Article(id=1319998798219296962, tenantId=1146029695717560320, journalId=1316781310003478668, issueId=1319998767835754596, articleNumber=null, orderNo=null, doi=10.11983/CBB24135, pmid=null, cstr=32102.14.CBB24135, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1725379200000, receivedDateStr=2024-09-04, revisedDate=null, revisedDateStr=null, acceptedDate=1730217600000, acceptedDateStr=2024-10-30, onlineDate=1792641837886, onlineDateStr=2026-10-22, pubDate=1757433600000, pubDateStr=2025-09-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1792641837886, onlineIssueDateStr=2026-10-22, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1792641837886, creator=13701087609, updateTime=1792641837886, updator=13701087609, issue=Issue{id=1319998767835754596, tenantId=1146029695717560320, journalId=1316781310003478668, year='2025', volume='60', issue='5', pageStart='669', pageEnd='853', issueExtLink='null', onlineDate='null', pubDate='1757433600000', pubDateStr='2025-09-10', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1792641830643, creator='13701087609', updateTime=1792641943258, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1319999240240222831, tenantId=1146029695717560320, journalId=1316781310003478668, issueId=1319998767835754596, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1319999240240222832, tenantId=1146029695717560320, journalId=1316781310003478668, issueId=1319998767835754596, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=816, endPage=830, ext={EN=ArticleExt(id=1319998798441595075, articleId=1319998798219296962, tenantId=1146029695717560320, journalId=1316781310003478668, language=EN, title=Functional Verification of GhDIR1 Gene Against Verticillium Wilt in Cotton, columnId=1317485691135706105, journalTitle=Chinese Bulletin of Botany, columnName=RESEARCH ARTICLES, runingTitle=null, highlight=null, articleAbstract=

INTRODUCTION: Verticillium wilt (VW), caused by Verticillium dahliae, severely reduces cotton yield and fiber quality. Previous transcriptomic analysis in V. dahliae-inoculated Arabidopsis thaliana identified the pathogen-induced DIR1-like gene AT3G53980.2. In cotton, we discovered a homologous gene, GhDIR1 (Gh_A09G180700.1), encoding a lipid transfer protein. This study investigates its role in cotton resistance to V. dahliae.

RATIONALE: We characterized GhDIR1’s molecular features, expression patterns under pathogen stress, and functional impact using bioinformatics, subcellular localization, qRT-PCR, and virus-induced gene silencing (VIGS) analyses. Transcriptomic analysis of wild-type and GhDIR1-silenced plants were conducted to unravel downstream regulatory networks, focusing on metabolic pathways linked to plant immunity.

RESULTS: The results showed that GhDIR1 contains a 351 bp ORF encoding 116 amino acids. Subcellular localization confirmed its presence on the cell membrane. qRT-PCR showed rapid induction of GhDIR1 by V. dahliae. Silencing GhDIR1 increased cotton susceptibility to the pathogen. Transcriptomic data revealed that differentially expressed genes in silenced plants were enriched in flavonoid biosynthesis, sesquiterpene/triterpene biosynthesis, and α-linolenic acid metabolism. Key genes (GhCHS, GhDFR, GhCAD, GhSEQ, GhLOX, and GhAOC) in these pathways were downregulated, suggesting impaired synthesis of protective metabolites.

CONCLUSION: It is speculated that GhDIR1 positively regulates cotton resistance to VW by modulating flavonoid and terpenoid biosynthesis and jasmonic acid-related signaling. Its silencing disrupts critical defense pathways, highlighting its role in coordinating immune responses. These findings propose GhDIR1 as a potential target for enhancing disease resistance in cotton.

The induced expression pattern of GhDIR1 and related genes after inoculation with Verticillium dahliae.

, authors=Huang Yuxin1, Xie Tao2, Wang Xingfen3, Guo Huiming2, Cheng Hongmei2, Ma Bojun1, Chen Xifeng1, *, Su Xiaofeng2, *, authorsList=Huang Yuxin, Xie Tao, Wang Xingfen, Guo Huiming, Cheng Hongmei, Ma Bojun, Chen Xifeng, Su Xiaofeng, authorCompany=null, correspAuthors=Chen Xifeng, Su Xiaofeng, authorNote=null, correspAuthorsNote=
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由大丽轮枝菌(Verticillium dahliae)引起的黄萎病是棉花(Gossypium hirsutum)生产中最主要的威胁之一, 其可导致棉花大幅减产和纤维品质严重下降。前期对接种大丽轮枝菌的拟南芥(Arabidopsis thaliana)进行转录组分析, 表明DIR1类蛋白基因AT3G53980.2受病原菌强烈诱导表达。该研究发现, 棉花脂质转移蛋白编码基因GhDIR1 (Gh_A09G180700.1)与AT3G53980.2表现出高度的同源性。生物信息学分析表明, GhDIR1开放阅读框(ORF)为351 bp, 编码116个氨基酸残基。亚细胞定位结果显示GhDIR1定位于细胞膜。分析GhDIR1在大丽轮枝菌V991侵染后的表达模式, 发现其能快速响应大丽轮枝菌侵染。利用病毒诱导的基因沉默(VIGS)技术下调该基因表达后, 棉花对黄萎病菌的抗性显著降低。野生型和GhDIR1沉默植株转录组测序结果表明, 差异表达基因主要在类黄酮生物合成、倍半萜和三萜生物合成以及α-亚麻酸代谢3个途径富集; 同时, 荧光定量PCR结果表明, 3个途径中的6个关键基因(GhCHS、GhDFR、GhCAD、GhSEQ、GhLOX和GhAOC)在GhDIR1沉默植株中均下调表达, 与转录组数据一致。推测GhDIR1可能通过介导类黄酮和萜类化合物的合成途径, 并调节茉莉酸(JA)等植物激素的次级代谢来激活相关信号通路, 进而影响植株抗病性。综上, GhDIR1作为棉花抗黄萎病的正向调控因子, 通过参与多种激素和抗病信号网络调控植物的免疫反应。

, authors=黄雨欣1, 谢涛2, 王省芬3, 郭惠明2, 程红梅2, 马伯军1, 陈析丰1, *, 苏晓峰2, *, authorsList=黄雨欣, 谢涛, 王省芬, 郭惠明, 程红梅, 马伯军, 陈析丰, 苏晓峰, authorCompany=null, correspAuthors=陈析丰, 苏晓峰, authorNote=

中国农业科学院生物技术研究所微生物智能设计与合成创新团队程红梅课题组, 长期致力于作物黄萎病综合防治及新型基因编辑器在棉花中的评估和应用研究。近年来, 该课题组成功建立了病原菌精准监测预警方法, 系统解析了病原菌致病力、植物防御反应和环境微生物三者之间的互作机制, 从而利用合成生物学及纳米技术进行作物黄萎病的综合防治。课题组先后主持国家转基因重大专项、973计划、国家自然科学基金、公益性行业科技专项和国家重点研发等项目。在Nature Communications、Plant Biotechnology Journal、Chemical Engineering Journal和International Journal of Biological Macromolecules等国际知名及中文核心期刊上发表论文80余篇。获国家发明专利授权20余项, 为作物黄萎病的综合防控相关研究做出了重要贡献

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中国农业科学院生物技术研究所微生物智能设计与合成创新团队程红梅课题组, 长期致力于作物黄萎病综合防治及新型基因编辑器在棉花中的评估和应用研究。近年来, 该课题组成功建立了病原菌精准监测预警方法, 系统解析了病原菌致病力、植物防御反应和环境微生物三者之间的互作机制, 从而利用合成生物学及纳米技术进行作物黄萎病的综合防治。课题组先后主持国家转基因重大专项、973计划、国家自然科学基金、公益性行业科技专项和国家重点研发等项目。在Nature Communications、Plant Biotechnology Journal、Chemical Engineering Journal和International Journal of Biological Macromolecules等国际知名及中文核心期刊上发表论文80余篇。获国家发明专利授权20余项, 为作物黄萎病的综合防控相关研究做出了重要贡献

