological Engineering of Zhejiang Province(CX2024017), fundOrder=null, country=null), Fund(id=1227303264425784063, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, awardId=CX2024017, language=CN, fundingSource=浙江省生物工程一流学科创新基金(CX2024017), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1227303255588385090, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, xref=null, ext=[AuthorCompanyExt(id=1227303255605162311, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, companyId=1227303255588385090, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo, Zhejiang, China), AuthorCompanyExt(id=1227303255613550919, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, companyId=1227303255588385090, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=浙江万里学院 生物与环境学院，浙江 宁波)])], figs=[ArticleFig(id=1227303260311171696, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=EN, label=Figure 1, caption=Distribution of orthologous proteins of five chitin lyases from Pseudoalteromonas arabiensis N1230-9 in Pseudoalteromonas. Phylogenomic tree based on genome sequences in the TYGS tree inferred with FastMe 2.1.6.1[22] from genome BLAST distance phylogeny approach (GBDP); Distances calculated from genome sequence. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values>50.0% from 100 replications. Different symbols indicate chic-lpmo-chiA cluster (CLC, filled circle), WOC27404 and its homologs (filled squares), and WOC27232 and its homologs (filled triangles). Blue branches and black branches indicate pigmented Pseudoalteromonas species and nonpigmented Pseudoalteromonas species, respectively., figureFileSmall=5Sm9oSKv1cp+jS6PYqUAWQ==, figureFileBig=Ot5stWVS8JQtOlps/M69vg==, tableContent=null), ArticleFig(id=1227303260416029300, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=CN, label=图1, caption=阿拉伯海假交替单胞菌N1230-9的5个几丁质裂解酶的直系同源蛋白在假交替单胞菌中的分布, figureFileSmall=5Sm9oSKv1cp+jS6PYqUAWQ==, figureFileBig=Ot5stWVS8JQtOlps/M69vg==, tableContent=null), ArticleFig(id=1227303260546052734, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=EN, label=Figure 2, caption=Phylogenetic analysis of orthologous proteins of four chitinases from Pseudoalteromonas. The tree was constructed by the neighbor-joining method with P-distance model. Bootstrap analysis of 1 000 replicates is conducted and values above 50% are shown., figureFileSmall=vUmPGZQqSaL36GqjA53Tzw==, figureFileBig=klrRK4fURF5RfVN5oSYvPg==, tableContent=null), ArticleFig(id=1227303260655104643, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=CN, label=图2, caption=阿拉伯海假交替单胞菌N1230-9中4个几丁质酶同源蛋白的系统发育分析, figureFileSmall=vUmPGZQqSaL36GqjA53Tzw==, figureFileBig=klrRK4fURF5RfVN5oSYvPg==, tableContent=null), ArticleFig(id=1227303262051807881, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=EN, label=Figure 3, caption=Phylogenetic analysis of WOC27404 and its homologs hydrolases. The tree was constructed by the neighbor-joining method with P-distance model. Bootstrap analysis of 1 000 replicates is conducted and values above 50% are shown., figureFileSmall=vV6kUz+MaYJ78POctZ/Eow==, figureFileBig=hNY2tmXbC2V0Mr2wDwJeeg==, tableContent=null), ArticleFig(id=1227303262198608529, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=CN, label=图3, caption=WOC27404及其同源蛋白的系统发育分析, figureFileSmall=vV6kUz+MaYJ78POctZ/Eow==, figureFileBig=hNY2tmXbC2V0Mr2wDwJeeg==, tableContent=null), ArticleFig(id=1227303262307660445, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=EN, label=Figure 4, caption=Transcriptional levels of five chitin lyase-encoding genes in strain N1230-9 at early stage (A) and later stage (B) of exponential phase growth in different substrate. Glc medium: Inorganic salt solution supplement with 2.5 g/L glucose; Chi medium: Inorganic salt solution supplement with 30 g/L colloidal chitin. ****: P<0.000 1; ns: No significant (P>0.05)., figureFileSmall=NygN/uGTtP+PpVQlOOwxDA==, figureFileBig=VG1skcTAkd3YezDcu3tn3A==, tableContent=null), ArticleFig(id=1227303262458655398, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=CN, label=图4, caption=五个几丁质裂解酶基因的转录水平分析, figureFileSmall=NygN/uGTtP+PpVQlOOwxDA==, figureFileBig=VG1skcTAkd3YezDcu3tn3A==, tableContent=null), ArticleFig(id=1227303262609650348, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=EN, label=Figure 5, caption=Colony PCR identification of the mutants. A-E indicated PCR detection of Δwoc28159, Δwoc28160, Δwoc28161, Δwoc27404 and Δwoc27232 double crossover mutants using three primer pairs, respectively. Lane M: DNA marker; Lanes 1-3: Three independent colonies after the second homologous recombination; WT: Wild-type strain N1230-9; NC: Negative control., figureFileSmall=EgiIYU+BXs/wUQYAV256yA==, figureFileBig=3oIfFMwiVu0BY6L5Cejrjg==, tableContent=null), ArticleFig(id=1227303262790005424, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=CN, label=图5, caption=缺失突变株的菌落PCR验证, figureFileSmall=EgiIYU+BXs/wUQYAV256yA==, figureFileBig=3oIfFMwiVu0BY6L5Cejrjg==, tableContent=null), ArticleFig(id=1227303262924223161, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=EN, label=Figure 6, caption=Growth phenotype of mutants in media with colloidal chitin (A, B) or granular chitin (C, D) as the sole carbon source. WT: Wild-type strain N1230-9., figureFileSmall=9EjhClNVELhE3hLR/y5gDQ==, figureFileBig=fqlhOBFS21y16vLhLA0Wyw==, tableContent=null), ArticleFig(id=1227303263058440894, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=CN, label=图6, caption=突变体在以胶体几丁质(A、B)或颗粒几丁质(C、D)为唯一碳源的培养基中的生长表型, figureFileSmall=9EjhClNVELhE3hLR/y5gDQ==, figureFileBig=fqlhOBFS21y16vLhLA0Wyw==, tableContent=null), ArticleFig(id=1227303263226213069, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=EN, label=Table 1, caption=

