Study On The Function Of Sex Determination Related Genes Bmhrp28 And BmPSI In Silkworm, Bombyx Mori

Sex determination is one of the scientific issues in the biosphere.As a lepidopteran insect, domesticated silkworm Bombyx mori,the researches of its sex determination have intercommunity and particularity compared with the Drosophila melanogaster which has the clearest research on sex determination mechanism,so the researches on sex determination mechanism of silkworm can provide us a new model for the better understanding of the insect sex determination,and has important practical value in the sericultural industry.Silkworm is unisexual insect,of which the economic value of male is notably higher than that of female.The average rate of cocooning, physical character,efficiency of feeding,and quality of cocoon are better in male.Therefore,the researches on sex determination of silkworm are always the important issue in the sericulture.The sex chromosomes of silkworm are ZW.In recent years,in the sex determination of silkworm the double sex gene Bmdsx which is in the downstream of cascade has been cloned and identified that it plays an important role in the male and female differentiation of silkworm. Recently,scientists has identified two regulational factors BmPSI and Bmhrp28 in the upstream of Bmdsx,but failed to clarify the role and relationship of them yet.Based on this,this study cloned and analyzed Bmhrp28 and BmPSI by using whole genome sequences,ESTs and microarray data. Meanwhile the study performed further functional research on the Bmhrp28 and BmPSI by RT-PCR,prokaryotic expression,RNAi,yeast two-hybrid,Co-Immunoprecipitation and so on. The main results are as follows:1.Cloning,sequence analysis and mRNA expression of Bmhrp28 and BmPSITwo pairs of primers were synthesized based on the Bmhrp28 and BmPSI which were reported in the former papers and GenBank.Two positive clones including complete ORF of the two genes were obtained.The ORF of Bmhrp28 is 771 bp,which codes 256 aa.Its putative molecular weight is 28.2 kD and pI is 8.72.It locates on nscaf3044 of the 21^st chromosome and contains 7 exons and 6 introns.The ORF of BmPSI is 2112 bp,which codes 703 aa.Its putative molecular weight is 76.4 kD and pl is 6.07.It locates on nscaf2789 of the 20^th chromosome and contains 14 exons and 13 introns.The boundaries of each exons and introns are consistent with the GT-AG rules. Domain prediction shows that both the Bmhrp28 and BmPSI are RNA binding proteins.Bmhrp28 contains two RRM domains while BmPSI contains four KH domains and two functional domains named as A and B.Both RRM and KH are RNA binding domains which are involved in RNA alternative splicing,processing,and transcription.The functional domains of Bmhrp28 and BmPSI are conserved with their homologous genes hrp48 and PSI in Drosophila melanogaster,which act as regulational factor in the alternative splicing of P element transposase.In order to discover the space-time expression profile of the two genes in Bombyx mori,we performed an expression analysis by using ESTs data,genome-wide microarray data and semi-quantitative RT-PCR.ESTs evidences of Bmhrp28 were found in BmN cell,embryo and ovary and that of BmPSI were found in testis,ovary,silkgland,and embryo.The genome-wide microarray data indicates that the expressions level of Bmhrp28 in each tissue of fifth-instar day 3 larvae are very low,except a gleam expression in testis.But the BmPSI exhibits a higher expression level in each tissue,especially in ovary and posterior silkgland and expression level is higher than Bmhrp28 as a whole.The expression data in developmental stages shows that Bmhrp28 slightly expresses from fifth-instar day 4 to moth stage.But BmPSI exhibits a stable expression level and there is no conspicuous difference between male and female.The expression data of early embryo indicates that Bmhrp28 has different expression patterns between male and female.In female the highest expression level is 48 h after oviposition and then begins to decrease,but in male there is no change at 48 h after oviposition and the expression reaches the peak at 60 h after oviposition and then begins to decrease.Compared to Bmhrp28,the expression pattern of BmPSI is similar in male but expression reaches the peak at 72 h after oviposition,while in female the expression is always at a low level and little change.Semi-quantitative