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中国农业科学院生物技术研究所微生物智能设计与合成创新团队程红梅课题组, 长期致力于作物黄萎病综合防治及新型基因编辑器在棉花中的评估和应用研究。近年来, 该课题组成功建立了病原菌精准监测预警方法, 系统解析了病原菌致病力、植物防御反应和环境微生物三者之间的互作机制, 从而利用合成生物学及纳米技术进行作物黄萎病的综合防治。课题组先后主持国家转基因重大专项、973计划、国家自然科学基金、公益性行业科技专项和国家重点研发等项目。在Nature Communications、Plant Biotechnology Journal、Chemical Engineering Journal和International Journal of Biological Macromolecules等国际知名及中文核心期刊上发表论文80余篇。获国家发明专利授权20余项, 为作物黄萎病的综合防控相关研究做出了重要贡献

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Int J Mol Sci 24, 5608., articleTitle=Virus-induced gene silencing (VIGS): a powerful tool for crop improvement and its advancement towards epigenetics, refAbstract=null)], funds=[Fund(id=1319998808050745636, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, awardId=2023ZD04039, language=CN, fundingSource=农业生物育种重大项目(2023ZD04039), fundOrder=null, country=null), Fund(id=1319998808130437413, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, awardId=LZ23C130004, language=CN, fundingSource=浙江省自然科学基金(LZ23C130004), fundOrder=null, country=null), Fund(id=1319998808201740582, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, awardId=21326314D, language=CN, fundingSource=河北省重点研发计划(21326314D), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1319998800895262933, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, xref=null, ext=[AuthorCompanyExt(id=1319998800899457238, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, companyId=1319998800895262933, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1College of Life Sciences, Zhejiang Normal University, Jinhua 321004, China), AuthorCompanyExt(id=1319998800907845847, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, companyId=1319998800895262933, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1浙江师范大学生命科学学院, 金华 321004)]), AuthorCompany(id=1319998802216468696, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, xref=null, ext=[AuthorCompanyExt(id=1319998802220663001, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, companyId=1319998802216468696, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2National Key Laboratory of Agricultural Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China), AuthorCompanyExt(id=1319998802229051610, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, companyId=1319998802216468696, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2中国农业科学院生物技术研究所农业微生物资源发掘与利用全国重点实验室, 北京 100081)]), AuthorCompany(id=1319998802308743387, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, xref=null, ext=[AuthorCompanyExt(id=1319998802317131996, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, companyId=1319998802308743387, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3College of Agriculture, Hebei Agricultural University, Baoding 071000, China), AuthorCompanyExt(id=1319998802329714909, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, companyId=1319998802308743387, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3河北农业大学农学院, 保定 071000)])], figs=[ArticleFig(id=1319998805039235344, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=EN, label=Figure 1, caption=Physicochemical properties of GhDIR1 and analysis of cis-acting element of the GhDIR1 gene promoter

(A) Hydrophilicity analysis of GhDIR1 protein; (B) GhDIR1 structural domain analysis; (C) Tertiary structure prediction of GhDIR1 protein; (D) Transmembrane structural domain prediction of GhDIR1 protein; (E) Analysis of cis-acting element of the GhDIR1 gene promoter

, figureFileSmall=kcoW50Zz7oCH8+sRjj9K/Q==, figureFileBig=89FHLfBZYEfXnwm9SfzVrg==, tableContent=null), ArticleFig(id=1319998805102149905, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=CN, label=图1, caption=GhDIR1蛋白质理化性质及基因上游启动子顺式作用元件分析

(A) GhDIR1蛋白亲疏水性分析; (B) GhDIR1结构域分析; (C) GhDIR1蛋白的三级结构预测; (D) GhDIR1蛋白跨膜结构域预测; (E) GhDIR1基因启动子顺式作用元件分析

, figureFileSmall=kcoW50Zz7oCH8+sRjj9K/Q==, figureFileBig=89FHLfBZYEfXnwm9SfzVrg==, tableContent=null), ArticleFig(id=1319998806888923414, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=EN, label=Figure 2, caption=Genome collinearity analysis of GhDIR1

Dark blue regions represent the chromosomes of Gossypium hirsutum. The gray lines depict all collinear pairs across the 26 chromosomes. The red lines highlight collinear gene pairs associated with segmental duplications of the target gene. The innermost and outermost light blue rings illustrate two distinct representations of gene density.

, figureFileSmall=H099eySUlAhLBuppRkXcBg==, figureFileBig=MDzA+FsK7vBicxKDNP3GXA==, tableContent=null), ArticleFig(id=1319998806968615191, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=CN, label=图2, caption=GhDIR1的基因组共线性分析

深蓝色区域为陆地棉染色体。灰线描绘了陆地棉26条染色体中的所有共线性对。红色线条为与目的基因存在片段重复的共线性基因对, 最内圈及最外圈浅蓝色区域为基因密度的2种不同表现形式。

, figureFileSmall=H099eySUlAhLBuppRkXcBg==, figureFileBig=MDzA+FsK7vBicxKDNP3GXA==, tableContent=null), ArticleFig(id=1319998807035724056, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=EN, label=Figure 3, caption=Expression of GhDIR1 in cotton inoculated with Verticillium dahliae

Heat map analysis of GhDIR1 gene expressions in different organs (roots, stems) and different inoculation times (0, 0.5, 1, 2, 4, 8, and 12 h) of Gossypium hirsutum with V. dahliae. The color from blue to yellow indicates low to high level of expression.

, figureFileSmall=2d88CcIkp+3ty7r+5BxdkQ==, figureFileBig=65UUJK+Nf+PFnxd2GVGKow==, tableContent=null), ArticleFig(id=1319998807107027225, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=CN, label=图3, caption=GhDIR1在接种大丽轮枝菌棉花中的表达

热图分析GhDIR1基因在不同器官(根、茎)和不同接种时间(0、0.5、1、2、4、8和12小时)棉花中的表达水平。从蓝色到黄色表示低表达到高表达。

, figureFileSmall=2d88CcIkp+3ty7r+5BxdkQ==, figureFileBig=65UUJK+Nf+PFnxd2GVGKow==, tableContent=null), ArticleFig(id=1319998807224467738, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=EN, label=Figure 4, caption=Subcellular localization of GhDIR1

The fusion proteins GFP and GhDIR1-GFP were co-localized with the PM-mCherry membrane localization marker in tobacco. The GFP and GhDIR1-GFP fusion proteins exhibited green fluorescence in the green fluorescence channel, while the PM-mCherry membrane localization marker displayed red fluorescence in the mCherry channel only. (A) GFP subcellular localization map (bars=20 μm); (B) GhDIR1-GFP subcellular localization map (bars=10 μm)

, figureFileSmall=cwxczlcodR3v2+SQ6e3Fyg==, figureFileBig=B/E/o5gPULGVmgfsqSRfLQ==, tableContent=null), ArticleFig(id=1319998807320936731, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=CN, label=图4, caption=GhDIR1的亚细胞定位

将GFP、GhDIR1-GFP融合表达蛋白分别与PM-mCherry膜定位marker共定位于烟草; GFP和GhDIR1-GFP融合表达蛋白在绿色荧光通道产生绿色荧光, PM-mCherry膜定位marker只在红色荧光通道mCherry中产生红色信号。(A) GFP亚细胞定位图(bars= 20 μm); (B) GhDIR1-GFP亚细胞定位图(bars=10 μm)

, figureFileSmall=cwxczlcodR3v2+SQ6e3Fyg==, figureFileBig=B/E/o5gPULGVmgfsqSRfLQ==, tableContent=null), ArticleFig(id=1319998807388045596, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=EN, label=Figure 5, caption=Detection of GhDIR1 gene silencing efficiency and the resistant analysis of GhDIR1 silenced seedlings against cotton Verticillium wilt

