家蚕双性基因dsx新拼接形式的鉴定及对A8腹节两性发育的调控
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摘要
家蚕作为鳞翅目昆虫典型代表,其性别调控机理的深刻阐释可为单养雄蚕和鳞翅目害虫防治提供理论依据。但遗憾的是,家蚕性别调控机制的研究还处于起步阶段,从上游性别决定初始信号,到中游性别决定关键基因,再到下游性别决定双性基因如何调控两性异型等等,都不十分清楚。基于比较基因组学和现代分子生物学实验方法鉴定家蚕性别决定信号通路中的重要基因,要么同源基因不存在,要么同源基因存在,但功能验证很可能不是性别决定信号通路上的关键基因,因此结果都不十分理想。到目前为止,唯一被证实参与家蚕性别调控的重要基因是Bmdsx。Bmdsx作为双性基因,处在性别调控信号通路的最下游,其雌雄差异表达既受中游家蚕性别决定未知关键基因的调控,同时又影响家蚕雌雄分化相关基因的表达,因此基于Bmdsx开展相应的研究工作,很可能是揭示家蚕性别决定和表型分化机制的突破口。据报道Bmdsx有6个外显子,经选择拼接产生2个雌拼接体和1个雄拼接体,但我们在对Bmdsx的雌雄表达模式进行调查时,发现该表达模式与理论推测的不一致。为了弄清Bmdsx的雌雄表达模式,并探索其对两性异型的调控机制,我们首先对Bmdsx可能存在的拼接体进行深入调查,并对其基因组结构和表达模式进行分析,然后对家蚕中可能存在几种具有潜在调控能力的Dsx蛋白进行分析,并用转基因技术对这些Dsx蛋白进行功能验证,最后探索Bmdsx对第8腹节(A8)两性发育的调控。获得的主要结果如下：
1. Bmdsx新拼接形式的鉴定
RT-PCR检测Bmdsx的表达情况时发现雌雄组织中有多条扩增条带,测序分析发现这些条带都是Bmdsx的不同拼接体。进一步3'RACE克隆发现Bmdsx确实有多种拼接形式存在,至此共鉴定到19种选择拼接体和10种反式拼接体。与基因组数据比对分析,发现Bmdsx共有9个外显子,其中外显子2n、3n和6n是新外显子,它们有可能是进化过程中新获得的外显子；第5内含子选择不同的5'或3'拼接位点,会导致不同拼接体中第3、4外显子的长度不同；进一步对其外显子内含子边界分析发现除6n外显子外,其它外显子5'端都是强的拼接位点,可能需要拼接抑制因子与之结合来调控雌雄差异拼接,只有6n外显子5'拼接受体位点是弱的拼接位点,类似Dmdsx第4外显子5'端,可能需要拼接增强因子与之结合。
2.反式拼接是Bmdsx新拼接方式
在分析拼接体Bmdsx-dsr1和Bmdsx-dsr2的结构时,发现这两个拼接体的3'端不是来自Bmdsx,前者的3'端与Bmdsx同位于第25号染色体上,且与Bmdsx紧密连锁；而后者的3'端位于第16号染色体上,其产生应该是反式拼接的结果。进一步克隆了Bmdsr1,发现它共有8个转录本,只有转录本Bmdsr1a可与BmdsxF1经反式拼接产生Bmdsx-dsr1反式拼接体。在调查Bmdsr1和Bmdsr2是否与其它Bmdsx拼接体发生反式拼接时,发现Bmdsx-dsr1的表达无法检测,可能是拼接噪音；而Bmdsx-dsr2在多个雌蚕组织,如表皮、脂肪体、气管和丝腺中都有表达,具有雌特异性特征,在两性生殖腺中虽都有表达,但进一步测序分析显示精巢中的两个反式拼接体是Bmdsr2与Bmdsx雌拼接体发生反式拼接的结果,且结构分析也显示Bmdsr2只与Bmdsx雌拼接体,如BmdsxF1、BmdsxF2、BmdsxF3发生反式拼接。细胞转染也暗示Bmdsr2与Bmdsx雌拼接体发生反式拼接后,改变了Bmdsx的3'UTR区,降低Bmdsx雌拼接体mRNA翻译蛋白的效率。因此,反式拼接是Bmdsx的新拼接方式,Bmdsr2可能是Bmdsx表达调控的新附加基因,通过反式拼接只影响雌蚕体细胞的发育。
3. Bmdsx的雌雄差异表达及其编码蛋白序列分析
在Bmdsx不同外显子上设计特异引物,检测不同拼接体或外显子在雌雄各组织中的拼接利用情况。结果显示：雄拼接体在雄蚕组织中特异表达,雌拼接体在雌蚕组织中高量表达,但在雄蚕组织中有极微弱表达。新外显子2n在雌雄各组织中都有拼接利用,是雌雄共用外显子；新外显子3n只在雌蚕组织中被拼接,是雌特异外显子；新外显子6n只在雄蚕的头、中肠、马氏管、气管、精巢及雌的卵巢中有拼接利用,具有一定的雄偏向性特征,进一步测序分析6n外显子的所有拼接条带,又发现另外3个新选择拼接体的存在,其中1个为雌拼接体,2个为雄拼接体,到此共在家蚕体内鉴定到22种选择拼接体；第3外显子3'端向外延伸15bp在雌蚕各组织中都存在,同第4外显子5'端向外延伸127bp,可能都受性别决定信号通路中游关键基因的调控。
为验证家蚕体内有多少种Dsx功能蛋白,进一步对22种选择拼接体可能编码多少种具有潜在调控能力的Dsx蛋白进行分析。发现Bmdsx的22种选择拼接体只可能编码5种雌Dsx蛋白、4种雄Dsx蛋白和4种未分类Dsx蛋白,存在多个选择拼接体共编码同一Dsx蛋白的情况,这些拼接体区别仅在3'UTR区,暗示选择利用不同的3'UTR可能是Bmdsx表达调控的一种方式。氨基酸序列分析显示上述13种Dsx蛋白中,只有4种雌Dsx蛋白、2种雄Dsx蛋白以及1种未分类Dsx蛋白可能对下游靶基因具有潜在的调控能力。4种雌Dsx蛋白具有不同C末端,可能影响Dsx蛋白对下游靶基因的激活或抑制效率。第2n外显子的获得导致在DBD/OD1和DBD/OD2之间插入27aa的多肽,但此多肽的插入并未改变两DNA结合结构域的完整性,可能只影响Dsx蛋白与下游靶基因启动子区的结合能力。
4. Bmdsx转基因品系的建立及对下游靶基因表达的影响
