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Mechanism Of Wing Development In Agrotis Ypsilon Rottemberg

Posted on:2015-07-06Degree:DoctorType:Dissertation
Country:ChinaCandidate:W WeiFull Text:PDF
GTID:1223330467950313Subject:Agricultural Entomology and Pest Control
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Black cutworm (Agrotis ypsilon Rottemberg) is a kind of underground insect with migratory habit. Based on the in-depth study on model insects including Drosophila melanogaster, many genes and pathways which are important for wing development have been revealed. In this thesis, the wing development of black cutworm has been systematically studied based on the hnown knowledge from model insects. The techniques of transcriptome sequencing analysis, gene cloning, qRT-PCR, cryosectioning, immunohistochemical staining, microsurgery, and nanomaterials-mediated RNA interference, have been used in this study and the results are summarized as follows:1. Black cutworm larval transcriptome is completed sequenced and analyzed. It gains a total of411739reads. The cumulative length of nucleotide is10,909,572554bp, GC content is50.32%and Cycle Q20reaches100%. A total of25698unigenes get comments by blasting with NR and the GO database, In NR database, for example,11509unigenes (47.4%) show high homology with other species,14509unigenes (59.7%) show more than60%of the similarity to the other insects. The functional proteins are distribution mainly in cells and organelles. Many wing development-related genes, such as decapentaplegic(dpp), wingless (wg), notch, hedgehog and so on, are identified and show high homology with Drosophila,.2. Aydpp and Aywg have been cloned which are the homologous of dpp and wg in Drosophila. Aydpp and Aywg expression level are detected during the developmental duration. Aydpp and Aywg are detectable in all tested developmental stages, with the highest expression level in embryonic stage and no apparent difference in larval stages. It indicates that they play important roles in embryonic and larval development.3. The embryonic origin and cell organization of the black cutworm wing have been revealed by the techniques of cryosectioning, immunohistochemical staining, and microsurgery. The wing originates from the wing primordium cell clusters on the2nd and3rd thoracic segment at the late embryonic stage. Unlike single cell layer structure of Drosophila wing disc, the black cutworm wing disc contains double cell layers. Plus the squamous cells of peripodal membrane around, the black cutworm wing disc is totally composed of four layers of cells. The wing disc grows rapidly and wing veins form during larval stage. During pupal stage, wing structure is similar with adult wing, and the double cell layers closely bonded, that is different from metamorphogenetic folding process of Drosophila wing.4. The regeneration ability of black cutworm’s wing disc is observed. Before the middle7th instar, either removing part or whole of the wing disc, the wing disc can be regenerated and develop to a complete one with smaller size compared with the control. After the middle7th instar, the regeneration ability is weakened. No or only incompleted adult wing is regenerated after the same treatments as before. Aydpp and Aywg expression levels in24hours micro injury wing disc are3-4times higer to controls. This indicates the remarkable roles of these two genes during the regeneration process. 5. The function of Aydpp and Aywg signaling pathway have been analyzed. Nanomaterials-mediated dsRNA are mixed with artificial diet, and feeding to black cutworm for RNA interference. The newly hatched larvae fed with above RNA interference of Aydpp and Aywg show apparent growth retardation and death. High instar stage larvae with above treatments show retard development with smaller the body size. During the wing regeneration, suppressing Aydpp and Aywg by above mothed leads to severe defects on adult wings. These data suggest that Aydpp and Aywg play important roles in black cutworm wing development and regeneration.
Keywords/Search Tags:Agrotis ypsilon Rottemberg, Wing disc, Tissue regeneration, RNA interference, Nanomaterial
PDF Full Text Request
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