【作者】 吕静；

【导师】 娄永根；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2011， 博士

【摘要】 植物诱导抗虫反应产生的分子机理一直是植物分子生物学领域的研究热点之一。植食性昆虫为害能够引起植物体内大量基因转录水平的变化,暗示转录因子在其中发挥了重要作用；此外,大量研究表明乙烯信号途径参与调控植物的诱导抗虫反应。为此,本文以水稻ERF (ethylene responsive factor)转录因子OsERF3及乙烯合成相关的ACC合酶(1-amino-cyclopropane-1-carboxylic acid synthase, ACS)基因OsACS2为研究对象,对其在水稻诱导抗虫反应中的作用及调控机理进行了详细研究,主要结果如下：1) OsERF3定位于细胞核,机械损伤、二化螟(Chilo suppressalis, rice striped stem borer, SSB)及稻纵卷叶螟(Cnaphalocrocis medinalis, rice leaf folder, LF)为害能够迅速诱导OsERF3基因表达,而褐飞虱(Nilaparvata lugens, rice brown planthopper, BPH)为害不诱导其表达,甚至有一定的抑制作用。利用农杆菌转化法,我们分别获得了OsERF3反义抑制及过量表达的水稻突变体植株。研究表明,OsERF3正调控2个MAPK基因OsMEK3, OsMPK3及2个WRKY转录因子OsWRKY53、OsWRKY70表达,并且正调控信号分子JA (jasmonic acid)、SA (salicylic acid)、乙烯的合成及胰蛋白酶抑制剂(trypsin protease inhibitors, TrypPIs)的产生。OsERF3正调控水稻对SSB的抗性,与野生型(wide type, WT)相比,取食反义品系后SSB幼虫生长加快,而过量品系则不利于SSB生长；SSB取食反义品系后植株提前死亡,过量品系则提高对SSB的耐害性。另一方面,OsERF3负调控水稻对BPH的抗性,反义品系降低了BPH雌成虫的取食、产卵选择性及初孵若虫存活率,过量品系则与之相反；进一步研究发现,OsERF3主要通过影响JA及H202合成分别调控水稻对SSB和BPH的抗性。综上所述,OsERF3是水稻诱导抗虫反应中的一个早期应答基因,位于信号途径的上游,在水稻防御不同取食习性昆虫为害中发挥了关键作用。2)机械损伤、SSB、LF及BPH为害能够迅速诱导OsACS2表达。反义抑制OsACS2表达减少SSB为害诱导的乙烯释放量、TrypPIs和挥发物含量,降低水稻对SSB的抗性；外源ACC (1-amino-cyclopropane-l-carboxylic acid)处理可以恢复水稻TrypPIs的合成及其对SSB的抗性。与此相反,反义抑制OsACS2降低BPH的取食和产卵选择性,而外源ACC处理后则可以恢复BPH对突变体的选择性。上述结果表明,OsACS2通过调控虫害诱导的乙烯合成影响水稻的直接防御和间接防御,从而对不同取食习性的昆虫产生不同的影响。更多还原

【Abstract】 Characterizing the mechanisms underlying herbivore-induced plant defense responses has always been one of hotspots in plant moleculer biology. Herbivore attack rapidly activates large-scale and specific transcriptional changes in plants, suggesting important roles of transcription factors in plant-herbivore interaction. Moreover, many studies have confirmed that ethylene signaling is important for manipulating herbivore-induced plant defense responses. Thus, we here chose a rice ERF (ethylene responsive factor) transcription factor gene OsERF3 and an ethylene biosynthesis-related ACS (1-amino-cyclopropane-l-carboxylic acid synthase) gene OsACS2 and characterized their biological functions in regulating herbivore-induced defense responses. The results are as follows:1) OsERF3 localizes nucleus, whose transcripts were rapidly up-regulated in response to mechanical wounding and feeding by the rice striped stem borer (SSB) Chilo suppressalis or rice leaf folder (LF) Cnaphalocrocis medinalis, but slightly suppressed by the rice brown planthopper (BPH) Nilaparvata lugens. Using Agrobacterium tumefaciens-mediated transformation, we generated OsERF3 antisense (as-erf) and over-expression (oe-ERF) transgenic rice plants. OsERF3 positively regulates transcript levels of two mitogen-activated protein kinases (MAPKs) genes, OsMEK3 and OsMPK3, two WRKY genes, OsWRKY53 and OsWRKY70, as well as concentrations of jasmonate (JA), salicylate (SA) and ethylene, and activity of trypsin protease inhibitors (TrypPIs). OsERF3 is also found to mediate rice resistance to SSB. SSB larval weight gain that fed on as-erflines was higher than those fed on WT plants; by contrast, overexpressing OsERF3 decreaced SSB larval growth. Moreover, as-erflines were more severely damaged by SSB than WT plants, whereas oe-ERF lines were less damaged. On the other hand, OsERF3 negatively mediate rice resistance to BPH: as-erf lines decreased feeding and oviposition preference of BPH female adults, and survival rates of BPH nymphae, whereas oe-ERF lines received more BPH female adults, eggs and caused higher survival rates of BPH nymphs. Our results also revealed that OsERF3 regulates rice resistance to SSB and BPH by influencing JA and H2O2 signaling pathways, respectavily. In summary, OsERF3 is an early responsive gene in herbivore-induced defense responses in rice, which can regulate herbivore-induced defense responses and plant resistance by modulating MAPK cascades, and JA, SA, ethylene and H2O2 pathways. Our results also illustrate that OsERF3 acts as a central switch that gears the plant’s metabolism towards an appropriate response to chewing or piercing/sucking insects.2) Transcript levels of OsACS2 were rapidly up-regulated in response to mechanical wounding and feeding by SSB, LF or BPH. OsACS2-silenced lines (as-acs) reduced SSB induced ethylene biosnythesis, TrypPI activity and volatile compounds, and decreased rice resistance to SSB. Exogenous application of ACC on as-acs lines can restore elicited TrypPI activity and plant resistance to SSB. By contrast, silencing OsACS2 decreased-the feeding and oviposition preference of BPH female adults, and ACC application can restore the preference. These results suggest that OsACS2 is involved in herbivore induced ethylene biosynthesis, and ethylene signaling plays an important role in rice resistance against chewing- and sucking-herbivores by modulating direct and indirect defenses.更多还原