(A) TRV2::CLA1 plants (bar=5 cm); (B) Gene expression of GhDIR1 in silenced cotton plants (three biological replicates (n=3); ANOVA was used for the statistical analysis; *** P<0.001); (C) Phenotype of TRV2::00 and TRV2::GhDIR1 plants at 14 days post inoculation (dpi) with Verticillium dahliae (bar=5 cm); (D) Stem phenotype of TRV2::00 and TRV2::GhDIR1 plants at 14 dpi with V. dahliae (bar=1 cm); (E) Statistical analysis of disease index in cotton plants of TRV2::00 and TRV2::GhDIR1 at 14 dpi (grades 1-4 represent disease severity classifications. 1: Cotyledons turn yellow, no symptoms on true leaves; 2: All cotyledons show symptoms, 1-3 true leaves show necrosis or chlorosis; 3: More than 5 cotton leaves, including cotyledons, show symptoms; 4: All leaves show symptoms, and leaves fall off/plant dies); (F) Fungal biomass of TRV2::00 and TRV2::GhDIR1 plants at 14 dpi with V. dahliae (three biological replicates (n=3); ANOVA was used for the statistical analysis; *** P<0.001)

, figureFileSmall=EVPAtGlwcm0+c0QCPc35kQ==, figureFileBig=cGTKT3fSaA5K+qbuq2Bvwg==, tableContent=null), ArticleFig(id=1319998807463543069, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=CN, label=图5, caption=GhDIR1基因沉默效率检测及其对棉花黄萎病抗性的影响

(A) TRV2::CLA1植株(bar=5 cm); (B) GhDIR1在沉默组棉花植株中的表达水平(3次生物学重复(n=3); 统计分析方法为ANOVA; *** P<0.001); (C) 大丽轮枝菌侵染14天后TRV2::00和TRV2::GhDIR1植株表型(bar=5 cm); (D) 大丽轮枝菌侵染14天后TRV2::00和TRV2::GhDIR1茎秆表型(bar=1 cm); (E) 接种14天后TRV2::00和TRV2::GhDIR1棉花植株病情指数统计(1-4分别为病情指数分级。1: 子叶变黄, 真叶无病症; 2: 子叶全部出现病症, 1-3片真叶出现病斑; 3: 包括子叶在内, 超过5片棉花叶片出现病症; 4: 所有叶片均表现病症, 叶片脱落, 植株枯死); (F) 接种14天后TRV2::00和TRV2::GhDIR1棉花植株相对真菌生物量测定(3次生物学重复(n=3); 统计分析方法为ANOVA; *** P<0.001)

, figureFileSmall=EVPAtGlwcm0+c0QCPc35kQ==, figureFileBig=cGTKT3fSaA5K+qbuq2Bvwg==, tableContent=null), ArticleFig(id=1319998807551623454, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=EN, label=Figure 6, caption=RNA-seq comparison between TRV2::00 and TRV2::GhDIR1 cottons

(A) Heat map of differentially expressed genes (DEGs) in each sample; (B) Volcano map of DEGs; (C) GO terms; (D) KEGG terms. WT: Wild type; VIGS: Virus-induced gene silencing plant; BP: Biological process; CC: Cellular component; MF: Molecular function

, figureFileSmall=ksWyvskB2jXknJXLXBvZ8w==, figureFileBig=d0GqHPSU7ee8SehKgxdzaQ==, tableContent=null), ArticleFig(id=1319998807631315231, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=CN, label=图6, caption=TRV2::00和TRV2::GhDIR1棉花RNA-seq分析

(A) 每个样本中差异表达基因(DEGs)热图; (B) DEGs火山图; (C) GO分析; (D) KEGG分析。WT: 野生型; VIGS: 病毒诱导的基因沉默植株; BP: 生物过程; CC: 细胞成分; MF: 分子功能

, figureFileSmall=ksWyvskB2jXknJXLXBvZ8w==, figureFileBig=d0GqHPSU7ee8SehKgxdzaQ==, tableContent=null), ArticleFig(id=1319998807694229792, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=EN, label=Figure 7, caption=qRT-PCR verification of the expression levels of six key differentially expressed genes (DEGs)

The blue bars represent the relative expression levels of genes in TRV2::00, normalized to 1, serving as the control group. The purple bars indicate the relative expression levels of genes in TRV2::GhDIR1. Three biological replicates (n= 3). ANOVA was used for the statistical analysis. ** P<0.01; *** P<0.001

, figureFileSmall=zNFIHqr2+vJFUixY8K/j8w==, figureFileBig=tLpAEVomI9/S76fjODKilA==, tableContent=null), ArticleFig(id=1319998807761338657, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=CN, label=图7, caption=qRT-PCR验证6个关键差异表达基因(DEGs)的表达水平

蓝色柱子表示TRV2::00中基因相对表达水平, 标定为1, 作为对照组。紫色柱子为TRV2::GhDIR1中基因相对表达水平。3次生物学重复(n=3)。统计分析方法为ANOVA。** P<0.01; *** P<0.001

, figureFileSmall=zNFIHqr2+vJFUixY8K/j8w==, figureFileBig=tLpAEVomI9/S76fjODKilA==, tableContent=null), ArticleFig(id=1319998807857807650, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=EN, label=Table 1, caption=

Primer sequences used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Primer names Primer sequences (5′-3′)
GhDIR1-F ATGGCAGCTGCAATGAAACTC
GhDIR1-R TCAAGGAAGTGTATATGCTCCAC
qGhDIR1-F CCTGGGAAAGGTGGTTGATG
qGhDIR1-R TCTCAGGCTTGATTCCAGATGC
qGhCHS-F GCTGCTGTTATAGTAGGTGCGGATC
qGhCHS-R CTCAGCTAGGCTCTTTTCAATGTTC
qGhDFR-F GCTCTGGGTTCATTGGTTCATGG
qGhDFR-R CCTCTTCAGCTAAATCTGCTTTCC
qGhCAD-F CCTGGCATTTGGGGAGATATCTTC
qGhCAD-R GACACCCAACCTTTGGACTGC
qGhSEQ-F GCAAAGGAGAATGCTCGCCTG
qGhSEQ-R CCTGGCTGCAACAGTTCTCCAAC
qGhLOX-F GATCGTGCTTGATGGTCTCACTG
qGhLOX-R CGTTCCCTCTCAATGCCTGC
qGhAOC-F CGGATAGGAATAACAGCAGGGATG
qGhAOC-R GCCAGATCCACCAGTAATAGCG
Vd-ITS-F TCCGTAGGTGAACCTGCGG
Vd-ITS-R TCCTCCGCTTATTGATATGC
GhDIR1-VIGS-F
TCTGTGAGTAAGGTTACC GAATTCG- TTCTGGGATTGATTGTGCTTATT
GhDIR1-VIGS-R
ACGCGTGAGCTCGGTACC GGAT C C-
ATGCTCCACATCTGTAACCGACT
p1132-GhDIR1-F
TCTGTGAGTAAGGTTACC GAATTCC- CTGGGAAAGGTGGTTGATG
p1132-GhDIR1-R
ACGCGTGAGCTCGGTACC GGATCC-
TCTCAGGCTTGATTCCAGATGC
UBQ-F AGCTCGGATACGATTGATAACG
UBQ-R GAAGACGAAGAACAAGGGGAAG
), ArticleFig(id=1319998807958470947, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998798219296962, language=CN, label=表1, caption=

