Study On Function Of Bmsxl On Sex Determination Pathway In Bombyx Mori

Studying the mechanism of sex determination in the silkworm has a great amplication value at industry. It has always been the research topic for most of the scholars for a long time. In the model organism Drosophila, Sxl acts as the key factor of sex determination. The excistence of protein SXL decides who can have the female development. At now, there aren’t any reports about the role of Sxl acting as the key factor of sex determination in the non-drosophila insects. In Bombyx mori, previous scholars just cloned the sequence of Sxl homologous gene and analysed its expression features and the results showed that the expression level of Sxl homologous gene (Bmsxl) in the reproductice system was highest. So, clarifing that whether Bmsxl acts as the key factor on sex determination pathway in silkworm has important reference significance to understand the mechanism of sex determination for Bombyx mori even for other insects.The main results are as follows:1> Cloning, prokaryotic expression, purification of Bmsxl’s domain and making the antibody of Bmsxl’s domainWe predicted the domain sequence of Bmsxl by SMART software. Then the specific primers were disigned to amplify the domain region by using the testis cDNA in the 3 days of the fifth instar of silkworm as the amplification template. We got the sequence which has the length of 675bp. This fragment was subcloned in pet 28(a+) vector and transformed into the expression strain BL21 (DE3)’fter sequenced successfully. The recombinant protein was expressed on the condition of 37℃,for 4 h. The soluble recombinant protein was purified through Ni-chelating affinity chromatography, and the target protein was eluted at the concentration of 100 mM imidazole. The purity of the eluted protein was tested by silver stain which has more sensibility for further validation.We measured the concentration of the recombinant protein by BCA and the quality of the protein was totally fullfilled with requirment for polyclonal antibody. The protein samples were sent to Nanjing GenScript comepany for the polyclonal antibody preparation of rabbit anti-BmSXL. The valence of antibody is 1:10000 and can be used for further research.2^. The regulation of Bmsxl to itselfWe cloned two splicing forms of Bmsxl and they were named by Bmsxl-PA、 Bmsxl-?B. In Drosophila melangaster, Sxl can bind to the specific strech of poly U sequence. The sequence analysis of Bmsxl showed that there was a special region contained poly U(UUUUUUUUUUUU) in the different region between Bmsxl-PA and Bmsxl-PB. According to the specific sequence we desinged probe to do EMS A (Electrophoretic Mobility Shift Assay) experiement. The results of EMS A showed that there were some unknown protein or compounds could bind to the poly U sequence. The result of RNA-immunoprecipitation (RNAIP) showe that the compound that binded to the specific poly U sequence contained BmSXL protein. In order to explore the effect of the poly U sequence on alternative splicing, this sequence was muted to UGUGUGUGUGUG and was cloned into the eukaryotic expression vector and in the same time, we constructed transformed plasmid contained normal poly U sequence. We transfected the recombinant plasmids into the cell lines of silkworm-BmE and BmNs. We compared the results between the experiermental group and blank group, and then found that in the treatmented BmE, the experiermental group didn’t amplify any form of Bmsxl, but the blank group can amplify the Bmsxl-PA. As for the BmNs, we found that the sequence of Bmsxl-PA could be examplified in the experiermental group, but the splicing form of Bmsxl-PB couldn’t be obtained. According to the results above, we can get a preliminary conclusion that Bmsxl protein could bind to the poly U region in itself and possibly the binding can promote splicing to produce Bmsxl-?B.3、The regulation of Bmsxl to BmdsxBmdsx which acted as the key gene located in the downstream of sex determination pathway. BmdsxF was the defauLt splicing form and the male specific splicing form BmdsxM was produced as the excistence of the splicing repressor. In Bombyx mori, sequence analysis showed that there was a strech of poly U sequece located in the third intron of Bmdsx and the other members in our group have proved that Bmsxl could surely bind to the specific poly U sequence located in Bmdsx.In order to explore the effect of the binding on the poly U sequence, we constructed minidsx plasmids that contained wild poly U and muted poly U eachly. The reconstructed plasmids were transfected into the ovary cell line of silkworm. We examplified the male splicing form of Bmdsx in the treatmented group that was transfected with minidsx plasmid contains muted poly U. This results showed that this sequence contained poly U was important to influence the alternative splicing, When the sequence was muted, the factor that promote splicing couldn’t bind to it, then the splicing form of BmdsxM was produced. Besides that, we also synthesized double strand RNA ds-Bmsxl as the injection mass for the male pre-pupa.72h later, part of the treated pre-pupas were used as raw materials to extract RNA and the RNA templates were reverse transcripted to cDNA. PCR results showed that in the male ones which were injected with double strand RNA ds-Bmsxl, the amount of BmdsxF splicing forms increased. The rest of the treated pre-pupa were raised up to the moth stage to observe the abnormal phenotypes of glands. Compared with the control group, the exogenous gonad of male silkworm in the experiermcntal group had pale colour, small size and the easing middle sage; as for the genital gland, two testis connected with each other. The exogenous gonad of female silkworm has the same trend of changing as the male silkworms, such as the lighter colour, small size and the fold edge tends to be gentle. As for female genital gonad, now it has developted to the fallopian tube, compared with the control group, length of the fallopian tupe in the experiermental group becomes shorter; the arrangement of eggs is slightly messy; single egg tends to be cuboid, rather than normal ellipsoid. The results above showed that Bmsxl could regulate Bmdsx alternative splicing.4、The regulation of Bmsxl to Bm-nanosIn Drosophila, nanos promoted the germ stem cells to renew. When binded with SXL protein in its 3’UTR poly U sequences, the expression level of Bm-nanos was down regulated, the formation of germ stem cells were inhibited and to be induced into differentiation. But whether silkworm has the same regulating pathway isn’t clear untilThrough the sequence analysis of Bm-nanos 3’UTR in Bombyx mori, we found that there were two segments of the poly U sequences. The two segments are designed to be the probe labeled with biotin, then incubated with cytoplasmic protein of gonads. EMAS results showed that there were hystersis bands existing. It implied that BmSXL protein could bind to the poly U sequence located in the 3’UTR of Bm-nanos in Bombyx mori. Together with RNAIP results, we can get a preliminary conclusion that Bmsxl protein could surely bind to the poly U sequence of 3’UTR ofBm-nanos. In order to test the funcion of the binding between BmSXL protein and Bm-nanos 3’UTR, we cloned the wild Bm-nanos 3’UTR sequence into the cell transformed plasmid and got Luc-nanos-Wild. In the same time, we constructed recombinant plasmid Luc-nanos-Mut contained muted poly U sequence. Each of these recombinant plasmids was co-transformed with PGL3-Bmsxl-PA into the BmE cell. Enzyme activity assay results showed that Bmsxl protein inhibited the expression level of Bm-nanos. This part of the expermental results illustrated that the bindation of BmSXL protein to the Bm-nanos 3’UTR could down regulate the expression level of Bm-nanos then further to control the differentiation of reproductive systerm.According to the results above, Bmsxl in Bombyx mori could regulate the alternative splicing of itself and Bmdsx, along with expression of Bm-nanos through the bindation of Bmsxl to the specific poly U sequence. These results were the first to report that the important role of Bmsxl on the sex determination in the non-Drosophila insects.