马铃薯内源JA-Ile升高对专食性昆虫块茎蛾和广食性昆虫甜菜夜蛾抗性的影响

	马铃薯内源JA-Ile升高对专食性昆虫块茎蛾和广食性昆虫甜菜夜蛾抗性的影响
	李奕
导师	王蕾
关键词	马铃薯，转录组学，JA-Ile羟基化酶，块茎蛾，甜菜夜蛾 potato, transcriptomics, StCYP94B3s, potato tuberworm, beet armyworm
摘要	专食性昆虫块茎蛾（Phthorimaea operculella）和广食性昆虫甜菜夜蛾（Spodoptera exigua）是两种严重影响栽培马铃薯产量和品质的害虫。为了研究马铃薯响应两种食性昆虫取食的异同，本课题首先利用RNA-seq研究了栽培马铃薯响应甜菜夜蛾和块茎蛾取食后的转录组变化。结果表明，昆虫取食后可诱导植物快速合成茉莉酸-异亮氨酸 (jasmonoyl-isoleucine, JA-Ile)，JA-Ile在植物中传递抗虫信号，最终开启植物抗虫防御体系。为了明确提升内源JA-Ile水平对两种食性昆虫抗性的影响，我们利用RNAi技术对栽培马铃薯鄂薯3号（ES3）的JA-Ile代谢基因StCYP94B3-1及其同源基因StCYP94B3-2、StCYP94B3-3进行了共沉默，获得了内源JA-Ile水平增加的irStCYP94B3s植物，但所获得的转基因植物对两种食性的昆虫抗性并不相同。为了探究其原因，我们通过测定irStCYP94B3s植物对块茎蛾及甜菜夜蛾的抗性变化、抗虫化合物胰蛋白酶抑制剂的活性、HGL-DTGs类化合物及苯丙烷类化合物的含量的变化，深入分析了内源JA-Ile 水平提高后马铃薯对两种昆虫抗性不同的原因。具体的研究结果如下： 1. 马铃薯响应甜菜夜蛾取食后共有7843个基因差异表达，响应块茎蛾取食后共有8706个基因差异表达。同时响应块茎蛾和甜菜夜蛾取食的基因多达7096个，这些基因多数富集在代谢、光合作用的天线蛋白、有机体系统、环境适应、植物病原体的相互作用、信号转导、植物激素的信号转导等信号通路中；特异性响应甜菜夜蛾取食的基因共有747个，主要富集在光合蛋白、能量代谢、光合作用、转运、分子伴侣和折叠催化等通路中；特异性响应块茎蛾取食的基因共有1610个，主要富集在卟啉代谢、脂肪酸生物合成、核糖体、辅因子和维他命代谢、植物激素的信号转导、响应信号和细胞过程的蛋白家族、氮代谢、转运、环境信息处理、同源重组和信号转导等通路中。 2. 栽培马铃薯ES3在模拟块茎蛾和甜菜夜蛾取食处理后，均能诱导茉莉酸通路中多个基因上调，这些基因的表达模式及表达量在两种处理间没有显著差异；此外，两种处理均能诱导马铃薯快速积累JA-Ile，诱导的JA-Ile峰值相当，没有显著差异。成功运用RNAi技术共沉默了栽培马铃薯ES3中的StCYP94B3-1, -2, -3基因，获得了irStCYP94B3s-1、irStCYP94B3s-2，这两个株系中StCYP94B3-1, 2, 3基因的沉默效率均超过60%。相比对照ES3，irStCYP94B3s植物对甜菜夜蛾抗性增强，而对块茎蛾抗性不变。 3. 模拟块茎蛾和甜菜夜蛾取食处理后，大部分具有胰蛋白酶抑制剂活性且参与抗虫的蛋白酶抑制剂编码基因上调，这些基因的表达模式及表达量在两种处理间没有显著差异，irStCYP94B3s植物中胰蛋白酶抑制剂活性在两种处理间也没有差异。抗虫物质HGL-DTGs类化合物合成相关基因在响应块茎蛾和甜菜夜蛾取食后的表达模式有较大差异；相较ES3，irStCYP94B3s植物被甜菜夜蛾取食后更多种类的HGL-DTGs化合物含量增加，包括lyciumoside I、 lyciumoside II、 nicotianoside I、attenoside，其中lyciumoside I增加的倍数最高；而块茎蛾取食能诱导更多的nicotianoside II和nicotianoside VII。参与抗虫的苯丙烷代谢途径基因在模拟块茎蛾和甜菜夜蛾取食处理后，引起的转录变化非常相似，两种处理间仅有PAL-9, -10、HCT基因的表达模式不同；相较ES3，irStCYP94B3s植物被甜菜夜蛾取食后五种苯丙烷类化合物O-Coumaroylquinic acid isomer 1 or 2、N-Caffeoylputrescine isomer 1、N',N''-Coumaroyl, sinapoyl spermidine isomer 1 / N',N''-Di-feruloyl-spermidine isomers 1,2,3、N',N''-Coumaroyl,sinapoyl spermidine isomer 1和rutin含量增加，被块茎蛾取食后仅有N',N''-Caffeoyl, feruloyl spermidine isomer 1 or 2、N',N''-Coumaroyl, sinapoyl spermidine isomer 1和rutin含量增加。综上，马铃薯响应专食性昆虫块茎蛾和广食性昆虫甜菜夜蛾取食后的转录组变化高度相似，两种昆虫取食均能诱导JA-Ile快速积累，但是通过转基因共沉默三个JA-Ile羟基化酶编码基因提高内源JA-Ile后，仅提高了马铃薯对甜菜夜蛾的抗性，抗性增加的原因可能与HGL-DTGs化合物lyciumoside I、attenoside以及苯丙烷类化合物O -Coumaroylquinic acid isomer 1 or 2、N -Caffeoylputrescine isomer 1、N',N''-Coumaroyl,sinapoyl spermidine isomer 1/N',N''-Di-feruloyl-spermidine isomers 1,2,3含量增加有关。本研究为利用JA-Ile羟基化酶编码基因培育抗虫性马铃薯奠定了理论基础，也为其它作物的抗虫性改良提供了参考和借鉴。