利用转基因技术对上述4种雌Dsx蛋白和2种雄Dsx蛋白进行功能验证。首先成功构建了6个转基因表达载体,经显微注射、荧光筛选、基因组反向PCR及Southern blot检测,建立了6个转基因品系,共获得12个不同插入位点、各发生1-2次转座事件的转基因。数据统计分析发现,6个转基因品系的G1代雌雄个体性别比例都未偏离1：1的性别比例。遗传判性分析显示所有G1代个体染色体组型与外部性表型一致,未发现性反转个体的存在。3'RACE及RT-PCR结果显示外源Bmdsx都被成功驱动表达。定量PCR分析显示：当在雄蚕中单一过表达雌Dsx蛋白时,可促进SP1和Vg在脂肪体中的表达,并降低PBP在触角中的表达水平；当在雌蚕中单一过表达雄Dsx蛋白时,可抑制SP1和Vg在脂肪体中的表达,并提高PBP在触角中的表达水平,表明具有潜在调控能力的4种雌Dsx蛋白和2种雄Dsx蛋白都能调控三个已知Bmdsx下游靶基因的表达,暗示4种雌Dsx蛋白和2种雄Dsx蛋白具有调控雌雄发育相关基因表达的能力,应该都参与家蚕体细胞的性别发育。
5. BmdsxM异位表达引起雌蛾第8腹节发育异常
家蚕雌雄蛾尾部形态呈现两性异型特征,即雌蛾尾部具有几丁质样锯齿板,附着于生殖节上,而雄蛾中对应部位则是被大量鳞毛覆盖的A8腹节。选取加秋、大09和301三个蚕品种,检测其雌雄蛾尾部Erk蛋白的磷酸化水平,发现不同蚕品种雌蛾尾部Erk蛋白磷酸化水平都显著低与雄蛾尾部,趋势都相同,暗示RTK信号通路的活性程度对家蚕幼虫第8腹节表皮处成组织细胞的两性发育有影响。
家蚕雌雄蛾交配时,只有雄蛾抱器钩住雌蛾锯齿板,交配才能顺利进行。在观察上述6个转基因品系时,发现ssd-6-2转基因雌蛾,从G1代到G6代,其所有雌蛾的交配行为出现异常,都交配不能,因此不能产生后代；细致观察发现ssd-6-2转基因雌蛾尾部生殖节发育异常,具有雄性化特征,锯齿板表面被大量鳞毛附着,暗示BmdsxMI在雌蚕中的异位表达,影响了雌蚕幼虫第8腹节表皮处成组织细胞向锯齿板方向的发育。检测四个蚕品种非转基因雌雄蛾尾部Erk蛋白的磷酸化水平差异,发现雄蛾尾部Erk蛋白磷酸化水平极显著高于雌蛾尾部；进一步检测Erk蛋白在转基因雌蛾尾部的磷酸化水平时,发现Erk蛋白的磷酸化水平被显著提高,趋向雄蛾,强烈暗示RTK信号通路的活性程度影响雌蛾A8腹节的正常发育。深入分析RTK信号通路相关基因在转基因雌蛾尾部的表达水平,发现EGFR信号通路上游配体基因spi,配体加工蛋白基因star和rho,调控二聚化受体内吞过程的三个基因cbI、mop和hrs,负反遗调节的一个基因kekl,以及下游5个细胞周期蛋白基因cyclinA、cyclinB、cyclinD、cyclinE和cyclinL的表达量都发生变化,都趋向于雄蛾,暗示EGFR信号通路的激活诱导了雌蛾A8腹节的雄性化发育。最后,我们也检测了Abd-B基因的表达水平,发现此基因在转基因雌蛾尾部的表达被诱导并趋向雄蛾尾部,暗示Hox基因也参与家蚕A8腹节的二向性发育。
The domesticated silkworm, Bombyx mori, is an important model of lepidopteran insects. The detailed decipherment of silkworm sex-determination mechanism is the theoretical basis of rearing male silkworm and lepidopteran pest insect control. Silkworm sex-determination cascade hasn't been described in detail. Problems about the primary element, the key gene, and how the doublesex gene regulates sexual dimorphism, haven't been decoded. Based on the methods of comparative genomics and mordern molecular biology, researchers have identified few candidate genes of silkworm sex determination, some of which were subsequently proved not to be the key genes of silkworm sex determination. Only the dsx gene, whose splicing should be regulated by a middlestream undiscovered key gene and which also modulates the expression of downstream sex differentiation genes, is confirmed to be the important gene of silkworm sex determination. Reportedly, Bmdsx has six exons, and it alternatively splice into two female-specific and one male-specific forms, which is different from the expression pattern indicated by our RT-PCR result. To clarify the expression of Bmdsx in two sexes and how Bmdsx controls the development of sexual dimorphism in silkworm, we cloned the possible splice forms of Bmdsx and analyzed its genomic organization and expression. Furthermore, we analysed the potential Dsx proteins and proved their regulation on three known downstream target genes by transgenic technique. Finally, we elucidated how Bmdsx patterns the sexually dimorphic development of eighth abdominal segment. The main results are as follows:
