| Bombyx is one of the important economic insects. It is a suitable model organism to study the sex regulation of the insects. It is easy to generate polyploids and gynandromorphs artificially in this insect .Therefore , Bombyx should be a suitable material for studies on sex determination. Because the sex determination of Bombyx is a model of epistatic systems in inverteberates, the clarification of the sex-determining mechanism of this insect would be helpful for a comprehensive understanding of sex determination in invertebrates. The mechanism of sex regulation of Bombyx is very complex and the molecular studying of it is in initial stage.For the sake of generating female-specific fragment, RAPD-PCR will be used and if s product will be clarified by cloning , sequence analyzing , and further PCR verifying .Finally, on the basis of the results of sequencing, sequence comparing and PCR verifying, it can be inferred whether the female specific fragment will be from W chromosome of Bombyx or related with the sex regulation Bombyx.dsx gene lies in the terminal of Drosophila sex regulation cascades , which controls sex differentiation in Drosophila. Bmdsx, a homologue of dsx in Bombyx , is a sex gene of Bombyx and was initially obtained from the EST database of Bombyx. So far, Bmdsx, which had been confirmed by the experiments, is the only gene related with the sex regulation of Bombyx .The studies on hand show that Bmdsx is a homologue of dsx not only in the sequence but also in the function, that is, Bmdsx is a key gene of controlling sex differentiation of Bombyx. Moreover, Bmdsx is a single copy gene which have been experimented.Another part of this study is that a pair of specific primer is designed on the basis of the sequence of Bmdsx cDNA . In order to analyze the expression of this gene in the different tissue of Bombyx, TaKaRa RNA LA PCR kit is used .Then we can analyze the quantitative expression ofthis gene and have a test of Bmdsx RNAi in the different tissues ofBombyx.1. selecting, cloning, sequencing and verifying the female-specific RAPD molecular markerThe genomic DNA was extracted from the Bombyx Y010 and C100 , including male and female individuals. A 355bp female-specific molecular marker was obtained. On the basis of a number of random primers used to select the different fragment between male and female the molecular maker was successfully cloned, sequenced, and was compared with other DNA sequences with the software blastx and blastn. Finally, the specific primer was designed according to the results of the sequence and the specific fragment was tested by genomic DNA amplified of 6 kinds of Bombyx as the templates , including Y010 and C100 with the specific primer. Therefore it is inferred that the fragment was from the W chromosome of Bombyx. However, the further study will be carried out in order to verify whether the fragment was really from the W chromosome of Bombyx.2.The expression analysis of Bmdsx gene in different tissues of BombyxThe primer which was designed by Shimada el during testing the Bmdsx expression in a tissue of Bombyx. As is expected, the final results show that a 475bp band is amplified from the female tissue and 226bp band is amplified from the midgut of male Bombyx C108(o , O ) in 6 and 7days during the 5th instar, which is in harmony with the results of Shimada. And the 226bp band had been clarified by cloning and sequence analyzing. At the same time, we have a new result that 475bp band and a Bmdsx 226bp band coexisted in Tashima gynandromorphs, which shows Bmdsx gene is expressed in Tashima gynandromorphs in female and male specific ways. According to both the results of this study and the previous result, so far, Bmdsx was expressed in all the tissues detected of Bombyx, so it probably will be expressed in tissues of Bombyx in most of or all the tissues of Bombyx, which needs further study to identify. Sex differentiation of Bombyx had been determined during the early embryo developing. But Bmdsx was expressed from the stage of larvae to... |
PDF Full Text Request