| Alfalfa is an important forage resource,and the large-scale outbreak of aphids on the clams will seriously affect the yield and quality of alfalfa.There have been many studies on cockroach resistance,but there is still no effective way to prevent and avoid the damage of mites.In addition,the research on the resistance of alfalfa is mostly focused on cold resistance,drought resistance and salt tolerance and there are few aspects related to insect resistance research and insect-resistant breeding at the molecular level.In this study,a very mature transcriptome sequencing technique was used to analyze the differentially expressed genes of two different resistant alfalfa cultivars after feeding stress at the transcriptome level.and the results as follows:1.The alfalfa cultivar Gan nong 5(insect-resistant)and Hunter River(the worm)were treated with pea licking feeding stress.The leaves were transcribed and sequenced after 48hours.Finally,it obtained 43.67 Gb Clean Data of 6 samples,the average Clean Data of per sample is about 7.3 Gb and each sample~’s Q30≥91.41%.The total number of Unigene was 100428,N50 was 1242 after assembly;the Transcript was 180150,and the N50 was1586 with high integrity.Mapped Reads accounted for more than 70%of the total Clean Reads after sequencing and assembly data comparison.The valid data in the sequencing library was sufficient and the gene expression abundance of each sample was higher.2.Differential expression analysis between the two samples was performed by DESeq2software and obtained 2315 differentially expressed genes(DEGs).Gannong 5 alfalfa has165 genes up-regulated and 2150 genes down-regulated compare to the Hunter River.Total of 2138 DEGs were be annotated and there are 996 comments in the database COG,1101 in the GO database,1148 in the KEGG database,1701 in the KOG database and 1916 in the Pfam database.Also 1318 DEGs in the Prot database,2038 in the eggNOG database and1780 in the NR database.The differential genes are mainly distributed in subclasses under three major categories:biological processes,molecular functions and cellular components after the GO function annotation.The functional classification of COG and eggNOG annotations mainly focuses on energy conversion and production,ribosome structure and formation,amino acid metabolism and transportation,signal transduction mechanism,defense mechanism,carbohydrate metabolism and metabolism of secondary metabolites.The main distribution in the KEGG metabolic pathway is plant hormone signal transduction,carbon metabolism,amino acid biosynthesis,eukaryotic ribosome biological origin and ribosome,etc.The c89088 and c89212 genes in the plant signaling metabolic pathway,the c73359 gene in the alpha-linolenic acid metabolic pathway,the c75375 gene in the ubiquitin-mediated protein hydrolysis pathway and the c89126 gene involved in the olefinic skeleton synthesis pathway and the c90924 gene in the synthesis of ketones.This genes were related to the degradation of resistance to insects.KEGG pathway enrichment analysis revealed that the gene involved in the regulation of glucosinolate metabolism which is higher in the up-regulated expression gene is c88736.graph_c0 and regulates the regulation ofα-linolenic acid metabolism in the expressed gene is c73359.graph_c0.This suggests that the genes involved in the metabolism of alpha-linolenic acid and glucosinolate are associated with insect resistance.Unigene’s annotated analysis showed that these genes play different biological functions during the growth and development of mites and against aphids.3.Twelve genes were selected for RT-qPCR verification.The results showed that the relative expression folds were different from the transcriptome results,but the overall expression trend was the same.Since c88736.graph_c0 has a high enrichment factor in the KEGG pathway,the related metabolite glucosinolate was selected to verify its effect on the growth and development of pea carp by artificial feed.The results showed that after feeding peas with artificial feed containing glucosinolates,the rate of winged mites increased and the survival rate decreased.This indicates that glucosinolates can significantly inhibit the growth and development of pea carp,and the related genes are related to the insect resistance of alfalfa.Above all the research results can provide theoretical support for the analysis of the molecular mechanism of cockroach cockroaches,the excavation of major cockroach genes,the interaction mechanism between ticks and ticks and the breeding of alfalfa transgenic insect-resistant varieties. |
