Transcriptional Regulation Of Thrips Resistance In Alfalfa(Medicago Sativa L.)

Alfalfa（Medicago sativa L.）is an important material basis for Agricultural industrial structure adjustment and grass and animal husbandry development.However,thrips damages will reduce the yield,cause the loss of nutrients,and seriously affect the development of alfalfa industry..In this study,two alfalfa varieties,Caoyuan No.4and Caoyuan No.2（as the control）,treated with and without thrips inoculation were used to perform the experiment.Based on the assessment of thrips resistance,comparative analysis was conducted at the physiological and biochemical,phytohormone and transcriptome levels to investigate the transcriptional regulatory network of thrips resistance in alfalfa.The results may provide theoretical reference for the study of transcriptional regulation of insect resistance in alfalfa.The main findings are as follows:（1）The insect index of Caoyuan No.4（0.378）was extremely significantly lower than that of Caoyuan No.2（0.841）（P<0.05）,confirming that Caoyuan No.4 is much more resistant to thrips than Caoyuan No.2.（2）The main physiological and biochemical indicators of thrips resistance in alfalfa showed that the PPO activity,POD activity,APX activity,SOD activity,LOX activity,H₂O₂activity,superoside anion and ABA content of Caoyuan No.4 were significantly higher than those of Caoyuan No.2 without thrips feeding（P<0.05）,indicating that these indicators may play an important role in the composition of thrips resistance in Caoyuan No.4.The PPO activity,POD activity,APX activity,SOD activity,total phenol content,PAL activity,superoside anion and JA content of the two alfalfa species significantly increased（P<0.05）after thrips feeding,indicating that these physiological and biochemical indicators may be involved in the induced thrips resistance in alfafla.（3）Transcriptome results showed that a total of 9731 differential genes were identified.The number of differentially expressed genes between Caoyuan No.2 and Caoyuan No.4（S＿CK vs R＿CK）without thrips treatment was 2204,of which 1303 were up-regulated and901 were down-regulated.KEGG enrichment analysis revealed that differential genes were enriched in genetic information processing,metabolic and organismal systems,including terpenoid metabolism,lipid metabolism,energy metabolism and phytopathogen interaction pathways.It is hypothesized that these metabolic pathways play an important role in the constitutive thrips defense process in Caoyuan No.4.（4）A total of 2517 genes were differentially expressed in Caoyuan No.2 treated with thrips（S＿CK vs S＿T）.Differentially expressed genes were mainly enriched in genetic information processing,metabolism,organismal systems and environmental information processing,including secondary metabolism（phenylalanine biosynthesis,isoflavone biosynthesis）,carbohydrate metabolism（amino acid sugar and nucleotide sugar metabolism）,lipid metabolism（linoleic acid metabolism,keratin,corkin and wax biosynthesis）,environmental adaptation（plant pathogen interactions）and signal transduction（MAPK signaling pathway-plant and phytohormone signaling）pathways.It is hypothesized that these pathways,especially secondary metabolite anabolic pathways,are associated with inducible thrips resistance defense in alfalfa.After thrips feeding,only 9 genes were differentially expressed in Caoyuan No.4（R＿CK vs R＿T）treatment,presumably Caoyuan No.4 itself is more resistant to insects and less sensitive to thrips feeding resulting in less transcript changes.（5）Further studies showed that SA and JA signaling,MAPK signaling pathway-plant,and PAPM-triggered immunity were significantly enriched in Caoyuan No.2（S＿T）treatment.where genes related to SA transduction such as NPR1,TGA,and PR-1 were significantly upregulated;genes related to JA such as JAZ and JAR1 were significantly induced after thrips feeding;plant-some key genes of pathogen interaction such as CDPK,MEKK1,MPK4 and CAMCM were significantly upregulated,and MAPK-related MEKK1,MPK4,NDPK2 and Chi B genes were significantly upregulated,presumably SA was associated with JA signaling,PAPM-triggered immunity,and MAPK signaling pathway-plant and alfalfa-induced thrips resistance signaling.（6）81 differential transcription factors related to thrips feeding induction in Caoyuan No.2 were mined,belonging to 18 families,among which WRKY and MYB＿superfamily families were the most abundant with 23 and 12 transcription factors,respectively.