Plasmids and bacterial strains used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Strains and plasmidsGenotype/Relevant characteristicsSource
Escherichia coli strains
DH5αF-Φ80lacZΔM15Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK-, mK+ ) phoA supE44 λ- thi-1 gyrA96 relA1Invitrogen
WM3064Donor strain for conjugation, diaminopimelate (DAP) auxotrophic[17]
Pseudoalteromonasarabiensis strains
N1230-9Wild-type strain isolated from Pacific seawater[16]
Δwoc28159N1230-9 with the chromosomal woc28159 gene deletedThis study
Δwoc28160N1230-9 with the chromosomal woc28160 gene deletedThis study
Δwoc28161N1230-9 with the chromosomal woc28161 gene deletedThis study
Δwoc27232N1230-9 with the chromosomal woc27232 gene deletedThis study
Δwoc27404N1230-9 with the chromosomal woc27404 gene deletedThis study
Plasmids
pK18mobsacB-EryKanR, EryR, suicide vector for gene knockout[17]

pK18mobSacB-Ery-

woc28159

pK18mobsacb-Ery with 1 478 bp upstream fragment and 719 bp downstream fragment of woc28159This study

pK18mobSacB-Ery-

woc28160

pK18mobsacb-Ery with 622 bp upstream fragment and 836 bp downstream fragment of woc28160This study

pK18mobSacB-Ery-

woc28161

pK18mobsacb-Ery with 760 bp upstream fragment and 1 300 bp downstream fragment of woc28161This study

pK18mobSacB-Ery-

woc27232

pK18mobsacb-Ery with 558 bp upstream fragment and 412 bp downstream fragment of woc27232This study

pK18mobSacB-Ery-

woc27404

pK18mobsacb-Ery with 441 bp upstream fragment and 417 bp downstream fragment of woc27404This study
), ArticleFig(id=1227303263393985238, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=CN, label=表1, caption=

本研究使用的菌株和质粒

, figureFileSmall=null, figureFileBig=null, tableContent=
Strains and plasmidsGenotype/Relevant characteristicsSource
Escherichia coli strains
DH5αF-Φ80lacZΔM15Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK-, mK+ ) phoA supE44 λ- thi-1 gyrA96 relA1Invitrogen
WM3064Donor strain for conjugation, diaminopimelate (DAP) auxotrophic[17]
Pseudoalteromonasarabiensis strains
N1230-9Wild-type strain isolated from Pacific seawater[16]
Δwoc28159N1230-9 with the chromosomal woc28159 gene deletedThis study
Δwoc28160N1230-9 with the chromosomal woc28160 gene deletedThis study
Δwoc28161N1230-9 with the chromosomal woc28161 gene deletedThis study
Δwoc27232N1230-9 with the chromosomal woc27232 gene deletedThis study
Δwoc27404N1230-9 with the chromosomal woc27404 gene deletedThis study
Plasmids
pK18mobsacB-EryKanR, EryR, suicide vector for gene knockout[17]

pK18mobSacB-Ery-

woc28159

pK18mobsacb-Ery with 1 478 bp upstream fragment and 719 bp downstream fragment of woc28159This study

pK18mobSacB-Ery-

woc28160

pK18mobsacb-Ery with 622 bp upstream fragment and 836 bp downstream fragment of woc28160This study

pK18mobSacB-Ery-

woc28161

pK18mobsacb-Ery with 760 bp upstream fragment and 1 300 bp downstream fragment of woc28161This study

pK18mobSacB-Ery-

woc27232

pK18mobsacb-Ery with 558 bp upstream fragment and 412 bp downstream fragment of woc27232This study

pK18mobSacB-Ery-

woc27404

pK18mobsacb-Ery with 441 bp upstream fragment and 417 bp downstream fragment of woc27404This study
), ArticleFig(id=1227303263490454234, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=EN, label=Table 2, caption=