RT-PCR was carried out to validate the microarray data.Bmhrp28 and BmPSI are widely expressed in both male and female fifth-instar day 3 larvae,while expression level of Bmhrp28 is lower than that of BmPSI.In early embryo they are almost the same,lowly expressed from unfertilized eggs to 24 h after oviposition.In the male early embryo,the expression gradually increases until it reaches the peak at 96 h after oviposition,and then begins to decrease. In the female early embryo,the expression reaches the peak at 48 h after oviposition and then begins to decrease.The developmental stage of early embryo is a key stage of sex-determination, and the similar expression patterns indicate that Bmhrp28 and BmPSI might be processed synergistically in this progress.2.Prokaryotic expression and polyclonal antibody preparation of Bmhrp28 and BmPSIThe cDNA of Bmhrp28 and BmPSI were ligated into modified p28 and pET-MBP plasmids to construct prokaryotic expression vector which named as Bmhrp28/p28 and BmPSI/pET-MBP, respectively.They were transformed into expression strain BL21(DE3) and Rosetta(DE3) and then induced by IPTG.A 31 kD and a 120 kD recombinant protein were obtained.The molecular weights were a little higher than the original protein because a 6×His-tag and a MBP-tag were added.Protein composition identification showed that the two recombinant proteins were expressed in dissoluble form.With the methods of affinity chromatograph and electroelution respectively,we purify recombinant BmHRP28 and BmPSI,and then immunized male rabbits with the two proteins to prepare polyclonal antibodies.Western blotting results show the antibodies are induced by the recombinant BmHRP28 and BmPSI,thus they are capable for the further researches.3.The functional study of Bmhrp28 and BmPSI with RNAiExisting studies have shown that both Bmhrp28 and BmPSI bound to the forth exon of Bmdsx. Bmdsx,the double sex gene in the sex determination pathway of Bombyx mori,as the positive regulation factor SP1 and Vg downstream,is predominantly expressed in the female.However,its negative regulation factor PBP is mainly expressed in the male.Based on these,we microinjected the in vitro synthesized siRNA into the 48 h embryo of white egg strains of sex-limited silkworm,and then the embryo was allowed to develop until 96 h.Real-time PCR was used to detect the expression difference of PBP,SP1 and Vg downstream Bmdsx to study the functions of Bmhrp28 and BmPSI in the silkworm sex determination pathway after the Bmhrp28 and BmPSI upstream Bmdsx were silenced.As a results,the expression of downstream PBP was dropped down,while the expression of SP1 and Vg increased,implying that BmPSI,the upstream regulation factor of Bmdsx,has an important function in the silkworm sex determination pathway. The expression of SP1 and Vg did not increase after Bmhrp28 was silenced,although the expression of the downstream gene PBP had a relatively small reduction.It is inferred that Bmhrp28 might have lower function than BmPSI although Bmhrp28 did participated in the silkworm sex determination pathway as the upstream regulation factor.4.Study on interaction between the upstream regulation fators of silkworm Bmdsx,Bmhrp28 and BmPSI The study used yeast two-hybrid and Co-Immunoprecipitation to identify the interaction between Bmhrp28 and BmPSI.First,in the absence of self-activation using yeast two-hybrid,the tested genes were co-transformed yeast in order to determine the relationship between target genes by detecting the three reporter genes(HIS3,URA3,LacZ).In this research,Bmhrp28 and BmPSI were connected into pPC86 and pDBLeu vectors,respectively.In the absence of self-activation, pPC86-Bmhrp28 and pDBLeu-BmPSI were co-transfected yeast.As a result,there were no positive clones on the SC/-Leu-Trp-His+3-AT and SC/-Leu-Trp-Ura plates and there was no positive result in the membrane detecting of LacZ.The expressions of the three reporter genes HIS3,URA3,LacZ were not detected.Thus there was no or little direct interaction between Bmhrp28 and BmPSI.In order to further validation of the result,we used Co-Immunoprecipitation to identify the relationship between Bmhrp28 and BmPSI.Anti-BmHRP28 antibody precipitated its antigen in the testis extract and at the same time protein which interacted with BmHRP28 was also precipitated.Then anti-BmPSI antibody was used to detect if BmPSI was in