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Article(id=1238813323685327454, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1238813307784712441, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250707, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1757952000000, receivedDateStr=2025-09-16, revisedDate=null, revisedDateStr=null, acceptedDate=1761580800000, acceptedDateStr=2025-10-28, onlineDate=1773285712404, onlineDateStr=2026-03-12, pubDate=1772553600000, pubDateStr=2026-03-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773285712404, onlineIssueDateStr=2026-03-12, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773285712404, creator=13701087609, updateTime=1773285712404, updator=13701087609, issue=Issue{id=1238813307784712441, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='3', pageStart='961', pageEnd='1466', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773285708614, creator=13701087609, updateTime=1773291912509, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1238839328915378858, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1238813307784712441, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1238839328915378859, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1238813307784712441, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1107, endPage=1118, ext={EN=ArticleExt(id=1238813324188643987, articleId=1238813323685327454, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Key amino acid sites of the TetR family transcription factor BcPDR1 in Botrytis cinerea, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

Objective To identify the key amino acid residues of the TetR family transcription factor BcPDR1 in Botrytis cinerea, thereby laying a foundation for elucidating the mechanism by which BcPDR1 regulates the growth, development, and pathogenicity of this pathogen. Methods The key amino acid sites of BcPDR1 were analyzed by bioinformatics methods, and four conserved regions (32-34 aa, 76-95 aa, 140-150 aa, and 189 aa) were selected for site-directed mutagenesis. On the basis of the knockout mutant ΔBcpdr1, the mutants BcPDR1-M1 (Δ32-34), BcPDR1-M2 (Δ76-95), BcPDR1-M3 (Δ140-150), and BcPDR1-M4 (mutation of Ile to Lys at 189 aa) were constructed. A comparative analysis of the phenotypic characteristics and pathogenicity was conducted on the four aforementioned mutants and the wild-type strain of B. cinerea, ΔBcpdr1, the complemented strain CE. Results The colony morphology, mycelial morphology, and growth rates of BcPDR1-M1, BcPDR1-M2, BcPDR1-M3, and BcPDR1-M4 were similar to those of ΔBcpdr1, but significantly different from those of BC22 and CE. These mutants could form lesions on tomato fruits and tobacco leaves, while their lesion areas were significantly smaller than those of BC22 and CE. Conclusion The regions 32-34, 76-95, 140-150, and the 189th amino acid are the regulatory sites for BcPDR1 to exert its functions.

, correspAuthors=Jihong XING, Jingao DONG, authorNote=null, correspAuthorsNote=
*E-mail: XING Jihong,
DONG Jingao,
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#These authors contributed equally to this work.

, authorsList=Dexuan QU, Xiaoying LIU, Yadi WEI, Jinping ZANG, Hongzhe CAO, Kang ZHANG, Jihong XING, Jingao DONG), CN=ArticleExt(id=1238813326797501213, articleId=1238813323685327454, tenantId=1146029695717560320, journalId=1192105938417971205, language=CN, title=灰葡萄孢TetR家族转录因子BcPDR1的关键氨基酸位点分析, columnId=1192149544164012138, journalTitle=微生物学报, columnName=研究报告, runingTitle=null, highlight=null, articleAbstract=

目的 明确灰葡萄孢TetR家族转录因子BcPDR1的关键氨基酸位点，为阐明BcPDR1调控病菌生长发育及致病力的机制奠定基础。 方法 运用生物信息学手段分析BcPDR1蛋白的关键氨基酸位点，选取4个保守区域(32-34、76-95、140-150和189位氨基酸)进行定点突变。在敲除突变体ΔBcpdr1的基础上，构建BcPDR1-M1 (Δ32-34)、BcPDR1-M2 (Δ76-95)、BcPDR1-M3 (Δ140-150)和BcPDR1-M4 (将189位Ile突变为Lys)突变体。对上述4个突变体与灰葡萄孢野生型菌株、ΔBcpdr1以及互补菌株CE进行表型和致病力的对比分析。 结果 突变体BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4的菌落形态、菌丝形态和生长速率与ΔBcpdr1相似，而与野生型BC22和互补菌株CE存在显著差异；各突变体均能在番茄果实和烟叶上形成病斑，但病斑面积显著小于野生型BC22和互补菌株CE。 结论 灰葡萄孢TetR家族转录因子BcPDR1的32-34、76-95、140-150位区域及189位氨基酸为其发挥功能的关键调控位点。

, correspAuthors=邢继红, 董金皋, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=ByNDmZXAnQz7fx+MzQ2fWg==, magXml=L3VrEo8sZ37iNU6dOmOtPQ==, pdfUrl=null, pdf=o3gOr0CkG6SGL8NGS7jI7w==, pdfFileSize=2777199, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=mQlG2TpOuZLoOR93mIMUnQ==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=3UfzFblvGKfXTtMWCIbZnA==, mapNumber=null, authorCompany=null, fund=null, authors=