Primers for RT-qPCR, the construction of mutant strains, and for DNA sequencing

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers namePrimer sequences (5′→3′)Restriction enzyme cutting site
woc28159-f-qPCRGCACAAGCTGCAGTTGATTGC
woc28159-r-qPCRGACAACGCTGTCACAAGC
woc28160-f-qPCRGTGGCAAGCACCATGTTGCTT
woc28160-r-qPCRTGCTGCTCGACAAGCTAG
woc28161-f-qPCRCAGTGGAATGCATGGAGTGGT
woc28161-r-qPCRTTCACTGCGCGCACAAGT
woc27404-f-qPCRGTTGCTCTTATCGCCTCTGCT
woc27404-r-qPCRATTGTCCCTCGTTTCAAGCGC
woc27232-f-qPCRGCTAACACAAGGTGCCTTACC
woc27232-r-qPCRGCAATGACTGCAATAAGCGTA
rpoD-f-qPCRGCACCTGATGCCGATGAATTA
rpoD-r-qPCRCATGTACATGCGGACAGGGTC
woc28159-UfAACTGCAGCGAATAATAATGCCTATGAAGCCAAGTGPst I
woc28159-UrCGCGTCGACGTAACAAGTAGCCTGAAGATGGAGAAGSal I
woc28159-DfCGCGTCGACAGGAACAGGCGTTGGTGAGAAGASal I
woc28159-DrGCTCTAGAGCCATTCACATCCACAGCCATACXba I
woc28159-wSGCAGTTGATTGCAGTAATCTTGAAGTATGG
woc28159-wACGATACCTAATGCTCCGTTACCACAT
woc28159-dSTTCTCCATCTTCAGGCTACTTGTTACG
woc28159-dAATACATCACTTGGGCATTTACGGTTTG
woc28160-UfCCCAAGCTTCTACAGCGAGAACACAACAATGAAHind III
woc28160-UrCGGAATTCGACCAGCAGGAATTGAGACACTEcoR I
woc28160-DfCGGAATTCGACGCTGGACCAACAGATTCAEcoR I
woc28160-DrCGCGGATCCTTGATAGGTTACTTCGGCACTGBamH I
woc28160-wSTTAACTCAATAAAGCGGAGCAAGGAGA
woc28160-wAGCCACCGACAACAGCGTCTT
woc28160-dSGGAATGACTTAGAGTTAGTTGCTGAATACG
woc28160-dAATACATCACTTGGGCATTTACGGTTTG
woc28161-UfCGCGTCGACGTTGAGGTGAATTTAGAAGGGACAGGATSal I
woc28161-UrCGGAATTCAGGCGACGAAGGTGTCACGATTEcoR I
woc28161-DfCGGAATTCGTGTAGATGCTGGTGGTGTTCCTTATEcoR I
woc28161-DrCGCGGATCCGTCACTTGGTCGCCGCTGTTATBamH I
woc28161-wSGCAGCCATTGGTGTTGCATTATTCG
woc28161-wATTGAATTGCGTTATCGGTTGTGTAAGC
woc28161-dSTCAGTGGGTGGTTGGACGTTATCA
woc28161-dACGGCTGCTTTAGAGGGAAATCACATTA
woc27404-UfAACTGCAGACGATTGTTGATGACCCGAATAACPst I
woc27404-UrCGCGTCGACCCATTCTTCGAGTTGTAATGGGTTGTCSal I
woc27404-DfCGCGTCGACCTCAGTGCGGGAATCGGATACSal I
woc27404-DrCGCGGATCCTGTTAGCGTGTCAACGACTGTTACTTGBamH I
woc27404-wSCTTGCTCTGCAAGTTGCTCTTATC
woc27404-wAGTAATATGCCCAGTCTGCTTCCCA
woc27404-dSGAAGCTATTACACCTAACAATGCAAGC
woc27404-dAACCTTGAGTCCACCACTTAGC
woc27232-UfCCGCTCGAGTGAATAGGTAAGGGTGTATTGACCAACXho I
woc27232-UrCGCGTCGACCAATTAACACAGCAAGGACAACCGSal I
woc27232-DfCGCGTCGACGCGAAGTGCGGCAGGTATAACSal I
woc27232-DrCGCGGATCCGGTTATTGGCACAATTGGTGTGATBamH I
woc27232-wSCCATTGGCTTGCACATAGTGT
woc27232-wACTTGAGCAAGCAGCCGTAACA
woc27232-dSTGTATCGACACTACCCGTGAC
woc27232-dAATGACTGCAATAAGCGTATCTGTACTC
pK18-fATTCCGCTGGCAGCTTAAG
PK18-rGGTAACGCCAGGGTTTTCC
), ArticleFig(id=1227303263586923227, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226598461756322661, language=CN, label=表2, caption=

