Characterization And Functional Analysis Of Sox Genes In The Silkworm, Bombyx Mori

Sox genes encode a class of transcription factor family with a high sequence similarity to sex-determining gene Sry (sex determining region of Y chromosome) in animals. This gene family is characterized by the presence of the conserved high-mobility group (HMG) box. Recently, studies on mouse, fruit fly and other model organisms have shown that Sox genes mainly involve in regulating sex differentiation and gonad development, early embryonic development, nervous system development and the formation of cartilage and other tissues and organs.The silkworm, Bombyx mori, is an economically important insect, and is also an excellent model for the research of Lepidoptera. Research projects about embryogenesis, sex determination and the formation of some tissues (such as silk gland, gonads and fat body) in silkworm are becoming as hot areas. Sox gene family should be regarded as the gene targets for investigating these biological stories in silkworm, and related progresses not only can be used to improve silkworm economic traits, but also provide a theoretical basis and key targets for biological control of lepidopteran pests.Based on the research achievements on Sox genes in fruit fly and other species, we used the fine genome sequence map and microarray data of genome-wide gene expression for silkworm to perform a genome-wide identification of Sox genes in silkworm, profile the spatio-temporal expression of Sox genes, and analyze the functions of BmSoxE gene at the cellular level and individual level. The main results obtained are as follows:1. Identification and expression profiling of Sox genes in silkwormBy BLASTP searches against the GLEAN gene collection predicted from silkworm genome sequences using the conserved SOX HMG box domain sequence as query, we identified nine Sox genes in silkworm, which include five members in group B, and one member each in groups C, D, E, and F. All Sox genes in silkworm can be mapped on the different chromosomes by silkworm SNP linkage map. The five group B genes, BmSox21a, BmSoxB3, BmDichaete, BmSox21b, and BmSoxNeuro, locate on chromosome 4, in which four genes, BmSox21a, BmSoxB3, BmDichaete, and BmSox21b, are tandemly organized in a 268-kb region on nscaf2589 scaffold. The other Sox genes were distributed separately on different chromosomes. We further compared the intron number in the HMG boxes of all Sox genes and found that the HMG boxes in five silkworm Sox genes, BmSoxC, BmSoxD, and three group B members including BmSox21a, BmSoxB3 and BmDichaete, had no introns. Two introns were checked out in the HMG box of BmSoxNeuro of group B, and one in BmSoxE.We further examined the SNPs in the coding sequences of silkworm Sox genes using 40 genomes for geographically different domesticated and wild silkworms. The numbers of SNPs in the Sox genes ranged from 4 to 32 in the domesticated silkworm group, and from 5 to 26 in wild silkworm group. The number of SNP per Sox gene was similar between the domesticated and wild groups. The SNP numbers in each Sox gene in the 40 silkworm varieties were also remarkably distinct, ranging from 6 to 33. Interestingly, we found that SNPs tended to occur frequently in SoxB3, Dichaete, SoxC, and SoxD during silkworm domestication. Moreover, some SNPs in six Sox genes, Sox21a, Sox21b, SoxB3, Dichaete, SoxE, and SoxF, could cause nonsynonymous substitutions. However, only one SNP in Sox21a, SoxE, or SoxF was located in the HMG box. In addition, using each Bombyx Sox gene sequence containing exons and introns to estimate the positive selection patterns of Sox genes during silkworm domestication by software package PAML, we found that SoxB3 and SoxD were significantly ascertained to be evolving under positive selection.Based on multiple sequence alignments of conserved HMG-box sequences of Sox proteins in silkworm and other surveyed insects, we constructed a phylogenetic tree of these Sox genes. All members from each of three subfamilies, SoxC, SoxD, and SoxF, grouped well. However, six members, Dichaete, Sox21a, Sox21b, SoxB1, SoxB2, and SoxB3 in B group and two members, SoxEl and SoxE2 in E group showed an irregular phylogeny, respectively. These above observations suggested that SoxC, SoxD, and SoxF in insects originated before the radiation of insects, and groups B and E evolved through gene duplication after the radiation of insects.The time-course expression patters of silkworm Sox genes during embryogenesis throughout just after egg lay (AEL) to just after hatching were profiled by RT-PCR. As a result, six Sox genes, BmSox21a, BmSox21b, BmDichaete, BmSoxNeuro, BmSoxC, and BmSoxF, were broadly and differently expressed at all selected time points during embryogenesis. Three Sox genes were expressed in the later embryo. BmSox21a expression was highly upregulated at 120 hours AEL. BmSoxB3 expression appeared from 144 hours AEL to hatching. BmSoxE was detected from 72 hours AEL to 216 hours AEL. These results indicate important roles of these three Sox genes in the formation of organs during later embryonic development. Based on the genome-wide expression profiles from silkworm multiple larval tissues on fifth-instar day 3, we found that only BmSoxE was exclusively enriched in ovary and testis, while the expression of other Sox genes were not detected in any tissues, suggesting BmSoxE is also involved in silkworm gonad development. In addition, microarray data for genome-wide expression during silkworm metamorphosis showed that four Sox genes, BmSoxB3, BmSoxNeuro, BmSoxC, and BmSoxE, exhibited a distinct expression pattern during metamorphosis. BmSoxB3 was expressed in male pupae at 216 hours after wandering and in male adults. BmSoxC was detected in male pupae at W60 (just