Novel Splice Forms Of Dsx Gene And Its Regulation On The Sexual Development Of Eighth Abdominal Segment In Silkworm,Bombyx Mori

The domesticated silkworm, Bombyx mori, is an important model of lepidopteran insects. The detailed decipherment of silkworm sex-determination mechanism is the theoretical basis of rearing male silkworm and lepidopteran pest insect control. Silkworm sex-determination cascade hasn’t been described in detail. Problems about the primary element, the key gene, and how the doublesex gene regulates sexual dimorphism, haven’t been decoded. Based on the methods of comparative genomics and mordern molecular biology, researchers have identified few candidate genes of silkworm sex determination, some of which were subsequently proved not to be the key genes of silkworm sex determination. Only the dsx gene, whose splicing should be regulated by a middlestream undiscovered key gene and which also modulates the expression of downstream sex differentiation genes, is confirmed to be the important gene of silkworm sex determination. Reportedly, Bmdsx has six exons, and it alternatively splice into two female-specific and one male-specific forms, which is different from the expression pattern indicated by our RT-PCR result. To clarify the expression of Bmdsx in two sexes and how Bmdsx controls the development of sexual dimorphism in silkworm, we cloned the possible splice forms of Bmdsx and analyzed its genomic organization and expression. Furthermore, we analysed the potential Dsx proteins and proved their regulation on three known downstream target genes by transgenic technique. Finally, we elucidated how Bmdsx patterns the sexually dimorphic development of eighth abdominal segment. The main results are as follows:1. Novel splice forms of Bmdsx RT-PCR and sequencing results showed that Bmdsx has more than three splice forms, which has also been proved by3’RACE result. By far, we have successfully cloned nineteen alternatively-spliced forms and ten trans-spliced variants. Nine exons and eight introns make up of the revised genomic structure of Bmdsx, and exon2n,3n, and6n are three novel exons, which might be gained during silkmoth evolution. The different choice of5’or3’splicing site of intron5leads to the different length of exons3and4in different splice forms. Only the5’acceptor splicing site of novel exon6n is the weak splicing site, which is the same as the5’acceptor splicing site of exon4in Dmdsx, and the splicing of exon6n may need the binding of some positive regulator. The rest of5’acceptor splicing sites of Bmdsx exons are canonical acceptor splicing sites, and their splicing may need the binding of some negative regulator.2.Trans-splicing is a novel splicing method of BmdsxThe3’ends of Bmdsx-dsrl and Bmdsx-dsr2didn’t derive from the Bmdsx. The3’ end of Bmdsx-dsrl positioned at chromosome25, and tightly linked with Bmdsx. The3’ end of Bmdsx-dsr2located chromosome16, and the presence of Bmdsx-dsr2was the trans-splicing result. The existence and structure of eight transcripts showed that Bmdsrl was a novel gene, and Bmdsx-dsrl was indeed the trans-splicing product between BmdsxF1and Bmdsr1a. Expression analysis suggested that Bmdsx-dsrl was splicing noise, whereas Bmdsr2, which trans-spliced with Bmdsx to generate five variants in female head, fat body, trachea, silkgland, ovary and male testis, might participate in regulating the expression of Bmdsx. Sequencing analysis indicated that the two trans-spliced variants in testis was the trans-splicing products between Bmdsr2and BmdsxFs. As a result, Bmdsr2only trans-spliced with Bmdsx female splice forms. Cell transfection further indicated that trans-splicing between Bmdsr2and BmdsxFs changed the3’UTR of BmdsxF mRNA, and suppressed the translation of BmdsxF mRNA. Our findings suggests that trans-splicing was a novel splicing method of Bmdsx, and Bmdsr2, a novel additional gene of Bmdsx, only participated in the somatic development of female silkworm by trans-splicing with BmdsxFs.3. Different expression of Bmdsx and the potential BmDsx in both sexesExpression analysis indicated that male splice forms specifically expressed in males, and female splice forms largely expressed in females, but also expressed in males at a very low level. Novel exon2n was a common exon and obtained in all tissues of two sexes. Novel exon3n was a female-specific exon and only spliced in a female-specific manner. Novel exon6n was only spliced in male head, midgut, malpighian tubule, trachea, testis and female ovary, and its splicing had male-biased characteristics. Sequencing all the splice bands of exon6n presented one novel female and two novel male splice forms. By far, we have cloned twenty-two alternatively-spliced forms of Bmdsx in all. Expression analysis also showed that the stetch of15bp at the3’end of exon3was common in female tissues, which was same as the situation of the stetch of127bp at the5’end of exon4.ClastalW alignment of BmDsx indicated that Bmdsx might decode five BmDsxF, four BmDsxM, four unclassified BmDsx. One protein was decoded by several splice forms, and the3’UTRs of these splice forms were different, suggesting that the choice of different3’UTRs might be a regulatory way of the