the precipitation product.The results showed that both of BmHRP28 and BmPSI were existed in the precipitation deposited by anti-BmHRP28 antibody.Considering the results of yeast two-hybrid,we presumed that between Bmhrp28 and BmPSI there were no direct interaction,but they might play a role in the sex determination of silkworm in a protein complex.5.Constructing yeast two-hybrid cDNA library of silkworm early embryo and screening other regulation factors in the regulator complexesIn order to explore whether there were other regulation factors to regulate Bmdsx in the Bmhrp28 and BmPSI protein complex,the present study constructed yeast two-hybrid cDNA library of early embryo.The white egg strain of sex-limited silkworm(male) was used to extract the mRNA for cDNA library construction.The cDNAs were connected to pDONR222 vector to generate gateway entry-library.And then entry-library was transferred to pDEST22 vector to gain yeast two-hybrid cDNA library.The total number of clones in library was 1.36×10⁷.Randomly we picked 40 colonies and extracted plasmid DNA to PCR detection.The results showed that the average size of insert fragments was larger than 1.3 kb and recombination rate was greater than 95%.1μL cDNA library was diluted 10⁶ to coat on plates and grew 77 colonies.After amplification the library titer was 7.7×10¹⁰ cfu/mL.Identification results showed that the library could be used for the library screening.In order to further identify other possible regulation factors in the regulator complex,we performed yeast two-hybrid.In the absence of self-activation,we used pDBLeu-BmPSI to screen the yeast two-hybrid cDNA library.After colonies grew on SC/-Trp/-Leu/-His plates added 30mM 3-AT for seven days,there were 22 transformations of yeast colonies.We crossed on the SC-Trp-Leu plates to save strain and picked a small colony to detect expression of report gene LacZ.LacZ positive clones were detected by the SC-Trp-Leu-Ura plates.Prey plasmid was extracted from LacZ and URA3 yeast co-transformed with the bait plasmid for validation.At last only one positive clone was tested.The cloning and sequencing of this positive clone showed that this gene was a spliceosomal protein on the X in silkworm.This gene ORF is 1029 bp,which encodes 342 aa.Its molecular weight is 37.0 kD and pI is 6.36.It locates on nscaf2827 of 8^th chromosome and contains 6 exons and 5 introns.The boundaries of each exons and introns are consistent with the GT-AG rules. Domain prediction shows that this gene,which has two same RRM domains as Bmhrp28,also is RNA binding protein.Compared with SPX protein sequence of Drosophila and SF3B4 of Aedes aegypti,Apis mellifera,Tribolium castaneum,Bos Taurus,Homo sapiens,Sus scrofa,Mus musculus, Equus caballus,Danio rerio etc.,BmSPX has homologe more than 85%and has very high identity in the RRM domains.Shown by genome-wide microarray expression data of tissues,Bmspx expresses in every tissue in fifth-instar day 3 but only has high expression level in testis that may have related to its potential function in sex determination of silkworm.However the expression data of each development period shows Bmspx is widely expressed in various periods and there is little difference between female and male.The expression data of early embryo indicates Bmspx has a similar expression pattern between female and male that expression reaches the peak at 48 h after oviposition,and then begins to decrease and the lowest expression level is at 96 h after oviposition. The function of this in the sex determination of silkworm still needs to be studied.Bmspx encodes an RNA binding protein which highly expresses only in testis.As one of the regulators of Bmhrp28-BmPSI protein complex,Bmspx can directly interact with BmPSI.Based on the results we speculate that upstream regulation of sex determination gene Bmdsx is controlled by BmPSI,Bmhrp28,Bmspx and so on.Bmspx interacts with BmPSI,but the relationships between Bmspx and Bmdsx,Bmspx and Bmhrp28 are not clear yet.If Bmspx can be binded to the Bmdsx and directly interact with Bmhrp28,then Bmspx could be a "bridge" between Bmhrp28 and BmPSI, therefore,the structure of upstream regulatory complex of Bmdsx would be more clearer.Further studies will be carried out to discover the relationship among Bmspx,Bmdsx and Bmhrp28,and also the specific role of Bmspx in the sex determination of silkworm as well.