1. Novel splice forms of Bmdsx
RT-PCR and sequencing results showed that Bmdsx has more than three splice forms, which has also been proved by3'RACE result. By far, we have successfully cloned nineteen alternatively-spliced forms and ten trans-spliced variants. Nine exons and eight introns make up of the revised genomic structure of Bmdsx, and exon2n,3n, and6n are three novel exons, which might be gained during silkmoth evolution. The different choice of5'or3'splicing site of intron5leads to the different length of exons3and4in different splice forms. Only the5'acceptor splicing site of novel exon6n is the weak splicing site, which is the same as the5'acceptor splicing site of exon4in Dmdsx, and the splicing of exon6n may need the binding of some positive regulator. The rest of5'acceptor splicing sites of Bmdsx exons are canonical acceptor splicing sites, and their splicing may need the binding of some negative regulator.
2.Trans-splicing is a novel splicing method of Bmdsx
The3'ends of Bmdsx-dsrl and Bmdsx-dsr2didn't derive from the Bmdsx. The3' end of Bmdsx-dsrl positioned at chromosome25, and tightly linked with Bmdsx. The3' end of Bmdsx-dsr2located chromosome16, and the presence of Bmdsx-dsr2was the trans-splicing result. The existence and structure of eight transcripts showed that Bmdsrl was a novel gene, and Bmdsx-dsrl was indeed the trans-splicing product between BmdsxF1and Bmdsr1a. Expression analysis suggested that Bmdsx-dsrl was splicing noise, whereas Bmdsr2, which trans-spliced with Bmdsx to generate five variants in female head, fat body, trachea, silkgland, ovary and male testis, might participate in regulating the expression of Bmdsx. Sequencing analysis indicated that the two trans-spliced variants in testis was the trans-splicing products between Bmdsr2and BmdsxFs. As a result, Bmdsr2only trans-spliced with Bmdsx female splice forms. Cell transfection further indicated that trans-splicing between Bmdsr2and BmdsxFs changed the3'UTR of BmdsxF mRNA, and suppressed the translation of BmdsxF mRNA. Our findings suggests that trans-splicing was a novel splicing method of Bmdsx, and Bmdsr2, a novel additional gene of Bmdsx, only participated in the somatic development of female silkworm by trans-splicing with BmdsxFs.