作者贡献声明

曲德轩：试验操作、数据分析及文章撰写；刘晓颖：数据分析及文章撰写；魏雅迪：部分试验操作、数据收集及分析；藏金萍：提供经费支持；曹宏哲：参与文章编辑和审阅；张康：参与技术指导及文章审阅；邢继红：提供技术指导，研究构思和设计及文章审阅与修改；董金皋：项目申请、资源提供等贡献。

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Stochastic activation of a family of TetR type transcriptional regulators controls phenotypic heterogeneity in Acinetobacter baumannii [J]. PNAS Nexus, 2022, 1(5): pgac231., articleTitle=Stochastic activation of a family of TetR type transcriptional regulators controls phenotypic heterogeneity in Acinetobacter baumannii, refAbstract=null), Reference(id=1238891114820858724, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, doi=null, pmid=null, pmcid=null, year=2013, volume=425, issue=18, pageStart=3289, pageEnd=3300, url=null, language=null, rfNumber=[16], rfOrder=15, authorNames=HAYASHI T, TANAKA Y, SAKAI N, OKADA U, YAO M, WATANABE N, TAMURA T, TANAKA I, journalName=Journal of Molecular Biology, refType=null, unstructuredReference=HAYASHI T, TANAKA Y, SAKAI N, OKADA U, YAO M, WATANABE N, TAMURA T, TANAKA I. 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Nature Communications, 2023, 14: 5622., articleTitle=High-efficiency green management of potato late blight by a self-assembled multicomponent nano-bioprotectant, refAbstract=null), Reference(id=1238891116905427844, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, doi=null, pmid=null, pmcid=null, year=2025, volume=73, issue=32, pageStart=20375, pageEnd=20384, url=null, language=null, rfNumber=[22], rfOrder=21, authorNames=FAN L, WEI YY, CHEN Y, JIANG S, XU F, ELSHERBINY EA, SHAO XF, journalName=Journal of Agricultural and Food Chemistry, refType=null, unstructuredReference=FAN L, WEI YY, CHEN Y, JIANG S, XU F, ELSHERBINY EA, SHAO XF. The antifungal ability of amino acid substitution of antimicrobial peptide epinecidin-1 to Botrytis cinerea in peach fruit[J]. Journal of Agricultural and Food Chemistry, 2025, 73(32): 20375-20384., articleTitle=The antifungal ability of amino acid substitution of antimicrobial peptide epinecidin-1 to Botrytis cinerea in peach fruit, refAbstract=null), Reference(id=1238891117035451272, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, doi=null, pmid=null, pmcid=null, year=2025, volume=15, issue=1, pageStart=194, pageEnd=null, url=null, language=null, rfNumber=[23], rfOrder=22, authorNames=SPADA M, PUGLIESI C, FAMBRINI M, PALPACELLI D, CANEO A, PECCHIA S, journalName=Agronomy, refType=null, unstructuredReference=SPADA M, PUGLIESI C, FAMBRINI M, PALPACELLI D, CANEO A, PECCHIA S. Spray-induced gene silencing (SIGS): nanocarrier-mediated dsRNA delivery improves RNAi efficiency in the management of lettuce gray mold caused by Botrytis cinerea [J]. Agronomy, 2025, 15(1): 194., articleTitle=Spray-induced gene silencing (SIGS): nanocarrier-mediated dsRNA delivery improves RNAi efficiency in the management of lettuce gray mold caused by Botrytis cinerea, refAbstract=null)], funds=[Fund(id=1238891110957904612, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, awardId=32072369, language=EN, fundingSource=National Natural Science Foundation of China(32072369), fundOrder=null, country=null), Fund(id=1238891112467854057, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, awardId=32072369, language=CN, fundingSource=国家自然科学基金(32072369), fundOrder=null, country=null), Fund(id=1238891112602071793, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, awardId=246Z6506G, language=EN, fundingSource=Central Guidance for Local Technology Development Funding(246Z6506G), fundOrder=null, country=null), Fund(id=1238891112744678138, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, awardId=246Z6506G, language=CN, fundingSource=中央引导地方科技发展资金(246Z6506G), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1238891103521403147, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, xref=1., ext=[AuthorCompanyExt(id=1238891103533986059, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, companyId=1238891103521403147, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology, Baoding, Hebei, China), AuthorCompanyExt(id=1238891103542374669, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, companyId=1238891103521403147, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.河北省植物生理与分子病理学重点实验室，河北 保定)]), AuthorCompany(id=1238891103634649363, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, xref=2., ext=[AuthorCompanyExt(id=1238891103643037972, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, companyId=1238891103634649363, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.State Key Laboratory of North China Crop Improvement and Regulation, Baoding, Hebei, China), AuthorCompanyExt(id=1238891103672398103, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, companyId=1238891103634649363, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.