Studies On The Histomorphology Of Wing Disc And In Situ Hybridization With BmSRF Gene Of Silkworm(Bombyx Mori.)

The silkworm (Bombyx mori) is the holometabolous insect and some of its adult organs come from imaginal discs. The wing disc is one of the imaginal discs of the silkworm, which locates the two sides of the 2nd and 3rd thoracic section and grows separately into adult wing. The wing disc changes greatly in the whole life of the silkworm, especially during the metamorphism of the larva to the pupae, as well as the pupae to adult. There are abundant mutant resources of in the silkworm. Although the morphology of the wing disc has been reported, it is very short in the developmental process and differentiation between wild type and mutants.Through dissecting and measuring the wild type (dazao) and mutant (cf) of wing disc, we obtained the macroscopical character of the wing disc development in the pre-wandering stage and identified the differences between two materials. Using paraffin sections technology, we obtained the microscopic entity photos of the silkworm wing disc in different stages and confirmed and supplied the studies of the silkworm wing disc. These results could be provided morphology reference for wing disc developmental gene function research in the silkworm. Then, we cloned the BmSRF gene, which may be related to cf mutant of the silkworm and in situ hybridized with BmSRF gene on day 3 of the 5th instar larvae.The main research contents and results are as follows:1. We dissected the wing disc of the wild type (dazao) and mutant (cf) and measured its length using imageJ software. The morphological and development-stage differences of wing disc were found, and the macroscopical character was obtained. The greatest changes of wing disc morphology in Dazao strain were found in the 2nd～3rd, 4th～5th day of the fifth instar and 8th day of the fifth instar larvae to wandering stage. While, in cf mutant, the stages of it's greatest changes are the 2nd～3rd, 4th～5th day of the fifth instar and 6th day of the fifth larvae to wandering stage.2. Through the paraffin sections for the wing disc of the fifth instar larvae, its entity photos in each day of the fifth instar larvae were obtained. The beginning time of wing disc extension was observed, and the sequential photos of extension course were obtained. And we differentiated between the wing disc and related organs, such as muscle, trachea, and connective tissue. We found that the wing disc had not natural differences between wild type and cf mutant, which both had many tracheas and extension of wing bud, etc. However, the wing bud of cf mutant was larger than that of wild type. On the other hand, for the normal wing, the wing sac became thinner and dissected with the wing bud on day three of the fifth instar, and the wing bud extended on day four of the fifth instar. But, for the cf mutant, the wing bud extended on day two of the fifth instar, and the wing sac became thinner on day fourth.3. The silkworm BmSRF gene was cloned from the wing disc on day three of the fifth instar larvae. Sequence analysis indicated that the length of coding sequence of BmSRF is 906 bp and codes a protein of 302 residues, which contained 3 exons. In our experiment, the expression signal of BmSRF was detected during the fifth instar larvae to pupae. The expression level was gradually decreased from the 1st day of the fifth instar larvae to wandering stage, but sharply increased in one-day pupae.4. Using the in situ hybridization, the silkworm BmSRF gene was located. The result indicated that the BmSRF expressed only in the wing disc. While the expression signal of BmSRF was not detected in trachea, the muscle.