【作者】 张战凤；

【导师】 刘同先；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2022， 博士

【摘要】 麦长管蚜Sitobion avenae(Fabricius)是小麦上的重要害虫,严重影响小麦产量和品质(有研究表明在我国的种群应为荻草谷网蚜Sitobion miscanthi,本文仍沿用麦长管蚜)。当前对麦蚜的防控仍主要依赖于化学农药,然而化学防治带来环境及生态方面的负面影响越来越受到关注。小麦的生产实践表明培育和推广抗蚜品种是一种低成本、环境友好的蚜虫防治手段,然而小麦抗蚜种质的稀缺及抗性机制研究的滞后严重影响了小麦抗蚜育种的进程。明确麦长管蚜与小麦间的互作机制,对鉴选抗蚜小麦种质资源及麦长管蚜的科学防治具有重要意义。游离氨基酸是小麦重要的初生代谢产物,也是麦长管蚜主要的氮素营养,然而游离氨基酸是否和小麦的抗蚜性相关还需进一步明确。苯并噁唑嗪酮类化合物是一类广泛存在于小麦中的次生代谢产物,对多种植食性昆虫具有防御功能,但其和麦长管蚜的抗性关系仍存在分歧。本研究以麦长管蚜为研究对象,以多种不同来源、不同抗性水平的小麦品种(系)为材料,研究小麦植株内初生代谢产物(游离氨基酸)及次生代谢产物(苯并噁唑嗪酮类化合物)的动态变化和蚜虫消长的关系,评估不同层次代谢产物对麦长管蚜的影响,探讨麦长管蚜对小麦防御反应的适应能力。主要研究结果如下:1.不同小麦品种(系)麦长管蚜抗性水平的苗期鉴定采用蚜量比值法对10个不同来源小麦品种(系)的抗性水平进行了连续多个世代的苗期鉴定,结果表明不同小麦品种(系)对麦长管蚜的抗性差异显著,其中表现为“中抗”品种(系)1个,“低抗”品种(系)3个,其余6个品种(系)表现为“低感”和“中感”。小偃22-3(小偃22的姊妹系)的抗性水平最强,西农979最适合麦长管蚜生长。通过对麦长管蚜在不同小麦品种(系)上连续世代的种群动态研究发现,寄主植物对麦长管蚜的影响具有世代累加效应,其中一些小麦品种(系)随着麦长管蚜世代的增加抗性水平依次增强,也有一些品种(系)随着麦长管蚜世代的增加抗生性水平逐渐减弱。2.小麦不同发育时期韧皮部游离氨基酸组成谱的变化及对麦长管蚜抗生性的影响对麦长管蚜在苗期及抽穗期小麦叶片上体重和生殖力进行了测定,并对不同发育时期小麦叶片的韧皮部游离氨基酸组成进行了分析。麦长管蚜在抽穗期小麦旗叶上的体重、生殖力及蜜露分泌量显著高于苗期小麦,在苗期对麦长管蚜有抗性的小麦品种小偃22在抽穗期抗蚜性丧失,韧皮部氨基酸分析结果表明抽穗期小麦叶片韧皮部谷氨酰胺、天冬酰胺的相对含量显著高于苗期,小偃22抽穗期抗蚜性的丧失可能与谷氨酰胺、天冬酰胺等非必须氨基酸含量的上升有关。3.苯并噁唑嗪酮类化合物与麦长管蚜的抗性关系研究对13个不同来源小麦品种(系)叶片中的苯并噁唑嗪酮类化合物的含量进行了定性和定量检测,发现小麦苗期叶片中的苯并噁唑嗪酮类化合物主要有以下6种:4-羟基-1,4-苯并恶嗪-3-酮-2-O-β-D-葡萄糖苷(DHBOA-Glc)、1,4-苯并恶嗪-3-酮-2-O-β-D-葡萄糖苷(HBOA-Glc)、2-羟基-4-甲氧基-1,4-苯并恶嗪-3-酮-2-O-β-D-葡萄糖苷(HMBOA-Glc)、2,4-二羟基-7-甲氧基-1,4-苯并恶嗪-3-酮(丁布,DIMBOA)、4-羟基-7-甲氧基-1,4-苯并恶嗪-3-酮-2-O-β-D-葡萄糖苷(DIMBOA-Glc)和4,7-二甲氧基-1,4-苯并恶嗪-3-酮-2-O-β-D-葡萄糖苷(HDMBOA-Glc)。小麦叶片中苯并噁唑嗪酮类化合物的含量在不同小麦品种(系)间差异显著,但苯并噁唑嗪酮类化合物的组成型含量与麦长管蚜的抗性水平无显著相关关系。一些苯并噁唑嗪酮类化合物的合成可以被麦长管蚜取食所诱导,该诱导作用在不同小麦品种(系)间表现各不相同。在一些小麦品种(系)中,麦长管蚜取食可以诱导DIMBOA-Glc转化为HDMBOA-Glc,从而使DIMBOA-Glc对胼胝质的诱导能力减弱,进而弱化了小麦对麦长管蚜的抗性。4.麦长管蚜取食诱导的小麦韧皮部游离氨基酸组成谱的变化及麦长管蚜对不均衡氨基酸营养的适应性研究麦长管蚜前期取食处理对继代麦长管蚜的取食行为影响不显著,在一些抗性小麦品种(系)上麦长管蚜前期取食处理后的植株反而更适合继代麦长管蚜的生长。韧皮部氨基酸分析结果表明,麦长管蚜取食诱导后小麦叶片韧皮部必需氨基酸的相对含量显著降低,而谷氨酰胺、天冬酰胺等非必需氨基酸的相对含量显著升高。用谷氨酰胺、天冬酰胺对小麦进行灌根处理能显著提高麦长管蚜的体重和生殖力,具有麦长管蚜抗性的小麦品种(系)经天冬酰胺及谷氨酰胺灌根处理后抗蚜性减弱。人工饲料结果表明,不同的氨基酸配比对麦长管蚜的体重及生殖力有显著影响,天冬酰胺及谷氨酰胺对麦长管蚜的生长发育起重要作用。5.麦长管蚜取食诱导的小麦叶片转录组学分析对小偃22-3的转录组学分析表明:麦长管蚜取食后小偃22-3叶片内有3657个基因的转录水平发生改变,其中上调基因2334个,下调基因1323个。这些基因主要富集于能量代谢、碳水化合物代谢、氨基酸代谢、次生代谢产物合成及信号传导等生物学通路。其中水杨酸信号通路有49个基因显著上调,这些基因主要为苯丙氨酸氨裂解酶和病程相关蛋白。丝裂原活化蛋白激酶(MAPK)通路有23个基因显著上调,主要为MAPK蛋白及WRKY转录因子。转录组数据表明麦长管蚜取食对小麦的光合作用产生了负面影响,有35个光合作用相关基因显著下调,下调基因主要包括光系统蛋白、叶绿素结合蛋白及光合作用中的重要酶类。麦长管蚜取食诱导小麦谷氨酰胺合成酶、天冬酰胺合成酶的转录水平显著提高,而谷氨酸合成酶的表达水平未受影响,这可能是导致谷氨酰胺和天冬酰胺在小麦叶片中积累的原因之一。通过转录组数据共鉴定到20个苯并噁唑嗪酮类化合物的合成基因,其中DIMBOA-Glc甲基转移酶基因(Bx10)的转录水平显著上调,其可以催化DIMBOA-Glc向HDMBOA-Glc转化。转录组数据说明麦长管蚜取食主要激活MAPK—SA级联信号通路,从而使小麦启动下游一系列调控反应,导致小麦初生及次生代谢产物发生重组,使小麦启动防御反应或增强耐害性。本研究从不同代谢产物层面研究了小麦和麦长管蚜的互作现象,并对其中的机理进行了必要探索。本研究表明,相对于苯并噁唑嗪酮类化合物,寄主植物的氨基酸营养水平对麦长管蚜的影响更为显著,麦长管蚜危害可以改变小麦叶片韧皮部的氨基酸组成谱,麦长管蚜能适应寄主不均衡的氮素营养,其可以利用天冬酰胺、谷氨酰胺等非必需氨基酸满足个体及种群的发展。本研究的结果将丰富小麦—麦长管蚜互作机制相关理论,同时为麦蚜防治和小麦抗蚜育种提供参考及切入点。更多还原