华北作物改良与调控国家重点实验室，河北 保定)])], figs=[ArticleFig(id=1238891109632504457, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, language=EN, label=Figure 1, caption=Construction of key amino acid mutation vector of BcPDR1 protein in Botrytis cinerea. A: Selection of site-directed mutagenesis sites of BcPDR1 protein; B: Construction of site-directed mutagenesis vector of BcPDR1 protein; C: From left to right are the colony PCR identifications of BcPDR1-M1, BcPDR1-M2, BcPDR1-M3, and BcPDR1-M4 vectors. Lane M: DNA marker; Lanes 1 and 2 are the corresponding bacterial samples of vectors BcPDR1-M1, BcPDR1-M2, BcPDR1-M3, and BcPDR1-M4., figureFileSmall=bqcRqB5CKpynwOAhGJygwg==, figureFileBig=+T4hxeuSDoxBwAHqh4Aj7w==, tableContent=null), ArticleFig(id=1238891109720584850, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, language=CN, label=图1, caption=灰葡萄孢BcPDR1蛋白关键氨基酸突变载体的构建。A：BcPDR1蛋白定点突变位点选择；B：BcPDR1蛋白定点突变载体的构建；C：由左至右分别是BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4载体的菌落PCR鉴定。泳道M：DNA marker；泳道1、2：由左至右分别为BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4载体的2个菌落。, figureFileSmall=bqcRqB5CKpynwOAhGJygwg==, figureFileBig=+T4hxeuSDoxBwAHqh4Aj7w==, tableContent=null), ArticleFig(id=1238891109959660188, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, language=EN, label=Figure 2, caption=Identification of key amino acid mutant transformants of BcPDR1 protein in Botrytis cinerea. A: Growth of transformants on resistant plate; B: PCR identification of transformants (Lane M: DNA marker; Lane 1: Genomic DNA of ΔBcpdr1; Lane 2 are the genomic DNAs of the BcPDR1-M1, BcPDR1-M2, BcPDR1-M3, and BcPDR1-M4 transformants, respectively); C: RT-qPCR identification of transformants. Different lowercase letters indicate significant differences between strains (P<0.05)., figureFileSmall=wGACaJHUuysNU5mS8bEXEg==, figureFileBig=ubpfTl+sjXTA3V8b+UK/1w==, tableContent=null), ArticleFig(id=1238891110098072227, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, language=CN, label=图2, caption=灰葡萄孢BcPDR1蛋白关键氨基酸突变转化子的鉴定。A：转化子在抗性平板上的生长情况；B：转化子的PCR水平鉴定(泳道M：DNA marker；泳道1：ΔBcpdr1基因组DNA；泳道2分别为BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4转化子基因组DNA)；C：转化子的RT-qPCR鉴定。, figureFileSmall=wGACaJHUuysNU5mS8bEXEg==, figureFileBig=ubpfTl+sjXTA3V8b+UK/1w==, tableContent=null), ArticleFig(id=1238891110211318441, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, language=EN, label=Figure 3, caption=Phenotypic analysis of key amino acid mutants of BcPDR1 protein in Botrytis cinerea. A: Colony morphology of mutants; B: Mycelial morphology of mutants; C: Length and width of the mycelial cells of mutants; D: Colony growth rate of mutants., figureFileSmall=y6VTw4W3g7u4q5EbFfR2uQ==, figureFileBig=HDuUA+2BnGp1she5L5kM/w==, tableContent=null), ArticleFig(id=1238891110299398833, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, language=CN, label=图3, caption=灰葡萄孢BcPDR1蛋白关键氨基酸突变体的表型分析。A：突变体的菌落形态；B：突变体的菌丝形态；C：突变体的菌丝细胞长度和宽度；D：突变体的菌落生长速率。, figureFileSmall=y6VTw4W3g7u4q5EbFfR2uQ==, figureFileBig=HDuUA+2BnGp1she5L5kM/w==, tableContent=null), ArticleFig(id=1238891110425227963, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, language=EN, label=Figure 4, caption=Pathogenicity analysis of key amino acid mutants of BcPDR1 protein in Botrytis cinerea. A: Pathogenicity of mutants to tomato fruits; B: Statistics of lesion areas of tomato fruits, different lowercase letters indicate significant differences between strains (P<0.05); C: Pathogenicity of mutants to tobacco leaves; D: Statistics of lesion areas of tobacco leaves, different lowercase letters indicate significant differences between strains (P<0.05)., figureFileSmall=AUINHv9LLnlMf7+AFLnsAA==, figureFileBig=+mBgBlOBY+KcllF5wR7fmw==, tableContent=null), ArticleFig(id=1238891110525891266, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, language=CN, label=图4, caption=灰葡萄孢BcPDR1蛋白关键氨基酸突变体的致病力分析。A：突变体对番茄果实的致病力；B：番茄果实发病部位的病斑面积统计[不同小写字母表示菌株间差异显著(P<0.05)]；C：突变体对烟草叶片的致病力；D：烟草叶片发病部位的病斑面积统计[不同小写字母表示菌株间差异显著(P<0.05)]。, figureFileSmall=AUINHv9LLnlMf7+AFLnsAA==, figureFileBig=+mBgBlOBY+KcllF5wR7fmw==, tableContent=null), ArticleFig(id=1238891110626554568, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, language=EN, label=Table 1, caption=

