Expression Profiles Of Two Spliced Variants Of Dorsal And Relish In Spodoptera Litura Larva Challenged With Microbes

Spodoptera litura is an important defoliator and an extremely destructive pest of many crops.In the present study,Rel/NF-kB family members,Dorsal and Relish spliced variants(Dorsal 1A,Dorsal 1B,Relish 1A and Relish IB)were cloned from the S.litura.Full sequences of Dorsal and Relish and their two spliced variants were obtained from single S.litura larvae.Dorsal 1A mRNA is 1,990 bp long,and encodes a putative 516 amino acid protein with a predicted molecular weight of 55.59 kDa and its theoretical pI was 8.18.Dorsal 1B is a differentially spliced version of Dorsal 1A,with 2,574 bp which turns to be different from Dorsal 1A from the 280 amino acid.Dorsal IB mRNA encodes for a putative 689 amino acid protein with a predicted molecular weight of 76.91 kDa and its theoretical pI was 5.67.Relish 1A mRNA is 3,467 bp long,and encodes a putative 951 amino acid protein with a predicted molecular weight of 107.05 kDa and its theoretical pI was 6.03.Relish 1B is a differentially spliced version of Relish 1A,with 3,211 bp which turns to be different from Relish 1A from the 869 amino acid.Relish IB mRNA encodes for a putative 938 amino acid protein with a predicted molecular weight of 105.26 kDa and its theoretical pI was 6.41.Analyzed with Conserved Domain Database,Dorsal and Relish shared two main common protein domains.The first one is the Rel homology domain(RHD),characteristic of eukaryotic Rel/NF-kB transcription factors.And the second conserved domain RHD is termed the immunoglobulin,plexin,transcription(IPT)domain which is an immunoglobulin-like fold domain,responsible for DNA binding.Dorsal 1A contains RHD and IPT domains,Fork head N-terminal region,NLS and LZ while Dorsal 1B lacks Fork head N-terminal region.Relish 1A involves RHD,IPT,ANK and Dead domains and Relish 1B contains the same domains with Relish 1A.To investigate the tissue distribution and developmental variation of Dorsal1ADorsal1B,Relish1A and Relish1B,the relative levels of the mRNA among various tissues and life stages were determined by Q-PCR.Each sample was normalized with UCCR.The two spliced variants of Dorsal both expressed in different tissues and development stages of S.litura.And their transcription levels in different stages were significantly different(Dorsal 1A:F10,32=162.7,P<0.001;Dorsal 1B:F10,32=22.4,P<0.001).Dorsal 1A had higher expression levels in egg and female pupa stages.And the transcription levels of Dorsal 1A in female pupa and adult were significantly higher than the levels in male pupa and adult(P<0.05),while there were no significantly differences of the expression levels of Dorsal 1B between female and male larva in pupa and adult stages(P>0.05).Between different tissues the transcription level of Dorsal 1A and Dorsal 1B had significantly differences(Dorsal 1A:F5,17=38.6,P<0.001;Dorsal 1B F5,17=33.3,P<0.001).Dorsal 1A had higher expression levels in fat body and body wall and had the lowest level in midgut.And Dorsal 1B had the highest transcription level in body wall and had the lowest level in midgut just as Dorsal 1A.Spatial expression profiles showed that Dorsal 1A and Dorsal IB had the highest expression level in body wall,while RelishlA and RelishlB had the highest transcription level in fat body.Dorsal and Relish expressed in all tissues and different development stages.Time expression profiles showed thatDorsal 1A had the highest transcription level in the stage of egg while Dorsal IB did not.Relish IB transcripts were relatively high in egg and larva stages but significantly low in pupas and adults which were quite different from Relish 1A.Both the spliced variants of dorsal and relish had high transcription levels in fat body which demonstrated that the fat body played an important role in innate immunity.Similarly,the two spliced variants of Relish both expressed in different tissues and development stages of S.litura.And their transcription levels in different stages were significantly different(RelishlA:F10,32=37.3,P<0.001;Relish 1B:F10,32=191.0,P<0.001)(Fig.4A).In the stages of pupa and adult the transcription levels of Relish 1A in female individuals were higher than the level in male individuals and the level in female adult was significantly high than male adult(P<0.05).But the expression levels of Relish 1B between female and male in stages of pupa and adult did not show significantly different(P>0.05).Between different tissues the transcription level of Relish 1A and Relish IB had significantly differences(Relish1A:F5,17=17.4,P<0.001;Relish IB:F5,17=306.0,P<0.001).Relish 1A had higher expression levels in fat body and haemolymph and had the lowest level in midgut.Relish1B had the highest transcription level in fat body and had the lowest level in midgut.And the expression level in fat body was significantly high(P<0.001)while the levels of other stages were quiet low.Insects react against pathogens through innate immunity,so the research of the mechanism of the immune response of S.litura to various pathogens can provide a theoretical basis for new approaches to biologically control this pest.After larva were injected with Escherichia coli,Isaria javanica and Beauveria bassiana,the transcription levels of Dorsal 1A,Dorsal IB,Relish 1A and Relish IB were measured at 1,2,6,12 and 24 h.DorsallA and Dorsal IB were only up-regulated by fungi and RelishlA and Relish 1B were only up-regulated by Escherichia coli(Gram-native).The expression time of Dorsal up-regulated by B.bassiana lasted longer than I.javanica.And the reaction of Relish 1B to E.coli injection was slower than Relish 1A.These four genes show different patterns responding to different microbes' injection.The present data clues that Dorsal 1A,Dorsal IB,Relish 1A and Relish IB might play different potential important parts in the innate immunity of S.litura.