CN114751971A - 一种牙鲆雄性相关Dmrt1重组蛋白及应用 - Google Patents
一种牙鲆雄性相关Dmrt1重组蛋白及应用 Download PDFInfo
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- CN114751971A CN114751971A CN202210356947.0A CN202210356947A CN114751971A CN 114751971 A CN114751971 A CN 114751971A CN 202210356947 A CN202210356947 A CN 202210356947A CN 114751971 A CN114751971 A CN 114751971A
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- dmrt1
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- paralichthys olivaceus
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Abstract
本发明属于分子生物学技术领域,具体涉及牙鲆Dmrt1重组蛋白及应用。本发明利用体外重组表达技术首次获得了牙鲆Dmrt1重组蛋白,提供了重组蛋白的制备方法,并利用重组蛋白孵育牙鲆性腺组织,表明其具有生物活性。本发明将重组Dmrt1蛋白注射至牙鲆雌鱼成体的腹腔,引起其卵巢组织中卵母细胞的细胞核染色质凝缩。进一步将其注射到性腺未分化鱼苗的腹腔中,分别引起幼鱼体内雌激素和雄激素水平的显著下降和升高(p<0.05),并且雄性率从0%增加至82%。表明所述重组蛋白能够用于牙鲆等鱼类的性别控制,可以为鱼类养殖中的性别控制和单性生产提供新的方法和途径。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及牙鲆Dmrt1重组蛋白及应用。
技术背景
Dmrt1(Doublesex and mab-3-related transcription factor 1)作为功能性保守因子,研究显示可以激活精巢特异性基因,降低体内雌激素水平,提示其在鱼类的性别决定和精巢分化中发挥关键作用。dmrt1基因序列已在多种鱼类中获得,并开展了性腺分化和发育过程的中mRNA转录表达研究,结果表明该基因在精巢分化早期开始表达,并在分化过程中持续高表达,研究者们由此推测dmrt1可能参与了雄性性别表型的形成。进一步的基因敲除和过表达研究发现,在模式鱼斑马鱼中敲除dmrt1,影响了精巢精原细胞的有丝分裂,表明其参与了生殖细胞的维持,但未实现雄鱼向雌鱼的完全转变;在另一种模式鱼青鳉中过表达dmrt1也未得到雌性向雄性的转变;而在淡水经济鱼类尼罗罗非鱼中过表达dmrt1,会导致遗传雌性个体的卵巢发育成类精巢样结构,由此认为该基因参与其精巢分化的过程,但只有不到5%个体出现完全性逆转。所以,dmrt1对鱼类雄性分化的准确作用尚待明确。与mRNA相比,蛋白是基因功能的直接执行者,在功能上与生物表型具有更高的相关性,但在鱼类性腺分化中Dmrt1蛋白水平研究也主要限于斑马鱼、青鳉以及尼罗罗非鱼等中,仅采用Western blot和免疫组化分析了Dmrt1蛋白表达图示,结果提示其对雄性分化起重要作用。真核细胞的转染实验表明,Dmrt1蛋白可以直接或间接抑制雌二醇合成关键酶基因cyp19a的转录,但该研究不能通过观察Dmrt1是否对雄性表型形成起直接作用来明确其功能。获得Dmrt1重组蛋白不仅可以更为直接的探究基因功能,也可作为诱导剂用于控制性别表型形成。目前,常用的体外蛋白表达载体主要有Blunt E2和pET等,对于不同的蛋白重组效果不同。
