| 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. |