用于实时RT-qPCR、突变体构建和DNA测序的引物

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers namePrimer sequences (5′→3′)Restriction enzyme cutting site
woc28159-f-qPCRGCACAAGCTGCAGTTGATTGC
woc28159-r-qPCRGACAACGCTGTCACAAGC
woc28160-f-qPCRGTGGCAAGCACCATGTTGCTT
woc28160-r-qPCRTGCTGCTCGACAAGCTAG
woc28161-f-qPCRCAGTGGAATGCATGGAGTGGT
woc28161-r-qPCRTTCACTGCGCGCACAAGT
woc27404-f-qPCRGTTGCTCTTATCGCCTCTGCT
woc27404-r-qPCRATTGTCCCTCGTTTCAAGCGC
woc27232-f-qPCRGCTAACACAAGGTGCCTTACC
woc27232-r-qPCRGCAATGACTGCAATAAGCGTA
rpoD-f-qPCRGCACCTGATGCCGATGAATTA
rpoD-r-qPCRCATGTACATGCGGACAGGGTC
woc28159-UfAACTGCAGCGAATAATAATGCCTATGAAGCCAAGTGPst I
woc28159-UrCGCGTCGACGTAACAAGTAGCCTGAAGATGGAGAAGSal I
woc28159-DfCGCGTCGACAGGAACAGGCGTTGGTGAGAAGASal I
woc28159-DrGCTCTAGAGCCATTCACATCCACAGCCATACXba I
woc28159-wSGCAGTTGATTGCAGTAATCTTGAAGTATGG
woc28159-wACGATACCTAATGCTCCGTTACCACAT
woc28159-dSTTCTCCATCTTCAGGCTACTTGTTACG
woc28159-dAATACATCACTTGGGCATTTACGGTTTG
woc28160-UfCCCAAGCTTCTACAGCGAGAACACAACAATGAAHind III
woc28160-UrCGGAATTCGACCAGCAGGAATTGAGACACTEcoR I
woc28160-DfCGGAATTCGACGCTGGACCAACAGATTCAEcoR I
woc28160-DrCGCGGATCCTTGATAGGTTACTTCGGCACTGBamH I
woc28160-wSTTAACTCAATAAAGCGGAGCAAGGAGA
woc28160-wAGCCACCGACAACAGCGTCTT
woc28160-dSGGAATGACTTAGAGTTAGTTGCTGAATACG
woc28160-dAATACATCACTTGGGCATTTACGGTTTG
woc28161-UfCGCGTCGACGTTGAGGTGAATTTAGAAGGGACAGGATSal I
woc28161-UrCGGAATTCAGGCGACGAAGGTGTCACGATTEcoR I
woc28161-DfCGGAATTCGTGTAGATGCTGGTGGTGTTCCTTATEcoR I
woc28161-DrCGCGGATCCGTCACTTGGTCGCCGCTGTTATBamH I
woc28161-wSGCAGCCATTGGTGTTGCATTATTCG
woc28161-wATTGAATTGCGTTATCGGTTGTGTAAGC
woc28161-dSTCAGTGGGTGGTTGGACGTTATCA
woc28161-dACGGCTGCTTTAGAGGGAAATCACATTA
woc27404-UfAACTGCAGACGATTGTTGATGACCCGAATAACPst I
woc27404-UrCGCGTCGACCCATTCTTCGAGTTGTAATGGGTTGTCSal I
woc27404-DfCGCGTCGACCTCAGTGCGGGAATCGGATACSal I
woc27404-DrCGCGGATCCTGTTAGCGTGTCAACGACTGTTACTTGBamH I
woc27404-wSCTTGCTCTGCAAGTTGCTCTTATC
woc27404-wAGTAATATGCCCAGTCTGCTTCCCA
woc27404-dSGAAGCTATTACACCTAACAATGCAAGC
woc27404-dAACCTTGAGTCCACCACTTAGC
woc27232-UfCCGCTCGAGTGAATAGGTAAGGGTGTATTGACCAACXho I
woc27232-UrCGCGTCGACCAATTAACACAGCAAGGACAACCGSal I
woc27232-DfCGCGTCGACGCGAAGTGCGGCAGGTATAACSal I
woc27232-DrCGCGGATCCGGTTATTGGCACAATTGGTGTGATBamH I
woc27232-wSCCATTGGCTTGCACATAGTGT
woc27232-wACTTGAGCAAGCAGCCGTAACA
woc27232-dSTGTATCGACACTACCCGTGAC
woc27232-dAATGACTGCAATAAGCGTATCTGTACTC
pK18-fATTCCGCTGGCAGCTTAAG
PK18-rGGTAACGCCAGGGTTTTCC
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阿拉伯海假交替单胞菌N1230-9几丁质裂解酶的功能
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兰肖敏 , 周敏婕 , 王景 , 朱四东 , 杨季芳 , 陈吉刚 *
微生物学报 | 微生物资源开发与应用 2025,65(4): 1774-1787
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微生物学报 | 微生物资源开发与应用 2025, 65(4): 1774-1787
阿拉伯海假交替单胞菌N1230-9几丁质裂解酶的功能
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兰肖敏, 周敏婕, 王景, 朱四东, 杨季芳, 陈吉刚*
作者信息
  • 浙江万里学院 生物与环境学院，浙江 宁波
Functions of five chitin lyases from Pseudoalteromonas arabiensis N1230-9
Xiaomin LAN, Minjie ZHOU, Jing WANG, Sidong ZHU, Jifang YANG, Jigang CHEN*
Affiliations
  • College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo, Zhejiang, China
出版时间: 2025-04-04 doi: 10.13343/j.cnki.wsxb.20240719
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【目的】几丁质裂解酶是细菌有效降解几丁质的关键酶。阿拉伯海假交替单胞菌(Pseudoalteromonas arabiensis) N1230-9拥有5个几丁质裂解酶编码基因(woc28159、woc28160、woc28161、woc27404和woc27232)，对这5个基因的功能进行鉴定是解析该菌株具有高效几丁质降解能力的关键。【方法】通过生物信息学方法分析菌株N1230-9中的5个几丁质裂解酶在假交替单胞菌44个模式菌株中的分布规律；采用实时荧光定量PCR技术(RT-qPCR)检测菌株N1230-9中5个几丁质裂解酶编码基因在以几丁质为唯一碳源培养基中的转录水平；利用同源重组策略构建菌株N1230-9中5个几丁质裂解酶编码基因的单基因缺失突变体，并分析突变体的几丁质降解能力。【结果】有33株假交替单胞菌模式菌株拥有与菌株N1230-9几丁质裂解酶同源的蛋白；几丁质可以强烈诱导woc28159、woc28160、woc28161和woc27404的转录水平上调，且这4个基因的单基因缺失均在不同程度上削弱了菌株的几丁质降解能力。然而woc27232的转录水平不受几丁质的诱导，且该基因的缺失对菌株的几丁质降解能力无影响。【结论】几丁质酶WOC28159和WOC28161是菌株N1230-9降解几丁质所必需的酶，WOC28160和WOC27404增强了菌株的几丁质降解效率，而WOC27232并不参与菌株N1230-9的几丁质降解过程。