3. Different expression of Bmdsx and the potential BmDsx in both sexes
Expression analysis indicated that male splice forms specifically expressed in males, and female splice forms largely expressed in females, but also expressed in males at a very low level. Novel exon2n was a common exon and obtained in all tissues of two sexes. Novel exon3n was a female-specific exon and only spliced in a female-specific manner. Novel exon6n was only spliced in male head, midgut, malpighian tubule, trachea, testis and female ovary, and its splicing had male-biased characteristics. Sequencing all the splice bands of exon6n presented one novel female and two novel male splice forms. By far, we have cloned twenty-two alternatively-spliced forms of Bmdsx in all. Expression analysis also showed that the stetch of15bp at the3'end of exon3was common in female tissues, which was same as the situation of the stetch of127bp at the5'end of exon4.
ClastalW alignment of BmDsx indicated that Bmdsx might decode five BmDsxF, four BmDsxM, four unclassified BmDsx. One protein was decoded by several splice forms, and the3'UTRs of these splice forms were different, suggesting that the choice of different3'UTRs might be a regulatory way of the expression of Bmdsx. Further analysis of the structure and key residues of the thirteen proteins found that four BmDsxF, two BmDsxM, one unclassified BmDsx, which had potential regulatory function on downstream target genes, might exist in silkworm. Four BmDsxF used different C-termini, which might affect the activation or repression of DsxF on downstream target genes. The insertion of27aa between DBD/OD1and DBD/OD2did not change the functional domains of BmDsx, and might affect the binding ability of BmDsx on the promoters of downstream target genes.
4. Ectopic expression of Bmdsx affects the expression of downstream targets
We subsequently validated the regulation of four BmDsxF and two BmDsxM on target genes. By microinjection, fluorescence choice, genomic inverse-PCR and Southern blot detection, we successfully constructed six transgenic strains including twelve transgenes, which have different insertion sites and transpositions from one to two. Statistic analysis indicated that the sexual ratios of G1generation from six transgenic strains conformed to normal sexual ratio1:1. Moreover, the chromosomal buildup of all G1individuals also corresponded with external sex phenotype, and no sex reversal had been found. However,3'RACE and RT-PCR confirmed the expression of exogenous Bmdsx. qPCR results also showed that the ectopic expression of BmDsxFs in males could induce the expression of SP1and Vg in fat body, and suppress the expression of PBP in antenna. Furthermore, the ectopic xpression of BmDsxMs in females could repress the expression of SP1and Vg in fat body, and induce the expression of PBP in antenna. All these results suggest that four BmDsxFs and two BmDsxMs could regulate the expression of three known downstream target genes, and they have the regulatory function on the expression of sexually development-related genes and shoud participate in the sexual development of silkworm soma.
5. Ectopic expression of BmdsxM1causes the abnormal development of female eighth abdominal segment.
A visible dimorphism exists in posterior abdomen of adult, that is:the naked chitin plate, a derivative of female gonosomite, only forms in the posterior abdomen of females but not in males, and its counterpart in males is the eighth abdominal segment (A8) with scaly hair. Three silkworm strains, Jia Qiu, Da09and301, had been chosen to detect the difference of phosphorylation level of Erk in posterior abdomens of two-sex adults, and the result showed that the phosphorylation level of Erk in the female posterior abdomens of three strains was all significantly lower than that in male posterior abdomen, indicating that RTK signaling activity had a key role in the development of histoblasts in the epidermis of eighth abdominal segment of larvae to the chitin plate of adult in females.
When the males courts the females, the double harpagones in male external genitalia must grasp the chitin plate, a derivative of gonosomite in female posterior abdomen. During the observation of the phenotype of transgenic strains, we obtained a fundamental line ssd-6-2. Observation of G1to G6generation of ssd-6-2indicated that all the females couldn't copulate with males and thereby produced no fertile offspring. The gonosomite of all transgenic females developed abnormally, having scaly-haired chitin plate attached, which affected the genital coupling of transgenic females with wild-type males, indicating that the ectopic expression of BmDsxM1in females affected the development of histoblasts in the epidermis of eighth abdominal segment of larvae to chitin plate. Subsequently, we investigated whether the RTK signaling activity was related with the abnormality of gonosomite of transgenic females. The results showed that the phosphorylation level of Erk in the posterior abdomen of wild-type males was markedly higher than that in wild-type females, and the phosphorylation level of Erk in the posterior abdomen of transgenic females was significantly elevated, which further mightily inplied that the lower activity of RTK signalings correlated with the development of female chitin plate. To survey how the ectopic expression of BmDsxM1in females induced the activation of RTK signalings, we checked the expression level of these genes related to RTK signalings by qPCR. The results showed that all the expression levels of the ligand gene of EGFR signaling, spi, the regulatory genes of Spi processing, star and rho, the regulatory genes of endocytsis of dimeric EGF receptors, cbl, mop and hrs, the gene participating in negative feedback loop of EGFR signaling, kekl, and the downstream cyclin genes, cyclinA, cyclinB, cyclinD, cyclinE and cyclinL, were markedly induced and virilescent, which indicated that the induction upstream, middlestream and downstream of EGFR signaling and the increase of EGFR signaling activity triggered the female chitin plate masculinized. Finally, we also detected the expression of Abd-B gene in the posterior abdomens of transgenic and wild-type females, and found that the expresion level of Abd-B gene was also induced and virilescent, which suggested that the Hox gene also participated in the dimorphic development of histoblasts in the eighth abdominal segment in two sexes.
