Cry1Ca-induced Differential Phosphorylation Proteome Analysis Of Chilo Suppressalis Midgut

Chilo suppressalis(Walker),a pest of Lepidoptera,is one of the most serious pests that harm rice production in China.The insecticidal protein(Cry)produced by Bacillus thuringiensis(Bt)has been successfully transferred into rice for the prevention and control of pests such as C.suppressalis,and the study of the defense mechanism of insects against Cry protein is conducive to the better application of transgenic crops of resistance insect.The cellular defense response of insects to Cry protein is mainly regulated by the mitogen-activated protein kinase(MAPK)pathway,and MAPK is only one of the emergency signaling pathways,and does not directly participate in the fight against pore-forming insecticidal proteins,and its downstream genes play a real defense role.Preliminary research in the laboratory found that p38 MAPK is involved in the defense process against Cry1 Ca by the stem borer,but the downstream genes of p38 MAPK that play a defensive role against Cry1 Ca are not clear.The activation of MAPK downstream genes may be regulated by p38 MAPK pathway phosphorylation.Therefore,this study first used differentially phosphorylated proteome to screen differentially phosphorylated proteins induced by Cry1 Ca,then identified the relationship between the candidate protein and the virulence of Cry protein by RNA interference technology,and finally analyzed the expression pattern of the target gene using techniques such as q PCR And its upstream and downstream relationship with p38 MAPK.The research results are of great significance to clarify the defense mechanism of insects against Cry protein,and provide new ideas and theoretical basis for increasing the toxicity of Cry protein to target insects and delaying the development of resistance of target insects to Bt-resistant crops.The main results are as follows:1.Using isobaric tags for relative and absolute quantification(i TRAQ)technology to measure Cry1Ca-treated and non-Cry1Ca-treated groups of C.suppressalis midgut phosphorylated proteome,a total of 523 unique peptides were obtained,of which 437 proteins were phosphorylated.There were 45 proteins with different phosphorylation levels in the midgut of C.suppressalis treated with Cry1 Ca and without Cry1 Ca,among which 21 were up-regulated and 24 were down-regulated.According to the phosphorylation difference factor and literature search,we selected 7 of these genes for the next experiment.According to the transcriptome annotation of C.suppressalis,these 7 genes are uncharacterized protein LOC101736359(Up101),uncharacterized protein LOC105391923 isoform X2(Up105),domain-binding glutamic acid-rich protein homolog(Gph),autophagy-related protein 2 homolog A-like(Arp),protein lethal(2)essential for life-like(Elf2),spectrin alpha chain isoform X3(Sac),thioredoxin-related transmembrane protein 1-like(Trtp).2.Synthesize the ds RNA of the target gene and use the feeding method for RNA interference(RNAi).The q PCR technology was used to detect the change in m RNA expression of the target gene after 48 hours of feeding ds RNA.The results showed that the expression levels of Up101,Up105,Gph,Arp,Elf2,Sac,Trtp genes were significantly down-regulated.After the suppression of gene expression,the C.suppressalis were transferred to T1C-19(expressing Cry1 Ca protein),TT51(expressing Cry1Ab/Cry1 Ac protein)and T2A-1(expressing Cry2 Aa protein)three kinds of transgenic rice.The results showed that only the Elf2 gene after being suppressed,the mortality of the rice borer larvae feeding on these three rice types was significantly higher than that of the control group,indicating that only the Elf2 gene was involved in the defense of the three Cry proteins by C.suppressalis.3.After obtaining the full-length sequence of Elf2,a search using NCBI blast revealed that the gene contains a typical small heat shock protein(s Hsp)functional domain.The Open Reading Frame(ORF)has a total of 507 bp,encodes 168 amino acids,and has a predicted protein molecular mass of 19.01 k Da.Phylogenetic analysis showed that the protein sequence of Elf2 had the highest homology with Hsp19.3 of Spodoptera litura.Elf2 spatio-temporal expression pattern analysis showed that the gene was expressed the highest in 4th instar larvae and in the midgut.Further using q PCR technology to verify the transcriptional regulation relationship between this gene and p38,the results showed that after feeding ds Csp38,the expression of Elf2 decreased significantly,and after feeding ds Elf2,the expression of p38 did not change significantly.This indicates that the Elf2 gene is located downstream of the p38 MAPK pathway..