假交替单胞菌  /  阿拉伯海假交替单胞菌  /  几丁质裂解酶  /  几丁质酶

[Objective] Chitin lyases are the key enzymes for bacteria to degrade chitin. Pseudoalteromonas arabiensis N1230-9 possesses five chitin lyase-encoding genes (woc28159, woc28160, woc28161, woc27404, and woc27232). Functional identification of these five genes is crucial for determining the ability of strain to degrade chitin. [Methods] The distribution patterns of five chitin lyases from strain N1230-9 in 44 model strains of Pseudomonas were analyzed by bioinformatics methods. The transcription levels of five chitin lyase-encoding genes of strain N1230-9 cultured in the medium with chitin as the sole carbon source were analyzed by RT-qPCR. The mutants with deletion of chitin lyase-encoding genes were constructed by homologous recombination, and their abilities to degrade chitin were evaluated. [Results] Thirty-three Pseudoalteromonas strains possessed chitin lyase homologous proteins of strain N1230-9. Chitin significantly upregulated the transcription levels of woc28159, woc28160, woc28161, and woc27404, and the deletion of each of the four genes weakened the chitin-degradation ability. The transcription level of woc27232 was not affected by chitin, and the deletion of this gene did not affect the chitin-degradation ability of the strain. [Conclusion] WOC28159 and WOC28161 are necessary for the degradation of chitin by strain N1230-9. WOC28160 and WOC27404 endow strain N1230-9 with efficient chitin-degrading ability, while WOC27232 does not participate in the chitin degradation process.

