【作者】 赵敏；

【导师】 夏庆友；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 性别决定是生物界基本的科学问题之一。家蚕属于鳞翅目昆虫,其性别决定机制与目前研究的最为清楚的模式昆虫果蝇相比,既有共同性,也有特殊性,因此家蚕性别决定机制的研究可为我们更好地理解昆虫性别决定提供一个新的模式。另外,其研究在产业上也具有重要的应用价值。家蚕泌丝结茧,具有很高的经济价值。家蚕为雌雄异体,而雄蚕的经济价值显著高于雌蚕。雄茧的平均出丝率要比雌茧高,并且雄蚕体质强健、好养、饲料效率高、茧丝品质好。因此,家蚕性别决定研究一直是蚕业的重要课题。家蚕性染色体类型为ZW型。近几年来,家蚕性别决定级联途径中的下游双性基因Bmdsx已克隆,并已经证明在家蚕雌雄分化中扮演了关键作用。最近,研究人员鉴定出Bmdsx上游的两个调控因子,分别为BmPSI和Bmhrp28。但对于这两个基因的具体作用及其相互关系尚未阐明。基于此,本研究利用家蚕的全基因组序列、表达序列标签（ESTs）数据以及基因芯片数据,对Bmhrp28和BmPSI进行了克隆和分析。同时采用了RT-PCR、原核表达、RNAi、酵母双杂交、免疫共沉淀等技术对Bmhrp28和BmPSI的功能进行了研究。获得主要结果如下:1.Bmhrp28和BmPSI的克隆、序列分析及mRNA表达根据文献报道与GenBank登录的Bmhrp28和BmPSI的基因序列,设计引物,进行克隆,经酶切和测序验证获得两个基因包括完整ORF序列的阳性克隆。在基因结构上,Bmhrp28的ORF长为771 bp,编码256个氨基酸残基,预测其蛋白质分子量为28.2 kD,等电点为8.72,位于第21号染色体nscaf3044上,含有7个外显子、6个内含子。而BmPSI的ORF长为2112bp,编码703个氨基酸残基,预测其蛋白质分子量为76.4kD,等电点为6.07,位于第20号染色体nscaf2789上,含有14个外显子、13个内含子。两个基因的外显子、内含子边界处均符合GT-AG规则。蛋白质结构域分析显示,Bmhrp28和BmPSI所编码蛋白均为RNA结合蛋白。Bmhrp28含有两个RRM结构域,BmPSI含有四个KH结构域和A、B两个功能域。RRM和KH结构域均为RNA结合的区域,此结构域在RNA的选择性剪接、加工、转录等过程中发挥作用。Bmhrp28和BmPSI蛋白质结构域部分与果蝇同源基因hrp48和PSI的结构域部分相似性较高,在果蝇中hrp48和PSI作为调控因子参与P元件转座酶的选择性剪接。为了探明这两个基因在家蚕组织时期的表达特征,我们利用家蚕ESTs数据、全基因组芯片数据以及半定量RT-PCR方法进行了表达谱分析。ESTs数据分析结果显示,Bmhrp28和BmPSI均具有EST证据。在家蚕卵巢细胞、胚胎、卵巢等组织器官中找到Bmhrp28的EST证据。在家蚕精巢、卵巢、丝腺、胚胎等组织器官中则具有BmPSI的EST证据。家蚕全基因组芯片的组织表达数据分析结果显示,Bmhrp28在幼虫5龄3天各组织中表达量非常低,只在精巢中有少量表达,在其它组织几乎不表达。而BmPSI在各个组织中均有表达,在卵巢和后部丝腺的表达较高,从整体上看,其表达量比Bmhrp28高。各个发育时期的表达数据表明,Bmhrp28从5龄4天到蛾期的表达量也非常低。而BmPSI在各个时期广泛表达,雌雄差异不大。早期胚胎的表达数据显示,Bmhrp28在雌雄间具有不同的表达模式,雌个体中在产卵后48 h表达水平最高,随后表达呈逐渐下降趋势;而在雄个体中,48 h几乎不表达,至60 h达到高峰,之后逐渐下降。而BmPSI与Bmhrp28相比,在雄个体中的模式类似,但在产卵后72 h达到高峰,而在雌中表达量几乎处于低水平,变化不大。为了进一步验证表达谱,我们进行了半定量RT-PCR检测。结果显示,Bmhrp28和BmPSI在家蚕幼虫5龄3天雌雄各个组织中广泛表达。