| Transforming growth factor beta (TGF(3) signaling pathway is involved in many cellular processes in all organism including embryo development, cell growth, cell differentiation, apoptosis, cellular homeostasis and other cellular functions. Based on the research of Drosophila melanogaster, the factors on the TGF beta signal pathway play roles in vein formation, muscle development of legs, antenna formation dorsal-ventral pattern formation and oogenesis.There're TGF beta signal pathway from insect to mammal.This paper makes use of the silkworm egg mutants to detect if the TGF beta signal pathway affect silkworm oogenesis.Silkworm is a kind of important economoic insects. The research of silkworm oogenesis can benefit not only the molecular mechanism but also the applification of such result.This paper make use of the assembly silkworm genome database, EST database, notation database, gene expression microarray database and TGF beta signal pathway imformation from other insect and RNAi techniques to find out important factors on TGF beta signal pathway in silkworm and its effection to egg formation.The main results are as following:1.Comparative analysis of important factors on TGF beta signal pathway of model organism and screening of key factors on TGF beta signal pathway in silkworm. Download submitted sequence of TGF beta signal pathway factors from Genbank, analyse their evolution relationship and similarity. By means of alignment of TGF beta signal pathway factors and silkworm genome database, EST database, notation database, gene expression microarray database, we find 2 candidate genes on silkworm TGF beta signal pathway, named Bmdpp and Bmsmad respectivelly.2.Gene clone of Bmdpp and Bmsmad in silkwormBased on the predicted candidate genes, PCR primers are designed to amplify them and sequence them.Bmdpp and Bmsmad are successfully cloned.Bmdpp is located on chromosome 12, which is scaffolded on silkworm genome nscaf2993.The coding sequence of Bmdpp is 1110 bp, translated amino acid are 370; intron is 1884 bp, and intron boundary obey the GT-AG rules.Bmdpp has a little difference from notation gene BGIBMGA010384 in silkworm genome database.Bmsmad gene is located on chromosome 12, which is scaffolded on silkworm genome nscaf2993.Bmsmad gene length is 6415 bp.There are 3 exons in coding region, and their length are 101bp,167bp and 1001bp respectivelly. 3.Bmdpp and Bmsmad sequencing and analysis of silkworm egg mutant.Silkworm line P50 is wild type control.6 egg mutant lines(col,ge,elp, sm,sp,grk) are sequenced to find out their variation of gene Bmdpp and Bmsmad.The result indicates that 2 lines:col and ge, each of them have one variation site on the amino acid level, GAG(E)→GGG(G), from glutine to glycine.Both of col and ge have such variation in its propeptide region.The sequences of Bmsmad are completely same in 7 different lines.4. Electronic microscope image of the surface structure and micropyle in silkworm egg mutants.The electronic microscope images of 7 different silkworm egg mutants indicates that the numbers of petals of the surrounding micropyle are slightly different. The numbers of petals are relatively small in 3 lines:elp,sm and sp, and 2 lines:col and ge are increased from line P50.5.Silkworm RNAi experiment of genes Bmdpp and BmsmadBy means of Bmdpp and Bmsmad dsRNA injection within the proper period, the interferenced individuals are tested in terms of Q-RT-PCR and electronic microscope image.After dsRNA injection, the expression of Bmdpp and Bmsmad are decreased.Even more, the more dsRNA are injected, the more decreased mRNA are observed. But electronic microscope images show no dramatically changes neither the surface structure or the "number" of petals of the surrounding micropyle.In conclusion, the TGF beta signaling pathway have limited effetion on oogenesis of silkworm, Bombyx mori. It's infered their functions are probably differentiated. |
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