Primers design of vector construction and RT-qPCR

, figureFileSmall=null, figureFileBig=null, tableContent=

基因名

Gene names

引物名称

Primer names

引物序列

Primer sequences (5′→3′)

BcPDR1BcPDR1-FATGGCTGAGAGGTTACCGACTG
BcPDR1-RTTCACGACGAATACTCGAATGG
BcPDR-M1BcPDR1-M1-FGATAAAAGCTGAAAAAAAGCAAAACGCAAACTCAG
BcPDR1-M1-RCTCCGATTTCCGGTTCTGAGTTTGCGTTTTGCTTT
BcPDR1-M2BcPDR1-M2-FCAAACCCGTGAATCATTGATGTACTTTGCATCACA
BcPDR1-M2-RGGCTTCGTTCTAATCCTCCGTAATAATGTGATGCA
BcPDR1-M3BcPDR1-M3-FTTTACATCGATTCAATCATTCATTTGCTCAGCATT
BcPDR1-M3-RCGTTCGATTGACGTGAATGCTGAGCAAATGAATGA
BcPDR1-M4BcPDR1-M4-FACTCACTGAAAATTTCGCTTACACTCCCCCATTCG
BcPDR1-M4-RTTCACGACGAATACTCGAATGGGGGAGTGTAAGCG
BcPDR1-M1RT-BcPDR1-M1-FTGGCTGAGAGGTTACCGACT
RT-BcPDR1-M1-RTACATCAGGAGTCTTGGCACC
BcPDR1-M2RT-BcPDR1-M2-FGGTGCCAAGACTCCTGATGTA
RT-BcPDR1-M2-RCGTGAATGCTGAGCCAAGTTT
BcPDR1-M3RT-BcPDR1-M3-FCAAAGACCTCGATGAAGGGGT
RT-BcPDR1-M3-RACCGAATGCCGAATGTGATG
BcPDR1-M4RT-BcPDR1-M4-FAGACCTCGATGAAGGGGTTC
RT-BcPDR1-M4-RCGAATGGGGGAGAAGCGAAA
BlpRBlpR-FTCAAATCTCGGTCACGGGCAGGACC
BlpR-RATGAGCCCAGAACGACGCCCGGC
GFPGFP-FAGTAAAGGAGAAGAACTTTTCACTG
GFP-RTTTGTATAGTTCATCCATGCCATGT
), ArticleFig(id=1238891110743995085, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1238813323685327454, language=CN, label=表1, caption=

载体构建和RT-qPCR的引物设计

, figureFileSmall=null, figureFileBig=null, tableContent=

基因名

Gene names

引物名称

Primer names

引物序列

Primer sequences (5′→3′)