1. Bmdsx新拼接形式的鉴定
RT-PCR检测Bmdsx的表达情况时发现雌雄组织中有多条扩增条带,测序分析发现这些条带都是Bmdsx的不同拼接体。进一步3'RACE克隆发现Bmdsx确实有多种拼接形式存在,至此共鉴定到19种选择拼接体和10种反式拼接体。与基因组数据比对分析,发现Bmdsx共有9个外显子,其中外显子2n、3n和6n是新外显子,它们有可能是进化过程中新获得的外显子；第5内含子选择不同的5'或3'拼接位点,会导致不同拼接体中第3、4外显子的长度不同；进一步对其外显子内含子边界分析发现除6n外显子外,其它外显子5'端都是强的拼接位点,可能需要拼接抑制因子与之结合来调控雌雄差异拼接,只有6n外显子5'拼接受体位点是弱的拼接位点,类似Dmdsx第4外显子5'端,可能需要拼接增强因子与之结合。
2.反式拼接是Bmdsx新拼接方式
在分析拼接体Bmdsx-dsr1和Bmdsx-dsr2的结构时,发现这两个拼接体的3'端不是来自Bmdsx,前者的3'端与Bmdsx同位于第25号染色体上,且与Bmdsx紧密连锁；而后者的3'端位于第16号染色体上,其产生应该是反式拼接的结果。进一步克隆了Bmdsr1,发现它共有8个转录本,只有转录本Bmdsr1a可与BmdsxF1经反式拼接产生Bmdsx-dsr1反式拼接体。在调查Bmdsr1和Bmdsr2是否与其它Bmdsx拼接体发生反式拼接时,发现Bmdsx-dsr1的表达无法检测,可能是拼接噪音；而Bmdsx-dsr2在多个雌蚕组织,如表皮、脂肪体、气管和丝腺中都有表达,具有雌特异性特征,在两性生殖腺中虽都有表达,但进一步测序分析显示精巢中的两个反式拼接体是Bmdsr2与Bmdsx雌拼接体发生反式拼接的结果,且结构分析也显示Bmdsr2只与Bmdsx雌拼接体,如BmdsxF1、BmdsxF2、BmdsxF3发生反式拼接。细胞转染也暗示Bmdsr2与Bmdsx雌拼接体发生反式拼接后,改变了Bmdsx的3'UTR区,降低Bmdsx雌拼接体mRNA翻译蛋白的效率。因此,反式拼接是Bmdsx的新拼接方式,Bmdsr2可能是Bmdsx表达调控的新附加基因,通过反式拼接只影响雌蚕体细胞的发育。
3. Bmdsx的雌雄差异表达及其编码蛋白序列分析
在Bmdsx不同外显子上设计特异引物,检测不同拼接体或外显子在雌雄各组织中的拼接利用情况。结果显示：雄拼接体在雄蚕组织中特异表达,雌拼接体在雌蚕组织中高量表达,但在雄蚕组织中有极微弱表达。新外显子2n在雌雄各组织中都有拼接利用,是雌雄共用外显子；新外显子3n只在雌蚕组织中被拼接,是雌特异外显子；新外显子6n只在雄蚕的头、中肠、马氏管、气管、精巢及雌的卵巢中有拼接利用,具有一定的雄偏向性特征,进一步测序分析6n外显子的所有拼接条带,又发现另外3个新选择拼接体的存在,其中1个为雌拼接体,2个为雄拼接体,到此共在家蚕体内鉴定到22种选择拼接体；第3外显子3'端向外延伸15bp在雌蚕各组织中都存在,同第4外显子5'端向外延伸127bp,可能都受性别决定信号通路中游关键基因的调控。
为验证家蚕体内有多少种Dsx功能蛋白,进一步对22种选择拼接体可能编码多少种具有潜在调控能力的Dsx蛋白进行分析。发现Bmdsx的22种选择拼接体只可能编码5种雌Dsx蛋白、4种雄Dsx蛋白和4种未分类Dsx蛋白,存在多个选择拼接体共编码同一Dsx蛋白的情况,这些拼接体区别仅在3'UTR区,暗示选择利用不同的3'UTR可能是Bmdsx表达调控的一种方式。氨基酸序列分析显示上述13种Dsx蛋白中,只有4种雌Dsx蛋白、2种雄Dsx蛋白以及1种未分类Dsx蛋白可能对下游靶基因具有潜在的调控能力。4种雌Dsx蛋白具有不同C末端,可能影响Dsx蛋白对下游靶基因的激活或抑制效率。第2n外显子的获得导致在DBD/OD1和DBD/OD2之间插入27aa的多肽,但此多肽的插入并未改变两DNA结合结构域的完整性,可能只影响Dsx蛋白与下游靶基因启动子区的结合能力。
4. Bmdsx转基因品系的建立及对下游靶基因表达的影响
利用转基因技术对上述4种雌Dsx蛋白和2种雄Dsx蛋白进行功能验证。首先成功构建了6个转基因表达载体,经显微注射、荧光筛选、基因组反向PCR及Southern blot检测,建立了6个转基因品系,共获得12个不同插入位点、各发生1-2次转座事件的转基因。数据统计分析发现,6个转基因品系的G1代雌雄个体性别比例都未偏离1：1的性别比例。遗传判性分析显示所有G1代个体染色体组型与外部性表型一致,未发现性反转个体的存在。3'RACE及RT-PCR结果显示外源Bmdsx都被成功驱动表达。定量PCR分析显示：当在雄蚕中单一过表达雌Dsx蛋白时,可促进SP1和Vg在脂肪体中的表达,并降低PBP在触角中的表达水平；当在雌蚕中单一过表达雄Dsx蛋白时,可抑制SP1和Vg在脂肪体中的表达,并提高PBP在触角中的表达水平,表明具有潜在调控能力的4种雌Dsx蛋白和2种雄Dsx蛋白都能调控三个已知Bmdsx下游靶基因的表达,暗示4种雌Dsx蛋白和2种雄Dsx蛋白具有调控雌雄发育相关基因表达的能力,应该都参与家蚕体细胞的性别发育。
5. BmdsxM异位表达引起雌蛾第8腹节发育异常
家蚕雌雄蛾尾部形态呈现两性异型特征,即雌蛾尾部具有几丁质样锯齿板,附着于生殖节上,而雄蛾中对应部位则是被大量鳞毛覆盖的A8腹节。选取加秋、大09和301三个蚕品种,检测其雌雄蛾尾部Erk蛋白的磷酸化水平,发现不同蚕品种雌蛾尾部Erk蛋白磷酸化水平都显著低与雄蛾尾部,趋势都相同,暗示RTK信号通路的活性程度对家蚕幼虫第8腹节表皮处成组织细胞的两性发育有影响。
