【作者】 高素红；

【导师】 高宝嘉；

【作者基本信息】 河北农业大学 ， 森林保护， 2022， 博士

【摘要】 本文探究了绿盲蝽Apolygus lucorum（Meyer-Dür,1843）取食刺激诱导赤霞珠葡萄Vitis vinifera L.’Cabernet Sauvignon’防御反应的动态变化规律及其防御机制。通过观察研究绿盲蝽刺吸为害、物理机械损伤诱导与赤霞珠葡萄防御互作中的表观响应,明确了绿盲蝽不同虫龄、不同密度、不同时间取食刺激下赤霞珠葡萄的防御反应变化规律。依据绿盲蝽不同取食时间刺激下赤霞珠葡萄防御反应中早期信号分子、酶系响应和生化代谢产物的变化规律,构建了赤霞珠葡萄-绿盲蝽典型自然生物反应模型和赤霞珠葡萄-针刺损伤典型物理反应模型。应用生物信息学研究技术,分析典型生物反应模型和物理反应模型的基因表达变化趋势特征,揭示赤霞珠葡萄响应绿盲蝽刺吸取食的相关代谢途径,探讨绿盲蝽诱导赤霞珠葡萄防御反应的分子基础与机制。主要研究结果如下:1.研究了赤霞珠葡萄对绿盲蝽不同虫龄在不同密度、不同取食时间下的防御反应规律。绿盲蝽连续刺吸4 d及以下,对赤霞珠生长无显著影响;连续取食4 d以上,对赤霞珠葡萄株高和叶宽造成显著和极显著影响。绿盲蝽连续取食赤霞珠葡萄4 d时死亡率增幅最大,死亡率超过50%。试验明确了赤霞珠葡萄嫩茎自上而下完全展开的第Ⅲ叶位叶片以上的部位受害,且以5头三龄若虫取食处理效应最为明显。2.利用“Y”型嗅觉仪观测了绿盲蝽对取食和针刺损伤不同时间赤霞珠幼苗的选择趋性。绿盲蝽对持续取食24 h内的赤霞珠植株表现出一定正趋性,对持续取食48 h及以后、物理损伤处理表现出负趋性,且于4 d左右时与赤霞珠健康植株达到极显著和显著差异。3.绿盲蝽取食和针刺物理损伤诱导赤霞珠葡萄防御反应过程中,不同信号分子和保护酶、防御酶的应激响应不同。绿盲蝽取食和针刺物理损伤后,赤霞珠葡萄叶片中JA含量迅速增加,均在6 h内达到最大值,比CK分别增加了 10.9倍和6.5倍,绿盲蝽取食刺激较针刺损伤诱导JA含量明显为高。试验发现外源JA诱导赤霞珠葡萄叶片中各防御酶活性变化规律与绿盲蝽取食诱导结果趋于一致。绿盲蝽取食和针刺损伤后赤霞珠叶片内SA内源含量没有发生明显变化。与抗性相关的酶系SOD、CAT、PPO、AOS、LOX活性在赤霞珠防御反应前期显著升高,PAL和POD活性于诱导第4 d显著升高。针刺损伤与绿盲蝽取食刺激相比,酶活性变化幅度较低。4.绿盲蝽取食和针刺物理损伤诱导赤霞珠葡萄叶片内蛋白酶抑制剂和初生次生代谢物质含量明显变化。绿盲蝽取食诱导两种蛋白酶抑制剂、黄酮、单宁、MDA及氨基酸含量在防御初期即显著升高,且持续高于CK。胰凝乳蛋白酶抑制剂活性和总酚含量在第4 d左右明显升高。而赤霞珠葡萄叶片内总糖、蛋白质及叶绿素含量在第4d左右显著降低。针刺物理损伤与绿盲蝽取食反应明显不同,除MDA外,针刺损伤诱导赤霞珠叶片中次生代谢物质含量较绿盲蝽取食为少,且未导致总糖等营养物质含量降低。5.绿盲蝽取食刺激导致赤霞珠葡萄叶片在转录组水平上发生明显变化。绿盲蝽取食刺激和针刺机械损伤与对照两组间差异表达基因（DEGs）分别为938和972个。其中绿盲蝽取食刺激与对照相比,上调基因明显多于下调基因,有762个基因表达量升高,176个基因表达量下降;而针刺机械损伤与对照相比,下调基因稍多于上调基因,具体为431个基因表达量升高,541个基因表达量下降;昆虫取食和针刺机械损伤两组间的DEGs为1605个,其中上调基因明显多于下调基因,有1008个基因表达量升高,595个基因表达量下降。上、下调DEGs在绿盲蝽取食、针刺机械损伤与对照两两处理间表达水平差异显著。绿盲蝽取食刺激赤霞珠葡萄叶片基因表达图谱与对照和针刺机械损伤相去甚远,形成独立的群体。6.通过转录组差异基因表达量分析筛选出与抗性相关的重要基因,如:MEKK激酶基因家族,WRKY转录因子基因家族,ERF1,JAZ,MYC2,CALM,CPK,PBS1,RBOH,CML,RPM1,EDS1,PP2C,SNRK2,PYL,ACS1₂₆基因等,这些功能基因在绿盲蝽取食和针刺损伤赤霞珠葡萄叶片抗性形成中起着重要作用。7.通过与GO和KEGG数据库对比,将所有DEGs进行pathway的显著富集分析,解析预测了差异表达基因的功能类别与其调控的不同代谢途径。本试验主要关注了信号转导过程、植物的抗氧化过程、植物与病原菌互作、MAPK激酶通路等免疫反应过程、次生物质代谢过程和细胞内过程等。这些与抗性相关的代谢通路均被绿盲蝽取食刺激诱导启动,富集到大量DEGs。但针刺物理损伤诱导赤霞珠葡萄叶片差异基因仅显著富集在类胡萝卜素生物合成通路上,而植物激素信号转导和植物病原菌互作代谢通路上DEGs富集不显著,分别被富集到3个和2个基因,MAPK植物信号通路上的DEGs为0。8.本试验明确了 GAPDH为赤霞珠葡萄叶片基因表达RT-qPCR分析中最为合适的内参基因。通过内参基因对转录组学中关注的显著上调或下调的20个关键基因的表达量进行了 RT-qPCR验证检测。所筛选20个明星基因表达量与转录组学结果一致。表明转录组RNA测序的结果真实而且可靠。更多还原

【Abstract】 The infestation of the pest Apolygus lucorum（Meyer-Dür,1843）induced changes in Vitis vinifera L.’Cabernet Sauvignon’ at the growth,physiological,and biochemical levels,early signal material,transcriptomics,which were recorded in this study.By observing and studying the apparent response of A.lucorum infestation,Puncture injury induced in the defense interaction of Cabernet Sauvignon,it was clear that the changes in defense response of Cabernet Sauvignon under the stimulation of different insect ages,different densities and different infestation times.According to the changes of early signal molecules,enzyme response and biochemical metabolites in the defense response of Cabernet Sauvignon stimulated by A.lucorum of different infestation times,the typical natural biological reaction model of grape-pest and the typical physical reaction model of grape-acupuncture were constructed.Bioinformatics technology was applied to analyze the variation characteristics of differential gene expression between typical biological response model and physical response model,revealed the related metabolic pathways of Cabernet Sauvignon response stimulated,explored