BcPDR1BcPDR1-FATGGCTGAGAGGTTACCGACTG
BcPDR1-RTTCACGACGAATACTCGAATGG
BcPDR-M1BcPDR1-M1-FGATAAAAGCTGAAAAAAAGCAAAACGCAAACTCAG
BcPDR1-M1-RCTCCGATTTCCGGTTCTGAGTTTGCGTTTTGCTTT
BcPDR1-M2BcPDR1-M2-FCAAACCCGTGAATCATTGATGTACTTTGCATCACA
BcPDR1-M2-RGGCTTCGTTCTAATCCTCCGTAATAATGTGATGCA
BcPDR1-M3BcPDR1-M3-FTTTACATCGATTCAATCATTCATTTGCTCAGCATT
BcPDR1-M3-RCGTTCGATTGACGTGAATGCTGAGCAAATGAATGA
BcPDR1-M4BcPDR1-M4-FACTCACTGAAAATTTCGCTTACACTCCCCCATTCG
BcPDR1-M4-RTTCACGACGAATACTCGAATGGGGGAGTGTAAGCG
BcPDR1-M1RT-BcPDR1-M1-FTGGCTGAGAGGTTACCGACT
RT-BcPDR1-M1-RTACATCAGGAGTCTTGGCACC
BcPDR1-M2RT-BcPDR1-M2-FGGTGCCAAGACTCCTGATGTA
RT-BcPDR1-M2-RCGTGAATGCTGAGCCAAGTTT
BcPDR1-M3RT-BcPDR1-M3-FCAAAGACCTCGATGAAGGGGT
RT-BcPDR1-M3-RACCGAATGCCGAATGTGATG
BcPDR1-M4RT-BcPDR1-M4-FAGACCTCGATGAAGGGGTTC
RT-BcPDR1-M4-RCGAATGGGGGAGAAGCGAAA
BlpRBlpR-FTCAAATCTCGGTCACGGGCAGGACC
BlpR-RATGAGCCCAGAACGACGCCCGGC
GFPGFP-FAGTAAAGGAGAAGAACTTTTCACTG
GFP-RTTTGTATAGTTCATCCATGCCATGT
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灰葡萄孢TetR家族转录因子BcPDR1的关键氨基酸位点分析
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曲德轩 1, 2 , 刘晓颖 1, 2 , 魏雅迪 1 , 藏金萍 1 , 曹宏哲 1 , 张康 1, 2 , 邢继红 1, 2, * , 董金皋 1, 2, *
微生物学报 | 研究报告 2026,66(3): 1107-1118
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微生物学报 | 研究报告 2026, 66(3): 1107-1118
灰葡萄孢TetR家族转录因子BcPDR1的关键氨基酸位点分析
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曲德轩1, 2, 刘晓颖1, 2, 魏雅迪1, 藏金萍1, 曹宏哲1, 张康1, 2, 邢继红1, 2, * , 董金皋1, 2, *
作者信息
  • 1.河北省植物生理与分子病理学重点实验室，河北 保定
  • 2.华北作物改良与调控国家重点实验室，河北 保定
Key amino acid sites of the TetR family transcription factor BcPDR1 in Botrytis cinerea
Dexuan QU1, 2, Xiaoying LIU1, 2, Yadi WEI1, Jinping ZANG1, Hongzhe CAO1, Kang ZHANG1, 2, Jihong XING1, 2, * , Jingao DONG1, 2, *
Affiliations
  • 1.Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology, Baoding, Hebei, China
  • 2.State Key Laboratory of North China Crop Improvement and Regulation, Baoding, Hebei, China
出版时间: 2026-03-04 doi: 10.13343/j.cnki.wsxb.20250707
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摘要
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目的 明确灰葡萄孢TetR家族转录因子BcPDR1的关键氨基酸位点，为阐明BcPDR1调控病菌生长发育及致病力的机制奠定基础。 方法 运用生物信息学手段分析BcPDR1蛋白的关键氨基酸位点，选取4个保守区域(32-34、76-95、140-150和189位氨基酸)进行定点突变。在敲除突变体ΔBcpdr1的基础上，构建BcPDR1-M1 (Δ32-34)、BcPDR1-M2 (Δ76-95)、BcPDR1-M3 (Δ140-150)和BcPDR1-M4 (将189位Ile突变为Lys)突变体。对上述4个突变体与灰葡萄孢野生型菌株、ΔBcpdr1以及互补菌株CE进行表型和致病力的对比分析。 结果 突变体BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4的菌落形态、菌丝形态和生长速率与ΔBcpdr1相似，而与野生型BC22和互补菌株CE存在显著差异；各突变体均能在番茄果实和烟叶上形成病斑，但病斑面积显著小于野生型BC22和互补菌株CE。 结论 灰葡萄孢TetR家族转录因子BcPDR1的32-34、76-95、140-150位区域及189位氨基酸为其发挥功能的关键调控位点。

灰葡萄孢  /  BcPDR1  /  关键氨基酸位点  /  生长和发育  /  致病力

Objective To identify the key amino acid residues of the TetR family transcription factor BcPDR1 in Botrytis cinerea, thereby laying a foundation for elucidating the mechanism by which BcPDR1 regulates the growth, development, and pathogenicity of this pathogen. Methods The key amino acid sites of BcPDR1 were analyzed by bioinformatics methods, and four conserved regions (32-34 aa, 76-95 aa, 140-150 aa, and 189 aa) were selected for site-directed mutagenesis. On the basis of the knockout mutant ΔBcpdr1, the mutants BcPDR1-M1 (Δ32-34), BcPDR1-M2 (Δ76-95), BcPDR1-M3 (Δ140-150), and BcPDR1-M4 (mutation of Ile to Lys at 189 aa) were constructed. A comparative analysis of the phenotypic characteristics and pathogenicity was conducted on the four aforementioned mutants and the wild-type strain of B. cinerea, ΔBcpdr1, the complemented strain CE. Results The colony morphology, mycelial morphology, and growth rates of BcPDR1-M1, BcPDR1-M2, BcPDR1-M3, and BcPDR1-M4 were similar to those of ΔBcpdr1, but significantly different from those of BC22 and CE. These mutants could form lesions on tomato fruits and tobacco leaves, while their lesion areas were significantly smaller than those of BC22 and CE. Conclusion The regions 32-34, 76-95, 140-150, and the 189th amino acid are the regulatory sites for BcPDR1 to exert its functions.