目前,鱼类中用来诱导雄性化的方法主要有外源雄激素、雌激素受体抑制剂和芳香化酶抑制剂,这些方法虽然能够人为调控鱼类的性别转化,得到具有功能性的雄鱼,但其应用都有一定的局限性。性激素及其受体抑制剂由于会污染水环境,养殖中不允许使用;芳香化酶抑制剂则由于使用成本相对较高或者引起外源诱导剂在体内堆积而带来潜在生物学影响,也不适合在养殖生产中应用。相比之下,鱼类的重组蛋白作为一种新型诱导剂,不仅能有效进行性别控制,也可以避免外源污染,因而具有潜在的应用前景。
牙鲆(Paralichthys olivaceus),隶属于鲽形目(Pleuronectiformes)、牙鲆科(Paralichthyidae)、牙鲆属(Paralichthys),为冷温性底栖鱼类,经济价值高,是我国重要的海水养殖鱼类。牙鲆生长存在明显的性别二态性,雌性生长显著快于雄性,为了实现全雌生产,获得更高的经济效益,有必要开展雌雄性别形成机制研究。因此,研究牙鲆性别相关基因功能,明确其在性别控制中的作用,进而应用于人工干预雌雄转换以进行单性养殖,具有重要的应用价值。
发明内容
本发明的目的在于一种牙鲆雄性相关Dmrt1重组蛋白及应用。
为实现上述目的,本发明采用的技术方案为:
一种牙鲆雄性相关Dmrt1重组蛋白,重组蛋白为在pET-30a-EGFP载体上插入dmrt1编码区,由此得牙鲆Dmrt1重组蛋白。
所述dmrt1基因核苷酸序列XM_020107068.1的编码区,参见SEQ ID NO:1所示氨基酸序列。
所述Dmrt1重组蛋白的获得:
(1)牙鲆精巢cDNA为模板进行PCR扩增;
(2)PCR产物经纯化后与大肠杆菌Escherichia coli表达载体pET-30a-EGFP进行连接,连接产物转化至大肠杆菌E.coli,测序鉴定重组子,得到重组质粒pET-30a-EGFP-Dmrt1;
(3)将重组质粒转入E.coli Transetta(DE3)化学感受态细胞,并接种于含0.1%卡那霉素的自动诱导复合培养基中,随后进行诱导培养、纯化、复性,即得到Dmrt1重组蛋白。
所述步骤(1)中PCR扩增采用的引物为
Dmrt1-F:5’-AAGGCCATGGCTGATATGAACAAGGACAAGCAGAG-3’;
Dmrt1-R:5’-TGAATCGATACGCATATTGATGCCATCCACGTCGA-3’。
所述重组蛋白的活性验证,利用含有1%抗生素的L15培养基进行牙鲆性腺预孵育,随后用含有重组Dmrt1蛋白和LipofectamineTM3000转染试剂的新鲜培养基进行性腺孵育,分析其下游基因cyp19a的表达变化。
一种牙鲆雄性相关Dmrt1重组蛋白的应用,所述Dmrt1重组蛋白在牙鲆性别控制或单性养殖中的应用。
所述Dmrt1重组蛋白引起了成鱼卵巢组织中卵母细胞的细胞核染色质凝缩,以及幼鱼性激素水平和雄性比例的变化。
本发明所具有的优点:
本发明首次利用体外重组表达技术获得了牙鲆Dmrt1重组蛋白,并利用体外性腺组织孵育验证了重组蛋白生物活性。本发明的牙鲆Dmrt1重组蛋白经雌性成体腹腔注射,能够引起其卵巢组织中卵母细胞的细胞核染色质的凝缩;而经遗传型雌性的分化期幼鱼腹腔注射,使得体内雌激素和雄激素水平的分别显著下降和升高(p<0.05),并且雄性率从0%增加至82%,说明重组蛋白可以应用于牙鲆性别控制和单性养殖。
本发明的技术方法可用于该基因在牙鲆雄性性别诱导研究,也可在其它鱼类上进行推广应用。
附图说明
图1为本发明实施例提供的获得的Dmrt1蛋白原核表达与纯化的SDS-PAGE检测;其中,A,蛋白表达(载体Blunt E2);B,蛋白表达(载体pET-30a-EGFP);C,蛋白纯化(载体pET-30a-EGFP)。箭头指示目的蛋白。M,Maker;SNI,未经诱导的上清;PNI,未经诱导的沉淀;S,诱导后上清;P,诱导后沉淀;F,清洗杂蛋白滤液;1-7,洗脱的目的蛋白。
