Study On The Regulation Mechanism Of Self-splicing Of Bmsxl Gene In Bombyx Mori

It has been five thousand years for mulberry growing and silkworm raising in China.In thousands of years of silkworm breeding process,people found that there is a big difference between silkworm male and female individuals no matter in body size or in silk quality and quantity,and male silkworm has higher economic value.At the same time,as an important lepidoptera insects,silkworm research also can be used as a model,provide a reference for basic scientific research.Taken together,the research on silkworm sex determination,not only is of great significance to improve the quality of the silk,but also to further understand the reproduction of insect reproduction and provide a valuable reference for prevention and control of plant diseases and insect pests.Found in previous research,BmSxl participated in the silkworm sex-determining pathway,it has two forms of mRNA,encoding two kinds of different forms of protein.In order to further understand BmSxl own splicing mechanism,this article design related experiment to explore the upstream regulatory factors of BmSxl splicing and regulation mechanism of itself,as well as with the other gender control gene BmPSI,Bm IMP and BmMasc interaction relations are studied.The main research results obtained as follows:1.The research of splicing-factor of BmsxlBmSxl in Silkworm has two kinds of splicing forms,BmSxl-PA and BmSxl-PB.The difference is that a short sequence of exon 8 retained in the BmSxl-PA and spliced in BmSxl-PB.In order to obtain splicing（or retain）regulation factor of upstream.This experiment designed primers firstly,and the corresponding RNA sequences were obtained in vitro transcriptio.Next,we prepared the above-mentioned sequence with marked biotin and extracted nucleoprotein from the testis of silkworm larvae at day 3 of the fifth instar.Then,through assay of RNA-Protein pull down and silver staining,it suggested that there were specific bands.Mass spectrometry assay showed that it contained 46 kinds of proteins.Intriguingly,an protein named heterogeneous nuclear ribonucleoprotein 87F-like were related to alternative splicing of BmSxl and reported specifically expressed in the testis of silkworm.While considering the BmSxl-PB only expressed in silkworm gonad,and the expression quantity in testis is higher than the ovaries.So the protein is likely to be the important candidate of splicing regulatory factors of BmSxl.2.the prokaryotic expression and purification of heterogeneous nuclear ribonucleoprotein 87F-likeThe heterogeneous nuclear ribonucleoprotein 87F-like of Silkworm on NCBI was numbered XP₀04930561.This gene encodes 317 amino acids,molecular weight is 34 kDa,and the isoelectric point is 9.2,which belongs to alkaline protein.The primers were designed according to the CDS sequence of hnRNP 87-like,and the CDS sequence was successfully cloned from the testis cDNA of fifth-instar silkworm（Bombyx mori）.Then I was constructed prokaryotic expression vector of hnRNP87-like-pET-28 a.The recombinant vector hnRNP87-like-pET-28 a was transformed into BL21（DE3）Escherichia coli competent cells.The results showed that the protein was highly expressed in the supernatant at 16 ℃,and the pure protein was obtained by affinity chromatography.3.The exploration of the key site where Bmsxl regulates its own splicingThe research indicate that some splicing factor was involved in alternative splicing,and Cis element of genetic sequence also related to alternative splicing.in order to explore the cis element which combined to Upstream regulatory factors,primers were designed to replace the mutations which is about 10-12 bases every time in the vicinity of the splice site within the 8th exon of BmSxl.And the setting of the mutation needs to avoid the necessary points required for regular splicing site.And 17 group mutations were received.Then the full length of the mutant exon was cloned into 1180 [Hrs-BmAct4-LUC-Ser1PA] and transfected into ovarian cells of silkworm.After 48 h,RNA was extracted for RT-PCR analysis.It was found that when GCCTTGGGCA / ACACGGACGA / ACGCTTCAAA / TAATCCAACA / CATCGCGGTT / GCTTAAAGGAAC these 6 sets of bases mutated,BmSxl-PB was disappeared which indicate that the six sets of mutant sequence is likely to be related to the alternative splicing of BmSxl.4.The binding verification between the splicing regulate sequence and the splicing factors of BmSxlTo confirm if splicing factor bind to regulate sequence,we devised an in virto electrophoretic mobility shift assay based on the theory that the protein-specific probe complex shift slowly during gel electrophoesis.We performed six sets of biotinylated RNA probe to detect binding of labeled-probe and splicing factor hnRNP 87-like.It was observed that heterogeneous nuclear ribonucleoprotein 87F-like could specifically bind to probe GCCUUGGGCA.These results showed that heterogeneous nuclear ribonucleoprotein 87F-like may regulate the splicing of BmSxl by binding to GCCUUGGGCA.5.Interaction between BmSXL and other genes regulating the sex-determinationAs a switch gene,Bmdsx is located at downstream of sex determination cascade.BmSXL bind to the sequence of poly U that located at Bmdsx,and then the interaction of BmSXL and poly U could facilitate male-specical alternative splicing of Bmdsx,prompting silkworm to develop male.BmPSI,Bm IMP and BmMasc can push Bmdsx to produce the male-specific form,too.Therefore,to identify the relationship of the BmSXL,BmPSI,Bm IMP and BmMasc,firstly,we successfully cloned Bmsxl,BmPSI and Bm IMP from the testis of silkworm larvae at day 3 of the fifth instar with specific primers.And we found that there existed a kind of interaction between BmSXL and BmPSI,but the interaction of BmSXL and Bm IMP,BmSXL and Bm Masc were non-existent.It can be safely concluded that it was probably for BmSXL to regulate the splicing of Bmdsx in the sex-determination of silkworm by interacting with BmPSI.