【作者】 周国鑫；

【导师】 程家安； 娄永根；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2009， 博士

【摘要】 解析植食性昆虫为害诱导的植物防御反应的分子机理一直是近几年来昆虫与植物互作关系研究中的一个热点。至今，已在拟南芥、烟草、蕃茄等双子叶植物中开展了较多的研究，但作为单子叶模式植物的水稻，在这方面的研究还很少。为此，本文利用Affymetrix公司的57 K GeneChip（?） Rice Genome Array，分析了二化螟取食24 h后水稻转录组的变化，并对二化螟诱导的一个差异表达基因水稻脂氧合酶基因OsHI-LOX在水稻诱导防御反应中的作用进行了的研究。结果如下：二化螟取食导致4545个（大约占水稻全基因的8％）水稻基因的转录水平发生变化，其中表达量上／下调的基因分别为2124和2421个。这些水稻基因涉及新陈代谢、能量生成、信号转导、植物激素合成、转录调控和逆境反应等18个功能类别。与在拟南芥、烟草等双子叶植物中所报道的相类似，二化螟取食抑制了绝大部分水稻叶绿体光合系统及光合磷酸化电子传递相关基因的表达，导致叶绿素降解，光合作用能力下降；而碳水化合物代谢途径，如糖酵解（glycolysis）、糖质异生（gluconeogenesis）、乙醛酸循环（glyoxylate cycle）和三羧酸循环（TCA）等相关基因的表达水平显著增强，即水稻的能量产生过程发生改变，由以光合磷酸化为主的ATP合成的方式转为以氧化磷酸化为主的ATP合成的方式。二化螟取食还调控了与众多植物激素如茉莉酸（JA）、水杨酸（SA）、乙烯（Ethylene）、生长素（Auxin）、脱落酸（ABA）、分裂素（CK）和赤霉素（GA）等合成及其信号转导途径的基因，但与在其它植物中发现的一样，参与二化螟诱导的水稻防御反应的植物激素主要也是茉莉酸、水杨酸和乙烯，这些激素的合成及其信号转导相关基因大多数是被诱导表达。二化螟取食诱导的水稻防御反应还涉及到很多胞内信号转导途径，如Ca²⁺信号途径（36个基因被调控）、G-蛋白信号途径（39个）、蛋白磷酸酯酶（21个）、蛋白激酶（217个）、磷脂酰肌醇信号（9个）等。此外，有42个家族的289个转录因子参与二化螟诱导的水稻防御反应，其中有13个家族如AS2、C2C2-Dof、EIL、GRAP-G2-Like、GRAS、HSF、MBF1、NAC、PLATZ、SRS、ULT、WRKY和ZIM的80％以上成员的表达水平是上调的。研究结果还发现，二化螟取食与很多其他逆境胁迫因子在诱导水稻防御反应方面存在复杂的相互作用，很多生物与非生物逆境因子（病原物侵染、氧胁迫、热胁迫、冷冻、营养缺乏等）所调控的基因，也被二化螟取食调控。因此，二化螟取食诱导的水稻防御反应是水稻整体在生理生化以及代谢等方面的一个重建过程，是由以生长发育为主向以防御为主的一种转移。OsHI-LOX基因全长2873 bp，包括2775 bp长的编码序列（ORF）以及一段14 bp 5’端非编码序列（5’-UTR）及84 bp的3’端非编码序列（3’-UTR），共编码924个氨基酸，预测分子量为102.82 kDa。OsHI-LOX蛋白定位于叶绿体，二化螟取食、JA处理和机械损伤均能增加OsHI-LOX基因的表达水平。反义抑制OsHI-LOX表达导致二化螟取食诱导的茉莉酸（JA）和胰蛋白酶抑制剂（TrypPI）含量下降，并降低水稻对两种咀嚼式口器害虫（二化螟和稻纵卷叶螟）的抗性；外用JA处理，可以恢复反义抑制品系中TrypPI的含量及对稻纵卷叶螟的抗性。与此相反，反义抑制OsHI-LOX表达却增强水稻对刺吸式口器害虫（褐飞虱）的抗性，导致褐飞虱成虫对稻株的选择性、产卵量以及取食量下降，取食反义抑制OsHI-LOX水稻的褐飞虱若虫成活率也下降。这种抗性的产生，与褐飞虱为害时反义抑制品系水稻产生的高浓度的H₂O₂和SA有关，从而引起水稻的类似过敏性细胞死亡（HR）。上述结果表明，OsHI-LOX基因参与了水稻虫害诱导的茉莉酸的合成，并且在调控水稻对咀嚼式口器和刺吸式口器害虫的防御反应中发挥了重要作用。更多还原

