Identification And Function Research Of The Key Genes In The Upper Part Of The Bombyx Mori Sex Determination Pathway

The Bombyx mori sex determination has long been greatly concerned, because it is not only afundamental biological problem, but also be closely related with the silk industry. The classicalgenetics researches showed a strong female determinator (Fem) presented on the W chromosomecontrols the sex development of the Bombyx mori. While, by far the putative Fern gene has not beenidentified. In 2001, Ohbayashi cloned Bombyx mori homologue of the Drosophila dsx gene lying inthe terminal of the sex determination cascade. Similar to dsx, the Bmdsx transcript is alternativelyspliced in male and female to produce male and female specific mature mRNAs, which code formale-specific (BmDSX^M) and female-specific (BmDSX^F) proteins to control the terminal sexdifferentiation in Bombyx mori. Even though, the alternative splicing mechanism of the Bmdsxpre-mRNA is very different from that of the dsx. The default splicing form of dsx is male specific, infemale, the TRA/TRA-2 activates the female-specific splicing. While in Bombyx mori, the defaultsplicing form is female specific, some inhibitors repress the splicing of the Bmdsx exons 3 and 4 inmale. By far, the regulation cascade between the Fem and the Bmdsx is still unclear. To make clearthe sex determination cascade upstream Bmdsx, we analyzed the differentially expressed genes inmale and female embryos by microarray, identified a splicing factor of Bmdsx by gel-shift and LCMS/MS, and we cloned two candidates of Fem and performed transgenic analysis. We obtained thefollowing conclusions:1. Microarray-based analysis of differentially expressed genes in male and female embryos The sex of silkworm is determined at the early stage of embryo development. The genesinvolved in sex determination should be differentially expressed in male and female embryos at theearly stage of embryo development. To identify genes involved in Bombyx mori sex determination,we performed microarray to identify those genes. The silkworm strain sex-limited white egg wasused as material. The 48 h, 60 h, 72 h and 96 h male and female embryos after fertilization wereused for microarray analysis, and 4 h embryos after fertilization were used as control.There are a total of 4049 genes differentially expressed at least at one time point in male andfemale embryos by analysis. GO analysis showed their functions are mainly catalytic activity,nucleic acid binding, protein binding and transcription regulation, involved in metabolic process,development process, the regulation of biological process and pigmentation. Of them, 2432 genesare zygotic expressed. 1062 genes are caused by the differential degradation of material transcripts.At 48 h, 60 h, 72 h and 96 h, the number of genes up-regulated compared with 4 h after fertilizationis larger than down-regulated. Clustering of all these up-regulated genes showed the expressionpatterns of these genes are different between male and female. In female, most of these genes forma peak at 48 h, the expression level of these genes gradually decreased from 48 h to 72 h, thenincreased at 96 h. While in male, most of them increased from 48 h to 72 h, then decreased at 96 h.It may be due to the pigmentation difference between male and female eggs during the early stagesof embryo development.We found 81 alternative splicing isoforms belonging to 43 genes, occupying 21.8% of all thealternative splicing isoforms. They are mainly enzymes with catalytic activity and DNA or RNAbinding proteins. Besides, we also found 17 splicing factors with expression difference in male andfemale embryos. They include 4 hnRNPs, 4 snRNPs and other RNA binding proteins.2. The function research about Bmsxl and Bmrbp1sxl is a key regulator in the Drosophila sex determination cascade. Previous researcher clonedtwo splicing variants of Bmsxl, Bmsxl-PA and Bmsxl-PB. They found Bmsxl-PB is specificallyexpressed in the testis. In Drosophila, SXL bind to a poly(U) sequence near the splicing site toinhibit the splicing of this site. By this way, it regulates the sex-specific splicing of the downstreamgene tra. While there is also a poly(U) sequence near the 3′splicing site of Bmdsx intron 3. Wecame to the speculation whether the Bmsxl-PB can bind to this sequence to inhibit the splicing ofthe Bmdsx exon 3 and exon 4 in male. Whether the Bmsxl-PB is the key regulator of themale-specific splicing of Bmdsx pre-mRNA?To make clear these two problems, the gel-shift assay was performed using the poly(U) sequencewith the male and female nuclear extracts. The result showed BmSXL-PB doesn't bind to the poly(U) sequence near the 3′splicing site of the Bmdsx intron 3. RT-PCR was performed withcDNAs from the embryos of silkworm strain sex-limited