after pupation). BmSoxNeuro and BmSoxE were similarly expressed in male pupae at 96 hours after wandering,144 hours after wandering,168 hours after wandering,216 hours after wandering,240 hours after wandering, and in male adults. BmSoxNeuro was also expressed in both male pupae at 36 hours after wandering,48 hours after wandering, and 60 hours after wandering and in female pupae at 48 hours after wandering and 60 hours after wandering. In summary, Sox genes were mainly expressed during the later stages of metamorphosis and may be associated with the transition from pupa to adult.2. Full-length cDNA cloning and expression profiling of BmSoxE geneThe full-length cDNA of BmSoxE gene was cloned by RACE approach. It contains 1398 bp encoding for a protein with 222 amino acid residues. Bioinformatic analysis showed that BmSoxE protein sequence has a typical HMG-box domain and several phosphorylation sites.We further investigated the spatio-temporal expression of BmSoxE genes during silkworm development using RT-PCR. The results showed that BmSoxE was highly expressed in the gonad of larvae on day 3 of fifth instar and were also continually detected from just hatching to adult. During silkworm metamorphosis, BmSoxE was differently expressed during gonad development. In particular, around prepupa from day 6 of fifth instar to 48 hours after wandering, BmSoxE was highly expressed in ovary but downregulated in testis. We speculated that BmSoxE may play roles from early embryo and exhibit different functions in gonad around prepupa. BmSoxE protein antibody-based immunohistochemical staining in adult testis showed that BmSoxE was localized in nucleus.3. Cellular functions of BmSoxE gene in the silkworm BmN4 cellsVenus fluorescent protein fusion plasmid Venus-BmSoxE and the control plasmids Venus-Dest were transfected into BmN4 cells. After 72 hours hoechst solution for staining nuclear DNA was added into the medium. Then the distribution of BmSoxE in cells was observed under a fluorescence microscope. The results showed that Venus fusion BmSoxE gene was expressed in the nucleus.We synthesized dsBmSoxE and dsEGFP, the dsRNA for the RNAi of the target gene BmSoxE and control gene EGFP, and added them into the different culture medium and then cultured BmN4-SID1 cells at 27℃. After 7 days we collected the cells to analyse the effects of BmSoxE gene RNAi. First, we observed that BmSoxE gene expression was significantly reduced after BmSoxE gene RNAi and could hardly be detected.Importantly, it showed that the cell numbers of BmN4-SID1 cells after BmSoxE gene RNAi were significantly less than those treated with dsEGFP. The cell proliferation curve showed that BmSoxE RNAi can seriously delay cell proliferation. A flow cytometry assay confirmed that, compared with the control treated with dsEGFP, the cell numbers of BmN4-SIDl cells after BmSoxE gene RNAi were significantly reduced in G2 phase and M phase, and most cells stayed in G1/S phase. This indicates that BmSoxE gene may play an important role during the conversion from G1 to S.Microarray analysis of gene expression changes in BmN4-SID1 cells after BmSoxE gene RNAi showed that, after dsBmSoxE treatment, the expression of 371 genes were changed, of which 142 genes were down-regulated and 227 genes were upregulated. Among these 371 genes,233 genes can be functionally annotated and the binding sites of the Sox protein conserved HMG box can be found in 157 genes upstream 2000 bp region of their transcription start sites. GO function analysis suggested the differentially expressed genes after BmSoxE gene RNAi mainly had catalytic and binding activity, and involved in development, metabolism, coloring and other biological processes. Most of differentially expressed genes with specific GO categories or with ten times change are related to cell fate determination including cell proliferation, cell death, cell differentiation, and so on.In addition, we also observed that the overexpression of BmSoxE in BmN4 cells can weakly promote cell proliferation. Fused Flag-based western blotting showed that the transfected BmSoxE gene can be expressed in BmN4 cells at protein level.4. Functional analysis of BmSoxE gene in the silkworm individualsGiven that BmSoxE gene was highly expressed in testis, we first used dsRNA mediated RNAi to investigate the potential functions of BmSoxE gene in male silkworm in vivo. Using the different dosage of dsRNA of target gene BmSoxE (dsBmSoxE) and control gene Red (dsRed) we injected into prepupae and then pupa at 2 days after pupation. The result showed that the injection of both 40μg per individual and 60μg per invidual, the silkworm prepupae can normally pupate and develop into adult (moth), and then mate with normal female moths. However, the eggs form control group at 48 hours after egg lay can normally change colors, while the eggs from 3 females mating with male moths from experimental group (60μg dsBmSoxE per male), including dsSoxE60-18, dsSoxE60-21 and dsSoxE60-23, failed to normally change color and finally failed to hatch, which were actually became into unfertilized eggs. After an anatomical analysis of these three male moths, we found that the testis and their attached tissues appeared some different varieties of developmental abnormalities. The left spermaduct and ejaculatary vesicle of dsSoxE60-18 male were connected directly with the external genitalia. The seminal vesicle of dsSoxE60-21 male expanded to vesicle-like. The accesory gland of dsSoxE60-23 male was abnormal. Its base and region near the end were hided by the seminal vesicle and formed a ring in the middle, so the exposed part of the entire gland was divided into short end and ring-like structures. These results showed that BmSoxE gene may involve in gonad development of the silkworm.