Positional Cloning Of Bpn Mutant Gene Of Black Pupa Mutant And Action Mechanism In The Silkworm,Bombyx Mori

Bombyx mori is an economically important lepidopteran and a model organism.It’s been domesticated and evolved over thousands of years,very rich genetic mutation materials have been accumulated.The mutants of silkworm mainly include markings,body color,body type,cocoon color mutants,and so on.A black pupa mutant with black pupa cuticle was found in the rearing of the silkworm strain Qiufeng N,named bpn.Some silkworm of the mutant had difficulty in molting during pupation,and some eggs produced by female moths could develop normally,but could not break out of the egg shell.The hatching rate was significantly lower than that of eggs produced by wild-type female moths,which had not been reported in other black pupa mutants,it’s a new black pupa mutant.Genetic analysis showed that the mutant character of bpn was controlled by a recessive gene located on the autosome and followed Mendelian inheritance.In this study,polymorphism SSR molecular markers were used to conduct linkage analysis and positional cloning of bpn gene,2-DE and transcriptome were used to analyze changes of total protein of the mutant pupal cuticle,so as to elucidate the formation of mutant bpn and the effect of the mutation on the pupal cuticle protein and BmHEL gene.It is of great significance to improve the formation mechanism of melanin and enrich the molecular mechanism of Bombyx mori growth and development.The main results obtained in this study are as follows:1.Phenotypic characteristics and genetic analysis of mutant bpnStatistical analysis showed that the hatching rate of mutant bpn is 50-60%,which is much lower than that of Qiufeng N（more than 90%）.F₁individuals of P50 and mutant bpn hybrid offspring showed normal pupa.There were two phenotypes of normal pupa and black pupa in F₂ generation,and the separation ratio was 3:1.The backcross population（BC₁F,BC₁M）had two phenotypes:normal pupa and black pupa,and the separation ratio was 1:1.According to Mendelian inheritance,it was determined that the gene controlling bpn mutant character was recessive and located in silkworm autosome.2.Linkage analysis and fine mapping of mutant gene bpnF₁ generation was obtained by crossing P50 and mutant bpn.According to the non-exchange property of silkworm female chromosomes,female moths of F₁ backcrossed mutant bpn male moths to produce BC₁F generation,which was used for linkage analysis of bpn gene,and male moths of F₁ backcrossed mutant bpn female moths produced BC₁M generation and raised normally until pupation,which was used for fine mapping of bpn gene.The genetic analysis showed that bpn gene was located in autosomes,so sex chromosome 1 was excluded.According to the constructed silkworm SSR marker linkage map,genomic DNA of P50,mutant bpn and its hybrid offspring F₁ were used as templates to screen SSR molecular markers on remaining 27 chromosomes,10 normal individuals and10 mutants of BC₁F were linked by polymorphic SSR markers,and bpn gene was determined to be located on chromosome 26.Fine mapping by using 200 mutants in BC₁M population.The results showed that the mutant gene bpn was located between two molecular markers S26-4-12 and S26-2-14,with a physical distance of about 211 kb,and contained 6 candidate genes:KWMTBOMO15782、KWMTBOMO15783、KWMTBOMO15784、KWMTBOMO15785、KWMTBOMO15786and KWMTBOMO15787.3.Screening and structural analysis of candidate genesIn order to determine the target genes,the expression of 6 genes in the candidate regions in pupal stage was analyzed.c DNA of Qiufeng N and mutant bpn pupal cuticle were used as templates for q RT-PCR.The results showed that:KWMTBOMO15783 was not expressed in pupal stage,and the relative expression level of KWMTBOMO15787 was not significantly different between Qiufeng N and mutant bpn pupal cuticle.The relative expression levels of KWMTBOMO15782,KWMTBOMO15784,KWMTBOMO15785 and KWMTBOMO15786 were significantly different in Qiufeng N and mutant bpn.The results showed that candidate genes could be identified as KWMTBOMO15782,KWMTBOMO15784,KWMTBOMO15785 and KWMTBOMO15786.According to the gene annotation information provided by KAIKObase,KWMTBOMO15782,KWMTBOMO15784and KWMTBOMO15785 were cilia-and flagella-associated protein 58-like genes,and KWMTBOMO15786 was ebony gene.Sequence analysis of the four candidate genes showed that sequences of KWMTBOMO15782、KWMTBOMO15784 and KWMTBOMO15785 in mutant bpn were not different from the reference sequences.A poly A structure with a length of 99 bp was inserted in the middle of exon 6 of the ebony sequence,and a stop codon TAA appeared in the functional region of ebony,exons 8 and 10 were partially missing,and exons 9 were completely missing.The results showed that the sequence of ebony had a nonsense mutation,which resulted in the loss of its function in inhibiting melanin and finally in the occurrence of melanization,resulting in melanization phenotype of mutant bpn pupal cuticle.4.2-DE analysis of total protein in pupae cuticle and expression of BmHEL in silkworm eggsIn order to determine whether the mutation affects the total protein of pupal cuticle,2-DE was used to analyze the total protein expression of Qiufeng N and mutant bpn pupal cuticle at different developmental times.The results of 2-DE were verified by transcriptome and q RT-PCR analysis.The results showed that the expression of Qiufeng N and mutant bpn pupal cuticle protein was stable,and there were three different proteins between them at different time points.The three different proteins were identified by mass spectrometry as KWMTBOMO11902,KWMTBOMO11904 and KWMTBOMO11906,which were all 30K proteins.The results showed that the expression of 30K protein in pupal cuticle of the mutant bpn was changed.BmHEL gene plays a role in softening egg shell and promoting egg hatching during egg hatching stage.Since the incubation rate of self-crossing silkworm eggs in Qiufeng N black pupa was significantly lower than that of Qiufeng N,it was speculated that it might be related to BmHEL gene.The expression of BmHEL gene in Qiufeng N and mutant bpn silkworm eggs was analyzed by q RT-PCR.The results showed that the expression of BmHEL gene at heading bluing stage was low in both Qiufeng N and mutant bpn eggs,which was due to the effect of the incubation enzyme to soften the egg shell,the expression of BmHEL gene was always low in the early stage of hatching.The expression of BmHEL gene in mutant bpn silkworm eggs was significantly higher than that in Qiufeng N silkworm eggs from heading bluing stage to hatching stage.The results showed that some eggs of the mutant bpn were difficult to hatch,and more hatching enzymes were needed to soften the egg shells,leading to high expression of BmHEL gene.These results indicated that the mutation of ebony gene not only caused the abnormal formation of melanin in pupae cuticle of Bombyx mori,but also might have an important effect on some genes closely related to growth and development,such as 30K protein and hatching enzyme.Therefore,the study of the mechanism of bpn mutant is of great significance for improving the formation mechanism of melanin and enriching the molecular mechanism of growth and development,which is worthy of further study.