| Silkworm (B. mori) after a long period of domesticated selection, has become an important agricultural economic insect. The silkworm moderate body, short growth cycle, the congenital advantage of the high reproduction rate, that makes it is an important model organism of Lepidoptera insects. With the development of molecular biology, the accomplishment of silkworm genome frame, fine mapping and important genetic resources sequencing, the researches of silkworm get further promoted. Second, silkworm has a large number of mutant gene resources, which involve many important inheritable characters including body model of development, metamorphosis, and morphology. It is an important material for studying genes and body development regulation pathways of insects.The insect body plan development is significance in survival and reproduction of silkworm. In silkworm, the mutants involving body plan development make a large share. Therefore, the study of silkworm body plan mutants can not only understand the development genes and regulation pathway of lepidoptera in depth, understanding the important regulation network of Hox genes in insect body development, also lay a foundation for functional use of insect genes. In this study, we chose the E mutant which is a representative of silkworm E group belonging to body plan mutations as the research object. We locked the candidate gene of E mutant using positional cloning technology, and then carried on the related researches of candidate gene according to the phenotype of E mutant. The main research results are as follows:1. The phenotype observation of Bombyx mori E mutant We observed the phenotypes of wild type Dazao and E mutant in postembryonic stage and on day3of the fifth instar with stereomicroscope and digital camera. After observation, we found that the morphological structure of abdominal appendages had formed fully along abdominal segments3to6(A3-A6) in postembryonic period. But in E mutant, there is a pair of superfluous legs which are smaller on A2except normal abdominal legs. The larvae E on day3of the fifth instar still have smaller legs on A2. But beyond that, the morphology of superfluous legs is similar to the distal part of the normal abdominal appendage. Different from the wild type, there were lesser number of crochets that were shaped into a’circle’in E mutant. In addition, Dazao have the paired crescent markings and stars on A2and A5respectively, but there is no marking on the back of E.2. The rough mapping analysis of E mutantUsing SSR markers on the6th linkage and designed markers in predicting regional, we carried out preliminary mapping for E loci in300BC1individuals. We carried on electrophoresis for analysis of PCR amplification products; and then drew the linkage map between E loci and molecular markers according to the results of electrophoresis band types. The candidate area was reduced to1.0cM between E3and E6. By contrasting the range that between E loci and its adjacent markers and the map distance in silkworm linkage map, we did the candidate gene screening combine with the bioinformatics analysis. In this region, there are5genes predicted by the Gene model program which are BGIBMGA006384, BGIBMGA006385, BGIBMGA006386, BGIBMGA006387, BGIBMGA006388on nscaf2853.3. The fine mapping of E locus and candidate gene screeningBased on the result of rough mapping, we designed new markers and expanded position groups to1115individuals. Using the new markers, it would make polymorphism screening, linkage analysis, PCR amplification, electrophoretic bands analysis and physical linkage map. The candidate region was shorter between E33and E39, which was0.2cM about209kb and had3genes, BGIBMGA006386, BGIBMGA006387, BGIBMGA006388. The marker E32had no crossing over individual and linkaged with E closely. It suggested that the candidate gene were located near E32. By bioinformatic analysis, the candidate gene was finally confirmed to be Bm-AbdA gene.4. Cloning multiple spliceosomes of candidate gene of EBy cloning and sequence alignment of Bm-AbdA between Dazao and E, we found there were two more spliceosomes in E compared with3spliceosomes in Dazao. In different alternative splice forms, the mutative sequences in spliceosomes all took place in the second exon. In addition, we found a new splicing form that its entire exon2is missing.5. Detecting the expressions of candidate gene and downstream target gene of EIn order to detect the impact of multiple different alternative splice forms in E, we measured the relative amounts of BmAbd-A mRNA and proteins between Dazao and E mutant. The expressions of BmAbd-A mRNA and proteins showed about twice in E as much as in Dazao. In order to study whether the higher expression of BmAbd-A influence can influence its regulation network, we measured the expression of Dll which is the downstream target gene and found that its expression also has doubled in E.6. Evolution analysis of Abd-A and its homologous gene Hox8From nematode to higher mammal, we gathered the gene numbers, the transcripts, the protein numbers and the gene apographs, and then we also analysed the diversity of alternative splice forms and evolution pattern of Abd-A and Hox8. With biological evolution, the functions of Abd-A gene become more complex. The spliceosomes of Abd-A also increase with more complex structure in organisms. In order to meet the requirements of development, Hox8increase itself apographs and encode more functional proteins. |
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