家蚕雌雄蛾交配时,只有雄蛾抱器钩住雌蛾锯齿板,交配才能顺利进行。在观察上述6个转基因品系时,发现ssd-6-2转基因雌蛾,从G1代到G6代,其所有雌蛾的交配行为出现异常,都交配不能,因此不能产生后代；细致观察发现ssd-6-2转基因雌蛾尾部生殖节发育异常,具有雄性化特征,锯齿板表面被大量鳞毛附着,暗示BmdsxMI在雌蚕中的异位表达,影响了雌蚕幼虫第8腹节表皮处成组织细胞向锯齿板方向的发育。检测四个蚕品种非转基因雌雄蛾尾部Erk蛋白的磷酸化水平差异,发现雄蛾尾部Erk蛋白磷酸化水平极显著高于雌蛾尾部；进一步检测Erk蛋白在转基因雌蛾尾部的磷酸化水平时,发现Erk蛋白的磷酸化水平被显著提高,趋向雄蛾,强烈暗示RTK信号通路的活性程度影响雌蛾A8腹节的正常发育。深入分析RTK信号通路相关基因在转基因雌蛾尾部的表达水平,发现EGFR信号通路上游配体基因spi,配体加工蛋白基因star和rho,调控二聚化受体内吞过程的三个基因cbI、mop和hrs,负反遗调节的一个基因kekl,以及下游5个细胞周期蛋白基因cyclinA、cyclinB、cyclinD、cyclinE和cyclinL的表达量都发生变化,都趋向于雄蛾,暗示EGFR信号通路的激活诱导了雌蛾A8腹节的雄性化发育。最后,我们也检测了Abd-B基因的表达水平,发现此基因在转基因雌蛾尾部的表达被诱导并趋向雄蛾尾部,暗示Hox基因也参与家蚕A8腹节的二向性发育。
The domesticated silkworm, Bombyx mori, is an important model of lepidopteran insects. The detailed decipherment of silkworm sex-determination mechanism is the theoretical basis of rearing male silkworm and lepidopteran pest insect control. Silkworm sex-determination cascade hasn't been described in detail. Problems about the primary element, the key gene, and how the doublesex gene regulates sexual dimorphism, haven't been decoded. Based on the methods of comparative genomics and mordern molecular biology, researchers have identified few candidate genes of silkworm sex determination, some of which were subsequently proved not to be the key genes of silkworm sex determination. Only the dsx gene, whose splicing should be regulated by a middlestream undiscovered key gene and which also modulates the expression of downstream sex differentiation genes, is confirmed to be the important gene of silkworm sex determination. Reportedly, Bmdsx has six exons, and it alternatively splice into two female-specific and one male-specific forms, which is different from the expression pattern indicated by our RT-PCR result. To clarify the expression of Bmdsx in two sexes and how Bmdsx controls the development of sexual dimorphism in silkworm, we cloned the possible splice forms of Bmdsx and analyzed its genomic organization and expression. Furthermore, we analysed the potential Dsx proteins and proved their regulation on three known downstream target genes by transgenic technique. Finally, we elucidated how Bmdsx patterns the sexually dimorphic development of eighth abdominal segment. The main results are as follows:
1. Novel splice forms of Bmdsx
RT-PCR and sequencing results showed that Bmdsx has more than three splice forms, which has also been proved by3'RACE result. By far, we have successfully cloned nineteen alternatively-spliced forms and ten trans-spliced variants. Nine exons and eight introns make up of the revised genomic structure of Bmdsx, and exon2n,3n, and6n are three novel exons, which might be gained during silkmoth evolution. The different choice of5'or3'splicing site of intron5leads to the different length of exons3and4in different splice forms. Only the5'acceptor splicing site of novel exon6n is the weak splicing site, which is the same as the5'acceptor splicing site of exon4in Dmdsx, and the splicing of exon6n may need the binding of some positive regulator. The rest of5'acceptor splicing sites of Bmdsx exons are canonical acceptor splicing sites, and their splicing may need the binding of some negative regulator.