white egg, the results showed theexpression of Bmsxl-PB is very weak, and showed no sex difference during the whole embryodevelopment. These results indicate that BmSXL-PB is not the key inhibitor of Bmdsx pre-mRNAsplicing.RBP1 is an important splicing factor involved in alternative splicing of the pre-mRNA ofDrosophila sex determining gene dsx. In this work, the Bombyx mori homologue of the rbp1 gene,Bmrbp1, was cloned. The pre-mRNA of Bmrbp1 gene is alternatively spliced to produce four maturemRNAs, named Bmrbp1-PA, Bmrbp1-PB, Bmrbp1-PC and Bmrbp1-PD, with nucleotide lengths of799 nt, 1,316 nt, 894 nt and 724 nt, coding for 142 aa, 159 aa, 91 aa and 117 aa, respectively.Bmrbp1-PA and Bmrbp1-PD contain a N terminal RNA recognization motif (RRM) and a Cterminal arginine/serine-rich domain, while Bmrbp1-PB and Bmrbp1-PC only share a RRM. Aminoacid sequence alignments showed that Bmrbp1 is conserved with its homologues in other insectsand with other SR family proteins. The RT-PCR results showed that Bmrbp1-PA was stronglyexpressed in all the examined tissues and development stages, but Bmrbp1-PB was weakly expressedin these tissues and stages. The expression of both Bmrb1-PA and Bmrbp1-PB showed no obvioussex difference. While the Bmrbp1-PC and Bmrbp1-PD were beyond detection by RT-PCR verylikely due to their tissue/stage specificity. These results suggested that Bombyx mori should be amember of SR family splicing factors, whether it is involved in the sex-specific splicing of Bmdsxpre-mRNA needs further research.3. Identification of splicing factor BmHrp28 upstream BmdsxSuzuki reported that the exon 4 of Bmdsx plays a critical role in the male-specific splicing ofBmdsx pre-mRNA. and there are three critical cis-elements on exon 4. BmPSI can bind to one ofthe cis-element (CE1) to regulate the splicing of Bmdsx. To identify other splicing factorsresponsible for male-specific splicing of Bmdsx pre-mRNA, we designed 5 probes on the exon 4 ofBmdsx to perform gel-shift and UV cross-linking assays. Results showed some nuclear factor canbind to the probe CE1+6. UV cross-linking assay showed this protein is about 28 kD. The shiftedband was incised from the gel and was identified by LC-MS/MS. After cloning of the coding region,the results showed it encodes 256 aa, homologous to the Drosophila Hrp48, a member of thehnRNP A/B family, named BmHrp28. It contains two RRMs at the N terminal and a glycine richdomain at the C terminal. Amino acid sequence alignment showed it is highly conserved at theRRM region with mammalian hnRNP A1 and Drosophila Hrp48. The BmHrp28 protein wasexpressed in Escherichia coli on pET50b vector and purified. The gel-shift and UV cross-linking results showed purified BmHrp28 protein can specifically bind to the CE1+6 probe. These resultsshowed that BmHrp28 can specifically bind to the exon 4 of Bmdsx, it might be one of the factorsinvolved in the male-specific splicing of Bmdsx pre-mRNA.4. Molecular cloning and function analysis of the Fem candidates BmWZF1 and BmWZF2In 2006, Mita referred to two zinc-finger domain containing genes on the W chromosome in theSeventh International Workshop on the Molecular Biology and Genetics of the Lepidoptera, andthese two genes have highly identical copies on the 25 linkage. According to this information, byanalysizing all the predicted zinc-finger domain containing genes, the ESTs and the genomeseqnence of the 25 linkage, we found a EST has obvious difference compared with the genomesequence. It might be transcribed from the W chromosome since the W chromosome is notsequenced in the silkworm genome shot-gun sequencing. Based on this EST, we cloned two mRNAmoleculars transcribed from the W chromosome, named BmWZF1-PA and BmWZF1-PB. Both ofthem contain a complete ORF, coding for 360 aa and 386 aa, respectively, speculating that thesetwo mRNAs come from the alternative splicing of the same gene. Domain prediction showed theyboth contain a proline rich domain and two CCCH zinc-finger domains. In general, the proline richdomains are involved in the protein-protein interaction. The CCCH domain can bind to both DNAand RNA, and involved in the splicing regulation of pre-mRNAs. Homologue search of BmWZF1in the NCBI showed that BmWZF1 is homologous to the mouse CHCR gene and Drosophilamuscleblind gene. The Drosophila Muscleblind protein regulates terminal muscle differentiation byregulating the alternative splicing of its targets. It implies that BmWZF1 may be a splicing factor,regulating alternative splicing of its downstream genes.Then we cloned another zinc-finger domain containing gene by extending to the 3′direction ofthe BmWZF1, named BmWZF2. It codes for a protein of 363 aa. containing 6 C₂H₂ zinc-fingerdomains. In general, the C₂H₂ domains are involved in DNA binding. Homologue search ofBmWZF2 in