【作者】 李文杰；

【导师】 梁海永；

【作者基本信息】 河北农业大学 ， 林木遗传育种， 2021， 硕士

【摘要】 柳树在我国的园林绿化中是比较常见的树木种类,其具有良好的防风固沙、固堤护岸、改良盐碱地、净化环境等作用,因此有很高的经济价值和观赏价值。但是柳树长期以来经常会受到柳蓝叶甲的侵害,在危害严重时可将整个植株叶片的叶肉吃光,从而导致柳树失去观赏价值,造成严重的经济损失。因此,筛选鉴定出对柳蓝叶甲抗性表现较强的柳树种类并探究其抗性机制,为柳蓝叶甲的有效防治以及对抗虫柳树的选育提供依据,在理论和生产实践方面均有重要意义。本研究对三蕊柳、蒿柳、黄花柳、红皮柳、垂柳、白柳6种柳树通过田间调查和室内饲虫试验进行了抗虫性鉴定,根据鉴定结果将三蕊柳确定为柳蓝叶甲的高抗性柳树。对供试柳树进行MDA含量以及POD、CAT、SOD活性的测定,通过非靶向代谢组与有参转录组确定差异代谢产物,差异基因与代谢通路的关系,对三蕊柳的抗虫机制进行了初步的探究。研究的主要结果如下:1.对六种柳树通过田间直接测定法和室内试验法进行抗虫性鉴定。三蕊柳在田间自然条件下叶片未受到柳蓝叶甲侵害,并且苗木上均未发现柳蓝叶甲成虫及幼虫。室内饲虫试验中三蕊柳的叶片受害面积、成虫存活率、幼虫存活率及蛹化率均显著低于其他柳树的。并且取食三蕊柳的柳蓝叶甲幼虫在饲喂第4d时全部死亡。综合评价这六种柳树对柳蓝叶甲的抗性高低,顺序依次为:三蕊柳、蒿柳、红皮柳、黄花柳、垂柳、白柳。2.对六种柳树分别进行了 SOD、POD和CAT三种抗氧化酶活性的检测以及MDA含量的测定,得到的实验结果为三蕊柳在POD、CAT、SOD三种抗氧化酶参与的催化作用中与其他柳树无显著差别;但是在丙二醛的含量上三蕊柳则显著高于其他柳树。3.将非抗虫柳树与三蕊柳进行非靶向代谢组测定,通过筛选条件共筛得55个显著差异代谢物,有37种为上调差异次生代谢产物,其中上调差异次生代谢产物中差异倍性log2FC>10的代谢物有:异泽兰黄素3-葡萄糖苷、异泽兰黄素-葡萄糖苷、二没食子酰葡萄糖、橙皮苷7-O-新橙皮苷(新橙皮苷)、没食子酰-β-D-葡萄糖、柠檬黄素-3-葡萄糖苷、槲皮素-3-O-桑布双糖苷、槲皮素-3-O-β-D-吡喃木糖(1→2)-β-D-吡喃葡萄糖苷2、槲皮素-3-O-葡萄糖-7-O-鼠李糖苷、异泽兰黄素、2a羟基-齐墩果酸、黄柏甙、木犀草素-香豆酰辅酶A己糖、儿茶素、甲基橙皮甙3-甲基-7-[鼠李糖-α-葡萄糖]橙皮甙、升麻酸A、异绿原酸B、槲皮素-O-戊糖苷-O-己糖苷-O-己糖苷、芥子酸甲酯、水杨酸、矢车菊黄素、2-O-二没食子酰基-β-D-吡喃葡萄糖苷-β-D-吡喃葡萄糖苷、6,8-二异戊烯基香豌豆苷元。抗虫组与非抗虫组中有18种为下调差异次生代谢产物,其中差异倍性log2FC>10的代谢物有:异鼠李素-3-O-芸香糖苷、异鼠李素-3-0-葡萄糖-7-O-鼠李糖苷、邻苯二酚、山奈酚葡萄糖醛酸苷、金圣草黄素-O-己糖-O-己糖-O-葡萄糖醛酸。4.三蕊柳与非抗虫柳树的差异代谢物主要涉及到代谢通路有:苯丙氨酸代谢、抗坏血酸和醛酸代谢、异黄酮的生物合成、谷胱甘肽代谢、植物激素信号转导、黄酮和黄酮生物合成。5.对非抗虫柳树与三蕊柳的转录谱做差异分析,结果表明非抗虫柳与三蕊柳之间共存在5636个差异表达基因,上调基因的数目为1677个,下调基因的数目为3959个。上调差异表达基因主要显著富集在以下几个通路中:苯丙烷生物合成、芪类化合物,二芳基庚烷和姜酚的生物合成、苯丙氨酸代谢、黄酮类生物合成、淀粉和蔗糖代谢、氨基糖和核苷酸糖代谢、半胱氨酸和蛋氨酸的代谢。下调差异表达基因主要显著富集在以下几个通路中:黄酮类生物合成、莨菪烷,哌啶和吡啶生物碱的生物合成、异喹啉生物碱的生物合成。6.差异代谢物与差异表达基因共同注释到的通路有抗坏血酸和醛酸代谢、苯丙氨酸代谢、谷胱甘肽代谢、黄酮类生物合成、植物激素信号转导6个途径。通过分析筛选得出可能对柳蓝叶甲产生毒害作用的代谢通路为苯丙氨酸代谢(ko00360)、黄酮类生物合成(ko00941)两个途径。苯丙氨酸代谢途径中水杨酸显著上调,参与此过程的基因蛋白有 AOC3、TAT、ASP5、PAT、MIF、PAL、CYP73A、4CL、E2.1.1.104。黄酮类生物合成代谢途径中的没食子儿茶素和绿茶儿茶酚显著上调,参与此过程的基因蛋白有 CYP73A、CHS、E5.5.1.6、HCT、CYP98A、E2.1.1.104、ANS、LAR。综上所述,分析推测三蕊柳对柳蓝叶甲表现出高抗性和毒害作用的原因为三蕊柳中水杨酸的较高含量以及相关基因表达量的变化。更多还原

【Abstract】 Willow is a relatively common tree species in my country.It has high economic value.It is an important tree species for embankment reinforcement,bank protection,wind and sand fixation,and improvement of saline-alkali land.However,willow trees have always been invaded by Plagiodera versicolora,which can eat up the whole leaf flesh when it occurs,causing the plant to lose its ornamental value in the garden,causing very serious economic losses.Therefore,screening and identifying willow varieties with strong resistance to Plagiodera versicolora and exploring their resistance mechanisms will provide a basis for the effective control of Plagiodera versicolora and the selection and breeding of insect-resistant varieties,it is of great significance in theory and production practice.In this