Botrytis cinerea  /  BcPDR1  /  key amino acid sites  /  growth and development  /  pathogenicity
曲德轩, 刘晓颖, 魏雅迪, 藏金萍, 曹宏哲, 张康, 邢继红, 董金皋. 灰葡萄孢TetR家族转录因子BcPDR1的关键氨基酸位点分析. 微生物学报, 2026 , 66 (3) : 1107 -1118 . DOI: 10.13343/j.cnki.wsxb.20250707
Dexuan QU, Xiaoying LIU, Yadi WEI, Jinping ZANG, Hongzhe CAO, Kang ZHANG, Jihong XING, Jingao DONG. Key amino acid sites of the TetR family transcription factor BcPDR1 in Botrytis cinerea[J]. Acta Microbiologica Sinica, 2026 , 66 (3) : 1107 -1118 . DOI: 10.13343/j.cnki.wsxb.20250707
正文
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灰葡萄孢(Botrytis cinerea)作为一种典型的死体营养型植物病原真菌，凭借其广泛的寄主范围和复杂的侵染策略，已成为全球农业经济中危害最为严重的病原真菌之一[1-2]。该菌可侵染葡萄、番茄、黄瓜、辣椒等多种经济作物，通过分泌果胶酶、纤维素酶等胞壁降解酶以及毒素类物质，在宿主植物组织中高效定殖与扩展，进而引发具有毁灭性的灰霉病，每年造成全球超百亿美元的经济损失[3]。相关研究表明，灰葡萄孢的侵染过程涉及菌丝穿透、分生孢子萌发、菌核形成等多形态转化机制，且需要精准调控次生代谢酶系及毒素合成基因网络[4-5]。因此，系统解析与灰葡萄孢生长发育及致病相关的关键基因功能不仅是揭示其致病分子机理的核心途径，还能为植物病原真菌的通用研究提供创新性研究范式，具有重大的科学价值。
TetR家族转录因子(TetR family transcriptional regulator, TFRs)在细菌中是一类高度保守的调控枢纽，在生理代谢、抗生素合成、群体感应以及致病抗药性调控等关键生物过程中发挥核心作用，已成为抗感染药物研发的重要靶标[6-8]。例如，在微白黄链霉菌(Streptomyces albidoflavus)中，TetR调节因子SCO3201通过抑制放线菌素的合成与形态分化对次级代谢过程产生影响[9]；在谷氨酸棒状杆菌(Streptomyces avermitilis)中，高度保守的TFR-FasR作为阻遏蛋白能够精细调控脂肪酸的合成过程[10]；在分枝杆菌属(Mycobacterium)中，TetR家族阻遏蛋白BkaR (也称Fad35R)通过特异性结合靶基因上游的回文基序，抑制自身以及参与支链酮酸代谢基因簇的转录[11]；此外，TetR家族转录调节因子SP_2854能够正向调节丁烯基-spinosyn的生物合成，同时影响菌株的生长、葡萄糖消耗以及菌丝形态[12]。截至目前，植物病原真菌中TetR家族成员的鉴定及功能研究仍处于空白状态，其在真菌特异性生理过程中的作用机制也尚未被揭示。
在本实验室的前期研究中获得了一个与灰葡萄孢致病相关的新基因BcPDR1。通过基因敲除与互补技术，证实该基因能够正向调控病菌的生长发育以及致病力，这提示其有望作为灰霉病防治药物筛选的新靶标。序列比对结果显示，BcPDR1与TetR家族典型成员SmcR [蛋白质结构数据库(Protein Data Bank, PDB)登录号3KZ9]、HapR (PDB登录号2PBX)的序列一致性分别为21%和29%，但其空间结构均含多个α-螺旋，这与TetR家族“低序列同源性但高结构保守性”的特征高度相符。为了验证BcPDR1的家族归属并深入解析其功能机制，本研究采用生物信息学方法，对BcPDR1与TetR家族的关键氨基酸序列进行对比分析，选取保守区域进行定点突变，并在敲除突变体ΔBcpdr1的基础上构建位点突变菌株。通过系统分析突变体的表型及致病力变化，明确BcPDR1的关键功能位点。本研究不仅为植物病原真菌TetR家族转录因子的功能与分子机制研究奠定基础，更提供了新型杀菌剂分子设计的新靶标，对灰霉病等植物病害防治具有重要理论价值与实践意义。
1 材料与方法
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1.1 菌株和载体
灰葡萄孢BC22野生型菌株、BcPDR1基因敲除突变体ΔBcpdr1以及互补菌株CE，pCR8克隆载体、pEarleyGate-103表达载体、农杆菌GV3101，均由河北省植物生理与分子病理学重点实验室保存并提供。
1.2 BcPDR1蛋白关键位点突变设计与载体构建
利用ClustalW 2软件(https://www.ebi.ac.uk/Tools/msa/clustalw2/)对BcPDR1蛋白及其同源蛋白的氨基酸序列进行比对，选择4个相对保守位点进行定点突变设计。其中，BcPDR1-M1敲除32-34位氨基酸，BcPDR1-M2敲除76-95位氨基酸，BcPDR1-M3敲除140-150位氨基酸，BcPDR1-M4将189位Ile突变为Lys。依据基因定点突变原理设计基因定点突变的引物(表1)。按照TRIzol (总RNA提取试剂，北京博迈德基因技术有限公司)及反转录试剂盒(TaKaRa公司)说明书，提取灰葡萄孢野生型BC22的总RNA，并反转录合成其cDNA。利用基因特异性引物(表1)进行PCR扩增。PCR反应体系(25 μL)：2×Super Pfx Master Mix 12.5 µL，上、下游引物(10 µmol/L)各1 µL，cDNA模板2 µL，ddH2O 8.5 µL。PCR反应条件：98 ℃预变性30 s；98 ℃变性10 s，60 ℃退火30 s，72 ℃延伸30 s，共29个循环；72 ℃终延伸5 min。电泳检测后，将其与pCR8克隆载体连接，对阳性克隆进行测序鉴定。将测序正确的定点突变基因与终载体pEarleyGate-103连接，经测序验证后，最终完成BcPDR1蛋白保守位点突变的载体构建。