图2为本发明实施例提供的获得的Dmrt1重组蛋白孵育卵巢和精巢组织的WB鉴定和cyp19a基因的表达变化;其中,A,WB鉴定;B,cyp19a基因的表达变化。蓝框,Dmrt1重组蛋白孵育的结果;绿框,EGFP蛋白孵育的结果。
图3为本发明实施例提供的获得的Dmrt1重组蛋白的牙鲆雌鱼成体腹腔注射后性腺组织学变化;其中,黑圈,发育异常的细胞形态。Og,卵原细胞;Oo,卵母细胞;Bar,5μm。
图4为本发明实施例提供的获得的Dmrt1重组蛋白的牙鲆雌核发育幼鱼腹腔注射后性激素水平变化;
图5为本发明实施例提供的获得的Dmrt1重组蛋白的牙鲆雌核发育幼鱼腹腔注射后性别比例和性腺组织学变化;其中,A,性别比例的变化;B(1),未经注射的卵巢;(2),注射EGFP后的卵巢;(3),注射Dmrt1重组蛋白的卵巢;(4),注射Dmrt1重组蛋白的精巢。红圈,发育异常的细胞形态。Og,卵原细胞;Oo,卵母细胞;Oc,卵巢腔;Sg,精原细胞;So,精母细胞;Bar,5μm。
具体实施方式
以下结合实例和附图对本发明的具体实施方式做进一步说明,应当指出的是,此处所描述的具体实施方式只是为了说明和解释本发明,并不局限于本发明。
本发明构建的牙鲆Dmrt1重组表达质粒,经诱导和纯化获得重组蛋白,所得蛋白具体是在特定pET-30a-EGFP载体上插入dmrt1编码区相连得到绿色荧光融合蛋白,所得蛋白可以明显提高重组蛋白的产量和表达稳定性,并且组织孵育结果表明重组蛋白具有生物活性,可用于牙鲆的腹腔注射,能够引起成鱼卵巢组织中卵母细胞的细胞核染色质的高度凝缩,成功改变幼鱼性别表型。
实施例1牙鲆Dmrt1蛋白的原核重组表达
1.重组质粒Blunt E2-Dmrt1与重组菌株的构建
提取牙鲆精巢组织的RNA,并反转录为cDNA,利用PCR扩增牙鲆Dmrt1的全部编码区,其编码的氨基酸序列如SEQ ID NO:1所示,再利用设计的特异性引物,其序列为:
Dmrt1-F:5’-ATGAACAAGGACAAGCAGAG-3’;
Dmrt1-R:5’-ATTGATGCCATCCACGTCGA-3’
进行PCR扩增,扩增后的片段经胶回收纯化测序验证后,与线性化Blunt E2载体进行连接,重组质粒转化至大肠杆菌DH5α感受态中,过夜培养后,挑取单克隆菌落进行菌液PCR扩增检测,并交由睿博兴科生物技术(北京)有限公司进行测序鉴定。
扩增条件为:
预变性:94℃2min;变性、退火和延伸:94℃30s,60℃30s,72℃1min 30s,共35个循环;终延伸:72℃5min。
SEQ ID NO:1为Dmrt1氨基酸序列为:
将重组质粒Blunt E2-Dmrt1转化至E.coli Transetta(DE3)中,涂布于含0.1%卡那霉素的LB固体培养基上,37℃倒置培养过夜。然后挑取单菌落进行菌液PCR检测鉴定。PCR产物经1%琼脂糖凝胶电泳后于凝胶成像仪下观察扩增产物目的条带大小,与预期片段大小相符的菌落为重组菌株。
2.重组菌株的诱导表达
2.1重组菌株的诱导表达:将重组菌种以划线方式接种于含0.1%卡那霉素的LB固体培养基平皿上,37℃倒置培养14-16h后,挑单菌落接种于含0.1%卡那霉素的LB液体培养基,置于37℃、200转/min培养12-16h作为一级种子;将一级种子以1%接种量接种于含0.1%卡那霉素的培养基中,加入IPTG(0.5mM),置于37℃、200转/min下诱导培养16h,收集菌液。
2.2Dmrt1重组蛋白的收集与检测:菌液经4℃下4,000g离心10min,收集菌体,PBS(137mM NaCl,2.7mM KCl,10mM Na2HPO4,1.8mMKH2PO4,pH 7.4)洗涤3次后,用含蛋白酶抑制剂的细菌裂解液进行重悬,冰上250W超声裂解25min。4℃下10,000g离心15min,并收集沉淀。Dmrt1重组蛋白用聚丙烯酰胺凝胶电泳(SDS-PAGE)分析(参见图1A)。