本研究所用引物

, figureFileSmall=null, figureFileBig=null, tableContent=
Primer names Primer sequences (5′-3′)
GhDIR1-F ATGGCAGCTGCAATGAAACTC
GhDIR1-R TCAAGGAAGTGTATATGCTCCAC
qGhDIR1-F CCTGGGAAAGGTGGTTGATG
qGhDIR1-R TCTCAGGCTTGATTCCAGATGC
qGhCHS-F GCTGCTGTTATAGTAGGTGCGGATC
qGhCHS-R CTCAGCTAGGCTCTTTTCAATGTTC
qGhDFR-F GCTCTGGGTTCATTGGTTCATGG
qGhDFR-R CCTCTTCAGCTAAATCTGCTTTCC
qGhCAD-F CCTGGCATTTGGGGAGATATCTTC
qGhCAD-R GACACCCAACCTTTGGACTGC
qGhSEQ-F GCAAAGGAGAATGCTCGCCTG
qGhSEQ-R CCTGGCTGCAACAGTTCTCCAAC
qGhLOX-F GATCGTGCTTGATGGTCTCACTG
qGhLOX-R CGTTCCCTCTCAATGCCTGC
qGhAOC-F CGGATAGGAATAACAGCAGGGATG
qGhAOC-R GCCAGATCCACCAGTAATAGCG
Vd-ITS-F TCCGTAGGTGAACCTGCGG
Vd-ITS-R TCCTCCGCTTATTGATATGC
GhDIR1-VIGS-F
TCTGTGAGTAAGGTTACC GAATTCG- TTCTGGGATTGATTGTGCTTATT
GhDIR1-VIGS-R
ACGCGTGAGCTCGGTACC GGAT C C-
ATGCTCCACATCTGTAACCGACT
p1132-GhDIR1-F
TCTGTGAGTAAGGTTACC GAATTCC- CTGGGAAAGGTGGTTGATG
p1132-GhDIR1-R
ACGCGTGAGCTCGGTACC GGATCC-
TCTCAGGCTTGATTCCAGATGC
UBQ-F AGCTCGGATACGATTGATAACG
UBQ-R GAAGACGAAGAACAAGGGGAAG
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棉花抗黄萎病相关基因GhDIR1的生物学功能分析
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黄雨欣 1 , 谢涛 2 , 王省芬 3 , 郭惠明 2 , 程红梅 2 , 马伯军 1 , 陈析丰 1, * , 苏晓峰 2, *
植物学报 | 研究论文 2025,60(5): 816-830
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植物学报 |研究论文 2025 , 60 (5) : 816 -830
棉花抗黄萎病相关基因GhDIR1的生物学功能分析
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中国农业科学院生物技术研究所微生物智能设计与合成创新团队程红梅课题组, 长期致力于作物黄萎病综合防治及新型基因编辑器在棉花中的评估和应用研究。近年来, 该课题组成功建立了病原菌精准监测预警方法, 系统解析了病原菌致病力、植物防御反应和环境微生物三者之间的互作机制, 从而利用合成生物学及纳米技术进行作物黄萎病的综合防治。课题组先后主持国家转基因重大专项、973计划、国家自然科学基金、公益性行业科技专项和国家重点研发等项目。在Nature Communications、Plant Biotechnology Journal、Chemical Engineering Journal和International Journal of Biological Macromolecules等国际知名及中文核心期刊上发表论文80余篇。获国家发明专利授权20余项, 为作物黄萎病的综合防控相关研究做出了重要贡献

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中国农业科学院生物技术研究所微生物智能设计与合成创新团队程红梅课题组, 长期致力于作物黄萎病综合防治及新型基因编辑器在棉花中的评估和应用研究。近年来, 该课题组成功建立了病原菌精准监测预警方法, 系统解析了病原菌致病力、植物防御反应和环境微生物三者之间的互作机制, 从而利用合成生物学及纳米技术进行作物黄萎病的综合防治。课题组先后主持国家转基因重大专项、973计划、国家自然科学基金、公益性行业科技专项和国家重点研发等项目。在Nature Communications、Plant Biotechnology Journal、Chemical Engineering Journal和International Journal of Biological Macromolecules等国际知名及中文核心期刊上发表论文80余篇。获国家发明专利授权20余项, 为作物黄萎病的综合防控相关研究做出了重要贡献

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黄雨欣1, 谢涛2, 王省芬3, 郭惠明2, 程红梅2, 马伯军1, 陈析丰1, * , 苏晓峰2, *
作者信息
  • 1浙江师范大学生命科学学院, 金华 321004
  • 2中国农业科学院生物技术研究所农业微生物资源发掘与利用全国重点实验室, 北京 100081
  • 3河北农业大学农学院, 保定 071000
通讯作者:
作者简介:

中国农业科学院生物技术研究所微生物智能设计与合成创新团队程红梅课题组, 长期致力于作物黄萎病综合防治及新型基因编辑器在棉花中的评估和应用研究。近年来, 该课题组成功建立了病原菌精准监测预警方法, 系统解析了病原菌致病力、植物防御反应和环境微生物三者之间的互作机制, 从而利用合成生物学及纳米技术进行作物黄萎病的综合防治。课题组先后主持国家转基因重大专项、973计划、国家自然科学基金、公益性行业科技专项和国家重点研发等项目。在Nature Communications、Plant Biotechnology Journal、Chemical Engineering Journal和International Journal of Biological Macromolecules等国际知名及中文核心期刊上发表论文80余篇。获国家发明专利授权20余项, 为作物黄萎病的综合防控相关研究做出了重要贡献

Functional Verification of GhDIR1 Gene Against Verticillium Wilt in Cotton
Huang Yuxin1, Xie Tao2, Wang Xingfen3, Guo Huiming2, Cheng Hongmei2, Ma Bojun1, Chen Xifeng1, * , Su Xiaofeng2, *
Affiliations
  • 1College of Life Sciences, Zhejiang Normal University, Jinhua 321004, China
  • 2National Key Laboratory of Agricultural Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • 3College of Agriculture, Hebei Agricultural University, Baoding 071000, China
  • Corresponding Author:
出版时间: 2025-09-10 doi: 10.11983/CBB24135
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由大丽轮枝菌(Verticillium dahliae)引起的黄萎病是棉花(Gossypium hirsutum)生产中最主要的威胁之一, 其可导致棉花大幅减产和纤维品质严重下降。前期对接种大丽轮枝菌的拟南芥(Arabidopsis thaliana)进行转录组分析, 表明DIR1类蛋白基因AT3G53980.2受病原菌强烈诱导表达。该研究发现, 棉花脂质转移蛋白编码基因GhDIR1 (Gh_A09G180700.1)与AT3G53980.2表现出高度的同源性。生物信息学分析表明, GhDIR1开放阅读框(ORF)为351 bp, 编码116个氨基酸残基。亚细胞定位结果显示GhDIR1定位于细胞膜。分析GhDIR1在大丽轮枝菌V991侵染后的表达模式, 发现其能快速响应大丽轮枝菌侵染。利用病毒诱导的基因沉默(VIGS)技术下调该基因表达后, 棉花对黄萎病菌的抗性显著降低。野生型和GhDIR1沉默植株转录组测序结果表明, 差异表达基因主要在类黄酮生物合成、倍半萜和三萜生物合成以及α-亚麻酸代谢3个途径富集; 同时, 荧光定量PCR结果表明, 3个途径中的6个关键基因(GhCHS、GhDFR、GhCAD、GhSEQ、GhLOX和GhAOC)在GhDIR1沉默植株中均下调表达, 与转录组数据一致。推测GhDIR1可能通过介导类黄酮和萜类化合物的合成途径, 并调节茉莉酸(JA)等植物激素的次级代谢来激活相关信号通路, 进而影响植株抗病性。综上, GhDIR1作为棉花抗黄萎病的正向调控因子, 通过参与多种激素和抗病信号网络调控植物的免疫反应。

陆地棉  /  棉花黄萎病  /  GhDIR1  /  病毒诱导的基因沉默  /  转录组分析

INTRODUCTION: Verticillium wilt (VW), caused by Verticillium dahliae, severely reduces cotton yield and fiber quality. Previous transcriptomic analysis in V. dahliae-inoculated Arabidopsis thaliana identified the pathogen-induced DIR1-like gene AT3G53980.2. In cotton, we discovered a homologous gene, GhDIR1 (Gh_A09G180700.1), encoding a lipid transfer protein. This study investigates its role in cotton resistance to V. dahliae.