2.Trans-splicing is a novel splicing method of Bmdsx
The3'ends of Bmdsx-dsrl and Bmdsx-dsr2didn't derive from the Bmdsx. The3' end of Bmdsx-dsrl positioned at chromosome25, and tightly linked with Bmdsx. The3' end of Bmdsx-dsr2located chromosome16, and the presence of Bmdsx-dsr2was the trans-splicing result. The existence and structure of eight transcripts showed that Bmdsrl was a novel gene, and Bmdsx-dsrl was indeed the trans-splicing product between BmdsxF1and Bmdsr1a. Expression analysis suggested that Bmdsx-dsrl was splicing noise, whereas Bmdsr2, which trans-spliced with Bmdsx to generate five variants in female head, fat body, trachea, silkgland, ovary and male testis, might participate in regulating the expression of Bmdsx. Sequencing analysis indicated that the two trans-spliced variants in testis was the trans-splicing products between Bmdsr2and BmdsxFs. As a result, Bmdsr2only trans-spliced with Bmdsx female splice forms. Cell transfection further indicated that trans-splicing between Bmdsr2and BmdsxFs changed the3'UTR of BmdsxF mRNA, and suppressed the translation of BmdsxF mRNA. Our findings suggests that trans-splicing was a novel splicing method of Bmdsx, and Bmdsr2, a novel additional gene of Bmdsx, only participated in the somatic development of female silkworm by trans-splicing with BmdsxFs.
3. Different expression of Bmdsx and the potential BmDsx in both sexes
Expression analysis indicated that male splice forms specifically expressed in males, and female splice forms largely expressed in females, but also expressed in males at a very low level. Novel exon2n was a common exon and obtained in all tissues of two sexes. Novel exon3n was a female-specific exon and only spliced in a female-specific manner. Novel exon6n was only spliced in male head, midgut, malpighian tubule, trachea, testis and female ovary, and its splicing had male-biased characteristics. Sequencing all the splice bands of exon6n presented one novel female and two novel male splice forms. By far, we have cloned twenty-two alternatively-spliced forms of Bmdsx in all. Expression analysis also showed that the stetch of15bp at the3'end of exon3was common in female tissues, which was same as the situation of the stetch of127bp at the5'end of exon4.
