类谷氨酸受体GLR3.3和GLR3.6调控拟南芥系统性抗虫响应 的机理研究

	类谷氨酸受体GLR3.3和GLR3.6调控拟南芥系统性抗虫响应的机理研究
	薛娜
导师	吴建强
关键词	类谷氨酸受体，系统性信号，桃蚜，斜纹夜蛾 Glutamate receptor-like genes, Systemic signaling, Myzus persicae, Spodoptera litura
摘要	植物和植食性昆虫是陆地生态系统的重要组成部分，在二者的共进化过程中，植物进化出了多种抵御植食性昆虫取食的方式。植物不仅会在昆虫取食部位（本地）做出响应而且还可以在昆虫未被取食的远端部位（系统）也产生抗虫响应，这一过程被称为系统性抗虫响应。活性氧（reactive oxygen species，ROS）信号、钙（Ca2+）信号和电信号参与了植物叶片与叶片间系统性抗虫信号的快速传递，而且三者的传递过程均依赖于类谷氨酸受体蛋白（glutamate receptor-like protein）GLR3.3和GLR3.6。尽管GLR3.3和GLR3.6在植物系统性信号传递中的功能较为明确，然而它们是否以及如何参与植物的系统性抗虫响应尚不清楚。本论文以刺吸式口器昆虫桃蚜（Myzus persicae）及咀嚼式口器昆虫斜纹夜蛾（Spodoptera litura）两种不同取食方式的植食性昆虫为研究对象，以不同基因型拟南芥为实验材料，通过抗虫表型分析、激素及次生代谢物检测，并利用转录组和代谢组关联分析，揭示了GLR3.3和GLR3.6在植物系统性抗虫中的作用机理，取得了以下研究成果： 1）植食性昆虫取食诱导的系统性叶片的Ca2+信号的增强依赖于GLR3.3和GLR3.6 基于GCaMP6s的钙离子体内成像检测系统发现：模拟斜纹夜蛾取食（W+OS）及桃蚜取食本地叶片均可以显著诱导拟南芥系统叶片的Ca2+荧光增强；然而GLR3.3和GLR3.6缺失后，则未检测到拟南芥系统性叶片中的Ca2+荧光信号，说明GLR3.3和GLR3.6在植食性昆虫取食诱导的系统性Ca2+信号传递中发挥了重要的作用。 2）GLR3.3和GLR3.6影响植物系统性抗虫响应分别对野生型拟南芥（WT）、glr3.3、glr3.6单突变体及glr3.3 glr3.6双突变体的抗虫能力进行检测发现：桃蚜及模拟斜纹夜蛾取食（W+OS）分别处理不同基因型的拟南芥本地叶片后，与对照组（拟南芥本地叶片无桃蚜或W+OS处理）相比，在WT系统叶片取食的桃蚜繁殖率及斜纹夜蛾体重显著降低；而在glr3.3 glr3.6双突变体中则均未发生明显变化，表明glr3.3 glr3.6双突变体的系统性抗虫响应丢失。而且与WT相似，在glr3.3和glr3.6单突变体的系统叶片中桃蚜繁殖率和斜纹夜蛾体重较对照组也降低，表明GLR3.3和GLR3.6是拟南芥系统性抗虫响应的关键基因，且存在功能冗余性。此外，与抗虫表型一致的是，桃蚜取食和模拟斜纹夜蛾取食（W+OS处理）都可以诱导WT、glr3.3、glr3.6单突变体系统叶片中植物激素茉莉酸（jasmonic acid，JA）和芥子油苷（glucosinolate，GS）含量的升高，但是JA和GS在glr3.3 glr3.6双突变体的系统叶片中则没有变化。 3）GLR3.3和 GLR3.6调控拟南芥系统叶片响应植食性昆虫取食的转录组变化转录组分析发现：桃蚜取食和模拟斜纹夜蛾取食（W+OS）诱导的系统叶片中的差异基因（DEGs）的表达相较于本地叶片，更依赖于GLR3.3和GLR3.6的调控，进一步KEGG分析发现，DEGs主要参与了氨基酸及碳水化合物的代谢；此外，JA和GS相关基因在系统叶片中的表达也部分依赖于GLR3.3和GLR3.6的调控。 4）植食性昆虫取食诱导的代谢物系统性积累依赖于GLR3.3和GLR3.6 代谢组分析发现：系统叶片中的代谢物，尤其是芥子油苷合成的关键前体氨基酸（色氨酸、缬氨酸和异亮氨酸）、糖类（葡萄糖、果糖、半乳糖和蔗糖）以及有机酸类物质（柠檬酸和苹果酸）对桃蚜取食及W+OS处理的响应依赖于GLR3.3和GLR3.6。 5）GLR3.3和 GLR3.6不参与拟南芥损伤信号由地下部分向地上部分的传递损伤拟南芥的本地叶片后，其系统叶片及根部的JA及GS的响应依赖于GLR3.3和GLR3.6；然而当拟南芥的根部遭受损伤后，glr3.3 glr3.6和WT叶片的JA和GS的含量均上调，表明GLR3.3和GLR3.6介导了损伤信号由叶片向根部的传递，而不影响叶片对根部损伤产生的系统性响应。综上所述，本论文深入地分析了类谷氨酸受体GLR3.3和GLR3.6在植物系统性抗虫响应中的功能，揭示了GLR3.3和GLR3.6不仅调控植物响应植食性昆虫取食产生的系统性Ca2+信号的传递，还调控植物系统性抗虫响应，包括抗性基因的表达以及抗虫代谢物的积累。本论文为从分子水平上了解植物系统性抗虫响应提供了重要的数据支持，也为培育抗虫农作物提供了参考。; Plants and insects are important components of terrestrial ecosystems, and plants have evolved a variety of ways to defend against insect herbivores during the coevolution of plants and insects. Plants accumulate defensive compounds not only in herbivore-damaged leaves but also in distal intact (systemic) leaves, which is known as systemic response. Reactive oxygen species (ROS), calcium (Ca2+), and electrical signaling are involved in the rapid transmission of systemic signals from leaf to leaf in plants, and the transmission of all these signals relies on the glutamate