RATIONALE: We characterized GhDIR1’s molecular features, expression patterns under pathogen stress, and functional impact using bioinformatics, subcellular localization, qRT-PCR, and virus-induced gene silencing (VIGS) analyses. Transcriptomic analysis of wild-type and GhDIR1-silenced plants were conducted to unravel downstream regulatory networks, focusing on metabolic pathways linked to plant immunity.

RESULTS: The results showed that GhDIR1 contains a 351 bp ORF encoding 116 amino acids. Subcellular localization confirmed its presence on the cell membrane. qRT-PCR showed rapid induction of GhDIR1 by V. dahliae. Silencing GhDIR1 increased cotton susceptibility to the pathogen. Transcriptomic data revealed that differentially expressed genes in silenced plants were enriched in flavonoid biosynthesis, sesquiterpene/triterpene biosynthesis, and α-linolenic acid metabolism. Key genes (GhCHS, GhDFR, GhCAD, GhSEQ, GhLOX, and GhAOC) in these pathways were downregulated, suggesting impaired synthesis of protective metabolites.

CONCLUSION: It is speculated that GhDIR1 positively regulates cotton resistance to VW by modulating flavonoid and terpenoid biosynthesis and jasmonic acid-related signaling. Its silencing disrupts critical defense pathways, highlighting its role in coordinating immune responses. These findings propose GhDIR1 as a potential target for enhancing disease resistance in cotton.

The induced expression pattern of GhDIR1 and related genes after inoculation with Verticillium dahliae.

