水稻NPR1基因的表达特征及基因（NPR1，AOS）的遗传转化

论文摘要

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。

论文目录

• ACKNOWLEDGEMENTS
• ABSTRACT
• 中文摘要
• CHAPTER 1 GENERAL INTRODUCTION AND PROJECT LAYOUT
• CHAPTER 2 REVIEW OF LITERATURE
• 2.1 PLANT DEFENSES
• 2.2 NON-EXPRESSOR OF PATHOGENESIS RELATED GENES 1 （NPR1）
• 2.2.1 NPR1 in SA mediated SAR
• 2.2.3 The role of NPR1 in induced systemic resistance （ISR） and JA mediated defense against herbivore （HIR）
• 2.2.4 Plant defense by NPR1
• 2.3 ALLENE OXIDE SYNTHASE （AOS）
• 2.3.1 Copy number of the gene
• 2.3.2 AOS in the JA signaling pathway
• 2.3.3 Expression of AOS
• 2.3.4 Plant defense by AOS
• CHAPTER 3 EXPRESSION PROFILE OF NPR1 IN RICE
• 3.1 INTRODUCTION
• 3.2 MATERIALS AND METHODS
• 3.2.1 Plant material
• 3.2.2 Insects
• 3.2.3 Plant treatments
• 3.2.4 Sample processing
• 3.2.5 Cloning of NPR1 and sequence analysis
• 3.2.6 Quantitative real-time PCR （qRT-PCR）
• 3.3 RESULTS
• 3.3.1 Cloning,identification of NPR1 and its sequence analysis
• 3.3.2 NPR1 protein nucleotide and amino acid sequence
• 3.3.3 Phylogenetic tree of NPR1 and its homologues
• 3.3.4 Relative expression levels of NPR1 in different rice tissues
• 3.3.5 Influence of signaling molecules on the expression levels of NPR1
• 3.3.6 Influence of herbivore infestation on expression levels of NPR1
• 3.4 DISCUSSIONS
• 3.4.1 Relative expression levels of NPR1 in different rice tissues
• 3.4.2 Influence of signaling molecules on the expression levels of NPR1
• 3.4.3 Influence of herbivore infestation on expression levels of NPR1
• 3.5 CONCLUSIONS AND FUTURE PROSPECTS
• CHAPTER 4 GENETIC TRANSFORMATION OF NPR1 （NON-EXPRESSOR OF PATHOGENESIS RELATED GENES-1） IN RICE
• 4.1 INTRODUCTION
• 4.2 MATERIALS AND METHODS
• 4.2.1 Cloning of NPR1 gene fragment into pCAMBIA 1301 vector
• 4.2.2 Transformation to Agrobacterium
• 4.2.3 Tissue culture/Callus induction
• 4.2.4 Bacterium activation and co-cultivation
• 4.2.5 Selection of the transformed calli
• 4.2.6 GUS assay of the resistant calli
• 4.2.7 Regeneration of the resistant calli
• 4.3 RESULTS AND DISCUSSION
• 4.3.1 Restriction enzyme digestion of DNA fragment
• 4.3.2 Successful extraction ofpCAMBIA 1301
• 4.3.3 Identification of the recombinant plasmid
• 4.3.4 Callus induction
• 4.3.5 Bacterium and Co-cultivation
• 4.3.6 Selection and plant regeneration
• 4.3.7 Transformation efficiency
• CHAPTER 5 CLONING AND GENETIC TRANSFORMATION OF A OS （ALLENE OXIDE SYNTHASE） IN RICE
• 5.1 INTRODUCTION
• 5.2 MATERIALS AND METHODS
• 5.2.1 Plant material
• 5.2.2 Isolation of total RNA
• 5.2.3 Reverse Transcription
• 5.2.4 Polymerase Chain Reaction
• 5.2.5 Cloning of AOS gene fragment into pMD19-T vector
• 5.2.6 Cloning of AOS gene fragment into pCAMBIA 1301 vector
• 5.2.7 Transformation to Agrobacterium
• 5.3 RESULTS AND DISCUSSION
• 5.3.1 Cloning and identification of AOS and its sequence analysis
• 5.3.2 AOS protein nucleotide and amino acid sequence
• 5.3.3 Phylogenetic tree of AOS and its homologues in different plant species
• 5.3.4 Successful extraction of pCAMBIA 1301
• 5.3.5 Restriction enzyme digestion of the DNA fragment
• 5.3.6 Identification of the recombinant plasmid
• 5.3.7 Callus induction
• 5.3.8 Bacterium and co-cultivation
• 5.3.9 GUS Histochemical Assay,election and plant regeneration
• 5.3.10 Transformation efficiency
• CHAPTER 6 CONCLUSIONS AND FUTURE PROSPECTS
• 6.1 CONCLUSIONS
• 6.2 RECOMMENDATIONS
• REFERENCES
• APPENDIX
• CHAPTER 7 DIFFERENTIAL ATTRACTION OF PARASITOIDS IN RELATION TO SPECIFICITY OF KAIROMONES FROM HERBIVORE AND ITS BY-PRODUCTS
• CURRICULUM VITAE