水稻NPR1基因的表达特征及基因（NPR1，AOS）的遗传转化
摘要
NPR1基因在各种植物包括水稻中的功能都非常保守，它在植物的诱导抗性中起着非常重要的作用，并且是介导各种防御信号途径互作的重要组分。茉莉酸是植物诱导抗虫反应中的一类重要信号分子，AOS基因编码蛋白是调控茉莉酸生物合成的关键酶。然而，NPR1和AOS在水稻诱导抗虫害性中的作用至今仍不清楚。为了阐明基因NPR1和AOS在虫害诱导的水稻防御反应中的功能，我们研究了NPR1在不同信号分子处理和虫害为害后的表达特征，并且获得了沉默基因NPR1及AOS的水稻品系。我们的研究计划及主要结果如下：
1)根据已经报道的水稻NPR1和AOS基因的序列设计引物，克隆了两个基因的片段。利用Realtime-PCR的方法，测定了NPR1在健康水稻体内不同部位的表达特征，同时测定了该基因在不同信号分子(水杨酸、茉莉酸、过氧化氢、乙烯ET)处理及不同虫害(二化螟、稻纵卷叶螟、褐飞虱)胁迫下的诱导表达特征。结果表明，茉莉酸、水杨酸和过氧化氢处理能在早期使NPR1的表达水平上升；ET处理则在24小时能显著诱导NPR1表达水平的变化。褐飞虱为害能诱导NPR1表达水平显著提高，并且是三种害虫为害中表达水平最高的；二化螟在为害只在早期诱导水稻茎中NPR1表达水平上升；但稻纵卷叶螟为害却降低了该基因的表达水平。这些结果表明，NPR1可能参与了水稻的防御反应。
2)为了分析NPR1和AOS在水稻防御反应中的作用，利用农杆菌转化系统获得了分别沉默NPR1和AOS基因的水稻品系，并且收获了这些品系T1代的种子，这为阐明基因NPR1和AOS在虫害诱导的水稻防御反应中的作用打下了基础。
通过基因工程技术可以帮助发展绿色的害虫控制技术。NPR1和AOS基因无疑是这方面的重要候选基因。通过阐明这些基因的生物学功能，可能为改进作物的抗虫性开启新的机会与可能性。
NPR1,functionally conserved in diverse plant species including rice Oryza sativaL.(Poeceae),plays a key role in induced plant resistance and mediates the cross talkbetween different defense pathways.Allene oxide synthase(AOS) is a focal point incontrolling biosynthesis of JAs,which are important signals in mediating plant resistanceagainst herbivores.However,the roles of NPR1 and AOS in induced resistance in ricecrop against herbivores remained unclear.To elucidate the biological functions of NPR1and AOS in herbivore-induced rice defenses,we observed the expression profile of NPR1following application of signaling molecules and herbivore feeding,and develop ricelines with silencing ofNPR1 orAOS gene.Our study layout and main results are:
1) According to the published sequences,fragments of rice genes NPR1 and AOS were PCRamplified and cloned.Expression profiles of NPR1 in different tissues of healthy riceplants,following treatment with signaling molecules(salicylic acid,SA;jasmonicacid,JA;H_2O_2;ethylene,ET) and herbivores feeding [stripped stem borer SSB,Chilosuppressalis Walker(Lepidoptera:Pyralidae);leaf folder LF,Cnaphalocrocismedinalis Guenee(Lepidoptera:Pyralidae);brown plant hopper BPH,Nilaparvatalugens Stal(Hemiptera:Delphacidae)] was observed using realtime RT-PCR,one ofthe enabling techniques of the genomic era.The expression of NPR1 was highest inroots followed by inflorescence,leaves and stem.Following treatment with JA,SA,and H_2O_2,an early NPR1 induction was observed,however,drastic changes in genelevels were observed by 24 h after ET treatment.Among herbivores,the maximumgene induction was observed after BPH feeding.An early gene induction by SSB instem tissues was also observed but LF decreased the gene levels.The results showthat NPR1 may participates in defense responses in rice.
2) To analyze the role of NPR1 and AOS in rice defense,rice lines silencing NPR1 and AOSexpression were obtained with an Agrobacterium-based transformation system.Theseeds of T_1 generation of these lines were collected,which lays the foundation forelucidating the functions of genes NPR1 and AOS in herbivore-induced rice defenseresponses.