Gossypium hirsutum  /  cotton Verticillium wilt  /  GhDIR1  /  virus-induced gene silencing  /  transcriptome analysis
黄雨欣, 谢涛, 王省芬, 郭惠明, 程红梅, 马伯军, 陈析丰, 苏晓峰. 棉花抗黄萎病相关基因GhDIR1的生物学功能分析. 植物学报, 2025 , 60 (5) : 816 -830 . DOI: 10.11983/CBB24135
Huang Yuxin, Xie Tao, Wang Xingfen, Guo Huiming, Cheng Hongmei, Ma Bojun, Chen Xifeng, Su Xiaofeng. Functional Verification of GhDIR1 Gene Against Verticillium Wilt in Cotton[J]. Chinese Bulletin of Botany, 2025 , 60 (5) : 816 -830 . DOI: 10.11983/CBB24135
棉花(Gossypium hirsutum)是中国种植业生产中最重要的经济作物之一, 且为国家经济和人民生活息息相关的战略物资(卢秀茹等, 2018)。大丽轮枝菌(Verticillium dahliae)是一种土传病原体, 通过根部侵入棉花体内, 并在植株维管束组织中大量增殖, 导致棉花黄萎病, 进而造成其平均产量损失高达35% (Song et al., 2020)。大丽轮枝菌的菌丝寄生于植株维管束组织中, 难以被消灭, 目前尚未开发出有效的杀菌剂来保护棉花免受大丽轮枝菌侵染(Fradin and Thomma, 2006)。因此, 挖掘棉花抗黄萎病相关基因, 选育新型棉花抗黄萎病品种是一种经济、高效且环保的策略(Mo et al., 2015)。
植物在长期进化过程中形成了多层面的防御机制来抵御病原体侵害。除了模式触发免疫(pattern- triggered immunity, PTI)和效应子触发免疫(effector triggered immunity, ETI), 植物还具备系统获得性抗性(systemic acquired resistance, SAR), 它使得植物在初次遭受病原体攻击后, 能够从整体上建立起一种增强、广谱且持久的抗性机制(王健等, 2018)。在SAR的激活过程中, 脂质转移蛋白DIR1 (defective in induced resistance 1)与甘油酯或脂质衍生物之间相互作用形成的复合物发挥至关重要的作用。DIR1能与脂质衍生分子相互作用, 促进长距离信号转导, 并在移动信号的产生、传递或感知中发挥关键作用(Maldonado et al., 2002)。此外, DIR1参与植物抗病。例如, 在拟南芥(Arabidopsis thaliana)中, 甘油-3-磷酸(glycerol-3-phosphate, G3P)、DIR1及另一个预测的脂质转移蛋白AZI1 (azelaic acid induced 1)相互作用形成精细的调控网络, 通过调节植物体内的信号分子和代谢物影响植物的免疫反应(Yu et al., 2013)。Liu等(2011)研究发现, 当烟草(Nicotiana tabacum)中的NtDIR1基因被沉默后, SAR机制无法正常激活。Maldonado等(2002)研究表明, 拟南芥dir1-1突变体无法从接种假性单胞菌的部位向未接种部位传递SAR信号, 使得植株整体抗病性降低, 说明在拟南芥中DIR1可以通过影响SAR信号途径来调节植株的抗病性。David等(2021)采用多组学方法对拟南芥气孔保卫细胞的SAR信号机制进行研究, 发现DIR1可能参与保卫细胞SAR过程中氨基酸的生物合成和碳代谢, 并作为连接植物基本代谢过程与免疫反应之间的桥梁。综上所述, DIR1基因在调控植物免疫系统中扮演着关键角色, 对植物的抗病性起至关重要的作用。此外, 植物可以合成各种次级代谢物分子, 包括类黄酮类、萜类和苯丙素类化合物以及生物碱等, 通过诱导相关基因的表达抵抗潜在的微生物侵害(Sanchez and Demain, 2008)。作为酚类化合物的主要类群和植物中最丰富的次生代谢产物之一, 类黄酮类化合物对植物生长及逆境适应具有显著影响, 其通过调控生长素的极性运输和参与自由基的清除过程, 帮助植物应对各类生物及非生物胁迫(Gautam et al., 2023; 谢靖雯等, 2024)。作为植物次生代谢物的重要组成部分, 萜类化合物参与构建植物的防御体系, 协助抵御病原体和植食性昆虫的攻击(Tholl, 2015)。类黄酮类和萜类化合物在植物生长发育以及防御和适应环境中发挥关键作用(董燕梅等, 2020; Huang et al., 2021)。植物激素及其信号串扰在植物的防御机制中扮演着核心角色(Waadt et al., 2022)。茉莉酸(jasmonic acid, JA)通过调节植物的抗病信号转导, 与SAR信号途径交叉作用, 共同调控植物防御基因的表达, 增强植物的抗病性(Anderson et al., 2004; Yang et al., 2015)。茉莉酸甲酯(methyl jasmonate, MeJA)作为JA的衍生物, 通过甲基化反应合成, 二者能够在植物体内相互转化(Seo et al., 2001), 它们作为植物体内的信号分子参与多种生理过程, 包括抑制病原体生长、传递抗性信号以及诱导防御化合物的合成(Harms et al., 1995; Gfeller et al., 2010; Liu et al., 2019)。亚麻酸作为合成JA和MeJA的关键前体(Huang et al., 2017), 其代谢过程对植物的免疫反应至关重要。
前期, 我们对接种大丽轮枝菌及对照组拟南芥进行转录组分析, 鉴定出一系列差异表达基因(Su et al., 2018), 其中DIR1类蛋白基因AT3G53980.2受大丽轮枝菌诱导表达变化显著。本研究中, 我们通过序列比对, 发现棉花脂质转移蛋白基因GhDIR1 (Gh_ A09G180700.1)与AT3G53980.2高度同源; 通过对GhDIR1基因的结构、染色体定位以及编码蛋白的理化性质等进行分析, 明确了其基本特征; 利用亚细胞定位揭示了该基因编码蛋白在细胞中的分布; 通过实时荧光定量PCR确定了GhDIR1在黄萎病菌侵染后的表达模式。此外, 结合病毒诱导的基因沉默(virus- induced gene silencing, VIGS)技术和RNA-seq分析, 进一步阐明了GhDIR1在棉花对黄萎病抗性中的分子作用机制。该研究丰富了对棉花抗病性分子机制的认识, 同时为棉花抗病育种提供了宝贵的基因资源。
本研究所用植物材料为陆地棉品种R15 (Gossypium hirsutum L. ‘R15’); 温室种植条件: 光周期为14小时光照/10小时黑暗, 相对湿度维持在60%-70%, 温度控制在22°C, 光照强度约为150 μmoL·m-2·s-1。棉花基因沉默所需载体TRV1、TRV2和TRV2-CLA1均由本实验室保存; 大肠杆菌DH5α和Trans1-T1感受态细胞及农杆菌GV3101感受态细胞购自北京全式金生物技术有限公司; 大丽轮枝菌强致病性菌系V991由中国农业科学院植物保护研究所简桂良研究员惠赠。
按照RNAprep Pure多糖多酚植物总RNA提取试剂盒(北京天根生化科技有限公司)说明书提取棉花根部组织RNA, 并参照反转录试剂盒PrimeScript™ RT reagent Kit (Perfect Real Time, 北京宝日医生物技术有限公司)说明书合成cDNA。通过blastP在NCBI中获得GhDIR1基因CDS序列(351 bp), 设计基因上、下游扩增引物GhDIR1-F和GhDIR1-R (表1), 利用2× Phanta Flash Master Mix (Dye Plus, 南京诺唯赞生物科技有限公司)扩增目的片段, PCR产物经1%琼脂糖凝胶电泳验证后, 使用通用型DNA纯化回收试剂盒(DP214, 北京天根生化科技有限公司)回收。之后送生工生物进行Sanger测序以确保目的基因的正确性。
利用在线软件SMART (http://smart.embl-heidelberg.de/)对GhDIR1蛋白保守结构域进行预测; 利用棉花数据库CottonFGD (https://cottonfgd.net//)获得陆地棉的染色体位置及注释信息; 利用在线工具SWISS-MODEL (https://swissmodel.expasy.org/)分析GhDIR1蛋白的三级结构; 利用Expasy在线工具(http://web.expasy.org/protparam/)对GhDIR1蛋白的分子量、等电点、氨基酸、原子组成、不稳定系数和亲水性等进行分析; 通过在线工具TMHMM 2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/)分析GhDIR1蛋白的跨膜结构; 利用在线软件PlantCARE (https://bioinformatics.psb.ugent.be/we- btools/plantcare/html/)对GhDIR1基因的启动子元件进行分析。
将2 mL大丽轮枝菌V991菌液加入40 mL含卡那霉素(kanamycin, Kan)和羧苄青霉素(carbenicillin, Car)抗性的CM培养基(6 g·L-1酵母提取物, 6 g·L-1酸水解酪蛋白, 10 g·L-1蔗糖)中, 置于28°C恒温摇床中220 r·min-1培养5-6天。用40 μm细胞滤器过滤菌液中的菌丝体, 获得孢子液。在光学显微镜下观察并统计孢子数量, 调整孢子浓度至107 cfu·mL-1。将生长至两叶一心期的棉花根部用清水洗净后完全浸入孢子悬浮液中, 浸染5分钟后取出, 包裹于湿纱布中。在0、0.5、2、4、8和12小时时间点分别取样根组织和茎组织。
