Positional Cloning And Functional Analysis Of The Gene Responsible For Black Dilute Mutant In Silkworm Bombyx Mori

On earth,approximately two million species organisms have been recored,and in which over 53% are insects.The silkworm Bombyx mori,an important economic insect,emerged as a result of domestication of the Chinese wild silkworm B.mandarina approximately 5000 years ago,and is model organism of Lepidoptera.From this century,following the whole genome sequencing of silkworm,meanwhile more and more silkworm biology research,it was considered as an emerged model organism.As a model organism,silkworm can be used for fundamental research,biological reactor,lepidoptera pest control and medical model research.The black dilute(bd),a spontaneous mutation,was frist reported by Japanese scholars,in 1941.The larvae exhibit a mlanism integument and the female moth was infertile.The bd mutants conform to autosomal recessive Mendel's laws,and classical genetic research on this mutant has identified it as a single-locus mutation linked to chromosome 9.And currently,the bd mutant is preserved in China and Japan.The coloration of insects,as one of the important characteristics,has important ecological significance.The molecular mechanism of pigmentation has well foundation,and the metabolic pathway was apt to comprehend.In additional,coloration pattern of insects was significantly diverged among species,it is particularly well-suited model for evolution analysis.Moreover,reproductive development of insects is an important basis for populations' survival and expansion.Sexual reproduction patterns can bring to organisms more evolutionary opportunities.Study on the molecular mechanism in reproductive development can promote the understanding of sexual somatic development,and provide potential targets for the lepidopteran pests' control.In this study,we used 1162 recombinant individuals to fine map the bd locus.We performed functional annotation,microarray data,difference expression of genes,cloning and sequencing to determine the candidate gene,Bmbd.Furthermore,using RNAi,over expression and knockout to identify the function of the candidate gene.In order to analyze the melanism mechanism,we sequenced the transcriptiome of larval integument of three strains,wild type strain Dazao,heterozygote mutant(+/bd)and homozygote mutant(bd/bd),at an early stage of fourth molting.The main results of this paper are below as:1.The phenotype survey of bd mutantsComparison of the color pattern among four strains,wild-type strain Dazao,homozygote mutant(bd/bd),heterozygote mutant(+/bd)and bdf strain,at the same period.The bd/bd larvae whole body are significant melanism,and deeper pigment at the markings.The color pattern of bdf larvae are similar with bd,which is a slightly lighter gray black.Moreover,the color pattern is no significant difference between bd mutants and control strains,at pupal and moth stage.The moths of bd/bd have abnormal wing morphology,which are crimple,and the adults are low activity.Male moths of bd/bd cannot mate independently.Moreover,the female moths are infertile,which laid unpigmented eggs.On contrary,the wings,activity,reproduction of +/bd and bdf mutant moths are normal.Comparative morphogenesis of the internal genital organs by anatomy,found that gonadal of bd female have no obvious defects,except the arrangement of eggs are irregular.We observed the microstructure of the bd eggs by scanning electron microscope,the result shows that micropyle and petals structure of egg are abnormal.We performed the parthenogenesis experiment,found that the treatment eggs of bd can not developed.2.Molecular mapping of bd locusDue to the bd female is infertile,we use bdf(bd fertile)and Dazao N as parents.The linkage groups were generated from female F1 backcross with bdf male,the recombinant groups were generated from male F1 backcross with bdf female.We selected SSR markers and designed genomic PCR primers of chromosome 9 for DNA polymorphic screening,and then linkage analysis of the polymorphic markers were preformed.Using BC1 M groups to analyze the recombination ratio among linked molecular markers.And the result shows that the bd locus was mapped in an about 600 Kb region,and in this region have 11 predicted genes.3.Screening analysis of candidate genesThe transcripts of 11 predicted genes were used in a BLASTX search against the non-redundant(nr)protein database.Expression pattern analysis of the 11 predicted genes base on silkworm microarray data.Combination of functional annotation,expression pattern,phenotype association and RT-PCR investigation to screen candidate gene of bd.The result shows that Can8 gene was silent in bd/bd strain,and the expression level of this gene in +/bd was half of that in wild-type strain Dazao.In additional,the expression level of other genes in this region were no signification difference among the three strains.Meanwhile Can8 encode a putative transcription factor protein,which have potential gene regulation action.Integrated,it propose that can8 as candidate gene of bd mutant for further research.4.Cloning and sequence analysis of candidate geneThe cDNA sequence of can8 gene was cloned in wild-type and bdf.The result indicated that can8 and can9 gene was one gene.We named it as Bmbd,which encode a BTB-zf protein.Two type ORF of Bmbd were detected,which are different in Zn-figer domain,and infer that may bind different DNA sequnces.We compared the CDS of Bmbd between wild-type and bdf,found that only several SNPs,and there is no obvious difference in higher structure of this gene.Moreover,we cloned the genome sequence in bd mutants.The result shows that