Pseudoalteromonas  /  Pseudoalteromonas arabiensis  /  chitin lyase  /  chitinase
兰肖敏, 周敏婕, 王景, 朱四东, 杨季芳, 陈吉刚. 阿拉伯海假交替单胞菌N1230-9几丁质裂解酶的功能. 微生物学报, 2025 , 65 (4) : 1774 -1787 . DOI: 10.13343/j.cnki.wsxb.20240719
Xiaomin LAN, Minjie ZHOU, Jing WANG, Sidong ZHU, Jifang YANG, Jigang CHEN. Functions of five chitin lyases from Pseudoalteromonas arabiensis N1230-9[J]. Acta Microbiologica Sinica, 2025 , 65 (4) : 1774 -1787 . DOI: 10.13343/j.cnki.wsxb.20240719
正文
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几丁质(甲壳素)是由N-乙酰氨基葡萄糖(GlcNAc)通过β-1,4糖苷键连接而成的天然聚合物。其以结晶纤维的形式广泛存在于硅藻、原生生物、真菌等单细胞生物，以及海绵、珊瑚、软体动物、节肢动物等多细胞生物中[1]。几丁质不仅是地球上最丰富的含氮多糖，也是许多细菌的碳源或氮源[2]。尽管全球海洋环境中每年几丁质的产量约为1011 t，但其在海洋沉积物中的积累却十分有限[3-4]，这主要归因于海洋细菌对几丁质的降解作用[5-6]。
几丁质降解菌通常通过多种几丁质酶的协同作用降解几丁质[7]，这些几丁质酶主要分布于糖苷水解酶(glycoside hydrolases, GHs)中的GH18、GH19和GH20家族中(CAZy，http://www.cazy.org)[8]。除了拥有GH家族几丁质酶外，某些几丁质降解菌还拥有隶属于辅助酶AA10家族的多糖裂解单加氧酶(lytic polysaccharide monooxygenases, LPMOs)。LPMOs通过氧化作用裂解多糖链(主要作用于C1和C4位点)，降低多糖表面氧化位点附近区域的结晶度，从而使多糖更易于被糖苷水解酶降解[9]。
假交替单胞菌(Pseudoalteromonas)不仅是海洋环境的特有种群，也是海洋环境中的主要几丁质降解者，其能通过降解甲壳动物的尸体或碎屑，为深海系统中的原核生物提供碳源和氮源[10]。基因组分析结果表明，假交替单胞菌属中的多个种群普遍拥有多个几丁质裂解酶编码基因，其中包括一个保守的几丁质降解基因簇chiC-lpmo-chiA (以下简称 “CLC”)[11-12]。尽管假交替单胞菌的多个几丁质裂解酶的酶学特性已被表征[13-15]，但仅凭这些酶的酶学数据无法准确判断它们的确切功能，尤其是无法确定位于基因簇CLC之外的几丁质裂解酶基因是否为 “冗余” 基因。因此，本研究以一株高效几丁质降解菌——阿拉伯海假交替单胞菌(Pseudoalteromonas arabiensis) N1230-9为研究对象，通过生物信息学方法、实时荧光定量PCR (RT-qPCR)技术和同源重组策略，对该菌株中的5个几丁质裂解酶的功能进行了分析。
1 材料与方法
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1.1 材料
1.1.1 菌株和质粒
实验菌株阿拉伯海假交替单胞菌(Pseudoalteromonas arabiensis) N1230-9是从太平洋表层海水中分离获得的一株几丁质降解菌[16]；本研究使用的其他菌株和质粒如表1所示。
1.1.2 引物
用于RT-qPCR扩增、基因缺失突变体构建和DNA测序的引物如表2所示。
1.1.3 培养基和菌株培养条件
大肠杆菌(Escherichia coli) DH5α培养采用LB培养基；E. coli WM3064培养使用含2,6-二氨基庚二酸(2,6-diaminopimelic acid, DAP)的LB培养基(LB-DAP)[17]；菌株P. arabiensis N1230-9及其基因缺失突变体培养使用Marine Broth/Agar 2216培养基(以下简称2216培养基)；菌株接合实验和二次同源重组菌株筛选分别使用LB-DAP固体培养基和2216-蔗糖培养基[17]。根据实验需求，在培养基中添加50 μg/mL的卡那霉素或30 μg/mL的红霉素。E. coli菌株的培养温度为37 ℃；P. arabiensis N1230-9及其基因缺失突变体的培养温度为25 ℃。
无机盐溶液(g/L)：NH4Cl 0.500，NaCl 30.000，MgCl2·6H2O 3.000，K2SO4 2.000，K2HPO4 0.200，CaCl2 0.010，FeCl3·6H2O 0.006，Na2MoO4·2H2O 0.005，CuCl2·2H2O 0.004，Tris 6.000。
几丁质培养基：在无机盐溶液中添加质量体积分数为3%终浓度的胶体几丁质或1%终浓度的颗粒几丁质。如需配制几丁质固体培养基，则在几丁质液体培养基中加入16 g/L的琼脂粉。
葡萄糖培养基：在无机盐溶液中添加质量体积分数为0.25%的葡萄糖。
LB培养基、Marine Broth/Agar 2216培养基，BD公司。
1.1.4 主要试剂和仪器
几丁质，Sigma-Aldrich公司；葡萄糖、T4 DNA连接酶、EcoR I、BamH Ⅰ、Pst Ⅰ、Sal Ⅰ、Hind III、Xho I、Xba I，宝生物工程(大连)有限公司；PCR扩增试剂盒、DNA回收试剂盒、RNA提取试剂盒RNApure Bacteria Kit (DNase I)、逆转录试剂盒HiFiScript gDNA Removal RT MasterMix、RT-qPCR试剂盒SuperStar Universal SYBR Master Mix，江苏康为世纪生物科技股份有限公司。
PCR仪，Eppendorf公司；小型台式高速冷冻离心机，Starorius公司；恒温振荡摇床，New Brunswick Scientific公司；蛋白核酸测定分光光度计(NanoDrop)，ThermoFisher Scientific公司；荧光定量PCR仪，Bio-Rad公司；紫外分光光度计，上海美谱达仪器有限公司。
1.2 几丁质酶编码基因生物信息学分析
使用MAFFT (http://mafft.cbrc.jp/alignment/server)进行氨基酸序列同源比对；使用SignalP 6.0 (https://services.healthtech.dtu.dk/service.php?SignalP-6.0)预测蛋白信号肽；使用Compute pI/MW (https://www.expasy.org/resources/compute-pi-mw)预测蛋白质分子量及等电点；使用InterPro数据库(https://www.ebi.ac.uk/interpro/)[18]预测结构域；使用NCBI BLASTp (https://blast.ncbi.nlm.nih.gov/)和JGI IMG数据库(https://img.jgi.doe.gov/)进行同源蛋白序列搜索与比对；从NCBI数据库下载44株假交替单胞菌属模式菌株的基因组序列，使用TYGS在线服务器(https://www.dsmz.de/services/online-tools/tygs)构建基因组系统发育树；使用MEGA v7.0.20构建假交替单胞菌几丁质酶系统发育树，并使用Chiplot (https://www.chiplot.online/)[19]进行美化；使用GraphPad Prism v9.5.1进行图表绘制。
