Molecular Base And Gene Regulation Of Body Developmental Mutants Wes, E^Cs-l And Ap In The Silkworm, Bombyx Mori

The morphological diversity of insects plays an important role in their survival and progenitive process and is the result of their adaption to different ecological environments. However, in essence it results from the differential expression of genes during the insect body development. With the development of life science, such as genetics, developmental biology and molecular biology, more and more people reveal the underlying reason for the diversity of insect morphology in the molecular level. The establishment of insect body plan is a very complicated process, including different levels of gene regulation network program. Specifically, the discovery of homeotic genes (Hox genes) represents a landmark in this research field. Hox gene is the master regulatory gene of the insect body plan development, and its importance also implies the complexity of the regulatory mechanism. Aside from Drosophila, studies on insect Hox genes are deficiency, especially in Lepidoptera which includes the main agriculture and forestry pests. Meanwhile, studies have shown that the expression patterns and regulatory mechanisms of the body development related genes are different between different insects. This suggests the necessity and essentiality of independent research of representative insects.As an important model of Lepidoptera, the silkworm (Bombyx mori) is not only abundant in genetic mutation resources but also a traditional experimental animal of genetic research, which provides a satisfactory platform for the research of molecular mechanisms for morphogenesis. In this study, we used three E group and similar mutants with different loci and related mutant phenotypes as research materials. Then, we identified the key genes involved in the establishment of silkworm body plan and studied their correlative functions. Finally, we made a comprehensive research on the regulatory mechanism of related genes during the development of silkworm through the relashionship among these three mutants. The results we obtained are as follows:1. Molecular analysis of the silkworm thoracic mutant Wes and functional studies on the candidate geneWedge eye-spot (Wes) is located at24.3cM on the sixth linkage in the silkworm genetic linkage map and controlled by a single dominant gene. The eye-spot of the heterozygotic larva (Wes/+Wes) looks like a narrower inverted triangle compared with the wild type and the Wes mutant was named based on the shape of the eye-spot. The homozygote Wes/Wes is embryonic lethal. In the Wes/Wes embryos, the thorax appeared to be fused and the first thoracic legs appeared under the mouthparts and were transformed into antenna-like appendages. The two remaining pairs of legs did not have the characteristics of thoracic legs, and exhibited partial homeotic transformation to antennae. The Wes adults exhibited smaller or abnormal wings.We mapped the Wes mutant using244BC1M individuals and concluded that the BmAntp gene was the candidate gene for the Wes mutant. In Wes mutant, there was a1570bp insertion in the third exon of BmAntp and destroyed the homeodomain in BmAntp. The inserted sequence was a non-LTR retrotransposon. In the Wes homozygous embryo (Wes/Wes) we found two coexisting transcripts, the normal type and abnormal type. The normal transcript was expressed at a much lower level in Wes/Wes compared with the wild type; meanwhile, the expression level of the abnormal transcript was much higher than that of the wild type in the Wes/Wes. We speculate that the Wes mutant phenotype should be owing to the loss-of-function of BmAntp gene, and the normal transcript is not enough to maintain its normal function. By contrast, the expression level of wild-type BmAntp gene was increased markedly in the heterozygous larvae compared with the wild type. It suggested that the mutant phenotype was owing to the BmAntp gene overexpression. This observation could reflect the fact that a negative feedback loop mechanism for BmAntp regulation has been disrupted with the disruption of a functional BmAntp protein. Taken together, the results suggest that the precise expression pattern of BmAntp gene is necessary for the thoracic development in the silkworm.In the dsBmAntp-RNAi embryos, the expression of the BmAntp gene was partially suppressed and BmAntp RNAi leads to fused thoracic segments and defects in the development of thoracic legs. This was consistent with the Wes/Wes mutant phenotypes. The results imply that BmAntp gene has an important role in thoracic segmentation. Considering the reduced thoracic legs in BmAntp-dsRNAi embryos and antennae-like appendages in the Wes homozygotes, we speculate that BmAntp gene is required for both development and identity of thoracic leg in Bombyx mori. In addition, the forewings and hindwings of Wes adult were much smaller compared to the wild-type wings and qRT-PCR showed that the expression levels of BmAntp gene in the forewings and hindwings are both higher than that in the wild-type wings. This implies that the BmAntp gene should be involved in the development and formation of wings in the silkworm, Bombyx mori.2. Molecular analysis of the silkworm abdominal mutant ECs-lThe ECs-l mutant exhibited a pair of supernumerary prolegs on the A2segment, extra crescents on the A3segment and normal stars on the A5segment. A mutant previously identified in the E homeotic complex, supernumerary crescents and legs (ECs), presented similar morphological characteristics, except for the absence of stars on the A5segment. Consequently, we designated the new mutant supernumerary crescents and legs-like (ECs-l).To identify the candidate ECs-l locus region, fine mapping was performed using1605BC1M individuals. As a result, the ECs-l locus was narrowed to a～68kb region between the Bmabd-A and BmAbd-B gene. There was no protein-coding genes in the candidate region except a non-coding RNA, miR-iab-4. However, genomic sequencing of pre-miR-iab-4revealed no sequence differences between the wild-type silkworm (Dazao) and ECs-l mutant. In addition, we identified9polymorphic markers in the candidate region and these markers cosegregated with the ECs-l locus. To determine whether these variable DNA fragments were unique for ECs-l mutant, we amplified these fragments in other18local strains. Genotypes of8markers displayed irregularly except the marker P39. The marker P39located about7.5kb upstream of miR-iab-4and based on