receptor-like 3.3 and 3.6 (GLR3.3 and GLR3.6). Although the functions of GLR3.3 and GLR3.6 in plant systemic signal transmission are well understood, whether and how they mediate plant systemic resistance to insects remain largely unexplored. In this dissertation, we used two insects with distinct feeding mode, the piercing-sucking insect Myzus persicae (green peach aphid, GPA) and the chewing insect Spodoptera litura (cotton leafworm, CLW), and multiple mutants of Arabidopsis as the experimental systems; by analyzing the resistance to insect feeding, measuring the contents of phytohormones and secondary metabolites, integrating transcriptome and metabolome profiling, our research revealed the mechanism underlying the regulation of GLR3.3 and GLR3.6 in plant systemic response to insect herbivores. The main results are listed as the following: 1) Insect feeding-induced systemic increase of [Ca2+]cyt depends on GLR3.3 and GLR3.6 Using the GCaMP6s Ca2+ sensor, we found both mimicking CLW feeding (W+OS) and GPA feeding on local leaves significantly induced systemic Ca2+ increases in Arabidopsis, while the Ca2+ fluorescence signal was not detected in Arabidopsis mutants with function loss of GLR3.3 and GLR3.6, suggesting that GLR3.3 and GLR3.6 play an important role in insect feeding-induced systemic Ca2+ signaling. 2) GLR3.3 and GLR3.6 are required for systemic resistance to insect herbivores By examining the insect resistance of wild-type (WT) Arabidopsis, glr3.3 and glr3.6 single mutant, and glr3.3 glr3.6 double mutant, we found that after GPA feeding or W+OS treatment on the local leaves, compared with the control group in which the local leaves were left untreated, the GPA nymph numbers and the masses of CLWs were significantly decreased when feeding on the systemic leaves of WT, but not on the systemic leaves of glr3.3 glr3.6 double mutants, indicating that the systemic resistance of glr3.3 glr3.6 double mutants was compromised. Furthermore, similar to the WT plants, compared with the control group, the GPA nymph numbers and the masses of CLWs were also reduced in systemic leaves of the glr3.3 and glr3.6 single mutants, indicating that GLR3.3 and GLR3.6 are key genes but played redundant role in systemic resistance to the GPA and CLW. Furthermore, consistent with the insect-resistant phenotype, GPA feeding and W+OS treatment elevated the JA (jasmonic acid) and GS (glucosinolate)
语种	中文
	2022-11
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75227
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	薛娜. 类谷氨酸受体GLR3.3和GLR3.6调控拟南芥系统性抗虫响应的机理研究[D]. 中国科学院大学,2022.

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