1.3 几丁质裂解酶编码基因的转录分析
挑取菌株N1230-9单菌落接种至2216培养基中，于25 ℃、180 r/min振荡培养至细菌对数生长期中期(OD600约为0.6)。取培养物，5 000 r/min离心3 min收集菌体。菌体用无菌海水洗涤2次后，用无菌海水将细胞浓度调整至OD600值为0.5。吸取菌悬液按照1:100的体积比分别接种至胶体几丁质培养基和葡萄糖培养基中，于25 ℃、180 r/min振荡培养至细菌生长的对数前期(OD600约为0.15)和对数后期(D600约为0.55)。利用RNApure Bacteria Kit (DNase I)提取细胞RNA，并通过逆转录试剂盒HiFiScript gDNA Removal RT MasterMix获得cDNA。利用1.5%琼脂糖凝胶电泳检测RNA质量，并用NanoDrop 2000对RNA和cDNA进行定量。利用SnapGene v6.0.2软件设计5个几丁质裂解酶基因和RNA聚合酶δ因子rpoD的特异性引物对(表2)，使用SuperStar Universal SYBR Master Mix配制RT-qPCR反应体系，以rpoD (GenBank登录号为WP_011327061)为内参基因进行RT-qPCR扩增反应。RT-qPCR反应体系(20 μL)：2×TransStart Green qPCR SuperMix 10 μL，cDNA模板(10 ng/μL) 1 μL，正、反引物(0.2 μmol/L)各0.4 μL，ddH2O 8.2 μL。反应条件：95 ℃ 30 s；95 ℃ 10 s，60 ℃ 30 s，42个循环；95 ℃ 15 s，绘制熔解曲线，采用2-ΔΔCt法进行数据分析[20]，利用GraphPad Prism v9.5.1软件对目标基因在胶体几丁质培养基和葡萄糖培养基中的相对表达量进行显著性差异分析。P<0.05为显著差异，P<0.000 1为极显著差异。每个样品均设置3个以上生物学平行。
1.4 基因缺失突变体构建
采用假交替单胞菌基因敲除系统[17]，按金佳凡等[21]的实验步骤进行菌株N1230-9的靶基因敲除。具体操作如下：首先，使用2对特异性引物对(wocX-Uf/wocX-Ur和wocX-Df/wocX-Dr，其中 “X” 代表靶基因编号，表2)分别扩增靶区上下游片段。接着，通过限制性内切酶对上下游片段进行单酶切，并使用T4连接酶连接上下游片段获得同源臂。随后，以同源臂为模板，利用引物对wocX-Uf和wocX-Dr进行PCR扩增，获得完整的同源臂片段。该片段依次经双酶切和割胶回收纯化后，将其亚克隆至自杀质粒pK18mobsacB-Ery中，获得重组自杀质粒。将重组质粒转化至大肠杆菌WM3064中，并利用引物对pK18-f/pK18-r对转化子进行DNA测序验证。之后，通过接合转移的方式将WM3064中的重组自杀质粒导入到目标菌株N1230-9中。基于同源重组的2次交换原理，实现靶基因的无痕敲除。在阳性克隆的筛选过程中，利用含有红霉素的2216培养基筛选发生一次交换的克隆，并通过引物对wocX-dS/wocX-wA对潜在的阳性克隆进行PCR验证；利用2216-蔗糖培养基筛选出双交换突变株，并利用3对引物对(wocX-wS/wocX-wA、wocX-dS/wocX-dA和wocX-dS/wocX-wA)对疑似阳性克隆进行PCR验证，以确保靶基因的成功敲除。
1.5 基因缺失突变体几丁质降解能力测试
按1.3节中描述的步骤分别制备野生菌株和各个突变体的培养物，并用无菌海水将细胞浓度调整至OD600值为0.5。吸取菌悬液按照1:100的体积比分别接种至2216培养基和几丁质液体培养基中，于25 ℃、180 r/min培养。培养期间定时取样，用分光光度法检测培养物的OD600值。每一种培养基均设3个生物学平行。同时取上述菌悬液各10 μL滴加至几丁质固体培养基中央，于25 ℃恒温培养箱正置培养36 h，观察透明圈产生情况并拍照留存。每个样品均设置3个以上生物学平行。
2 结果与分析
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2.1 菌株N1230-9几丁质裂解酶在假交替单胞菌中的分布规律
菌株N1230-9的基因组编码5个几丁质裂解酶，包括3个GH18家族的几丁质酶(GenBank登录号分别为WOC28159、WOC28161和WOC27232)，1个GH19家族的几丁质酶(GenBank登录号为WOC27404)，以及1个AA10家族的多糖裂解单加氧酶(LPMO，GenBank登录号为WOC28160)，其中WOC28159、WOC28160和WOC28161的编码基因位于小染色体上，形成一个几丁质降解基因簇(CLC)，而WOC27232和WOC27404的编码基因则散在地分布于大染色体上。信号肽预测结果表明，菌株N1230-9中的5个几丁质裂解酶均含有信号肽序列，提示它们可能均为分泌酶。蛋白结构域预测结果显示，WOC27404含有一个未知的GH19家族结构域，而其他3个几丁质酶则均拥有一个GH18家族的糖苷水解酶催化结构域。此外，4个几丁质酶均含有1-2个第5家族的几丁质结合模块。基因组系统发育树分析显示，45株假交替单胞菌形成2个大的分支，其中15株有色菌株聚为一支，而无色菌株和另外2株有色菌株则聚为另一支(图1)。同源对比结果显示，33株假交替单胞菌含有1-2个CLC，其中17株菌株同时拥有WOC27404的直系同源蛋白，且这17株菌株均为有色菌株(图1)。此外，通过同源比对仅发现1株假交替单胞菌模式菌株拥有WOC27232的直系同源蛋白，且该菌株同样为有色菌株(图1)。
2.2 四个几丁质酶的系统发育分析
系统发育树分析显示，4个几丁质酶及其假交替单胞菌来源的同源蛋白形成了4个分支，其中隶属于GH19家族的WOC27404及其同源蛋白形成了1个独立的分支(图2)。NCBI BLASTp同源比对结果表明，WOC27404与一些弧菌和部分陆源细菌的GH19家族几丁质酶具有同源性，且与弧菌的GH19家族几丁质酶的一致性较高(73.86%-91.61%)，而与陆源细菌的GH19家族几丁质酶的一致性较低(26.17%-41.71%)。系统发育树显示，WOC27404与非假交替单胞菌来源的GH19家族几丁质酶形成了2个分支，其中WOC27404与陆源细菌的GH19家族几丁质酶明显分离(图3)。
2.3 五个几丁质裂解酶基因的转录水平分析
在几丁质培养基和葡萄糖培养基中，对菌株N1230-9中5个几丁质裂解酶基因的转录水平进行了检测，结果如图4所示。相较于葡萄糖培养基，除woc27232在几丁质培养基中的转录水平未发生显著上调外，其余4个几丁质裂解酶基因的转录水平均显著上调，其中位于CLC基因簇内的3个裂解酶基因(woc28159、woc28160和woc28161)在2个检测时间点均表现出显著的转录上调，而woc27404则仅在对数生长期前期表现出显著的转录上调。
2.4 几丁质裂解酶编码基因缺失突变体的几丁质降解能力