study,6 species of willows were tested for insect resistance through field investigation and indoor feeding experiments,and the highly resistant species of Plagiodera versicolora was identified as Salix triandra.The MDA content and POD,CAT,SOD activity of the tested varieties were determined,and the differential metabolites were determined through the non-targeted metabolome and the parametric transcriptome,and the relationship between the differential genes and metabolic pathways was used to preliminary research on the insect-resistant mechanism of willow.The main results of the research are as follows:1.The six species of willow,Salix triandra,Salix viminalis var.viminalis,Salix purpurea,Salix caprea L,Salix babylonica,and Salix alba L.,were identified for their resistance to insects through direct field determination and laboratory testing.Under the natural conditions of the field,the leaves of the Plagiodera versicolora were not invaded,and the adult and larvae of the Plagiodera versicolora were not found on the Salix triandra.In the indoor insect feeding experiment,the leaf damage area,adult survival rate,larval survival rate and pupalization rate of Salix triandra lower than those of other varieties.And the larvae of Plagiodera versicolora feeding on the Salix triandra all died on the 4th day of feeding.Comprehensive evaluation of the resistance of these six willow species to Plagiodera versicolora in order from high to low were Salix triandra,Salix viminalis var.viminalis,Salix purpurea,Salix caprea L,Salix babylonica,and Salix alba L.2.Determination of MDA content and SOD,POD,CAT three antioxidant enzyme activities on six willows.The experimental results obtained are that the catalysis of the Salix triandra enzymes of POD,CAT,and SOD is not significantly different from other willows.But the content of malondialdehyde is significantly higher than that of other varieties.3.Non-targeted metabolome assays were performed on non-insect-resistant willows and Salix triandra,and a total of 55 significantly different metabolites were screened through screening conditions.There are 37 kinds of up-regulated differential secondary metabolites.Among them,the metabolites with differential ploidy log2FC>10 among the differential secondary metabolites up-regulated are:Eupatilin 3-glucoside、Eupatilin 3-glucoside、Digalloylglucose、Hesperetin 7-O-neohesperidoside(Neohesperidin)、1,6-Bis-O-galloyl-β-D-glucose、Limocitrin 3-glucoside、Quercetin 3-sambubioside、Quercetin-3-O-β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside、Quercetin-O-rhamnoside-O-Hexoside-O-rhamnoside、Eupatilin、2-Hydroxyoleanolic acid、Phellamurin、C-Hexosyl-luteolin O-p-coumaroylhexoside、Gallocatechin-catechin、Kaempferol-3,7-O-α-L-rhamnoside、Shomaside