Positional Cloning And Molecular Mechanism Analysis Of The Body Shape Mutant Tubby(tub) In The Silkworm, Bombyx Mori

Body shape is one of the most basic and significant features of the organism.The size of body affects many aspects of animal biology,from its external morphological and physiological characteristics to the behavioral and ecological characteristics.So the body shaping is an important process of biological development.Although there are some studies on the regulation of the morphological size of cells,organs and individuals in Drosophila,nematode and other model organisms,but unfortunately,there is only a preliminary understanding of this.It is known that the basic process of body contour formation is the expression regulation and interaction of morphological factors and transcription factors in the development process.Obviously,the difference in body shape is far more than the diversity in body contour,and its molecular regulation mechanism is also quite complex,there are still many unsolved mysteries on this.Silkworm is a representative insect of Lepidoptera,numerous body shape mutants have appeared during the long domestication and breeding process,and there are also many artificially induced body shape mutants to obtain research materials.The tubby?tub?mutant is on the list.It is a natural mutant with typical phenotype-a stout body,and it is recessively inherited.In the 1980s,Japanese scholar Doi Haruo reported that tubby's genetic gene was located on the 23rd chromosome.In 1995,the author's tutor began to introduce this mutation into the commonly used experimental strain-Dazao through hybridization,and cultivated the near isogenic line of tubby?Dazao-tub?,which has been backcrossed with the recipient parent Dazao for more than 30 generations.Dazao-tub has become a precious model material for studying the formation mechanism of body shape?fat and thin?.In our research,we precisely located the tub locus on the genome by molecular marker mapping methods.Combining gene function annotation,expression differences analysis,and cloning and sequencing method,we identified the candidate genes successfully.RNAi and other methods were used to study the function of genes and transcriptome sequencing were used to analyze the regulatory mechanisms of the tubby mutation in the silkworm.The main research results obtained are as follows:1.Phenotype observation and measurement of tub mutationIn order to adequately grasp the phenotypic characteristics of tubby?tub?,we conducted continuous observation and analysis.It was found that the tub mutant exhibite a short and fat phenotype during the entire development period?embryo,larvae,cockroach,adult?,and the larval phenotype is most obvious,the abdomen expands approximately to a spindle shape.The body length and width?the second,third,and fourth abdominal segments?of the wild-type and Dazao-tub larvae were measured and compared.The results showed that the body length of Dazao-tub was significantly shorter than that of wild-type Dazao?p=0.002?,and the width of the body segments?the second,third and fourth ventral segments?was prominently greater than that of the wild-type Dazao?mean p=0.001?;the body weight of 5th instar larvae was measured and displayed that Dazao-tub was significantly heavier than wild-type Dazao.At the same time,we observed and measured the body length and width of the wild-type and Dazao-tub during the embryonic stage.The results showed that the body length was significantly shorter?p<0.0001?and the body width was notably wider?p<0.001?in mutant than those in wild-type Dazao.Moreover,the ratio of body length to body width in the embryonic period was similar to the ratio in the larval period,that is to say,the mutant has a similar body shape pattern in the embryonic and larval stages,suggesting the fusiform body shape has already been determined in the early embryonic stage.2.Molecular mapping of tub locusA hybrid segregating population was prepared.Using the existing SSR markers and designed polymorphic markers,388 mutated individuals in BC1M populations were genotyped,and the tub locus was initially narrowed to² Mb region on the nscaf3026of chromosome 23 between marker P1 and A1.For fine mapping,new polymorphic markers were designed,and the localization population was expanded to 1766 BC1M mutant individuals,finally,the tub locus was locked to a range of approximately 208 kb between marker M1 and M6.There are 4 markers?M2,M3,M4 and M5?closely linked?not exchanged?with tub locus in this interval.9 predicted genes were found in this region in the silkworm genome database.3.Screening analysis of candidate genesBioinformatics analysis combining sequence amplification,it was found that there were only four genes instead of nine,including BGIBMGA011360,BGIBMGA011364,BmFGF and Bmscarface,in the²08 kb region.qRT-PCR analysis were performed to investigate the expression level of the four genes between Dazao and Dazao-tub.The result showed that the expression levels of Bmscarface and BGIBMGA011364 were significantly down regulated in Dazao-tub compared with Dazao,whereas no significant change was detected in the expression levels of the other two genes.The primers were designed to amplify the cDNA sequences of the four genes and found that only the gene Bmscarface has polymorphic difference between wild-type Dazao and mutant Dazao-tub.Further,genotyping analysis was performed,which showed that no recombination existed between this gene and the tub locus.The cDNA