1.3 突变体的构建与鉴定
利用根癌农杆菌介导的转化(Agrobacterium tumefaciens-mediated transformation, ATMT)技术将构建成功的载体分别转化到ΔBcpdr1中，经筛选获得阳性转化子。采用CTAB法提取转化子的基因组DNA，根据终载体上的GFP和BlpR基因设计特异性引物(表1)，对转化子进行PCR鉴定。PCR反应体系(20 μL)：2×Hieff® Ultra-Rapid HotStart PCR Master Mix 10 µL，上、下游引物(10 µmol/L)各0.5 µL，cDNA模板2 µL，ddH2O 7 µL。PCR反应条件：95 ℃预变性3 min；95 ℃变性30 s，65/60 ℃退火30 s，72 ℃延伸10 s，共34个循环；72 ℃终延伸5 min。同时，以转化子的cDNA为模板，利用基因特异性定量引物(表1)进行Real-time PCR鉴定，具体步骤根据通用荧光定量PCR试剂盒(Biosharp公司)说明书进行。最终获得BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4菌株。
1.4 突变体的生长发育表型分析
将BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4菌株以及野生型BC22、ΔBcpdr1和CE分别接种在PDA平板上，25 ℃培养3-14 d，分别对各菌株的菌落形态和菌丝形态进行细致观察，同时测量菌落的生长直径，统计其生长速率。
1.5 突变体的致病力分析
采用番茄果实和烟草叶片作为试验材料，对BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4菌株以及野生型BC22、ΔBcpdr1和CE进行致病力检测。将接种病原菌的材料置于黑暗条件下，保湿处理24-48 h后在正常条件下培养，每个菌株设置3个重复，观察各菌株的致病情况，并对各菌株的病斑面积进行统计分析。
2 结果与分析
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2.1 BcPDR1蛋白关键氨基酸突变的载体构建
为确定BcPDR1蛋白的关键氨基酸位点，将BcPDR1蛋白的氨基酸序列与TetR家族转录因子SmcR (PDB: 3KZ9)、HapR (PDB: 2PBX)、DesT (PDB: 3LSR)进行多序列比对。结果发现，BcPDR1与TetR家族转录因子之间存在多个相对保守区域，本研究选择32-34 aa、76-95 aa、140-150 aa和189 aa位点进行进一步地鉴定(图1A)。利用基因定点突变技术，构建BcPDR1蛋白定点突变的载体BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4 (图1B)。菌落PCR鉴定均得到了目的条带(图1C)，经测序鉴定后，成功获得了BcPDR1蛋白的关键氨基酸位点突变载体BcPDR1-M1、BcPDR1-M2、BcPDR1-M3、BcPDR1-M4。
2.2 BcPDR1蛋白关键氨基酸突变体的鉴定
通过ATMT转化和抗性筛选，获得了BcPDR1蛋白关键氨基酸位点突变的转化子BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4。在草铵膦抗性PDA上转化子均能正常生长，而ΔBcpdr1则不能正常生长(图2A)。利用重组载体上GFP、BlpR基因的特异性引物对转化子的基因组DNA进行PCR鉴定，发现转化子均能扩增出目的条带，而突变体ΔBcpdr1则未检测到目的条带，表明重组载体成功转入突变体ΔBcpdr1中并能稳定表达(图2B)。对转化子中BcPDR1的转录水平进行分析，发现与ΔBcpdr1相比，转化子中BcPDR1的表达水平均显著升高(图2C)，表明BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4突变体构建成功。
2.3 BcPDR1蛋白关键氨基酸突变体的表型分析
本研究对BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4点突变体菌株与野生型BC22、敲除突变体ΔBcpdr1以及互补菌株CE的表型进行对比观察。结果发现，与ΔBcpdr1的表型相比，4个点突变体菌株在菌落形态、菌丝形态以及生长速率方面均未呈现明显差异，但均显著区别于野生型BC22和互补菌株CE。相较于野生型BC22和互补菌株CE，4个点突变体以及敲除突变体ΔBcpdr1的菌落颜色更浅，气生菌丝相对更薄，在培养基上的生长情况呈现特定特征(图3A)；在菌丝形态方面，突变体的菌丝更为纤细，其菌丝细胞的长度和宽度均有所减小(图3B、3C)；在生长速率上，突变体明显慢于野生型BC22。其中，野生型BC22在第4天就能长满整个培养皿，而4个突变体均是在第5天才长满培养皿(图3D)。
2.4 BcPDR1蛋白关键氨基酸突变体的致病力分析