the molecular basis and mechanism of defense response induced by A.lucorum infestation.The main results were as follows:1.The defense response of Cabernet Sauvignon to different insect ages,different density and different infestation time was studied.The growth of Cabernet Sauvignon was not significantly affected by the continuous injection of 4 d and below by A.lucorum.But continuous infestation for more than 4 days had significant and extremely significant effects on plant height and leaf width of Cabernet Sauvignon.The death rate increased the most when A.lucorum continuously ate Cabernet Sauvignon for 4 days,the death rate was more than 50%.The experiment confirmed that the damage occurred above the Ⅲth leaf of the tender stem of Cabernet Sauvignon,and the infestation effect was most obvious in 5 third-instar nymphs.2.The selection trend of Cabernet Sauvignon seedlings at different time of infestation and acupuncture injury was observed by Y type olfactory instrument.It showed a certain positive tendency to cabernet Sauvignon plants within 24 h of continuous infestation,and a certain negative tendency to cabernet Sauvignon plants after 48 h of continuous infestation and treatment of puncture injury.Moreover,at about 4 d,it was significantly different from cabernet Sauvignon healthy plants.3.The stress responses of different signal molecules,protective enzymes and defensive enzymes were different in the process of the Cabernet Sauvignon defense response induced by acupuncture physical injury and A.lucorum infestation.After injury caused by A.lucorum infestation and puncture injury,JA content in grape leaves of Cabernet Sauvignon increased rapidly,reached its maximum value within 6 h,which increased by 10.9 times and 6.5 times respectively compared with CK.The content of JA induced by A.lucorum infestation was significantly higher than that induced by acupuncture.It was found that the variation of the defense enzyme activity in grape leaves induced by exogenous JA was consistent with that induced by the green leaf bug fed.The content of SA in Cabernet Sauvignon leaves did not change obviously after the injury of infestation and acupuncture.The activities of SOD,CAT.PPO,AOS and LOX related to resistance were significantly increased in the early stage of cabernet Sauvignon defense reaction,and the activities of PAL and POD were significantly increased on the 4th day after induction.The change range of enzyme activity in puncture injury was lower than that in infestation.4.Puncture injury induced and by A.lucorum infestation induced significant changes in protease inhibitors and primary secondary metabolites in grape leaves of Cabernet Sauvignon.The contents of protease inhibitors,flavones,tannins,MDA and amino acids were significantly increased at the initial stage of defense induced by infestation of A.lucorum,and it was consistently higher than CK.The activity of chymotrypsin inhibitor and the content of total phenols were significantly increased on the 4th day.However,the contents of total sugar,protein and chlorophyll in grape leaves of Cabernet Sauvignon were significantly decreased around the 4th day.The physical injury of acupuncture was obviously different from the infestation reaction of A.lucorum.In addition to MDA,the content of secondary metabolites in the Cabernet Sauvignon