the marker P39, the ECs-l mutant had a～210bp deletion compared with other strains. Moreover, this sequence was a single copy in the silkworm genome and non-transposable element. We speculate that this sequence may play an important role in the gene regulation as the regulatory element of Bmabd-A gene or miR-iab-4.In this study, considering that the candidate region of ECs-l mutant located approximately11kb upstream of Bmabd-A gene, we performed study on Bmabd-A gene as the candidate gene for the ECs-l mutant. The expression profiles of Bmabd-A showed a marked increase in mRNA as well as protein level in the ECs-l mutant. Subsequently, immunocytochemistry experiments revealed that the Bmabd-A protein is ectopically over-expressed in the A2segment, indicating that supernumerary proleg development is driven by variable expression patterns of Bmabd-A. Therefore, we assume that the Bmabd-A gene play a largely permissive role in promoting proleg formation and development in silkworm.3. Mapping of the apodal mutant ap in silkwormThe apodal (ap) mutant is located at22.3cM on the third linkage in the silkworm genetic linkage map. The ap mutant exhibited degraded thoracic legs and that made their crawling and eating mulberry leaves difficult, and also showed female sterility.Through the analysis of the reproductive system of ap mutant female moths, we confirmed that the infertility of ap female moths was due to the degradation of the bursa copulatrix. Meanwhile, we excluded the influence of the ability to make eggs, the egg holes and the egg contents of female moths through the investigation of the quantity of eggs, observation of egg holes and the parthenogenetic experiment, respectively. Because of the female sterility of ap mutant, we used the F2generation produced by F1selfing for mapping the ap locus. We mapped the ap locus using384F2individuals with ap mutant phenotype and the ap locus was narrowed to a region between the markers A60and A73. The markers A54and A85were tightly linked with ap locus. In the candidate region, there were two predicted genes, BGIBMGA008843and BGIBMGA008844. We found that the two genes actually should be one gene, Bmsob gene, and concluded that the Bmsob gene was the candidate gene for ap mutant. Bmsob gene belongs to the odd skipped gene family, involved in a series of process during the embryonic development and the establishment of body plan. We obtained the full-length cDNA of Bmsob gene in the normal and ap mutant using RACE technology,1923bp and1924bp, respectively. The length of coding sequence region was1311bp, encoding436amino acids. The5’untranslated region was both85bp long and the3’untranslated region was527bp and528bp, respectively. Both of them had two exons. There were fifteen single base mutations in Bmsob gene ORF between the Dazao and ap mutant, thirteen of which were synonymous mutations and two were missense mutations (Pâ†'A, Sâ†'P). In addition, there were eight single base mutations and one base insertion in the Bmsob gene in ap mutant. The prediction of Bmsob protein showed that it was a transcription factor including five C2H2type zinc finger structures.Analysis of the spatio-temporal expression pattern of Bmsob gene revealed strong expression in the silkworm larval genital gland and in the third day of wandering stage and the first day of pupa. This period is the key stage from larvae to pupae metamorphosis and also the important stage of genital gland development, implying that Bmsob gene plays an important role in the development of silkworm genital gland. In addition, analysis of Bmsob expression pattern during the wing development showed that the expression level of Bmsob increased gradually with the wing primordium development. The expresson level of Bmsob was highest in the final stage of5th instar, implying that the Bmsob gene is involved in the development and formation of silkworm wings. Quantitative RT-PCR (qRT-PCR) revealed that the expression of Bmsob was both significantly reduced in the ap mutant compared with the wild-type in the embryo and1st day pupa. We found that there were sequence differences in the Bmsob promoter region between Dazao and ap mutant. Then we detected the promoter activity in wild-type and ap mutant using the dual luciferase report system. The results showed that the activity of the Bmsob promoter in ap mutant was decreased significantly. Hence, we speculated that the sequence variation of Bmsob promoter region may be a significant reason for the decreased expression level of Bmsob gene.4. Study on the regulatory interactions between candidate genesAccording to the studies on Wes and ECs-l mutants and the functions of Hox genes, we speculate that three kinds of mutant phenotypes of ap, no thoracic legs, abnormal wings and degenerate bursa copulatrix, may be related to the Bombyx mori Hox genes, such as BmAntp, BmUbx and BmAbd-B. Quantitative RT-PCR showed that the expression levels of these three genes were all up-regulated expression in the ap mutant compared with the wild-type. The wings in Wes and ap were both small and abnormal, while BmAntp gene also showed overexpression. Therefore, we assume that Bmsob is the upstream regulatory gene of Hox gene.First we obtained the Bmsob protein by prokaryotic expression and the protein was purified. We validated the interactions between Bmsob protein and BmAntp, BmUbx, BmAbd-B genes through the EMSA experiment. The results show that the Bmsob protein can bind directly to Bm Ubx gene. Then, in order to verify whether Hox gene can be regulated indirectly by Bmsob gene, we screened the differentially expressed genes in wild-type and ap mutant using the gene chip. The results indicate that BmDsp gene may play an important role in the regulatory network. We confirmed that the Bmsob protein can bind to BmDsp gene. We obtained the BmDsp protein by prokaryotic expression and the protein was purified. At the same time, we analyzed the binding ability of BmDsp protein to BmPc, BmAntp, BmUbx and BmAbd-B gene, and the results showed that BmDsp. protein can bind to all of these genes, In summary, we draw the following regulatory relationships. The BmDsp gene can promote the expression of Hox gene. Bmsob gene not only can directly inhibit Hox gene expression, but also can indirectly inhibit Hox gene through suppressing the expressions of BmDsp and BmPc genes. We speculate that the regulatory network plays an important role in the precise expression pattern of Hox gene during the development of silkworm body plan.