amplification products of the other three genes were identical in size,after sequencing,we found that the amino acid sequences of BGIBMGA011360 and BmFGF are identical in Dazao and Dazao-tub,whereas there was an amino acid substitution within the open reading frame of BGIBMGA011364.This amino acid substitution was verified in multiple strains without tub phenotype and found to be not unique to the mutant.Integrated,it enlighten us take Bmscarface as the candidate gene of tub mutant for further research.4.Cloning and sequencing analysis of candidate geneThe cDNA sequences of Bmscarface were cloned in wildtype Dazao and mutant Dazao-tub.The ORF is 5451bp in length and contains 13 exons encoding 1817 amino acids with a Tryp-SPc domain in wild-type.In contrast,the Bmscarface gene cloned from the mutant Dazao-tub lacks 3 bp and 312 bp in the first exon and 48 bp in the sixth exon.Although the deletion does not destruct the ORF,it results in the loss of 121amino acids,which changes the spatial structure of the protein.The 312bp-deletion on the first exon was unique to the mutant which was verified by multi-strains analysis.Both homologous and phylogenetic analysis showed that the Bmscarface gene is only conserved in Lepidoptera,and only paralogous genes of scarface were identified in many species.And its function remains to be studied.5.Temporal and spatial expression patterns of Bmscarface gene and effects of EcdysoneThe tub mutant has an abnormal short and fat body shape throughout the development stages,including embryo,larva,pupae and moth,we investigated the temporal expression profiles of Bmscarface in Dazao at all developmental stages.The results revealed that Bmscarface was not expressed in the early stage of the embryo until the trachea formation stage.During the larval stage,the expression profile of Bmscarface exhibited a pulse-like pattern with high expression at the mid-molting stage in each instar but very low expression at the initial stage of molting,there was still expression during the feeding period.Additionally,the expression of Bmscarface was high at 42 h after the beginning of wandering,but decreased thereafter and turned up a small peak in the mid-pupal stage.The spatio-expression pattern of Bmsacrface showed that it was expressed at a high level in the head,epidermis and trachea,and a much lower level in the other organs.In the 20E induction experiment on the 5^th instar larval,the expression of Bmscarface was significantly lower at 24h after 20E injection than the control.Similarly,when treated cells with 20E,the expression of Bmscarface was significantly reduced after 12 h of 20E treatment compared to the control.These results showed that the expression of Bmscarface was negatively correlated with the titer of20E.6.Functional validation of Bmscarface in the silkwormIn order to verify the molecular function of the Bmscarface,we performed RNAi experiments.To decrease the expression level of Bmscarface mRNA in Dazao,the dsRNA was synthesized and injected into eggs at 2–6 h after oviposition.The results showed 31%of newly-hatched individuals exhibited a small body size like tub phenotype.After measuring the body length and body width of each tub-like individual,it was found that these individuals are shorter and wider,meanwhile the ratio of body length to body width is not significant compared with the Dazao-tub mutant.These results suggested that Bmscarface is responsible for the phenotype of tub mutant,indicating that Bmscarface gene involved in regulating the body shape of silkworm.7.Transcriptome sequencing and data analysisBy RNAi we validated the function of the Bmscarfce.Due to the body shape is determined at the embryonic stage and Bmscarface has high expression level at the trachea formation period,transcriptome sequencing of the wildtype Dazao and the mutant tub was performed at the trachea formation period.We analyzed and compared the differentially expressed genes in the two transcriptome data,and got 696differentially expressed genes.Among them,201 genes were up-regulated,and 495genes were down regulated.The differentially expressed genes in the positional interval are only the candidate gene Bmscarface.We focus on the genes involved in the signaling pathways that modulate body shape,such as insulin signaling pathway,mTOR signaling pathway,Hippo signaling pathway,and so on.The changes in the genes on these pathways can affect the body size,this may provide important clues for studying the molecular mechanism of the body shape regulation in silkworm.8.Molecular mechanism of the tub mutant in the silkwormIn Drosophila melanogaster,it is reported that scarface gene as a new gene of JNK signaling pathway plays a negative role in the process of epidermal morphogenesis.Any change in the JNK pathway may affect the expression of dpp.So,we investigated the expression level of dpp in the trachea formation stage when Bmscarface has a high expression level,it is found that the expression level of dpp in the Dazao-tub mutant was significantly higher than that in the wild-type.At the same time,we investigated the expression level of dpp after down regulating the expression of Bmscarface,the result was that the expression of dpp was significantly higher than the control group,this is in accordance with the expression of this gene in tub individuals.We speculated that the high expression of the dpp in mutant caused excessive extension of the lateral epidermis,leading to a abnormal phenotype of broader somatotype.