【作者】 肖玉涛；

【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】 植物的诱导防御反应是一个从诱导信号识别到防御化合物合成、最终产生防御反应的复杂的生理生化与分子调控过程。在这一过程中,可能起到信号识别作用的植物类受体蛋白激酶(Receptor like kinase,RLK)以及作为防御化合物的萜类挥发物发挥着重要作用。然而,究竟植物类受体蛋白激酶中哪些家族成员参与了这一过程,以及各萜类挥发物起着什么防御作用,目前还了解得不多。为此,本文选择合成水稻萜类挥发物中最主要成分之一的芳樟醇的合成酶基因OsLIS和受二化螟取食诱导的水稻LRR-RLK(Leucine rich repeat receptor like kinase)基因OsHI-LRR-RLKl (OsHI-LKl)为研究对象,结合利用反向遗传学、化学分析、分子生物学以及生物测定等研究方法,剖析了OsLIS调控的芳樟醇和OsHI-LKl在水稻诱导防御反应中的作用。OsLIS是典型的单萜合成酶,定位在叶绿体。组织表达谱分析发现OsLIS在水稻茎和叶片中表达量较高,在根中表达较低。二化螟取食、机械损伤和茉莉酸(jasmonic acid,JA)处理可以显著诱导OsLIS的表达,褐飞虱危害也能诱导OsLIS表达,但与二化螟取食相比表达水平较低,水杨酸(salicylic acid,SA)对OsLIS表达无影响。利用RNAi方法结合遗传筛选,获到了沉默OsLIS的单拷贝插入的水稻突变体纯合子(ir-lis)。突变体表型分析结果表明,转基因品系中OsLIS的转录水平比野生型水稻中的下降90%以上,芳樟醇的释放量仅有野生型的约2-5%,而其他挥发物不受影响。生测结果表明,褐飞虱更喜欢在ir-lis品系上取食和产卵,而褐飞虱的天敌稻虱缨小蜂更多地选择能够合成释放芳樟醇的野生型水稻植株。田间实验表明,褐飞虱更多地分布在ir-lis品系上,而在野生型水稻上稻虱缨小蜂对褐飞虱卵的寄生率更高。另外天敌蜘蛛、稻纵卷叶螟、稻蚜等昆虫在突变体与野生型水稻上的分布也存在差异。表达谱分析表明OsHI-LKl在水稻叶和根中的表达水平高于在茎杆中的,机械损伤、JA处理、二化螟和褐飞虱为害后期(1天以后)显著诱导OsHI-LKl表达,而SA对其无显著影响。RNAi沉默OsHI-LKl的纯合品系ir-lk中,OsHI-LKl的表达水平仅为野生型水稻中的15～30%。与野生型水稻相比,ir-lk品系的虫害诱导JA、SA和绿叶性气味物质(green leaf volatiles, GLVs)含量有所下降,并且对二化螟和褐飞虱的抗性亦降低。更多还原

【Abstract】 Herbivore induced plant defense response is regulated by complex physiological and biochemical processes, which starts from the recognition of signals from herbivores, followed by activation of signaling pathways, expression of defense-related genes and the production of defense chemicals. Receptor like kinases (RLKs) that may recognize elicitors from herbivore and plant terpenoids, one kind of plant defense chemicals, play important roles in this response. However, which members of RLK are involved in this process and what is the function of terpenoids are little known. Thus, we here cloned a rice gene OsLIS encoding linalool synthase, whose product linalool is one of the most important terpenoids in rice, and a LRR-RLK (Leucine rich repeat receptor like kinase) gene OsHI-LRR-RLK1 (OsHI-LK1), which is up-regulated by SSB (rice stem stripped borer, Chilo suppressalis Walker) infestation and characterized their roles in herbivore induced rice defense responses with combined research methods of reverse genetics, chemical analysis, molecular biology and bioassay.OsLIS is a monoterpene synthase, which is localized in chloroplasts. Tissue specific analysis showed that OsLIS expressed high in rice stems and leaves while low in roots. Expression levels of OsLIS could be significantly enhanced by mechanical wounding, SSB infestation and JA treatment, whereas weakly induced by BPH infestation and not by SA. RNA interference was elected to silence OsLIS to obtain OsLIS silencing (ir-lis) lines. qRT-PCR proved that OsLIS expression levels in ir-lis lines was only about 10% of those in WT (wild type) plants, and the emission of linalool in ir-lis was only 2～5% of that in WT plants. Moreover, silencing OsLIS had no effect on other herbivore-induced plant volatiles. When the various rice genotypes were exposed to the BPH colony, BPH female adults were more often found on ir-lis plants than on WT plants, and eggs were more on ir-lis lines. Compared with ir-lis plants, Anagrus nilaparvatae, which is an egg parasitoid of BPH, preferred the odors from WT plants. The abundance of BPH was found more on ir-lis lines in field experiments whereas parasitism of Anagrus nilaparvatae was higher in WT plants. ir-lis lines also had an influence on the population densities of other herbivores and natural enemies, such as spider, RLF(Cnaphalocrocis medinalis Guenee) and rice aphid.Tissue specific analysis showed that OsHI-LK1 expressed high in rice roots and leaves while low in stems. Transcript levels of OsHI-LK1 were up-regulated by mechanical wounding, JA treatment, and SSB and BPH infestation (1 day after infestation), but not by SA. RNA interference was elected to silence OsHI-LK1 and we finally got silencing OsHI-LK(ir-lk) lines. qRT-PCR analysis showed that OsHI-LK1 expression levels in ir-lk lines were only 15～30% of those in WT rice plants. JA, SA and GLVs levels were lower in ir-lk lines than in WT rice plants after herbivore infestation. Silencing OsHI-LK1 decreased plant resistance to SSB and BPH.更多还原