the NCBI showed that BmWZF2 is homologous to the transcription factors in otherspecies. BmWZF2 might code for a transcription factor regulating the transcription of its target.BLASTN search of BmWZF1 and BmWZF2 against the silkworm genome sequence showed thatboth genes have two copies on the autosome. The copies of BmWZF1 were named BmAZF1a andBmAZF1b, those of BmWZF2 were BmAZF2a and BmAZF2b. All the BmAZFla, BmAZF1b,BmAZF2a and BmAZF2b form a cluster on the 25 linkage. Of them, the transcription direction ofBmAZF1a and BmAZF2a is tail to tail.The autosome zinc-finger domain containing genes BmAZF1a, BmAZF1b, BmAZF2a andBmAZF2b were cloned based on the information of the genome sequence, predicted CDS and ESTs. The BmAZF1a is alternatively spliced to mainly produce two kinds of mRNAs, coding for 360 aaand 386 aa, respectively. Both two isoforms contain one proline rich domain and two CCCHdomains. Meanwhile, the BmAZF1a contains many SNP sites, which lead to the formation ofseveral different BmAZF1a isoforms. Sequence alignment showed the amino acid sequence ofBmWZF1 and BmAZF1a is identical. But the BmAZF1a codes for multiple isoforms because of theexistence of SNPs. It Implies that BmWZF1 and BmAZF1a may have the same function in Bombyxmori.BmAZF1b encodes 456 aa, like BmWZF1 and BmAZF1a, it also contains one proline rich domainand two CCCH domains, and comprises of 11 exons and 10 introns. Both nucleotide sequence andamino acid sequence alignments showed that BmAZF1b has a larger difference with the BmWZF1and BmAZF1a, suggesting the function of BmAZF1b was different from that of BmWZF1 andBmAZFla. The pre-mRNA of BmAZFla is also alternatively spliced to produce two kinds ofmature mRNAs, BmAZF2a-PA and BmAZF2a-PB. Domain prediction showed BmAZF2a-PBcontains 12 C₂H₂ domains, and BmAZF2a-PB contains 6 C₂H₂ domains. BmAZF2a-PA contains 12exons and 11 introns. BmAZF2b encodes 599 aa, containing 11 C₂H₂ domains. It contains 11 exonsand 10 introns. Homologue search of BmAZF2a and BmAZF2b in the NCBI showed that they arehighly conserved with transcription factors in other species. It implies that the proteins encoded bythese two genes are transcription factors, regulating the transcription of their downstream genes.Amino acid sequence alignment between BmAZF2a and BmAZF2b showed they are highlyidentical. The amino acid sequence of BmWZF2 is also highly identical with BmAZF2a andBmAZF2b, but it lacks part of N terminal sequence existed in BmAZF2a and BmAZF2b.The expression of each zinc-finger domain containing genes during embryo development weredetected by RT-PCR. The silkworm strain sex-limited white egg was used as material. The resultsshowed that BmWZF1 begins to be transcribed at the time about 36 h after fertilization. Moreover,it is just transcribed in female embryo while not in male, suggesting that BmWZF1 might be theFern gene predicted by previous researchers. The PCR results showed BmAZF1a, BmAZF1b,BmAZF2a and BmAZF2b are transcribed in the unfertilized eggs and during the development of theembryo, and showed no difference between male and female embryos.Then RNAi was performed to reudce the BmWZF1 expression. The results showed thearrangement of the eggs in female oviduct and the mucous gland are abnormal after RNAi. Thentransgenic analysis of BmWZF1 was performed. The results showed it has no affect on the sexwhen BmWZF1 was transferred to male or female, while the RT-PCR result showed the exogenousBmWZF1 was transcribed with a higher level. These results indicated that BmWZF1 hasn't changedthe sex of silkworm after transgene expression, it might not effect the sex of male and female if just BmWZF1 is transferred into the male or female.5. The speculation of Bombyx mori sex determination cascadeThe sex of Bombyx mori is determined by a predicted Fern gene, but the molecular structure ofFern hasn't been identified by now. The researches performed by Suzuki et al showed that Bmdsx isthe key gene lying in the terminal of the Bombyx mori sex determination cascade. But theregulatory cascade upstream Bmdsx seems to be different between Bombyx mori and Drosophila.Meanwhile, Suzuki identified the Bombyx mori homologue of Drosophila PSI gene, BmPSI, as asplicing regulator of Bmdsx pre-mRNA male-specific splicing. In Drosophila, PSI is the keyregulator of the tissues specific splicing of P element transposase. Therefore, it might be the keyregulator of the Bmdsx pre-mRNA. In this research, we identified another splicing factor(BmHrp28) of the Bmdsx pre-mRNA male-specific splicing. BmHrp28 specifically bind to theCE1+6 cis-element on the Bmdsx exon 4. We also cloned two zinc-finger domain containing genes,BmWZF1 and BmWZF2, from the W chromosome. Based on these results, we speculate that thesex determination cascade in Bombyx mori is as follows:...