通过番茄果实和烟草叶片离体接种试验检测各菌株的致病力差异，结果发现4个突变体菌株(BcPDR1-M1、BcPDR1-M2、BcPDR1-M3和BcPDR1-M4)、野生型BC22以及互补菌株CE在接种后均能产生较为明显的病斑；与之形成鲜明对比的是，敲除突变体ΔBcpdr1在相同试验条件下均未产生肉眼可见的病斑。进一步对病斑面积进行量化分析发现，野生型BC22和互补菌株CE所引发的病斑面积最大，而4个点突变体菌株所造成的病斑面积均显著小于野生型BC22和互补菌株CE。在4个点突变体中，BcPDR1-M4引发的病斑面积相对最大，且明显大于BcPDR1-M1、BcPDR1-M2和BcPDR1-M3所产生的病斑面积(图4)。
3 讨论
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TetR家族转录因子具有独特的分子结构特征：其N端为DNA结合结构域，C端为配体结合结构域。这种独特的结构赋予了该家族成员双重功能，使其既能与DNA发生特异性结合，又可与多种配体分子相互作用[13]。当小分子配体与之结合时能够诱导TFRs发生构象变化，进而对靶基因的表达产生抑制或激活的调控作用[14]。正是基于这种结构-功能紧密耦合的特性，TFRs得以广泛参与形态分化、氮代谢、脂类代谢、群体感应、细菌毒力调控以及抗生素生物合成等多样化的生理过程，通常作为阻遏蛋白发挥关键的调控作用[15-16]。例如，在糖多孢红霉菌(Saccharopolyspora erythraea)中TetR家族转录因子SACE3446通过负调控红霉素生物合成相关基因抑制抗生素的产生[17]；而SACE_0012则通过抑制amfC同源基因SACE_7115调控形态发生[18]。在霍乱弧菌(Vibrio cholerae)中，HapR与LuxT作为群体感应调节剂[19]，其中LuxT可直接抑制HapR的转录，解除毒力级联抑制[20]。作为细菌体内核心转录因子，TFRs在信号转导和转录调控中发挥着不可替代的关键作用，被视为极具开发潜力的广谱新药靶点[21]。
然而，在植物病原真菌领域目前尚未有关于TetR家族转录因子的相关报道。本研究在灰葡萄孢中揭示了TetR成员的功能特征。尽管BcPDR1与经典的TetR成员SmcR、HapR的序列一致性极低，仅分别为21%和29%，但通过高级结构预测发现，它具有典型的TetR特征——由多个α-螺旋构成的同源二聚体构象，这为BcPDR1的家族归属提供了有力的结构生物学证据。本研究通过定点突变分析，精准定位了BcPDR1的4个关键功能区域：M1 (32-34 aa)可能参与DNA结合活性，M4 (189 aa)可能参与配体结合，而M2-M3 (76-95 aa和140-150 aa)则可能在维持蛋白构象稳定性方面发挥作用。表型分析显示，突变体在菌落形态、菌丝生长等方面与敲除菌株ΔBcpdr1表现一致，但致病力显著减弱，证实这些位点直接参与了致病调控过程。
本研究不仅在植物病原真菌中验证了TetR家族成员的功能位点，更通过结构-功能关联分析揭示了真菌特异性致病调控网络的关键节点。结合当前分子生物学与遗传学的最新进展，本研究后续将基于BcPDR1的关键位点开展靶向药物设计工作，例如设计配体小分子、抑菌肽[22]，以及运用RNA干扰技术(如采用外源dsRNA/siRNA的喷雾诱导基因沉默，即SIGS[23])。通过沉默BcPDR1或其下游效应因子，并充分利用外源dsRNA在植物-病原体系统中的传递特性，为实现灰霉病的高效精准防控以及开发新型杀菌剂提供全新思路，有望推动植物病理学与抗菌药物研发领域的交叉创新发展。
4 结论
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灰葡萄孢TetR家族转录因子BcPDR1的32-34 aa、76-95 aa、140-150 aa和189 aa位点突变导致菌落形态、生长速率与敲除株ΔBcpdr1相似，且致病力显著下降，表明BcPDR1的4个关键氨基酸位点(32-34 aa、76-95 aa、140-150 aa、189 aa)是其调控灰葡萄孢生长发育和致病力的关键调控位点。本研究结果为阐明TetR家族转录因子在植物病原真菌中的作用提供关键理论支撑，并为基于该家族的新型杀菌剂研发开辟了分子靶标新方向。
基金
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  • 国家自然科学基金(32072369)
  • 中央引导地方科技发展资金(246Z6506G)
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doi: 10.13343/j.cnki.wsxb.20250707
  • 接收时间：2025-09-16
  • 首发时间：2026-03-12
  • 出版时间：2026-03-04
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  • 收稿日期：2025-09-16
  • 录用日期：2025-10-28
基金
National Natural Science Foundation of China(32072369)
国家自然科学基金(32072369)
Central Guidance for Local Technology Development Funding(246Z6506G)
中央引导地方科技发展资金(246Z6506G)
作者信息
    1.河北省植物生理与分子病理学重点实验室，河北 保定
    2.华北作物改良与调控国家重点实验室，河北 保定

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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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