【Abstract】 The English grain aphid Sitobion avenae(Fabricius)is one of the major pests of wheat,which seriously affects the quality and yield of wheat(In recent years,this insect species is also considered as Sitobion miscanthi in China.In this paper,we still used the name S.avenae).Currently,chemical control is the main strategy of controlling S.avenae,but the negative effects of pesticides have brought more serious challenges upon our ecosystem and environment.Growing aphid-resistant wheat varieties are considered as the most economical,safe,and environment-friendly method to control S.avenae,while the lack of aphid-resistance germplasm has seriously hindered the process of breeding aphid-resistant wheat variety.Understanding the interaction mechanism between wheat and S.avenae is of great significance to the control of S.avenae.Amino acids play crucial roles in plant-aphid interactions,not only as regulation factors of nutrition status for host plant but also as key nitrogen nutrition for aphid development.However,the fitness benefits of amino acids metabolism to host plant and the subsequent effects to aphid remain poorly understood.Benzoxazinoids are important secondary metabolites in wheat and have inhibitory and toxic effects against a wide range of herbivore pests,while the relationship between benzoxazinoid level and wheat resistance to aphids remains controversial.In this study,we evaluated the dynamic changes of the primary metabolites(amino acids),secondary metabolites(benzoxazinoids)and S.avenae performance in different wheat varieties/lines,and investigated the interactions between S.avenae and wheat host plant,Triticum aestivum.The main results are as follows:1.Evaluation of wheat resistance to S.avenae at the seedling stageWe investigated the aphid quantity ratio(AQR)of 10 different wheat varieties/lines based on the number of S.avenae that had colonized the plants.By comparing the differences of S.avenae performance among continuous generations on different wheat varieties/lines,we found that the AQR was significantly affected by the wheat varieties/lines.One wheat variety/line exhibited median resistance(MR)and 3 wheat varieties/lines exhibited low resistance(LR)to S.avenae.The other six varieties/lines exhibited low susceptible(LS)and median susceptible(MS)to S.avenae.Line xiaoyan22-3(sib-line of variety xiaoyan22)exhibited the strongest resistance to S.avenae,while xinong979 was the most susceptible.Besides,the antibiosis to S.avenae showed cumulative effects with the increase of aphid generations in some resistance varieties/lines.2.Changes in free amino acid composition of phloem sap and the antibiosis resistance to S.avenae on wheat at different developmental stagesWe investigated the performance of S.avenae on wheat at different developmental stages and analyzed the amino acid composition of phloem sap of host plant leaves.We found that S.avenae performed better on wheat leaves at heading stage in terms of weight gain and fecundity increase compared with those at seedling