【作者】 王晓峨；

【导师】 宋凤鸣；

【作者基本信息】 浙江大学 ， 植物病理学， 2008， 博士

【摘要】 在植物识别病原物后激活防卫反应的过程中，转录因子起着重要的作用，它通过激活或抑制防卫基因的表达来实现防卫反应的调控。NAC转录因子是一个植物特有的庞大家族。最近研究表明，NAC转录因子在植物生长发育、激素应答及抗逆反应中起重要作用。本研究中，我分离鉴定了2个与拟南芥抗病反应相关的NAC型转录因子，其中一个是新鉴定的抗病相关NAC基因DRN1(defense-relatedNAC1)，另一个是先前鉴定的抗逆相关NAC基因ATAF1，并研究了这两个NAC转录因子在抗病反应调控中的功能。在分析基因芯片数据中，发现DRN1基因在灰霉病菌(Botrytis incerea)、Pseudomonas syringae pv．tomato(Pst)DC3000侵染及水杨酸(salicylic acid，SA)、flg22等处理后上调表达，暗示DRN1可能与抗病反应有关。DRN1编码一个含275个氨基酸的蛋白，含有一个高度保守的NAC结构域。GFP融合蛋白分析显示，DRN1定位于细胞核内。转录激活活性分析结果表明，DRN1是一个转录激活因子，而且其转录激活区域位于C-端。promoter：：GUS染色结果显示，DRN1在正常拟南芥植株茎、叶、花内均有表达。RT-PCR分析结果表明，Pst DC3000和B．cinerea侵染后DRN1表达增强，其中24～48 hr时表达水平最为显著；SA、ACC(l-aminocyclopropane-1-carboxylic acid)和苯并噻二唑(benzothiadiazole，BTH)处理后均不同程度地诱导DRN1的表达。为了研究DRN1在抗病反应中的功能，筛选获得一个DRN1基因的T-DNA插入突变体drn1-1。在drn1-1植株中，RT-PCR检测不到DRN1基因表达，但是drn1-1对Pst DC3000和B．cinerea侵染仅有轻微的表型改变。考虑到NAC家族中基因冗余现象，构建了过量表达的chimeric repressor载体35S：：DRN1-SRDX和过量表达载体35S：：DRN1，并利用根癌农杆菌介导花序浸蘸法导入拟南芥中，分别获得7个35S：：DRN1和6个35S：：DRN1-SRDX的单拷贝株系，转代选取各3个纯合株系(DRN1-OE#2、#6和#11及DRN1-SRDX#1、#3和#8)。比较了野生型、drn1-1、DRN1-OE和DRN1-SRDX植株对B．cinerea、Pst DC3000和早疫病菌(Altenariabrassicicola)的抗病性变化。喷雾接种B．cinerea、注射接种Pst DC300或离体叶片滴注接种A．brassicicola后，drn1-1的发病略重于野生型，DRN1-SRDX#1、#3和#8植株病害明显重于野生型，而DRN1-OE#2、#6和#11植株上病害则显著轻于野生型。接种后，DRN1-SRDX#1、#3和#8植株叶片中B．cinerea生长量和PstDC3000菌量显著高于野生型对照，而DRN1-OE#2、#6和#11植株叶片中B．cinerea生长量和Pst DC3000菌量显著低于野生型对照。B．cinerea侵染后，DRN1-SRDX#1和#8植株中PR-1和PR-5基因表达上调，DRN1-OE#2和#11植株中PDF1．2和BIK1基因表达上调；而Pst DC3000侵染后，DRN1-SRDX#1和#8植株中PDF1．2基因表达上调，DRN1-OE#2和#11植株中PR-1和PR-5以及NPR1基因表达上调。此外，DRN1转基因拟南芥植株增强了对ABA的敏感性。这些结果表明，DRN1在拟南芥对不同类型病害的抗病反应中起正调控作用。ATAF1基因编码一个含289个氨基酸的蛋白，含有高度保守的NAC结构域。RT-PCR结果表明，B．cinerea和Pst DC3000侵染后，野生型植株中ATAF1表达受到抑制，表达水平逐渐下降；同样，SA、BTH、ACC和JA处理也不同程度抑制ATAF1的表达。在接种B．cinerea和Pst DC3000后，ATAF1的T-DNA插入突变体ataf1植株上病害水平略低于野生型，但不显著。为了克服NAC家族基因冗余引起功能研究上的困难，构建过量表达的chimeric repressor载体35S：：ATAF1-SRDX和过量表达载体35S：：ATAF1，利用根癌农杆菌介导花序浸蘸法导入拟南芥中，经转代筛选，分别获得2个35S：：ATAF1(ATAF1-OE#1和#7)和2个35S：：ATAF1-SRDX(ATAF1-SRDX#3和#10)的单拷贝株系。分析了野生型、ataf1、ATAF1-OE和ATAF1-SRDX植株对B．cinerea、Pst DC3000和A．brassicicola的抗病性变化。喷雾接种B．cinerea、注射接种Pst DC300或离体叶片滴注接种A．brassicicola后，ataf1的发病略轻于野生型，ATAF1-SRDX#3和#10植株病害症状明显轻于野生型，而ATAF1-OE#1和#7植株上病害则显著重于野生型。接种后，ATAF1-SRDX#3和#10植株叶片中B．cinerea生长量和Pst DC3000菌量显著低于野生型对照，而ATAF1-OE#1和#7植株叶片中B．cinerea生长量和Pst DC3000菌量显著高于野生量显著高于野生型对照。B．cinerea侵染后，ATAF1-SRDX#3和#10植株中PR-1、PR-5和NPR1基因表达上调，ATAF1-OE#1和#7植株中BIK1基因表达上调；而Pst DC3000侵染后，ATAF1-SRDX#3和#10植株中PR-1、PR-5和NPR1基因表达上调，ATAF1-OE#2和#11植株中PDF1．2基因表达上调。在灰霉菌侵染后，ATAF1-OE植株叶片中产生并积累了更多的活性氧，而在ATAF1-SRDX植株叶片中则仅有少量活性氧积累。这些结果表明，ATAF1在拟南芥对不同类型病害的抗病反应中起负调控作用。更多还原

【Abstract】 Activation of defense responses against pathogen infection is usually regulatedthrough modulation of a large set of defense genes at transcriptional level by specifictranscription factors.A large body of evidence has implicated that several groups oftranscription factors have functions directly or indirectly in regulating plant defenseresponses to pathogen attack.Recently,transcription factors of NAC family have beenreported to be involved in various biological processes of plant growth anddevelopment and in responses to hormones and abiotic stresses.In this study,Iidentified two defense-related NAC genes in Arabidopsis thaliana.One is a newlyidentified NAC gene DRN1（defense-related NAC1） and another one is the previouslyreported stress-responsive ATAF1.I studied in detail by functional analysis of these twoNAC genes for their biological