Identification And Analysis Of Cuticle Morphological Mutation Related Gene Of C8 Silkworm,Bombyx Mori

Silkworm(Bombyx mori),Lepidoptera,Bombycidae,is a typical example of Lepidoptera insect.By the accomplishment of silkworm genomic exquisite draft,the research of silkworm entered the post genome stage.Silkworm has more than 400 morphological characters mutations,including many cuticle mutant forms,like stick mutation,bamboo mutation,stony mutation and so on.Research of genes,which caused the cuticular morphological mutation and its regulation mechanism,is still at initial stage,a lot of cuticular morphological mutation mechanisms need to be researched.In this study,we used a near-isogenic line(NIL)silkworm based on morphological mutation,C8 and its backcross parent C108 for material(compared to its backcross parent C108,C8 has several characters: the bodyshape is thin and short,the cuticule is stiffness),we studied the cuticular morphology differences by using scanning electron microscopy(SEM);utilized SDS-PAGE and mass spectrometry,MALDI-TOF-MS,to identified the different expressed cuticular proteins between C8 and C108.Furthermore,we evaluated and compared these genes cDNA sequences to screen the candidate mutant gene,and analyzed its expression model by RT-qPCR;furthermore,analyzed the linkage genetic of the candidate gene.The results were listed as follows:1.The protrudes of C8 cuticular surface are more apparent than C108,but the cuticle of C8 is more compact,which maybe the reason to induce the stiffness of C8 cuticle.2.We screened 8 different expressed protein bands between C8 and C108 by using SDS-PAGE,and identified 9 proteins of these 8 bands through MALDI-TOF-MS,including 3 differential expressed cuticular proteins: BmCPH34,Bm CPG4 and BmCPR41,protein BmCPH34 expression quantity in C8 is higher than C108,but BmCPG4 and BmCPR41 are reverse to BmCPH34.3.We cloned and sequenced the cDNAs of these 3 cuticular protein genes:BmCPH34,BmCPG4,BmCPR41 and BmCPR2,which we had obtained in earlier study.The results indicated: the amino acids sequence coded by gene BmCPR2 and BmCPR41 in C8 and C108 are consensus with P50-loaded in SilkDB database;the cDNA sequences of gene BmCPG4 have larger difference between C108 and P50,but only one base pair replaced in C8 compared to P50 and result in one amino acid mutation.So we speculated these 3 genes are not the direct factors refer to C8 morphological mutation.Otherwise,compared to C108 and P50,the cDNA sequenceof BmCPH34 has 15 bp repeat and 9 bases substitution in C8,and in its amino acids sequence has 5 residues duplication,and lead to 2 amino acids residues mutation.We speculated gene BmCPH34 may relevant of the construction of C8 cuticular morphological mutation.4.Detected the expression discrimination of all of 4 cuticular protein genes between C8 and C108 by RT-qPCR,results demonstrated: all of 4 cuticular protein genes expression levels in C8 were higher than C108,and reached significant different levels,indicating these 4 cuticular protein genes all involved in the formation of the C8 cuticular morphological mutation.In further study of the expression model of gene BmCPH34 during the developmental phase indicated that BmCPH34 main expressed in the head,cuticle,fat body,and trachea of silkworm,the expression peck at fourth instar.Therefore,we speculated gene BmCPH34 is relevant to the formation of the C8 cuticle.As described above,we identified 4 cuticular protein genes relevant to C8 cuticular morphological mutation: BmCPR2,BmCPH34,BmCPG4 and BmCPR41.Our paper suggests that future research may explore the formation of C8 cuticular morphology mutation in more detail.