提取经大丽轮枝菌侵染的不同时间点棉花各组织RNA, 经1%琼脂糖凝胶电泳检测其完整性后反转录合成cDNA, 作为模板用于后续的扩增反应。以棉花持家基因Polyubiquitin (LOC107918137)作为内参基因, 设计检测引物UBQ-F/R及qGhDIR1-F/R (表1), 并利用Taq Pro Universal SYBR qPCR Master Mix qRT-PCR试剂盒(南京诺唯赞生物科技有限公司)检测棉花根和茎中GhDIR1基因在接种菌液后的相对表达水平。每个样本设置3个重复, 采用2-ΔΔCT方法计算基因的相对表达量(Livak and Schmittgen, 2001)。
设计引物p1132-GhDIR1-F/R (表1)特异性扩增GhDIR1基因的开放阅读框(ORF)序列, 将其插入含绿色荧光蛋白(green fluorescent protein, GFP)的表达载体pYBA1132中, 将重组质粒转化农杆菌感受态GV3101, 培养2-3天后, 挑取阳性转化子进行扩繁。菌液在28°C恒温摇床中180 r·min-1培养至OD600为0.4-0.6, 用重悬液重悬菌体至其OD600=0.8, 室温静置3小时后对培养至5-6片真叶的烟草进行注射, 注射完成后暗处理48小时; 剪取烟草叶片下表皮, 以PM-mCherry膜定位标记蛋白为对照, 在LSM980共聚焦激光扫描显微镜(Zeiss, Jena, Germany)下观察荧光亚细胞定位情况。
设计引物GhDIR1-VIGS-F/R (表1)特异性扩增GhDIR1基因VIGS片段, 利用限制性核酸内切酶EcoRI和BamHI (NEB北京有限公司)酶切表达载体TRV2, 将扩增片段插入TRV2酶切位点EcoRI/BamHI之间, 使用Uniclone One Step Seamless Cloning Kit (北京金沙生物有限公司)连接酶连接; 构建重组载体TRV2::GhDIR1, 通过MolPure® Plasmid Mini Kit质粒小量提取试剂盒(翌圣生物科技上海股份有限公司)对阳性克隆进行质粒抽提, 并转化质粒至农杆菌感受态细胞GV3101中。待棉花植株生长到2片子叶完全展开而真叶尚未露出时, 活化空载体TRV1、TRV2::00、TRV2::CLA1 (棉花白化对照)及TRV2::GhDIR1农杆菌菌液。吸取1 mL活化菌液加入含Kan和利福平(rifampin, Rif)抗性的LB培养基中培养至OD600为0.4-0.6, 用重悬液重悬菌体至其OD600=0.8。将重悬液TRV1分别与重悬液TRV2::00、TRV2::CLA1和TRV2::GhDIR1等体积混合, 室温静置3小时以上。将静置处理后的混合菌液注射入棉花子叶, 对注射后的棉花暗处理24小时(Fan et al., 2021)。待第2片真叶出现明显白化现象时, 从对照组TRV2::00及沉默组TRV2::GhDIR1中选取生长状况相似的植株, 提取真叶RNA, 反转录为cDNA后利用引物qGhDIR1-F/R (表1)经qRT-PCR检测GhDIR1基因的沉默效率。
选取生长状况一致的TRV2::00和TRV2::GhDIR1棉花材料各50株, 同时接种大丽轮枝菌V991。将棉花从土壤中完整拔出, 洗净根部泥土, 然后在浓度为107 cfu·mL-1的孢子悬浮液中浸泡5分钟, 之后重新种植回新的营养土中。接种14天后观察各组植株表型并拍照记录, 依据病情指数分级方法对植株病情进行详细统计(李名江, 2021)。同时, 对沉默植株和阴性对照植株进行剖秆检测(胡子曜等, 2022)。剪取不同处理组棉花植株根部组织, 提取DNA, 以棉花持家基因Polyubiquitin (LOC107918137)作为内参基因, 设计检测引物Vd-ITS-F/R (表1), 通过qRT-PCR检测接菌后棉花植株所含真菌的相对生物量。
对TRV2::00和TRV2::GhDIR1棉花植株进行取样, 每个样本设置3个生物学重复, 提取RNA并进行RNA- seq。原始数据经过滤筛选后, 得到用于后续分析的精确数据, 使用|Log2(Fold Change)|≥1和padj≤0.05的标准得到差异表达基因(differentially expressed genes, DEGs) (Thayale Purayil et al., 2020)。利用GO、KEGG和GSEA对DEGs进行分析和功能注释。为了进一步确定GhDIR1是否参与JA生物合成和相关信号通路, 对JA合成和信号通路相关的6个关键基因GhCHS (Gh_D02G036800)、GhDFR (Gh_A05G175900)、GhCAD (Gh_A01G225700)、GhSEQ (Gh_A02G180100)、GhLOX (Gh_D05G070200)和GhAOC (Gh_D08G042500)进行qRT-PCR检测。引物序列见表1。
对GhDIR1蛋白进行理化性质分析, 预测得到该蛋白的分子式C512H867N145O154S12, 分子量为11.90312 kDa, 理论等电点为8.84, 丙氨酸(Ala)在氨基酸残基中出现频率最高(达17.2%), 而酪氨酸(Tyr)含量最低(仅为1.7%)。不稳定系数为33.35, 小于40, 说明该蛋白相对稳定。脂肪族氨基酸指数为9.76, 平均亲水性为0.308, 根据Expasy-ProScale对蛋白质亲疏水性进行进一步预测(图1A), 发现GhDIR1氨基酸多肽链的亲水性最大值为4.50, 最小值为-3.90, 表明该蛋白为疏水性蛋白。对GhDIR1蛋白结构域进行分析, 结果显示在其第41-101位氨基酸之间含有1个AAI保守结构域(图1B)。对GhDIR1三级结构进行预测(图1C), 发现其氨基酸序列中无规则卷曲(Cc)含量最高(达44.83%), 其次为α-螺旋(Hh)(含量为38.79%), 延伸链(Ee)含量为12.07%, β-转角(Tt)含量最低(仅为4.31%)。GhDIR1蛋白跨膜结构域预测结果显示, 该蛋白不存在跨膜结构域(图1D)。利用NCBI数据库在线软件选取GhDIR1基因启动子上游大约2 Kb序列, 将序列提交至PlantCARE进行顺式作用元件预测分析, 整理分析结果后通过Tbtools的Simple BioSequence Viewer功能绘图。结果显示, 该基因启动子区除具有CAAT-box和TATA-box等植物常见的顺式作用元件以外, 还具有多项参与调控植物发育和抗逆过程的元件(如MYC、MYB和O2-site)。此外, 启动子中还含有与水杨酸(salicylic acid, SA)应答相关的元件as-1以及CGTCA-motif, MeJA应答相关元件TGACG-motif, 光响应元件AE-box、LAMP-element和l-box等(图1E)。推测在大丽轮枝菌胁迫下, GhDIR1的表达可能受到这些激素诱导元件的调控从而对黄萎病产生抗性。
从参考棉花数据库CottonFGD中获得陆地棉的染色体位置及注释信息。利用TBtools中的多重共线性可视化工具(MCS-scanX)分析GhDIR1基因的共线性(Yang et al., 2023)。使用TBtools (v2.019)中的Advance Circos绘制共线性图。结果显示, GhDIR1位于染色体A09上, 与另2个基因之间分别存在共线性关系(Gh_A09G180700.1和Gh_A11G376500.1, Gh_A09G180700.1和Gh_D09G171800.1) (图2), 说明这2组基因对中的基因存在片段重复, 揭示了基因重复在基因数量增加及其进化中的关键作用, 对于种内基因家族的扩张与功能分化具有重要影响。
利用qRT-PCR检测接种大丽轮枝菌V991后不同时间点棉花植株根和茎中GhDIR1的相对表达水平。结果显示, 在接菌后0.5小时, 该基因在棉花根部的表达水平显著上调, 并在1小时达到峰值, 随后逐渐下降。相比之下, GhDIR1在茎部几乎不受诱导表达(图3)。上述结果表明, 棉花根部GhDIR1受到大丽轮枝菌诱导表达, 推测其可能在调控棉花对黄萎病免疫应答中发挥重要作用。
生物信息学预测GhDIR1定位于细胞膜。为了进一步验证预测结果, 构建重组质粒pYBA1132-GhDIR1- GFP, 并通过农杆菌介导转化法将其导入烟草叶片。利用激光共聚焦显微镜进行观察, 结果表明, 作为对照, 在GFP和PM-mCherry共转化的细胞中, 荧光均匀且分散地分布在细胞质、质膜和细胞核中(图4)。而GhDIR1-GFP蛋白和PM-mCherry在细胞膜中共定位, 这一结果与生物信息学预测结果一致。
为进一步鉴定GhDIR1的功能, 利用VIGS技术在棉花中沉默该基因。对棉花植株注射TRV2::CLA1菌液, 14天后观察到植株真叶出现明显的白化表型(图5A), 说明已成功建立了VIGS技术体系。通过qRT-PCR检测GhDIR1基因沉默效率, 结果显示GhDIR1基因表达下调70%左右(图5B), 说明基因沉默效果显著, 可进一步对GhDIR1相关功能进行分析。依据病情指数计算公式对棉花病情指数进行统计, 发现在接种大丽轮枝菌后, 与对照组相比, 沉默组棉花植株整体表现出更严重的黄萎病症, 叶片出现大面积萎蔫黄化脱落(图5C)。将沉默组和对照组棉花茎秆进行剖秆处理, 发现与对照组相比, 沉默组植株茎秆出现更明显的黄褐色纹(图5D), 病情指数及相对真菌生物量显著提高(图5E, F)。以上结果表明, GhDIR1沉默导致棉花抵抗黄萎病菌入侵和定殖的能力减弱, 该基因正向调控棉花对黄萎病的抗性。
分析TRV2::00和TRV2::GhDIR1植株转录组数据, 共获得15 938个DEGs (9 755个上调和6 183个下调)。为了直观地展示这些DEGs的表达模式及其在平行样本组中的可重复性, 构建了用于聚类分析的热图(图6A)。火山图进一步揭示了15 938个DEGs之间的差异程度(图6B)。DEGs反映了GhDIR1沉默引起的变化。GO聚类分析显示, 在生物过程方面, DEGs在抗病性密切相关的代谢途径中高度富集; 细胞成分方面, 这些DEGs与核小体、细胞壁、外部封装结构和质外体等有关; 在分子功能层面, DEGs涉及微管动力蛋白活性、萜烯合酶活性和裂解酶活性等, 进一步凸显了它们在植物抗病机制中的重要性(图6C)。KEGG富集分析显示, TRV2::GhDIR1棉花中的DEGs富集于多个抗病相关途径, 如类黄酮生物合成、倍半萜类和三萜类生物合成和α-亚麻酸代谢(图6D)。上述功能注释表明, GhDIR1沉默影响植物激素和次生代谢物合成相关途径, 这可能进一步改变棉花对大丽轮枝菌的抗性。综上表明, GhDIR1沉默可能导致相关生理和分子途径发生变化, 从而在调控棉花抗病性方面发挥关键作用。