并且Bmhrp28的表达量比BmPSI的表达量更低。在早期胚胎各发育时间点的表达情况显示,Bmhrp28和BmPSI较为相似,从未受精卵到产卵后24 h表达量都很低。在雄性早期胚胎里,基因的表达量逐渐增加,到产卵后96 h表达量达到高峰,而随后表达量降低。在雌性早期胚胎里,产卵后48 h表达量最高,随后开始降低。早期胚胎发育过程是性别决定的关键阶段,而较为相似的表达模式暗示Bmhrp28和BmPSI在此过程中可能是协同作用行使功能。2.Bmhrp28和BmPSI的原核表达以及多克隆抗体的制备为了进一步研究基因功能,我们将Bmhrp28和BmPSI分别连入经改造的p28和pET-MBP表达载体进行原核表达。将构建好的重组表达质粒命名为Bmhrp28/p28和BmPSI/pET-MBP,并将其分别转化到BL21（DE3）表达菌株和Rosetta（DE3）表达菌株。通过IPTG诱导表达,分别得到了分子量约为31 kD和120 kD的重组蛋白,目的蛋白分子量约为28.2 kD和76.4 kD,加上6×His标签序列和MBP标签序列,大小约为31 kD和120 kD,与预测分子量相吻合。经蛋白组分鉴定发现,诱导表达的两个重组蛋白均主要以可溶形式表达。通过亲和层析和电洗脱的方法分别纯化原核表达的BmHRP28和BmPSI蛋白,将纯化的蛋白免疫成年健康公兔,制备多克隆抗体。利用western blotting分析表明,BmHRP28和BmPSI的抗体是由其蛋白作为抗原而产生的,可以用做后续实验。3.Bmhrp28和BmPSI的RNAi研究已有研究表明,Bmhrp28和BmPSI均能结合到Bmdsx的第四外显子上,而Bmdsx作为家蚕性别决定路径中的双性基因,下游有其正调控因子贮藏蛋白SP1和卵黄原蛋白Vg,主要在雌性中表达;负调控因子信息素结合蛋白PBP,主要在雄性中表达。基于此特征,本研究采用体外合成短链siRNA介导RNAi的方式,利用家蚕限性白卵早期胚胎（雄）为材料,在产卵后48 h进行显微注射,后让其继续发育到96 h进行荧光定量PCR检测在干扰Bmdsx上游基因Bmhrp28和BmPSI后,Bmdsx下游基因PBP、SP1和Vg的表达差异,进而对Bmhrp28和BmPSI在家蚕性别决定中的功能进行研究。研究结果显示,干扰BmPSI,下游基因PBP表达量降低,SP1和Vg表达量增加,暗示BmPSI作为Bmdsx上游调控因子在家蚕性别决定路径中具有重要作用。而干扰Bmhrp28,下游基因PBP表达量虽然也有所降低,但SP1和Vg的表达量并未增加。我们推测Bmhrp28作为Bmdsx的上游调控因子虽然同样参与了家蚕性别决定路径,但是功能可能弱于BmPSI。4.家蚕Bmdsx上游调控因子Bmhrp28与BmPSI相互关系的研究为确定Bmhrp28与BmPSI的相互关系,我们采用酵母双杂交和免疫共沉淀的方法进行研究。首先利用酵母双杂交技术,在无自激活的前提下,将待检测的目标基因共转酵母,通过检测三个报告基因（HIS3、URA3、LacZ）是否表达从而确定目标基因之间的相互关系。本研究中,首先将Bmhrp28连入pPC86载体（AD）,BmPSI连入pDBLeu载体（BD）。在检测无自激活后,将pPC86-Bmhrp28和pDBLeu-BmPSI共转酵母。结果显示,在SC/-Leu-Trp-His+3-AT平板与SC/-Leu-Trp-Ura平板上均无可生长的阳性克隆,在LacZ膜检中无显蓝色的阳性结果,即HIS3、URA3、LacZ三个报告基因均未检测到表达。由此可见Bmhrp28与BmPSI之间没有直接相互作用或者相互作用极弱。为了进一步验证该结果,我们采用免疫共沉淀检测Bmhrp28与BmPSI之间的相互关系。首先以精巢组织抽提物为材料,加入BmHRP28抗体去沉淀其抗原,与BmHRP28相互作用的其它蛋白也会一并沉淀下来,再用BmPSI抗体检测沉淀蛋白产物中是否存在BmPSI。