【Abstract】 Characterizing the molecular mechanism of herbivore induced plant defense responses isone of the most important aspects of revealing relationship between insect and plant So far,much has been done in this area in dicotyleon plants such as Arabidopsis, tobacco and tomato.However, little to nothing is known in this aspect in rice, a model monocot species. Therefore,we first analyze the transcriptional response of rice plants 24 h after they were infested by larvaof rice stem stripped borer（SSB）, Chilo suppressalis Walker, using Affymetix’s 57 K GeneChip（?） Rice Genome Arrays, and then one SSB feeding up-regulated gene OsHI-LOX, whichencoded a rice lipoxygenase was isolated and its role in herbivore-induced rice defenseresponses was investigated. The results are as follows:Transcriptional levels of 4545 genes（about 8% of the total rice genes） in rice were alteredafter plants were infested by SSB larvae, in which the numbers of upregulated anddownregulated genes were 2124 and 2421 respectively. These SSB-regulated genes can beclassified into 18 functional groups, including genes involved in metabolism, energy generation,signal transduction, plant hormones biosynthesis, transcription regulation, stress responses, andso on. Similar to those found in dicotyledon such as Arabidopsis and tobacco, SSB feedingdown-regulated expression levels of most of genes involved in photosynthesis system andelectron transport in photophosphorylation were down-regulated, leading to degradation ofchlorophyll and decreasing the efficiency of photosynthesis, on the other hand, metabolism ofcarbohydrates, such as glycolysis, gluconeogenesis, glyoxylate cycle, TCA were enhancedsignificantly, it is obvious that main energy generation pathway have been changed, ATPsynthesis was generated mainly by photophosphorylation originally, now mainly by oxidativephosphorylation. Lots of genes involved in synthesis and signaling transduction of planthormones such as jasmonic acid（JA）, salicylic acid（SA）, Ethylene, IAA, ABA, and GA wereregulated by SSB feeding, but as founded in other plants, JA, SA and Ethylene are the mainplant hormones that playing roles in induced defense resistance against SSB. Many intracellularsignaling transduction pathways are involved in SSB activated rice defense resistance, forexamples Ca²⁺ signaling pathway（36 genes were regulated by SSB）, G-protein signaling pathway（39 genes）, protein phosphatases（21 genes）, protein kinases（217 genes）, andphosphatidylinositol signal pathway（9 genes）. In addition to, expression levels of 289transcription factor genes in 42 transcription factors families were changed, among them 8transcription factor families including AS2, C2C2-Dof, EIL, GRAP-G2-like, GRAS, HSF,MBF1, NAC, PLATZ, SRS, ULT, WRKY, and ZIM, in which 80% genes were up-regulated.Results also provide new information on the interactions between SSB feeding and other stressfactors, many biotic and abiotic stress factors as pathogen attack-, oxygen-, heat-, cold-,nutrition lacking-stress regulated genes are also regulated by SSB feeding. It is indicated thatSSB induced defense resistance in rice was reconstruction process involved in physiology,biochemical, and metabolism, and other areas of rice plants, the process that rice focuses ondefense other than growth and development.In this paper, one of SSB induced genes, OsHI-LOX cDNA（2873 bp） was isolated fromSSB wounded rice stems including ORF sequence（2775 bp）, a 5’UTR sequence（14 bp） and a3’UTR sequence（84 bp）, and encodes a protein of 924 residues with a predicted molecular weithof 102.82 kDa. OsHI-LOX is a chloroplast located protein, and SSB feeding, wounding and JAtreatment all induce its expression. Antisense inhibition OsHI-LOX expression in rice reduceSSB induced JA and TrypPI synthesis, and decrease rice resistance to two chewing herbivoreSSB（Chilo suppressalis Walker）and RLF（Cnaphalocrocis medinalis Guenee）. Treated withJA, antisense transgenic lines could restore the TrypPI activity and resistance to RLF. Incontrast, antisense inhibition OsHI-LOX expression enhance resistance to BPH（Niaparvatalugens St（?）l）, resulting in selectivity, Eggs, feeding, nymphae survival ratio of BPH ontransgenic rice plant to declined. The enhanced resistance is related to generating high levels ofH₂O₂ and SA in transgenic lines while infested by BPH, which could cause hypersensitiveresponse（HR） along the infested site. 1"he results indicated that OsHI-LOX was involved inherbivore induced JA biosynthesis, and played the important roles in rice resistance againstchewing-and sucking-herbivore.更多还原