expression of Bmdsx. Further analysis of the structure and key residues of the thirteen proteins found that four BmDsxF, two BmDsxM, one unclassified BmDsx, which had potential regulatory function on downstream target genes, might exist in silkworm. Four BmDsxF used different C-termini, which might affect the activation or repression of DsxF on downstream target genes. The insertion of27aa between DBD/OD1and DBD/OD2did not change the functional domains of BmDsx, and might affect the binding ability of BmDsx on the promoters of downstream target genes.4. Ectopic expression of Bmdsx affects the expression of downstream targetsWe subsequently validated the regulation of four BmDsxF and two BmDsxM on target genes. By microinjection, fluorescence choice, genomic inverse-PCR and Southern blot detection, we successfully constructed six transgenic strains including twelve transgenes, which have different insertion sites and transpositions from one to two. Statistic analysis indicated that the sexual ratios of G1generation from six transgenic strains conformed to normal sexual ratio1:1. Moreover, the chromosomal buildup of all G1individuals also corresponded with external sex phenotype, and no sex reversal had been found. However,3’RACE and RT-PCR confirmed the expression of exogenous Bmdsx. qPCR results also showed that the ectopic expression of BmDsxFs in males could induce the expression of SP1and Vg in fat body, and suppress the expression of PBP in antenna. Furthermore, the ectopic xpression of BmDsxMs in females could repress the expression of SP1and Vg in fat body, and induce the expression of PBP in antenna. All these results suggest that four BmDsxFs and two BmDsxMs could regulate the expression of three known downstream target genes, and they have the regulatory function on the expression of sexually development-related genes and shoud participate in the sexual development of silkworm soma.5. Ectopic expression of BmdsxM1causes the abnormal development of female eighth abdominal segment.A visible dimorphism exists in posterior abdomen of adult, that is:the naked chitin plate, a derivative of female gonosomite, only forms in the posterior abdomen of females but not in males, and its counterpart in males is the eighth abdominal segment (A8) with scaly hair. Three silkworm strains, Jia Qiu, Da09and301, had been chosen to detect the difference of phosphorylation level of Erk in posterior abdomens of two-sex adults, and the result showed that the phosphorylation level of Erk in the female posterior abdomens of three strains was all significantly lower than that in male posterior abdomen, indicating that RTK signaling activity had a key role in the development of histoblasts in the epidermis of eighth abdominal segment of larvae to the chitin plate of adult in females.When the males courts the females, the double harpagones in male external genitalia must grasp the chitin plate, a derivative of gonosomite in female posterior abdomen. During the observation of the phenotype of transgenic strains, we obtained a fundamental line ssd-6-2. Observation of G1to G6generation of ssd-6-2indicated that all the females couldn’t copulate with males and thereby produced no fertile offspring. The gonosomite of all transgenic females developed abnormally, having scaly-haired chitin plate attached, which affected the genital coupling of transgenic females with wild-type males, indicating that the ectopic expression of BmDsxM1in females affected the development of histoblasts in the epidermis of eighth abdominal segment of larvae to chitin plate. Subsequently, we investigated whether the RTK signaling activity was related with the abnormality of gonosomite of transgenic females. The results showed that the phosphorylation level of Erk in the posterior abdomen of wild-type males was markedly higher than that in wild-type females, and the phosphorylation level of Erk in the posterior abdomen of transgenic females was significantly elevated, which further mightily inplied that the lower activity of RTK signalings correlated with the development of female chitin plate. To survey how the ectopic expression of BmDsxM1in females induced the activation of RTK signalings, we checked the expression level of these genes related to RTK signalings by qPCR. The results showed that all the expression levels of the ligand gene of EGFR signaling, spi, the regulatory genes of Spi processing, star and rho, the regulatory genes of endocytsis of dimeric EGF receptors, cbl, mop and hrs, the gene participating in negative feedback loop of EGFR signaling, kekl, and the downstream cyclin genes, cyclinA, cyclinB, cyclinD, cyclinE and cyclinL, were markedly induced and virilescent, which indicated that the induction upstream, middlestream and downstream of EGFR signaling and the increase of EGFR signaling activity triggered the female chitin plate masculinized. Finally, we also detected the expression of Abd-B gene in the posterior abdomens of transgenic and wild-type females, and found that the expresion level of Abd-B gene was also induced and virilescent, which suggested that the Hox gene also participated in the dimorphic development of histoblasts in the eighth abdominal segment in two sexes.