Modifying plant responses through manipulating expression of genes involved indefense pathways can greatly help to control pests in an environmental friendly way.NPR1 and AOS are no doubt an important such candidates to work on.Knowing their rolein rice plant resistance may open new opportunities and possibilities for improving cropresistance against herbivores.
1)根据已经报道的水稻NPR1和AOS基因的序列设计引物，克隆了两个基因的片段。利用Realtime-PCR的方法，测定了NPR1在健康水稻体内不同部位的表达特征，同时测定了该基因在不同信号分子(水杨酸、茉莉酸、过氧化氢、乙烯ET)处理及不同虫害(二化螟、稻纵卷叶螟、褐飞虱)胁迫下的诱导表达特征。结果表明，茉莉酸、水杨酸和过氧化氢处理能在早期使NPR1的表达水平上升；ET处理则在24小时能显著诱导NPR1表达水平的变化。褐飞虱为害能诱导NPR1表达水平显著提高，并且是三种害虫为害中表达水平最高的；二化螟在为害只在早期诱导水稻茎中NPR1表达水平上升；但稻纵卷叶螟为害却降低了该基因的表达水平。这些结果表明，NPR1可能参与了水稻的防御反应。
2)为了分析NPR1和AOS在水稻防御反应中的作用，利用农杆菌转化系统获得了分别沉默NPR1和AOS基因的水稻品系，并且收获了这些品系T1代的种子，这为阐明基因NPR1和AOS在虫害诱导的水稻防御反应中的作用打下了基础。
通过基因工程技术可以帮助发展绿色的害虫控制技术。NPR1和AOS基因无疑是这方面的重要候选基因。通过阐明这些基因的生物学功能，可能为改进作物的抗虫性开启新的机会与可能性。
NPR1,functionally conserved in diverse plant species including rice Oryza sativaL.(Poeceae),plays a key role in induced plant resistance and mediates the cross talkbetween different defense pathways.Allene oxide synthase(AOS) is a focal point incontrolling biosynthesis of JAs,which are important signals in mediating plant resistanceagainst herbivores.However,the roles of NPR1 and AOS in induced resistance in ricecrop against herbivores remained unclear.To elucidate the biological functions of NPR1and AOS in herbivore-induced rice defenses,we observed the expression profile of NPR1following application of signaling molecules and herbivore feeding,and develop ricelines with silencing ofNPR1 orAOS gene.Our study layout and main results are:
1) According to the published sequences,fragments of rice genes NPR1 and AOS were PCRamplified and cloned.Expression profiles of NPR1 in different tissues of healthy riceplants,following treatment with signaling molecules(salicylic acid,SA;jasmonicacid,JA;H_2O_2;ethylene,ET) and herbivores feeding [stripped stem borer SSB,Chilosuppressalis Walker(Lepidoptera:Pyralidae);leaf folder LF,Cnaphalocrocismedinalis Guenee(Lepidoptera:Pyralidae);brown plant hopper BPH,Nilaparvatalugens Stal(Hemiptera:Delphacidae)] was observed using realtime RT-PCR,one ofthe enabling techniques of the genomic era.The expression of NPR1 was highest inroots followed by inflorescence,leaves and stem.Following treatment with JA,SA,and H_2O_2,an early NPR1 induction was observed,however,drastic changes in genelevels were observed by 24 h after ET treatment.Among herbivores,the maximumgene induction was observed after BPH feeding.An early gene induction by SSB instem tissues was also observed but LF decreased the gene levels.The results showthat NPR1 may participates in defense responses in rice.
2) To analyze the role of NPR1 and AOS in rice defense,rice lines silencing NPR1 and AOSexpression were obtained with an Agrobacterium-based transformation system.Theseeds of T_1 generation of these lines were collected,which lays the foundation forelucidating the functions of genes NPR1 and AOS in herbivore-induced rice defenseresponses.
Modifying plant responses through manipulating expression of genes involved indefense pathways can greatly help to control pests in an environmental friendly way.NPR1 and AOS are no doubt an important such candidates to work on.Knowing their rolein rice plant resistance may open new opportunities and possibilities for improving cropresistance against herbivores.
引文
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