leaves induced by acupuncture injury was less than that of the leaves fed by green leaf bug,and the content of total sugars and other nutrients did not decrease.5.The transcriptome changes of Cabernet Sauvignon grape leaves were caused by the stimulation of A.lucorum infestation.There were 938 DEGs and 972 DEGs between the infestation and acupuncture groups,respectively.Among them,compared with the control group,the up-regulated genes were significantly more than the down-regulated genes on infestation reaction,with 762 genes expressing increased and 176 genes expressing decreased.Compared with the control group,the down-regulated genes were slightly more than the up-regulated genes in physical injury,specifically,the expression levels of 431 genes were increased and 541 genes were decreased.The DEGs between the two groups of insect infestation and puncture injury were 1605,among which the up-regulated genes were significantly more than the down-regulated genes,with 1,008 genes expressing increased and 595 genes expressing decreased.The expression levels of up-regulated and down-regulated DEGs were significantly different among the treatment of acupuncture.infestation and the control.The gene expression profile of Cabernet Sauvignon leaves stimulated by A.lucorum infestation was far different from that of control and mechanical injury caused by acupuncture,formed an independent group.6.Star genes related to resistance were selected through transcriptome differential gene expression analysis,such as:MEKK kinase gene family,WRKY transcription factor gene family,ERF1,JAZ,MYC2,CALM,CPK,PBS1,RBOH,CML,RPM1,EDS1,PP2C,SNRK2,PYL,ACS1₂₆ genes,etc.These functional genes played an important role in the defense response of Cabernet Sauvignon by A.lucorum infestation and needling.7.Through comparison with GO and KEGG databases,significant enrichment analysis of all DEGs was carried out to analyze and predict the functional categories of differentially expressed genes and the different metabolic pathways they regulate.This experiment mainly focused on signal transduction process,antioxidant process of plants,interaction between plants and pathogenic bacteria,immune reaction process such as MAPK kinase pathway,secondary substance metabolism process and intracellular process,etc.These resistance-related metabolic pathways were induced and activated by the stimulation of A.lucorum infestation and enriched to a large number of DEGs.However,the leaf differential genes of Cabernet Sauvignon were only significantly enriched in the carotenoid biosensation pathway induced by puncture injury.However,DEGs enrichment was not significant in the metabolic pathways of plant hormone signal transduction and plant pathogen interactions,which were enriched to 3 and 2 genes respectively,and DEGs on MAPK plant signal pathway was 0.8.This experiment confirmed that GAPDH was the most suitable internal reference gene for RT-qPCR analysis of gene expression in grape leaves of Cabernet Sauvignon.RT-qPCR was used to verify the expression levels of 20 key genes that were significantly up-regulated or down-regulated in transcriptomics with reference genes.The expression levels of 20 star genes screened were consistent with the results of transcriptome.It showed that the results of transcriptome RNA sequencing were true and reliable.更多还原