I-TRAQ Analysis Of Intestinal Immune Responses Against Serraria Marcescens In Spodoptera Exigua

Serratia marcescens Bizic PS-1,a Gram-negative bacteria,is a highly efficient entomopathogenic bacteria.S.marcescens can infect many agricultural pests,such as Spodoptera exigua,via intestineby feeding.Intestinal immune system of insectsis the first defense line againstinfection of PS-1,but the intestinal defending mechanism of insects againstPS-1 is not clear.In order to clarify the mechanism of intestinaltract ofS.exigualarvae defending themselves from the PS-1 infecton,in the present study,the intestinal protein libraries of the fifth instar S.exigualarvae day 3treated by PS-1 were constructed and sequenced by i-TRAQ.Based on the depth sequence of transcriptome of S.exigua,211 differential proteins were identified,including 86 up-regulated proteins and 125 down-regulated proteins.In order to understand the expression pattern of diferential proteins from the mRNA level,significant differential proteins were selected from protein library and combined with unigenes from transcriptome.11 significant diferential proteins,inlcuding JNK,Afadin,Hemolin,Unigene19139,Unigene50,Unigene25585,Unigene29765,Unigene4584,CL1044,Unigene14488 and CL2840,were selected and analysed by quantitative real time polymerase chain reaction?q RT-PCR?.The qRT-PCR results showed that the mRNA expression levels of JNK,Unigene19139and Unigene29765 were upregulated separately 3 times,2 times,4 times more than their control no feeding on PS-1 significantly?p<0.05?.While Afadin,Hemolin,Unigene25585,CL1044,Unigene14488 and CL2840 are downregulated,especially the Hemolin,Unigene25585,CL2840 are downregulated significantly?p<0.05?.The verification results of q RT-PCR are almost identical to i-TRAQ results.Because of the highly differential protomic level,five immune related genes,IMD,relish,IKK,TAB and Cecropin,are also selected and analyzed by q RT-PCR,the results also indiacted that the expression patterns of these immunity related genes are identical to iTRAQsequencing results.In order to further clarify the function of JNK and Afadin in S.exigua,the fragment of JNK and Afadin with 1569bp and 2729bp was cloned respectively by RAC E technology,according tothe JNK and Afadin unigenes obtained from S.exiguatranscriptome.Phylogenetic tree analysis showed that S.exigua JNK is in the same branch with the JNK from Amyelois transitella,Danaus plexippus,Bombyx mori.And S.exiguaAfadinis in the same branch with the Afadin from A.transitella.q RT-PCR results indiacted that both thetranscrips of JNK and Afadin are significantly upgraded in intestine of S.exigua feeded by PS-1.q RT-PCR results showed that the JNK was mainly expressed in larva,while the transcript of JNK was almost no expressed in pupa,the expression trend of JNK in developmental stages is:larva>egg>adult>pupa.SDS-PAGE and Western blot were used to testify the JNK protein level in S.exiguaintestinal tract after feeding thePS-1,the results showed that the protein level of JNK were increased both in the5^{t h} S.exigua day 2 and the day 3 feeding on PS-1,it indicated that the PS-1 can induce the expression of JNK in the S.exiguaintestinal tract.In conlcusion,in the present study,the proteomics of S.exigua intestine feeded with S.marcescensPS-1 were sequenced byiTRAQ,anddifferential proteins were screened from S.exiguaintestinal protein library.The mRNA level of the differential proteins was analysed by q RT-PCR.The preliminary results indicated that JNK is involved in the immune response of S.exiguaintestine against S.marcescens PS-1.The results of this research provided a theoretical basis for the development of biopesticides targeting the intestine immune of the S.exigua.