genomic vartion was happened in Bmbd locus,in here,deleted about 160 kb genomic fragment which from second intron to upstream of the transcription start site,and insert a sequence of approximately 3.6kb.The deletion and insertion result in complete silencing of the Bmbd gene.And the indel bring to a potential new gene,which encode a sodium/potassium/calcium exchanger 4-like protein,we name it Bm NCKX4-like.We conducted phylogenetic analysis of Bmbd protein in insects,it is found that highly conserved homologous gene can be detectd in many species.5.Expression pattern analysis of Bmbd in wild-type strainWe investigate the spatiotemporal expression pattern of Bmbd gene in wild-type Dazao strain.The transcript of Bmbd were detected in all larvae tissues at early stage of fourth molting,the result shows that higher expression level was detected in head,gonad,integument and silk gland,and low erexpression level in the blood.Meanwhile,temporal expression pattern experiment indicadite thatfrom newly-hatched to 1 day of moth,the expression level of Bmbd present a fluctuation pattern.The expression level was up-regulated at each molting and late stage of pupa,a moderate expression at mulberry leaves eating period,and lower expression level at wandering stage.6.Knocking down Bmbd using RNAiThree si RNAs were designed and synthesized of Bmbd gene for RNA silencing.The si RNA was injected through the lateral spiracle of the larval abdomen into haemolymph at the foruth instar 2 day.Immediately after injection,PBS droplets were placed nearby and 20 volts voltage was applied.The phenotype were observed and quantified at 5th instar stage.There are about 47% treatment larvae appeared melanism epidermis,and molecular detection experiment showed that the expression level of Bmbd was significantly down-regulated.7.Transgene expressionTwo type transcripts of Bmbd were cloned,and recombined into expression vector.The expression plasmid,which contains the Bmbd gene and the enhanced GFP marker,and helper plasmid A3 Helper,in which piggybac transposase is encoded,were mixed to a final concentration of 1 ?g/?L.The mixture plasmid was injected into haemolymph of the 2nd or 3rd larvae by a glass needle connected to a microinjector.Soon after the injection,electrical stimulation was applied.The treatment larvae were placed under 25?,75% relative humidity conditions,and fed fresh mulberry leaves.At the 5 instar,we observed that there have some white patches on the larval epidermis,and which were co-located with the EGFP.8.Knocking outThe g RNAs of Bmbd gene were designed and synthesized,and mixed with RNA of Cas9 endonuclease enzyme.We injected 10 n L of the mixed RNA into each eggs of wild-type strain N4,at spawning 1-4 hours.Soon after the injection,seal the injection hole with glue.The treatment embryos were placed under 25?,85% relative humidity conditions for incubation.And the epidermis melanism individuals have be found,following the phenotypic observation in the offspring.9.RNA-seqWe validated the function of the Bmbd gene,as a transcription factor protein,which can inhibit the pigmentation.In this paper,we reveal a new color pattern regulation protein in insects.Due to Bmbd is high expression at early stage of molting,and insects will form a new skin and color pattern following the molting.It is infer that Bmbd regulated early expression genes,which are involved in pigmentation,to affect the body color.We performed integument transcriptome sequencing of the wild-type strain Dazao and the mutant strains +/bd and bd/bd during which feeding stops,an early stage of the fourth molt.Genome-wide,approximately 51% of genes were expressed(RPKM ?1)in each strain,and a comparison of the transcriptome data revealed 31 differentially expressed genes(DEGs)as potential candidates for bd larval melanism,including Bmbd.In the DEGs,15 cuticular protein genes were remarkably up-regulation expression in the bd/bd mutant.Cuticular proteins are very important components of the epidermis,and more than 200 cuticular protein genes have been reported in B.mori.Report indicated that many cuticular proteins display marking specificity in papilio xuthus larvae,which suggestion that cuticular proteins may bind with pigments in larval epidermis.In this research,the 15 remarkable up-regulation cuticular proteins may as a synergistic protein of melanin,and suggestion that cuticular proteins contributed to larval melanism.It provides a new factor for the development of colored pattern.In this paper,we cloned the Bmbd gene which responsible for bd mutants,and verified the function of inhibiting melanization.In additional,comparative transcriptome and q RT-PCR analysis of integument among the three strains,Dazao,bd/bd and +/bd,the result shows that the melanization promote gene,laccase2,were down-regulated in bd/bd individuals.And reveals the 15 cuticular protein genes,which were very high expressed in bd/bd larvae,and indicated that contributed to larval epdermis melanism.The BGIBMGA000988 gene was significant down-regulation in bd mutants,as an orthology gene of ovo in fruit fly,which play a key role for egg development.It suggestion that the the down-regulation of Bmovo may associate with infertile of bd female.It is propose that the Bmbd as a new regulation gene for pigmentation pattern and reproduction development,in silkworm.Together with the present results,we propose that the Bmbd is a new color pattern gene.Due to this gene was highly conserved among insects,and which can both control the color pattern and reproductive development,therefor we suggestion that it can be used as a potential target for lepidopteran pest control research.