ClastalW alignment of BmDsx indicated that Bmdsx might decode five BmDsxF, four BmDsxM, four unclassified BmDsx. One protein was decoded by several splice forms, and the3'UTRs of these splice forms were different, suggesting that the choice of different3'UTRs might be a regulatory way of the expression of Bmdsx. Further analysis of the structure and key residues of the thirteen proteins found that four BmDsxF, two BmDsxM, one unclassified BmDsx, which had potential regulatory function on downstream target genes, might exist in silkworm. Four BmDsxF used different C-termini, which might affect the activation or repression of DsxF on downstream target genes. The insertion of27aa between DBD/OD1and DBD/OD2did not change the functional domains of BmDsx, and might affect the binding ability of BmDsx on the promoters of downstream target genes.
4. Ectopic expression of Bmdsx affects the expression of downstream targets
We subsequently validated the regulation of four BmDsxF and two BmDsxM on target genes. By microinjection, fluorescence choice, genomic inverse-PCR and Southern blot detection, we successfully constructed six transgenic strains including twelve transgenes, which have different insertion sites and transpositions from one to two. Statistic analysis indicated that the sexual ratios of G1generation from six transgenic strains conformed to normal sexual ratio1:1. Moreover, the chromosomal buildup of all G1individuals also corresponded with external sex phenotype, and no sex reversal had been found. However,3'RACE and RT-PCR confirmed the expression of exogenous Bmdsx. qPCR results also showed that the ectopic expression of BmDsxFs in males could induce the expression of SP1and Vg in fat body, and suppress the expression of PBP in antenna. Furthermore, the ectopic xpression of BmDsxMs in females could repress the expression of SP1and Vg in fat body, and induce the expression of PBP in antenna. All these results suggest that four BmDsxFs and two BmDsxMs could regulate the expression of three known downstream target genes, and they have the regulatory function on the expression of sexually development-related genes and shoud participate in the sexual development of silkworm soma.
5. Ectopic expression of BmdsxM1causes the abnormal development of female eighth abdominal segment.
A visible dimorphism exists in posterior abdomen of adult, that is:the naked chitin plate, a derivative of female gonosomite, only forms in the posterior abdomen of females but not in males, and its counterpart in males is the eighth abdominal segment (A8) with scaly hair. Three silkworm strains, Jia Qiu, Da09and301, had been chosen to detect the difference of phosphorylation level of Erk in posterior abdomens of two-sex adults, and the result showed that the phosphorylation level of Erk in the female posterior abdomens of three strains was all significantly lower than that in male posterior abdomen, indicating that RTK signaling activity had a key role in the development of histoblasts in the epidermis of eighth abdominal segment of larvae to the chitin plate of adult in females.
When the males courts the females, the double harpagones in male external genitalia must grasp the chitin plate, a derivative of gonosomite in female posterior abdomen. During the observation of the phenotype of transgenic strains, we obtained a fundamental line ssd-6-2. Observation of G1to G6generation of ssd-6-2indicated that all the females couldn't copulate with males and thereby produced no fertile offspring. The gonosomite of all transgenic females developed abnormally, having scaly-haired chitin plate attached, which affected the genital coupling of transgenic females with wild-type males, indicating that the ectopic expression of BmDsxM1in females affected the development of histoblasts in the epidermis of eighth abdominal segment of larvae to chitin plate. Subsequently, we investigated whether the RTK signaling activity was related with the abnormality of gonosomite of transgenic females. The results showed that the phosphorylation level of Erk in the posterior abdomen of wild-type males was markedly higher than that in wild-type females, and the phosphorylation level of Erk in the posterior abdomen of transgenic females was significantly elevated, which further mightily inplied that the lower activity of RTK signalings correlated with the development of female chitin plate. To survey how the ectopic expression of BmDsxM1in females induced the activation of RTK signalings, we checked the expression level of these genes related to RTK signalings by qPCR. The results showed that all the expression levels of the ligand gene of EGFR signaling, spi, the regulatory genes of Spi processing, star and rho, the regulatory genes of endocytsis of dimeric EGF receptors, cbl, mop and hrs, the gene participating in negative feedback loop of EGFR signaling, kekl, and the downstream cyclin genes, cyclinA, cyclinB, cyclinD, cyclinE and cyclinL, were markedly induced and virilescent, which indicated that the induction upstream, middlestream and downstream of EGFR signaling and the increase of EGFR signaling activity triggered the female chitin plate masculinized. Finally, we also detected the expression of Abd-B gene in the posterior abdomens of transgenic and wild-type females, and found that the expresion level of Abd-B gene was also induced and virilescent, which suggested that the Hox gene also participated in the dimorphic development of histoblasts in the eighth abdominal segment in two sexes.
引文
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