A、Isochlorogenic acid B、Quercetin-O-Pentoside-O-Hexoside-O-Hexoside、Methyl sinapate、Salicylic acid、Centaureidin、2-O-Di-gallic acyl-β-D-Glucopyranoside-β-D-Glucopyranoside、5,7,4’,5’-Tetrahydro-3’,6-dimethoxyflavone.In the insect-resistant group and the non-insect-resistant group,18 species were down-regulated differential secondary metabolites.Among them,the metabolites with differential ploidy log2FC>10 are:Isorhamnetin-3-O-rutinoside、Isorhamnetin glc-rha、Pyrocatechol、Kaempferol glc-rha、Chrysoeriol O-hexosyl-O-hexosyl-O-Glucuronic acid.4.Differential metabolites are mainly related to metabolic pathways:Phenylalanine metabolism,Ascorbic acid and aldehyde acid metabolism,Isoflavone biosynthesis,Glutathione metabolism,Plant hormone signal transduction,Flavonoids and flavonoid biosynthesis.5.Analysis of the difference between the transcription profiles of non-insect-resistant willow and Salix triandra.The results showed that there were 5636 differentially expressed genes between non-insect-resistant willow and Salix triandra.The number of up-regulated genes was 1677,and the number of down-regulated genes was 3959.The up-regulated differentially expressed genes are mainly significantly enriched in the following pathways::Phenylpropanoid biosynthesis、Stilbenoid,diarylheptanoid and gingerol biosynthesis、Phenylalanine metabolism、Flavonoid biosynthesis、Starch and sucrose metabolism、Amino sugar and nucleotide sugar metabolism、Cysteine and methionine metabolism.Down-regulated differentially expressed genes are mainly significantly enriched in the following pathways:Flavonoid biosynthesis、Tropane,piperidine and pyridine alkaloid biosynthesis、Isoquinoline alkaloid biosynthesis.6.Differential metabolites and differentially expressed genes are jointly annotated to the pathways:Ascorbate and aldarate metabolism、Phenylalanine metabolism、Glutathione metabolism、Flavonoid biosynthesis、Plant hormone signal transduction.Through analysis and screening,the metabolic pathway that may have a toxic effect on the beetle were phenylalanine metabolism(ko00360)and Flavonoid biosynthesis(ko00941)two ways.Salicylic acid is significantly up-regulated in the metabolic pathway of phenylalanine,the gene proteins involved in this process are AOC3,TAT,ASP5,PAT,MIF,PAL,CYP73A,4CL,E2.1.1.104.The gallocatechin and green tea catechol in the biosynthetic pathway of flavonoids are significantly up-regulated,the gene proteins involved in this process are CYP73A,CHS,E5.5.1.6,HCT,CYP98A,E2.1.1.104,ANS,LAR.The analysis speculated that the high resistance and toxic effects of Salix triandra against Plagiodera versicolora might be due to the higher content of salicylic acid in Salix triandra and the changes in the expression of related genes.更多还原