Identification Of Immune System Genes And Analysis Of Their Induced Expression Patterns In The Silkworm, Bombyx Mori

Insect is the largest animal groups on the earth with more than one million species, and play an important role in ecological systems.In the process of evolution,insects have formed a highly effective defense system to resist the invasion and parasitization of microbes.Besides the protection of physical barriers such as cuticle,scale and carapace,insects have immune related molecules to recognize and kill invaders. Immune system is one of the most important issues in insect studies and has great significance for clarifying the growth-development and origin.The silkworm is a typical model system for Lepidoptera with abundant mutants, and is also an important economy insect kept by human.Studies on the silkworm immune-related genes will help us understand not only the molecular mechanisms of silkworm immune response,but also the diversity of insect immunity.Using the fine silkworm genome map,gene chips and EST,we(1)identified the immune-related genes by bioinformatic method,(2)analyzed the evolutionary relationships of crucial gene families,(3)analyzed the induced expression pattern on the basis of the silkworm gen0me microarray,and(4)presumed reasonable molecular mechanism of silkworm immunity.The main results were as follows:1.Immune-related genes in the silkworm genomeComparing with all immune-related genes of Drosophila melanogaster and Anopheles gambiae,we have identified 218 immune-related genes in the silkworm genome,including pattern recognition receptors:such as PGRP,GNBP,lectin and scavenger receptor gene family;and components of four signaling pathways,including Toll pathway:Spz,Toll,MyD88,Pelle,Tube,TRAF2 and Cactin;Imd pathway:FADD, Dredd,TAK1,TAB,IKKβ,IKKγand Relish;and JAK/STAT pathway:Domeless, Hopscotch and STAT;and many genes coding antibacterial peptides.About 65%of these genes have the expression evidence of EST;about 90%of these genes can be located on the silkworm chromosomes.Evolutionary analysis showed that these genes are very conservative within insects.Furthermore,genes encoding pattern recognition receptors and antibacterial peptides showed significant species specificity,and have multiple copies in the silkworm genome.The phenomenon of obvious sequence divergence and species-specific gene copies implied that they undergo smaller Darwinian selection pressure.At the same time,we found 45 silkworm-fruitfly -mosquito-honeybee orthologous genes with the ratio of 1:1:1:1,especially,all components of signal transduction pathways showed 1:1:1:1 orthologous relationship, which can be presumed that the immune response mechanism of the silkworm is similar to the fruit fly and other insects.2.Genome-wide analysis of the silkworm immune response by microarrayUsing the silkworm oligonucleotide microarray containing 22,987 probes,we analyzed the induced transciptome of fat body of 5^thinstar Day-3 silkworm larvae after infecting of Escherichia coli(Gram-negative bacteria)and Bacillus thuringiensis (Gram-positive bacteria).The results indicated that 9,961 probes which is about 43.3% of all probes have been expressed(signal value＞400),which was similar to previous results.When setting a standard of Ratio＞2 folds for screening differentially expressed genes,we obtained 210 genes with up-regulated expression and 200 genes with down-regulated expression.By Gene Ontology(GO),up-regulated genes are mainly involved in immune response,including pattern recognition receptors,antimicrobial peptides,serine proteases and its inhibitors,components of melanization cascade and iron metabolism.Whereas,the down-regulated members are some of genes encoding energy metabolism and biochemical enzymes genes,including several cytochrome P450, cuticle proteins,low-density lipoprotein,components of hormone metabolism and dynamic protein,and many common metabolism enzymes.This expression pattern may reflect the characteristics of the fat body as an important organ of insect immune response. Comparison of the induced expression profile of antimicrobial peptide genes such as attacin,enbocin,cecropin,moricin,lebocin and gloverin,we found that these genes were more quickly induced after challenge with E.coli than B.thuringiensis.The induction level of these genes was significantly higher in the initial 2 hours after E.coli infection,whereas,the highest peaks of the induced expression of B.thuringiensis were obviously higher.For example,enbocin was more than 99-fold up-regulated after 12 hours of induction by B.thuringiensis,which is about the twice of the ratio by E.coli at the same time.Attacin was more than 93-fold induced by B.thuringiensis at 12 hours, which is more than four times of the ratio by E.coli.It is presumed that the difference of expression profiles might be controlled by different recognition receptors and/or pathways in early immune response.As a whole,the induced expression patterns are very similar when the silkworm larvae were challenged by E.coli and B.thuringiensis, suggesting that the mechanism of immune response to Gram-negative andGam-positive bacteria is similar.3.Toll-related genes in the silkwormToll receptors play an important role in innate immune and embryonic development.Toll family is very conservative in both insects and vertebrates,and contains extracellular leucine enrichment domain and intracellular TIR domain. Comparing with Drosophila Toll receptors,the silkworm genome contains 13 Toll-like receptors,named as BrnToll,BmToll-2 to-11,BmToLK-1 and BmToLK-2,respectively. Of these genes,eight members were located on the 23 chromosome and of which BmToll,BmToll-2 and BmToll-7 compose of a silkworm-specific subset.By phylogenetic analysis,the silkworm Toll receptors were divided into seven subgroups with other insects Toll receptors,of which BmToll-6,BmToll-8 and BmToll-9 were obvious homologs with D.melanogaster and A.gambiae.Through mircoarray analysis of spatial and temporal expression profile,the results showed that BmToll and BrnToll-2 were highly expressed in the ovary,suggesting that they may play an important role in embryogenesis.Whereas,BrnToll-2 and BmToll-4 were highly expressed in the midgut,presuming that they may be involved in immune defense.In addition,BmToll-lO,BmToll-ll and BmToLK-2 had testis-specific and male-specific expression profiles,suggesting that they may be possess sex-specific biological function.When injecting with E.coli,S.aureus and B.bassiana,we tested the induced expression of 10 Toll genes by RT-PCR.The results indicated that the expression profiles of BmTolls were obviously distinct