Positional Cloning Of The Homeotic Mutant E^Kh And Analysis Of The Modified Genes For Ventral Appendage In Silkworm, Bombyx Mori

The emergence of appendage takes great convenience to better adapt to the complex environment in the evolution of insect. The insect appendages are divided into dorsal and ventral appendages. The dorsal appendages include wing and derivation, but ventral appendage possess multiple types, containing the antenna located on the head, the locomotive organs distributed on thoracic and abdominal segments, external genitals for copulation insemination and oviposion, and so on. Hox genes, the main regulatory factors in the development of body plan, are essential for the development of ventral appendages. However the previous researches suggested that the functions of Hox genes on the development of ventral appendage are various in different insects. Therefore it is essential to study the role of certain genes in ventral appendage phenotype modification in insects.Silkworm, Bombyx mori, is crucial economic insect, possessing abundant materials for genetics research, and includes a variety of mutants exhibiting obviously structural variation in ventral appendage patterning compared to wild type. In consequence, we can survey the genes for modifying ventral appendage by comparing the mutants owning similar phenotype. In this study, utilizing EKh mutant, we are aware of the relation between Hox gene and the ventral appendage modification, through analysis of candidate genes of that mutant. Then we use two similar mutants, named ENk and EN, to perform the Digital Gene Expression Profiling (DGE) sequence. Genes, showing the same expression variation in the two mutants compared to the wild type, are probably the candidates for ventral appendage modification. The main results in this study are as follow:1. The phenotype investigation of silkworm EKh mutantIn wild type silkworm, there are three pairs of legs on the thoracic segments T1-T3, and four pairs of abdominal legs on the A3-A6 segments respectively. Furthermore the thoracic and abdominal legs are obviously different with each other. The former are subdivided into different sub-segments, but the latter are fleshy protuberances. Both of them are apparently different and co-existed in the larval period. Only three pairs of thoracic legs are reserved in the adult period.Ekh mutant was one member of the E group, and the mutation phenotype appeared in the embryo, larval and adult period in silkworm, Bombyx mori. In the EKh embryo, except for the normal ventral appendages, there are extra abdominal legs on the Al segment, and its phenotype was similar to the normal thoracic leg. The extra legs occasionally present on the A2 segment. The extra appendages are existed through the larval period. In the adulthood, very few individuals possess extra appendages, the 4th pair of appendages on the ventral side.2. The positional cloning analysis of the silkworm EKh mutantThe EKh was located on the position of 21.1 cM of the sixth linkage. Screening the existed SSR markers on the sixth linkage, we found that they are not polymorphism between Dazao and EKh mutant. We designed new primers to discover new polymorphism markers. We synthesized results of PCR using the polymorphism markers as primer, and discovered that three markers are completely linked to the EKh mutant, named L1779, L1011 and L1153. Further, we targeted the candidate region between L1048 and L1391, about 355kb region. The candidate region includes the first exon of BmUbx and BmabdA gene through bioinformatics. Therefore we suggested that BmUbx and BmabdA genes are the candidate genes for EKh mutant.3. The expression profile detection of the candidate genes for EKhWe cloned the candidate genes, BmUbx and BmabdA. There are only few single-base nucleotide mutations in the full-length cDNA, no any amino acid mutation caused in the open reading frame. QRT-PCR and western blotting results suggested that BmUbx were obviously down-regulated and BmabdA were apparently up-regulated not only on the mRNA, but also on the protein level. Through the immunoflurescence of the 60h embryo, we found that BmUbx was normally strong expressed on the A1 segment, but was down-regulated on the same segment of the EKh mutant. However the BmabdA gene was expressed on the abdominal segments A3-A6 to promote the development of abdominal proleg in the wild type Dazao, and the EKh mutant appeared ectopic expression of BmabdA gene on Al and A2 segments. Based on the above results and the pre-existed research, we supposed that the ectopic expression of BmabdA gene promotes the development of extra legs on A1 and A2, and the down-regulated expression of BmUbx on the A1 segment caused the extra legs to be thoracic leg-like appendage.4. The screening of genes for ventral appendage modification in silkwormAccording to the above research, we supposed that the BmUbx was important for the modification of the ventral appendages. Therefore we used two mutants ENk, EN and the wild type N4 to investigate the genes for the ventral appendages modification using DGE sequencing. They exhibited similarly phenotype that both have thoracic leg-like appendage on every abdominal segment in the homozygote embryos. Through dissection, we observed that the phenotype of normal thoracic and abdominal legs could be easily distinguished by the morphology around 60h old embryos. We took the abdominal segments of the embryos in that period, and extracted RNA to conduct DGE sequencing. Compared to the wild type N4, we found 102 upregulated and 123 downregulated genes co-excisted in the ENk and EN mutants. Through function analysis, these differentially expressed genes included genes associated with body and organ segmentation, genes has been proved with the development of hands and feet in vertebrate, genes has been identified to the development of distal end in the thoracic appendage development, etc. We hypothesized that these genes may be involved in the process of appendage modification in thoracic and abdominal legs.5. The identification of Dll gene for ventral appendage modification in silkwormDll genes involved in the distal end development of the thoracic legs in Drosophila, and the expressed stage and model are both different in the thoracic and abdominal legs. The quantitative RT-PCR in the same material of DGE sequencing, suggest that compared to wild type N4, the expression level of Dll gene have up-regulated tendency in both of the mutants. Through the immunoflurescence of the 48h embryo, we found that the expression profile and period are obviously different in the normal thoracic and abdominal legs. In addition, the expression model of Dll gene in the mutant abdominal legs changed to the nomal thoracic legs. Therefore we speculated that the Dll gene may be involved in the formation of phenotypic differences between thoracic and abdominal legs in silkworm, Bombyx mori.