roles in plant defense responses against necrotrophicand biotrophic pathogens.Analyses of public microarray data revealed that expression of DRN1 gene wasup-regulated after infection with Botrytis incerea,Pseudomonas syringae pv.tomato（Pst） DC3000 and by treatment with salicylic acid（SA）,flg22,suggesting that DRN1may be involved in regulation of defense response in Arabidopsis.The DRN1 geneencodes a 275 amino acid protein containing a highly conserved NAC domain.RT-PCRanalysis showed that expression of DRN1 was induced in Arabidopsis plants afterinoculation with Pst DC3000 or B.cinerea and that the expression level wassignificantly up-regulated during 24～48 hr after inoculation.Likely,the expression ofDRN1 was also induced in Arabidopsis plants after treatment with some well-knowndefense-related chemical inducers such as salicylic acid（SA）,benzothaidiazole（BTH）,jasmonic acid（JA） and 1-aminocyclopropane-1-carboxylic acid（ACC,a precursor ofethylene biosynthesis）.These results provide preliminary evidence that DRN1 is involved in Arabidopsis defense response.Subcellular localization analysis using GFP fusion protein showed that the DRN1protein is localized in the nucleus of cells.Transactivation analysis demonstrated thatDRN1 can activate.reporter gene expression in yeast,indicating that DRN1 is atranscriptional activator.Furthermore,deletion analysis of transactivation revealed thattransactivation activity of the DRN1 protein is localized in a region of 168-275 aminoacids at its C-terminal domain,and that the N-terminal region containing the conservedNAC domain does not have transactivation activity.Therefore,DRN1 is atranscriptional activator and its C-terminal region contributes to the transactivationactivity.To study the function of DRN1 in disease resistance response,a T-DNA insertion linedrn1-1 was identified from genotyping screening.RT-PCR analysis indicated thattranscript of the DRN1 gene was undetectable in drn1-1 plants,suggesting that drn1-1is a knockout mutant of the DRN1 gene.However,the phenotypes of the drn1-1 plantsafter inoculation with Pst DC3000 or B.cinerea showed slight but not significanthigher levels of diseases than those in wild-type plants.Considering redundancy ofgene function in NAC family,I constructed a chimeric repressor vector35S::DRN1-SRDX and an overexpression vector 35S::DRN1,and introduced them intowild-type plants using floral dip transformation protocol.Seven 35S::DRN1 and six35S::DRN1-SRDX transgenic lines with single copy of the transgene were obtained.Homozygous lines were identified from the 3rd generation through screening and threehomozygous lines for 35S::DRN1（DRN1-OE#2,#6 and #11） and 35S::DRN1-SRDX（DRN1-SRDX#1,#3 and #8） were chosen for further work.Disease phenotypes of thewild-type,drn1-1,DRN1-OE and DRN1-SRDX plants were compared afterinoculation with B.cinerea,Pst DC3000 or Altenaria brassicicola.As compared withthe wild-type plants,the drn1-1 plants showed a slight but not significant increasedsusceptibility to B.cinerea,Pst DC3000 or A.brassicicola.However,the transgenicDRN1-SRDX#1,#3 and #8 plants showed significant increased susceptibility to theabove-mentioned pathogens;while the DRN1-OE#2,#6 and #11 plants showed asignificant reduced susceptibility to the patogens.Furthermore,growth of B.cinerea and Pst DC3000 in leaves of the DRN1-SRDX#1,#3 and #8 plants was much greaterthan in the wild-type plants;whereas growth of