Regulation Of BmPSI And Its Interaction Protein BmSPXx On Downstream Bmdsx

Bombyx mori is a lepidopteran model organism,and its male and female individuals have obvious value differences.Therefore,the study on the sex determination of silkworm is not only a scientific basis for the prevention of lepidopteran pests,but also an important issue for the economic development of agricultural mulberry.The Bmdsx gene located downstream of the silkworm sex-determination pathway is the key to the determination of silkworm,and the different alternative splicing forms of Bmdsx determine the direction of sex differentiation of silkworm individuals.Previous studies found that BmPSI?Byxbom mori P-element somatic inhibitor?protein can specifically bind to the exon 4 of the Bmdsx pre-mRNA in CE1,weaken the activity of nearby splice sites,and promote the male-specific splicing of Bmdsx.When the BmPSI gene was knocked out,many sex-regulated key genes in the transgenic silkworm individuals showed disorder,and the most critical Bmdsx splicing disorder also appeared.Female-specific splicing of Bmdsx appeared in male individuals.The researchers constructed a cDNA library of silkworm yeast two-hybrid early embryos and screened the library with BmPSI as a bait.Then they found a splicing protein,BmSPX,which interacts with BmPSI.We speculate that BmSPX may be involved in the alternative splicing of Bmdsx.In order to study the conservation of PSI-regulated dsx alternative splicing process in lepidopteran and the regulation of BmSPX on the alternative splicing of downstream Bmdsx,we designed experimental protocols and obtained the following results:1.Binding activity of KH₁ domain in BmPSI to the pre-mRNA of BmdsxThe BmPSI protein contains four KH₁ domains,which are RNA-binding domains.In this study,we purified maltose binding protein?MBP?,wild-type BmPSI and four KH₁ region truncation proteins of BmPSI.After the concentrations of these six proteins were adjusted consistently,they were used for an electrophoretic mobility shift assay?EMSA?.Compared with the wild-type BmPSI as a positive control,the four mutant proteins showed gaint lost in their ability to combine with CE1,especially the truncation proteins KH₁-1 and KH₁-2.We concluded that these four KH₁ motifs participate in the combination of BmPSI and CE1.Additionally,the KH₁-1 and KH₁-2 motifs play important roles in the binding.2.Discovery and verification of key amino acid sites in BmPSI protein to bind CE1Through the comparison of the four KH₁ motifs of BmPSI,seven potential key amino acids were predicted.Based on previous reports,KH₁ activity is mainly affected by the internal conserved zinc peptide repeat Ile-Gly-X2-Gly-X2-Ile.After cloning these amino acid mutant of BmPSI proteins,the proteins were purified with the wild-type BmPSI for EMSA.As suggested by the result that five types of mutant lost most of theri combining ability compared with the wild-type BmPSI,especially I116 G,L127G,and the IGGI mutant.To determine whether these three amino acid mutant proteins transform the secondary structure of BmPSI,circular dichroism?CD?spectroscopy was performed on the wild-type BmPSI and these three mutant proteins.There is no significant differencese among there three mutant proteins and wild-type BmPSI in there secondary structure.To verify that these three amino acid mutants do lose their ability to bind CE1 to BmPSI,isothermal titration calorimetry?ITC?was performed.From the ITC results,we found that these three amino acid mutant proteins do lose their ability to combine with CE1.3.The Conservation study of the binding between PSI and pre-mRNA of dsx in Spodoptera lituraIn order to study the conservation of the binding between PSI protein and CE1 regulatory elements,an evolutionary trees analysis and the conservative analysis of KH₁ motifs were performed with ten sequences of homologous BmPSI proteins in Lepidoptera.Based on the genome project of Spodoptera litura,this study analyzed the sequence of Sldsx-F with CE1.The results showed that the sequence of CE1 element stay conservative in Sldsx-F.Then,the SlPSI gene was cloned and the SlPSI protein was purified to be used in the EMSA experiment which showed that the binding between SlPSI and CE1 existed.According to the key sites of BmPSI to bind CE1,the same amino acid position of SlPSI was mutated,and then the binding ability of the mutant protein to CE1 probe was compared by EMSA.The ability of I116 G and mut IGGI proteins to bind CE1 was found reduced sharply.But the mutation at the L127 site has no significant effect on the ability of SlPSI to bind CE1.This result confirmed that the binding of PSI protein to CE1 element in the dsx pre-mRNA is conserved among Lepidopteran insects.4.Functional study of BmSPX which has intereaction with BmPSIPrevious studies have shown that the silkworm sex-determining key protein BmPSI can bind BmSPX.For the functional study of BmSPX,we used the piggyBac transgene incremental expression vector to construct the piggyBac-BmSPX recombinant vector,and obtained the transgenetic silkworm strain by embryo microinjection.The genome of the over-expression strain Over-BmSPX was extracted,and the transgene insertion was detected.It was found that the BmSPX insertion site was in the intergenic region of chromosome 11.Real-time quantitative PCR analysis of transgenic individuals revealed that BmSPX expression was up-regulated about 30-fold in transgenic male individuals,while BmSPX expression was up-regulated by about 4-fold in female transgenic individuals.Taken together,we obtained a stable genetically transgenic BmSPX incremental expression line.Phenotypic expression of BmSPX silkworms in the transgenic growth stage of moth development was observed,and developmental disorders of the internal genitalia and external genitalia were observed.Therefore,we conclude that the BmSPX gene plays an important role in the sex differentiation of both female and male silkworms.In order to study the physiological function of BmSPX in silkworm sex determination,we performed molecular level detection on Over-BmSPX transgenic lines,and the expression levels of BmMasc,BmIMP and Bmdsx-F changed significantly.At the same time,the BmSPX gene was cloned into the pSL1180 overexpression vector,and the silkworm BmE cells were used to carry out CO-IP?immunoprecipitation?experiments on the silkworm BmSPX and BmPSI proteins,which confirmed the interaction between BmPSI and BmSPX in the silkworm cell environment.Subcellular localization using BmE cells from the silkworm embryonic cell line revealed that the BmSPX protein belongs to the nuclear shuttle protein.Because BmSPX has the specific expression of testis tissue,which is consistent with the high-specific expression of BmMasc in the silkworm sex-determining key gene,we use CO-IP experiments to prove that BmSPX protein interact with BmMasc protein.Combined with the transgenic line Over-BmSPX assay and other experimental results above,we concluded that the BmSPX protein is involved in the alternative splicing of Bmdsx as a protein component in the Bmdsx male-specific inhibitor complex.At the same time,the accumulation of BmSPX protein expression can cause the regulation of sex determination cascade,causing the up-regulation of BmMasc and BmIMP to promote the male-specific splicing of Bmdsx,and then promote the male development of silkworm individuals.In summary,this paper identified the key amino acid locus of BmPSI involved in the selective splicing of Bmdsx.It was a new discovery that I116 has a significant regulatory effect on the ability of the KH₁ domain to bind RNA.And we discovered that the binding between PSI and CE1 is conserved in lepidopteran insects;The method proved that the silkworm splicing protein BmSPX which binds to BmPSI has an important role in the sex determination of silkworm,which makes the silkworm sex determination cascade more complete.