stage.The wheat varieties that were resistant to S.avenae at seedling stage lost resistance at heading stage.Nutritional quality of phloem sap analysis showed that higher proportion of glutamine and asparagine seemed to favor the performance of S.avenae on wheat.3.Impacts of constitutive and induced benzoxazinoids levels on wheat resistance to the Sitobion avenaeWe analyzed the content of benzoxazinoids of 13 wheat varieties/lines qualitatively and quantitatively.Overall,six benzoxazinoids were detected and identified by mass spectrometry-based metabolites profiling,including three lactams,two hydroxamic acids,and one methyl derivative.These compounds were as follows:2-O-β-D-glucopyranosyloxy-7-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one(DHBOA-Glc),2-O-β-D-glucopyranosyloxy-1,4-benzoxazin-3(4H)-one(HBOA-Glc),and 2-O-β-D-glucopyranosyloxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one(HMBOA-Glc),2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one(DIMBOA)and 2-O-β-D-glucopyranosy-loxy-4-hydroxy-7-(2H)-methoxy-1,4-benzoxazin-3(4H)-one(DIMBOA-Glc)and2-O-β-D-glucopyranosyloxy-4,7-dimethoxy-(2H)-1,4-benzoxazin-3(4H)-one (HDMBOA-Glc).The constitutive levels of these benzoxazinoids were significantly different among the wheat varieties/lines.However,none of these benzoxazinoids exhibited considerable correlation with aphid resistance.S.avenae feeding elevated the level of HDMBOA-Glc and reduced the level of DIMBOA-Glc in some of the wheat varieties/lines.Converting DIMBOA-Glc to HDMBOA-Glc suppressed callose deposition and subsequently reduced aphid resistance.4.Changes in free amino acid composition of phloem sap induced by S.avenae feeding and the adaptation of S.avenae to unbalanced amino acid nutritionWe evaluated the performance and feeding behavior of S.avenae on non-infested(control)and previously infested(pre-infested)seedlings of two wheat cultivars.The amino acid composition in the phloem of wheat leaves was examined and its potential effects on aphid’s performance were evaluated by artificial diets based on the amino acids ratio of phloem.Aphid performance results showed that S.avenae performed better on pre-infested seedlings in terms of weight gain and fecundity increase compared with those on the control seedlings especially in the resistance varieties/lines.Furthermore,the electrical penetration graph(EPG)recordings of aphid probing behavior revealed that pre-infested wheat had no obvious effects on feeding behavior of S.avenae.Nutritional quality of phloem sap analysis showed that the relative contents of essential amino acids significantly decreased after aphid feeding,whereas the relative content of non-essential amino acids such as glutamine and asparagine were significantly increased.Wheat seedlings root-drenched with glutamine and asparagine significantly increased weight and fecundity of S.avenae.We evaluated the potential effects of amino acid