结果显示,BmHRP28抗体沉淀的蛋白产物中,既存在BmHRP28,也存在BmPSI。结合之前酵母双杂交的结果表明,Bmhrp28与BmPSI并不是直接相互作用,而是共存于一个蛋白复合体在家蚕性别决定路径中行使功能。5.构建家蚕酵母双杂交早期胚胎cDNA文库并筛选Bmhrp28、BmPSI蛋白复合体中其它调控因子为了进一步筛选Bmhrp28、BmPSI蛋白复合体中的其它调控因子,我们构建了酵母双杂交早期胚胎cDNA文库。此文库以家蚕限性白卵品种（雄）为材料,将cDNA连入pDONR222载体,产生Gateway入门文库。随后将入门文库转到pDEST22载体上,获得酵母双杂交cDNA文库。经鉴定,该文库的总克隆数为1.36×10⁷。从平板上随机挑取40个转化子菌落,提取文库质粒DNA进行PCR检测。结果显示,该文库插入的平均片段人小大于1.3 kb,重组率大于95%。取文库1μL稀释10⁶倍后涂板检测,共长出77个菌落。扩增后的文库效价为7.7×10¹⁰cfu/mL。鉴定结果表明,该文库达到构建文库的标准,可以用做下一步文库的筛选。我们采用酵母双杂交的方法,在无自激活的前提下,将pDBLeu-BmPSI筛选已构建的家蚕酵母双杂交早期胚胎cDNA文库。菌落在SC/-Trp/-Leu/-His+30mM 3-AT平板上生长7天后,共长出22个酵母转化菌落。将酵母菌落在SC-Trp-Leu二缺平板上划线保种,并挑取少量菌落检测LacZ报告基因的表达。通过LacZ检测的阳性克隆在SC-Trp-Leu-Ura平板上划线。从通过LacZ和URA3检测的酵母菌中抽提出猎物质粒与诱饵质粒做共转化验证。最终1个阳性克隆通过检测。对该阳性克隆进行测序并分析发现,该基因为家蚕的一个剪接体蛋白（Bmspx,spliceosomal protein on the X in silkworm）。在基因结构上,该基因的ORF长为1029 bp,编码342个氨基酸残基,预测分子量为37.0 kD,等电点为6.36,位于第8号染色体nscaf2827上,含有6个外显子、5个内含子,外显子、内含子边界处均符合GT-AG规则。蛋白质结构域分析显示,该基因所编码的蛋白具有两个RRM结构域,同样为RNA结合蛋白。与其他物种中的同源基因的蛋白质序列比对结果显示,BmSPX与果蝇SPX,以及伊蚊、蜜蜂、赤拟谷盗、牛、人、猪、老鼠、马、斑马鱼等的SF384（splicing factor 3b subunit 4）同源性达85%以上,特别是在RRM结构域处非常保守。应用家蚕全基因组芯片的组织表达数据分析显示,Bmspx在幼虫5龄3天各组织均有表达,但仅在精巢中高量表达,这可能与其在家蚕性别决定中的潜在功能相关。而各个发育时期的表达数据表明,从5龄4天到蛾期,Bmspx在各时期广泛表达,雌雄差异不大。早期胚胎的表达数据显示,Bmspx在雌雄中表达模式较为类似,在产卵后48 h表达水平最高,但自48 h开始表达呈逐渐下降趋势,到产卵后96 h表达水平最低。Bmspx编码RNA结合蛋白,且只在精巢中高量表达,作为Bmhrp28、BmPSI蛋白复合体中的一个调控因子能与BmPSI直接相互作用。综合研究结果我们推测,家蚕性别决定双性基冈Bmdsx上游的调控受BmPSI、Bmhrp28、Bmspx等控制。Bmspx与BmPSI相互作用,但Bmspx与Bmdsx以及复合体中的另一成员Bmhrp28之间的关系尚未研究清楚。如果Bmspx能结合到Bmdsx上并与Bmhrp28直接相互作用,那么Bmspx则是Bmhrp28与BmPSI之间的桥梁,使Bmdsx上游调控复合体组分之间的关系更为清晰。对于该基因与Bmdsx、Bmhrp28的相互关系以及在家蚕性别决定路径中的具体作用,我们将进一步研究。更多还原

【Abstract】 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.更多还原