B.cinerea and Pst DC3000 in leaves ofthe DRN1-OE#2,#6 and #11 plants was significantly reduced.After inoculation with B.cinerea,expression of PR-1 and PR-5 genes was markedly up-regulated in theDRN1-SRDX#1 and #8 plants,and expression of PDF1.2 and BIK1 genes wasup-regulated in the DRN 1-OE#2 and # 11 plants.By contrast,after inoculation with PstDC3000,the expression of PDF1.2 was up-regulated in the DRN1-SRDX#1 and #8plants,and the expression of PR-1,PR-5 and NPR1 genes was up-regulated in theDRN1-OE#2 and #11 plants.Moreover,DRN1-overexpressing transgenic plantsshowed an enhanced ABA sensitivity.These results demonstrate that DRN1 is apositive regulator of defense responses in Arabidopsis against both necrotrophic andbiotrophic pathogens through activation of defense gene expression.ATAF1 is one of the first identified NAC genes in Arabidopsis thaliana and waspreviously reported to be involved in abiotic stress response.Because ATAF2,a NACgene that is closely related to ATAF1,plays an important role in defense responseagainst vascular Fusarium wilt pathogen,I therefore studied the possible involvementof ATAF1 in Arabidopsis defense responses against necrotrophic and biotrophicpathogens.The ATAF1 gene encodes a 289 amino acid protein containing a highlyconserved NAC domain.RT-PCR analysis showed that expression of the ATAF1 genewas suppressed in Arabidopsis plants after inoculation with Pst DC3000 and B.cinerea,or treatment with several well-known defense-related chemical inducers including SA,BTH,ACC and JA.To study the function of ATAF1 in defense response,I constructeda chimeric repressor vector 35S::ATAF1-SRDX and an overexpression vector35S::ATAF1 and introduced them into Arabidopsis through floral dip transformationprotocol.Two independent transgenic lines with a single copy of the transgene wereidentified for 35S::ATAF1（ATAF1-OE#1 and #7） and 35S::ATAF1-SRDX（ATAF1-SRDX#3 and #10）,respectively,and chosen for further studies.Diseasephenotypes of the wild-type,atafl（a T-DNA insertion mutant ofATAF1）,ATAF1-OEand ATAF1-SRDX plants were compared after inoculation with B.cinerea,Pst DC3000and A.brassicicola.After inoculation,the atafl plants showed a slight but not significant reduced disease level than the wild-type plants.The disease levels in theATAF1-SRDX#3 and #10 plants were significantly reduced,whereas the disease levelsin the ATAF1-OE#1 and #7 plants were markedly increased.Furthermore,whencompared with those in the wild-type plants,growth of B.cinerea and Pst DC3000 inleaves of the ATAF1-SRDX#3 and #10 plants was significantly decreased,whilegrowth of the pathogens in leaves of the ATAF1-OE#1 and #7 plants was significantlyincreased.After inoculation with B.cinerea,expression of PR-1,PR-5 and NPR1 wasup-regulated in the ATAF1-SRDX#3 and #10 plants,and the expression of BIK1 wasup-regulated in the ATAF1-OE#1 and #7 plants.However,expression of PR-1,PR-5and NPR1 was up-regulated in the ATAF1-SRDX#3 and #10 plants,and the expressionof PDF1.2 was up-regulated in the ATAF1-OE#1 and #7 plants after inoculation withPst DC3000.Moreover,significant accumulation of reactive oxygen species（H₂O₂ andsuperoxide anion） in leaves of the ATAF1-OE#1 and #7 plants,but not in leaves ofATAF1-SRDX#3 and #10 plants after Botrytis infection.These results clearlydemonstrate that ATAF1 is a negative regulator of defense responses in Arabidopsisagainst necrotrophic and biotrophic pathogens.更多还原