composition on the performance of S.avenae by artificial diets.The results showed that amino acid composition had significant effects on the weight gain and fecundity of S.avenae and the contents of glutamine and asparagine had a significant impact on the performance of S.avenae.5.Transcriptome analyses of wheat in response to S.avenae feedingWe investigated the wheat transcriptomic changes in response to S.avenae feeding by high-throughput RNA sequencing.The expression patterns of genes involved in defense signaling pathway and photosynthesis process were also evaluated.A total of 3,657 genes were either upregulated(2,334 genes)or downregulated(1,323 genes)and differed significantly in response to aphid feeding.KEGG enrichment analysis showed that these genes were mainly involved in the energy metabolism,carbohydrate metabolism,amino acid metabolism,biosynthesis of other secondary metabolites and signal transduction.The transcriptome data showed that 49 genes involved in salicylic acid signaling pathways were significantly upregulated in response to S.avenae feeding.These genes mainly included phenylalanine ammonia-lyase(PAL)pathogenesis-related protein(PR),and non-expresser of pathogenesis-related gene 1(NPR1).Furthermore,23 genes involved in MAPK-WRKY pathways were significantly upregulated by S.avenae feeding and most of genes were mitogen-activated protein kinases(MAPKs)and WRKY transcription factors(TFs).S.avenae feeding negatively affected the photosynthesis process of wheat.Thirty-five genes associated with photosynthesis process,such as photosystem I/II proteins,chlorophyll a/b binding proteins and photosynthetic NDH were significantly downregulated.S.avenae feeding can affect the balance of amino acids metabolism of wheat.Glutamine and asparagine synthetase genes were significantly upregulated by S.avenae feeding,while the expression level of glutamate synthase genes was not affected.This may be the reason for the accumulation of glutamine and asparagine in wheat leaves.S.avenae feeding can also affect the balance of benzoxazinoids metabolism in wheat plants.DIMBOA-Glc O-methyltransferase genes(Bx10),which converts DIMBOA-Glc to HDMBOA-Glc were significantly upregulated by S.avenae feeding.In summary,S.avenae feeding mainly activated the MAPK-SA signaling pathway and initiated the recombination of primary and secondary metabolites in wheat.The defense response and the tolerance to aphids would be enhanced by these mechanisms in wheat plants.In conclusion,this study demonstrated that host plant nutrition condition was more important than the levels of benzoxazinoids in the wheat plants and S.avenae generally preferred to obtain more nutrition rather than avoiding host plant defense.This study also showed that feeding by S.avenae could alter host plant nutritional conditions and S.avenae could adapt to unbalanced nitrogen nutrition by utilizing non-essential amino acids.更多还原