; Potato tuberworm (Phthorimaea operculella, zeller(PTM)) and beet armyworm (Spodoptera exigua, BAW) are two major pests of potato causing severe economical and quality lost every year. To examine how potato responses to the generalist BAW and the specialist PTM, we performed RNA-seq using potato (cultivar ES3) leaves after simulated BAW and PTM feeding treatments. Insect feeding rapidly induces the accumulation of jasmonoyl- isoleucine (JA-Ile), which is the most bioactive jasmonate and functions as a signal for the defensive responses. To examine whether the resistances to the two insects can be enhanced by endogenous JA-Ile increasement, we generated the three JA-Ile hydroxylases simultaneously silenced transgenic potato, irStCYP94B3s, in ES3 background. The irStCYP94B3s plants accumulated higher levels of JA-Ile after simulated BAW and PTM feedings, but their resistances to the two insects are different. We further compared the levels of the well-known anti-herbivory secondary metabolites including trypsin proteinase inhibitor, HGL-DTGs and phenylpropanoid metabolites between BAW and PTM damaged irStCYP94B3s plants. The following results were obtained: 1. In response to simulated BAW and PTM feeding, totally 7843 and 8706 genes differently expressed, respectively. There are 7096 genes co-regulated by BAW and PTM, and these genes were enriched in metabolism, photosynthesis-antenna proteins, organismal systems, environmental adaptation, plant-pathogen interaction, signal transduction, plant hormone signal transduction, environmental information processing, photosynthesis proteins, metabolism of terpenoids and polyketides, alpha-linolenic acid metabolism, not included in pathway or brite, transporters, zeatin biosynthesis, lipid metabolism, biosynthesis of other secondary metabolism, cytochrome P450, enzymes with EC numbers, MAPK signaling pathway-plant; 747 genes were specifically regulated by BAW, and were enriched in photosynthesis proteins, energy metabolism, photosynthesis, transporters, chaperones and folding catalysts pathways; 1610 PTM-specific genes were enriched in porphyrin metabolism, fatty acid biosynthesis, ribosome, metabolism of cofactors and vitamins, plant hormone signal transduction, protein families: signaling and cellular processes, nitrogen metabolism, transporters, environmental information processes, homologous recombination, signal transduction pathways. 2. No differences of the expression patterns and transcription levels of JA pathway gene were found between simulated BAW and PTM feeding. The levels of JA-Ile induced by simulated BAW and PTM feeding are also equivalent in ES3 plants. We generated StCYP94B3-1, -2, -3, three genes encoding JA-Ile hydroxylases, co-silenced irStCYP94B3s plant in ES3 background, and the silencing efficiencies of the three genes are all over 60%. After simulated BAW and PTM feeding, the levels of JA-Ile in irStCYP94B3s plant were about 30% higher than those in wild type plant. The irStCYP94
语种	中文
	2022-05
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/151853/75110
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	李奕. 马铃薯内源JA-Ile升高对专食性昆虫块茎蛾和广食性昆虫甜菜夜蛾抗性的影响[D]. 中国科学院大学,2022.

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