采用同源重组策略成功构建了菌株N1230-9中5个几丁质裂解酶编码基因的单基因缺失突变体(图5)。生长能力测试结果显示，5个基因缺失突变体在2216培养基中的生长状态与野生菌株(WT)无显著差异，说明这5个基因的缺失未对菌株产生极性效应(图略)。在几丁质培养基中对这5个基因缺失突变体的生长状态进行了观察，结果除了woc27232基因缺失未对菌株的几丁质降解能力产生显著影响外，其他4个几丁质裂解酶基因的缺失均对菌株的几丁质降解能力产生了不同程度的影响(图6)。Δwoc28159和Δwoc28161在胶体或颗粒几丁质液体培养基中均无法生长(图6A、6C)，且这2个突变体在胶体或颗粒几丁质平板上也无法产生透明圈(图6B、6D)，表明它们已丧失了几丁质的降解能力。与野生菌株相比，Δwoc28160和Δwoc27404在胶体几丁质或颗粒几丁质液体培养基中的延滞期明显延长，最高生物量降低(图6A、6C)，且在胶体几丁质平板上产生的透明圈也明显减小(图6B)。此外，相较于Δwoc28160，Δwoc27404在胶体几丁质液体培养基中的延滞期更长，生物量更低(图6A)。
3 讨论与结论
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同源比对结果显示，菌株N1230-9中的5个几丁质裂解酶在其他几丁质降解菌中均存在直系同源蛋白，且部分同源蛋白的酶学活性已得到了初步表征。普里兹湾假交替单胞菌(Pseudoalteromonas prydzensis) ACAM 620的ChiC (PpChiC)和ChiA (PpChiA)分别是WOC28159和WOC28161的直系同源蛋白。PpChiC和PpChiA分别具有外切几丁质酶和内切几丁质酶的活性，均能切割胶体几丁质和结晶几丁质产生还原糖[23]。海摩替亚氏菌(Moritella marina)的MmChi60是WOC27232的同源蛋白，该酶属于低温胞外几丁质内切酶，能够有效降解几丁质和几丁寡糖[(GlcNAc)n，n>2][24]。长衫假交替单胞菌(Pseudoalteromonas tunicata) CCUG 44952的PtChi19是WOC27404的直系同源蛋白，该酶对胶体几丁质和结晶几丁质均有降解活性[25]。P. prydzensis ACAM 620的LPMO (PpLMPO)是WOC28160的直系同源蛋白，该酶介导氧化几丁质中糖苷键的C1碳，并产生具有末端氧化糖2-(乙酰氨基)-2-脱氧-d-葡萄糖酸(GlcNAc1A)的壳寡糖[23]。
荧光定量PCR结果显示，在菌株N1230-9对数生长期的2个检测时间点，葡萄糖对该菌株中5个几丁质降解酶的表达均无诱导能力，而几丁质却可强烈持续诱导该菌株CLC基因簇内的3个几丁质裂解酶基因的表达，这一结果与P. prydzensis ACAM 620转录组测序结果相吻合[23]，说明这3个几丁质裂解酶均参与了几丁质的降解过程，并在几丁质的降解过程中发挥了关键作用。RT-qPCR检测结果还显示，菌株N1230-9 CLC基因簇外的2个几丁质酶编码基因对几丁质的响应存在差异：几丁质不能诱导woc27232的表达，但却可以显著诱导woc27404的表达，但其诱导能力并不持续，说明woc27232很可能并未参与几丁质的降解过程，而woc27404可能参与了几丁质的前期降解过程。
基因敲除是验证基因功能的 “金标准”，为此本研究构建了目标菌株的5个几丁质酶编码基因的单基因缺失突变体，并分析了突变体的几丁质降解能力。研究结果显示，位于CLC基因簇内的woc28159或woc28161缺失均导致菌株丧失了胶体和颗粒几丁质的降解能力，这不仅说明WOC28159和WOC28161是菌株降解几丁质所必需的，也提示该菌株所拥有的其他几丁质酶并不能替代这2个酶的功能。相比之下，同样位于CLC基因簇内的woc28160缺失并不会导致菌株完全丧失几丁质降解能力，这进一步说明WOC28160主要在解聚复杂几丁质的起始过程中发挥作用[19]。本研究结果显示，位于CLC基因簇外的woc27232和woc27404对菌株降解几丁质的贡献度存在明显差异。woc27232缺失对菌株降解利用胶体和颗粒几丁质均无影响，这与RT-qPCR获得的实验结果相吻合，说明该基因对假交替单胞菌几丁质降解而言是功能冗余的。此外，如前文所述，WOC27232同源蛋白编码基因在假交替单胞菌模式菌株中并不保守，说明woc27232是通过基因水平转移获得的无功能几丁质酶基因。与WOC27232相比，WOC27404赋予了菌株更强的几丁质降解能力，因为该基因的缺失导致菌株在几丁质培养基中的延滞期延长，最高生物量降低，以及对几丁质的降解能力明显减弱，这一结果不仅与RT-qPCR结果相吻合，且进一步佐证了P. tunicata CCUG 44952的PtChi19 (WOC27404的同源蛋白)的酶学特性[25]。值得注意的是，在本研究分析的45个假交替单胞菌模式菌株中，所有有色菌株均拥有WOC27404同源蛋白，而所有无色假交替单胞菌模式菌株均缺失WOC27404同源蛋白，提示woc27404可能是通过基因水平转移方式从有色假交替单胞菌中获得的。
基金
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  • 全球变化与海气相互作用专项(二期)(GASI-04-HYDZ-02)
  • 宁波市自然科学基金(2023J301)
  • 浙江省生物工程一流学科创新基金(CX2024017)
文献
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2025年第65卷第4期
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doi: 10.13343/j.cnki.wsxb.20240719
  • 接收时间：2024-11-13
  • 首发时间：2026-02-06
  • 出版时间：2025-04-04
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  • 收稿日期：2024-11-13
  • 录用日期：2025-01-24
基金
National Program on Global Change and Air-sea Interaction (Phase Ⅱ)(GASI-04-HYDZ-02)
全球变化与海气相互作用专项(二期)(GASI-04-HYDZ-02)
Ningbo Natural Science Foundation(2023J301)
宁波市自然科学基金(2023J301)
The First-class Discipline of Biological Engineering of Zhejiang Province(CX2024017)
浙江省生物工程一流学科创新基金(CX2024017)
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    浙江万里学院 生物与环境学院，浙江 宁波

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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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