after being induced by different pathogens.For example,BmToll was significantly suppressed after being injected by S. aureus and E.coli.By contrast,BmToll-2 was obviously down-regulated after challenge with E.coli and B.bassiana,whereas,BmToll-3 was significantly up-regulated after infection of each treatment.The identification and expression analysis of these Toll genes will help to explore their biological functions and the mechanism of Toll signaling pathway.4.Antimicrobial peptides of the silkwormA local BLASTP search of the silkworm genome database identified that 39 amino acid sequences were similar to other insect antimicrobial peptides(AMPs),including 12 Moricins,12 Cecropins,7 Gloverins,3 Attacins,2 Lebocins,2 Enbocins and 1 Defensin. Of these genes,Moricin and Gloverin are two Lepidoptera-specific gene families.The analysis of sequence similarity and chromosome location suggested that the silkworm AMP genes were clustered in the chromosome and may be produced by gene duplication from the ancestors of their families.The species-specific gene duplication may reflect that these AMP genes play a key role in resisting various pathogenic microorganisms by multiple copies in the immune system.Upstream regions of the AMP genes contain sequence motifs are similar to the binding sites of NF-κB/Rel and GATA transcription factors in mammals and play a key role in controlling their downstream genes expression.In the course of evolution,these regulatory sequences also endure the selection pressure.We searched upstream sequences of 29 AMP genes in the silkworm genome and identified clusters ofκB-like, GATA,R1,and CATT motifs.The upstream regulatory sequences were obviously different among the members of Cecropin and Moricin family.The number and location ofκB-like and GATA elements among these genes were also significantly different. Whereas,the upstream regulatory sequences of Gloverin family were very similar for all of the members containingκB-like motif,GATA and R1 motifs near the transcription start site.By comparative analysis,we found that the consensus sequences of Moricin and Cecropin families are GGKWNNRRNR and GGRRANYMMC,respectively.To compare relative abundances of AMP transcripts,we performed the semi-quantitative RT-PCR to analyze total RNA samples of fat body at different time points after the LPS injection.The results showed that Cecropin and Gloverin families were up-regulated highly,whereas Moricin B subfamilies were not induced.Even in the same family,the members were expressed with different peaks at different times.For example,Cecropin E was expressed lower than other Cecropin genes and its expression peak was also later than other genes.On the basis of the induced expression profile and the upstream sequence characteristics,we presumed that the difference of expression profiles may be relative to the number and location of their corresponding regulatory motifs.Interestingly,attacin and enbocin can be induced by LPS,but the expression quantity is lower than Cecropin and Gloverin.However,attacin and enbocin were more highly up-regulated by E.coli and B.thuringiensis than other AMP genes,suggesting that the mechanism of immune response for LPS and bacteria may be different.5.The evolution and induced expression patterns of the silkworm AMPsThe silkworm genome contains more AMP genes than other insects.The Moricin, Cecropin and Gloverin are multiple gene families.By calculating the synonymous/non-synonymous mutation rates(d_N/d_s),we found that all d_N/d_s rates of the three gene families are less than 1,and the d_N/d_s rates of Moricin,Cecropin and Gloverin is 0.637,0.487 and 0.202,respectively.Moreover,the rates of signal peptides are obviously larger than mature peptides of these families,suggesting that the mature peptides tend to stabilizing selection.According to comparative analysis,we found that the antibacterial spectrum and induced expressive activity of the members of Moricin and Cecropin families were more significient than those of Gloverin family.On the basis of secondary and tertiary architecture prediction,Moricin and Cecropin are comprised ofα-helix and the positive-selection sites are mainly located onα-helix region.Through correlation analysis of antibacterial spectrum and induced expressive activity with sequence divergence,we can draw the following deduction:these genes with greater antibacterial activity will be induced greater expression.In the silkworm, the AMP families may be produced by unequal crossingover and were co-evoluted with microbes by natural selection.Their coding sequences may be mutated and lead to the changes of the structures and electrostatic potential of AMPs.These sequence divergences eventually lead to the functional differentiation of the members of AMP families.For example,the Cecropin family and Lepidoptera-specific Moricin family are the typical families,of which the sequence divergence will cause obviously different function.Three genes,BmcecB1,BmcecD and Bmmor A1 have the characteristics of high antibacterial activity and high induced expression,suggesting that they might play main functional roles for killing microbes and other members might have a synergistic function.Whereas,the Lepidoptera-specific Gloverin family is divergent less than the above two families and accord with above principle unperfectedly.In the gene family, BmglvA2 is a gene according with high antibacterial activity and high-induced expression,whereas BmglvB has high antibacterial activity but the induced expression is lower than other members of this gene family.The comparative analysis of silkworm AMPs function will provide a good research model the evolution and function divergence of insect AMPs.6.Molecular mechanism of immune response in the silkwormAccording to our results,we initially presumed that the silkworm possess a conservative network of gene expression and regulation of antibacterial peptide.Here are about four steps for this regulatory network:First,the pattern recognition receptors (PGRPs and/or GNBPs)recognize the infection signal of bacteria and activate the cytokine Sp(?)tzle by extracellular serine protease cascade.Second,the infection signal is transferred into cells by the ligand of activated Sp(?)tzle binding to the extracellular domain of Toll receptor or PGRP-L receptor which activates intercellular Imd protein. Third,BmRel and/or BmRelish transcription factors are activated through the intracellular signaling pathways.Finally,these activated transcription factors enter into the nucleus and bindκB-like motifs of the upstream of AMP genes to efficiently trigger these genes expression.