为验证转录组数据的可靠性, 从DEGs中筛选了6个关键基因: GhCHS和GhDFR (类黄酮生物合成途径), GhCAD和GhSEQ (倍半萜类化合物和三萜类化合物生物合成途径), GhLOX和GhAOC (α-亚麻酸代谢途径), 对TRV2::00和TRV2::GhDIR1植株进行qRT- PCR检测, 结果显示这6个与植物抗病性密切相关的基因均下调表达(图7)。qRT-PCR验证结果与RNA- seq结果一致, 进一步证实了转录组数据的真实性和可靠性。上述结果表明, GhDIR1通过调控这3个富含DEGs的代谢途径, 正向调节陆地棉对大丽轮枝菌的抗性。
本研究从陆地棉R15中克隆到GhDIR1基因, 生物信息学分析发现在其编码蛋白第41-101位氨基酸之间含有1个AAI保守结构域。在拟南芥非特异性脂质转移蛋白AtLTP2 (Jacq et al., 2017)和棉花GhAAI66 (Qanmber et al., 2019)蛋白中均存在该结构域, 表明其可能是植物脂质转移蛋白特有的结构域。AAI家族在保护植物免受昆虫和病原体侵害、细胞内膜之间的脂质运输和营养储存方面发挥重要作用(Qanmber et al., 2019)。亚细胞定位结果显示, GhDIR1定位于细胞膜, 与水稻脂质转移蛋白nsLTP144 (汪笑峰, 2007)和OsLTP47 (Chen et al., 2022)定位结果一致, 符合脂质转移蛋白定位特征。对GhDIR1表达模式进行分析, 发现在大丽轮枝菌V991侵染棉花植株后, 该基因在根部的表达水平呈显著的先上升后下降趋势。这一表达模式与已有研究的棉花抗黄萎病基因GhNAC (张曼等, 2023)和GhRAR1 (胡晓倩等, 2023)表达模式相似, 因此推测GhDIR1可能在参与棉花抗黄萎病中起到关键作用。
VIGS技术是20世纪90年代开始兴起的一种由植物双链RNA (double stranded RNA, dsRNA)介导的反向遗传学技术, 已发展成为基因功能分析不可或缺的方法(Zulfiqar et al., 2023)。代培红等(2022)研究表明, 利用VIGS技术沉默棉花抗黄萎病相关基因GhMYB6后, 沉默组棉苗表现出比对照组更明显的黄化、萎蔫和落叶等黄萎病典型症状。Chen等(2021)通过VIGS技术在棉花中沉默GhGPA后, 棉花对黄萎病的抗性显著降低。本研究中, 采用VIGS技术沉默棉花GhDIR1后接种大丽轮枝菌V991, 观察到沉默植株的叶片出现更严重的萎蔫黄化, 茎秆褐变程度加剧。这些表型变化表明, GhDIR1沉默后植株对黄萎病菌的抗性降低, 从而推测GhDIR1在棉花中作为正向调控因子参与对黄萎病的防御反应。
通过转录组学分析对照组和GhDIR1沉默植株, 发现了15 938个DEGs。KEGG分析显示, DEGs在类黄酮、萜类化合物生物合成和α-亚麻酸代谢等生物途径中富集。为了进一步验证转录组结果, 从3个途径中进一步筛选获得6个关键基因: GhCHS、GhDFR、GhCAD、GhSEQ、GhLOX和GhAOC。qRT-PCR检测发现其在沉默GhDIR1植株中表达均下调(图7), 从而证实了转录组数据的可靠性。这些结果表明, GhDIR1参与棉花的类黄酮和萜类化合物生物合成以及α-亚麻酸代谢等生物途径的调控。类黄酮生物合成代谢途径是植物中最重要的代谢过程之一, 其协助植物适应环境并提高对病原体的耐受性(Mansfeld et al., 2017)。在大豆(Glycine max)中, 查尔酮合酶(chal- cone synthase, CHS)作为类黄酮合成的关键调节酶, 其编码基因的表达对于植物抗毒素的产生至关重要(Yamaguchi et al., 2011)。在烟草和油菜(Brassica napus)中, 二氢黄酮醇4-还原酶基因(dihydroflavonol 4-reductase, DFR)过表达导致叶片中的类黄酮含量增加, 进而提高了转基因植物对各类胁迫的耐受性(Kumar et al., 2013; Kim et al., 2017)。此外, 萜类化合物是植物防御病原体的重要化合物, 具有免疫调节作用(Wang et al., 2021)。例如, 萜烯合酶基因Tps6/Tps11通过调控玉米(Zea mays)倍半萜类植物抗毒素家族的生物合成途径对多种植物病原菌表现出抗真菌活性(Huffaker et al., 2011)。棉花中的(+)-δ-cadinene合酶((+)-delta-cadinene synthase isozyme, CAD)是一种倍半萜类环化酶, 它催化合成包括棉酚在内的植物抗毒素, 增强植物对病原真菌的抗性(Xu et al., 2004)。大豆中角鲨烯单加氧酶(squalene monooxygenase, SQE)过表达显著提升大豆的胁迫耐受性, 并提高单株产量; 相反, 抑制SQE表达使得耐受性被削弱(Yu et al., 2024)。已有研究表明, JA信号的改变可影响植株对黄萎病菌的抗性(高升旗等, 2023), 在植物对病原菌侵染的先天性免疫应答中扮演关键角色(Zhu et al., 2022)。在水稻(Oryza sativa)中, 脂氧合酶(lipoxygenase, LOX)通过催化由JA介导的植物发育和防御途径, 增强植物对褐飞虱(Nilaparvata lugens)的抗性(Liao et al., 2022)。此外, 与JA生物合成密切相关的丙二烯氧化物环化酶(allene oxide cyclase, AOC)基因突变会降低水稻对稻瘟病菌的抗性(Wang et al., 2023)。
本研究推测GhDIR1可能通过调节类黄酮和萜类化合物的生物合成途径及JA激素信号转导过程, 影响棉花对黄萎病的抗性, 但GhDIR1与其它植物激素和信号途径的相互作用, 以及这些激素如何协同作用于植物免疫反应还有待进一步研究。后续我们将通过挖掘GhDIR1互作蛋白及其功能, 测定植株相关的生理生化指标(如类黄酮和萜类化合物的含量及相关酶活性变化), 深入探究其在细胞内的生物学功能和作用机制。
实验室前期对接种大丽轮枝菌的拟南芥进行转录组分析, 鉴定获得了一系列差异表达基因, 其中DIR1类蛋白基因AT3G53980.2显著受大丽轮枝菌诱导表达。本研究中, 我们通过同源比对得到陆地棉GhDIR1 (Gh_A09G180700.1)。进一步研究表明GhDIR1基因能快速响应大丽轮枝菌侵染。亚细胞定位结果显示GhDIR1定位于细胞膜。采用VIGS技术下调GhDIR1表达, 使得棉花对大丽轮枝菌的抗性显著降低。转录组分析表明DEGs主要富集在类黄酮生物合成、倍半萜类和三萜类生物合成和α-亚麻酸代谢等途径, 进一步检测从这些途径中筛选到的6个关键基因的表达水平, 发现其在沉默组棉花植株中表达均下调, 与转录组数据相符。一系列实验结果表明, GhDI- R1是棉花抗黄萎病的正向调控基因, 可能通过介导类黄酮和萜类化合物的生物合成途径以及JA等激素次级代谢途径和相关信号通路增强棉花对大丽轮枝菌的抗性, 对GhDIR1的功能验证和研究可为分子遗传育种提供潜在的候选基因。
黄雨欣: 实验操作、数据收集分析以及论文撰写; 谢涛、王省芬和郭惠明: 协助整理及分析数据; 程红梅和马伯军: 提供实验设备及技术支持; 陈析丰和苏晓峰: 实验方案设计和规划指导, 参与文章讨论及修订。
  • 农业生物育种重大项目(2023ZD04039)
  • 浙江省自然科学基金(LZ23C130004)
  • 河北省重点研发计划(21326314D)
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2025年第60卷第5期
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doi: 10.11983/CBB24135
  • 接收时间:2024-09-04
  • 首发时间:2026-10-22
  • 出版时间:2025-09-10
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  • 收稿日期:2024-09-04
  • 录用日期:2024-10-30
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农业生物育种重大项目(2023ZD04039)
浙江省自然科学基金(LZ23C130004)
河北省重点研发计划(21326314D)
作者信息
    1浙江师范大学生命科学学院, 金华 321004
    2中国农业科学院生物技术研究所农业微生物资源发掘与利用全国重点实验室, 北京 100081
    3河北农业大学农学院, 保定 071000

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2种不同金属材料的力学参数
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属数
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genus
种数
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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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