上述由Blunt E2载体获得重组蛋白的表达产量较低,使得重组蛋白不能进一步分析和应用,可见选用载体可能不适用。
实施例2
1.重组质粒pET-30a-EGFP-Dmrt1与重组菌株的构建
提取牙鲆精巢组织的RNA,并反转录为cDNA,利用PCR扩增牙鲆Dmrt1的全部编码区,其编码的氨基酸序列如SEQ ID NO:1所示,再利用设计的特异性引物,其序列为:
Dmrt1-F:5’-AAGGCCATGGCTGATATGAACAAGGACAAGCAGAG-3’;
Dmrt1-R:5’-TGAATCGATACGCATATTGATGCCATCCACGTCGA-3’
进行PCR扩增,扩增后的片段经胶回收纯化测序验证后,与线性化pET-30a-EGFP载体进行同源重组,重组质粒转化至大肠杆菌DH5α感受态中,过夜培养后,挑取单克隆菌落进行菌液PCR扩增检测,并交由睿博兴科生物技术(北京)有限公司进行测序鉴定。
扩增条件为:
预变性:94℃ 2min;变性、退火和延伸:94℃ 30s,60℃ 30s,72℃1min 30s,共35个循环;终延伸:72℃ 5min。
SEQ ID NO:1为Dmrt1氨基酸序列为:
将重组质粒pET-30a-EGFP-Dmrt1转化至E.coli Transetta(DE3)中,涂布于含0.1%卡那霉素的LB固体培养基上,37℃倒置培养过夜。然后挑取单菌落进行菌液PCR检测鉴定。PCR产物经1%琼脂糖凝胶电泳后于凝胶成像仪下观察扩增产物目的条带大小,与预期片段大小相符的菌落为重组菌株。
2.重组菌株的诱导表达与Dmrt1重组蛋白的纯化
2.1重组菌株的诱导表达:将重组菌种以划线方式接种于含0.1%卡那霉素的LB固体培养基平皿上,37℃倒置培养14-16h后,挑单菌落接种于含0.1%卡那霉素的LB液体培养基,置于37℃、200转/min培养12-16h作为一级种子;将一级种子以1%接种量接种于含0.1%卡那霉素的自动诱导复合培养基中,置于37℃、200转/min培养16h,收集菌液。
2.2Dmrt1重组蛋白的收集与纯化:菌液经4℃下4,000g离心10min,收集菌体,PBS洗涤3次后,用含蛋白酶抑制剂的细菌裂解液进行重悬,冰上250W超声裂解25min。4℃下10,000g离心15min,并收集沉淀。
按每g蛋白沉淀用10mL平衡缓冲液(5mM咪唑,0.5mM NaCl,8M尿素,20mM三羟甲基氨基甲烷盐酸盐(Tris-HCl),pH 7.9)的比例进行重悬,室温下涡旋振荡溶解10min后,4℃下10,000g离心20min,收集上清。将上清负载上柱(Ni2+柱),用15倍柱体积的平衡缓冲液冲洗,洗去杂蛋白。使用适量洗脱缓冲液(500mM咪唑,0.5mM NaCl,8M尿素,20mM Tris-HCl,pH7.9)洗脱,收集洗脱峰。
3.Dmrt1重组蛋白的透析复性
将纯化好的蛋白置于MD34(Mw50,000)透析袋,按蛋白样品与透析缓冲液的体积比1:100进行浸泡,透析缓冲液的主要成分是50mM Tris、24mM NaCl、1mM KCl、0.9mM L-谷胱甘肽还原(GSH)、0.1mM L-谷胱甘肽氧化(GSSG)和不同浓度的咪唑(250mM、100mM、0mM)。每个梯度的透析时间为6h,最后一个梯度重复2次,透析后的蛋白储存于-80℃。透析后的Dmrt1重组蛋白及未经诱导的重组菌蛋白用SDS-PAGE分析,测定其分子量大小(图1B,C),符合预期蛋白大小。这表明Dmrt1重组蛋白以不溶性的包涵体形式存在,且经纯化获得了较高纯度的重组蛋白。由此得到牙鲆Dmrt1重组蛋白,蛋白长度:302个氨基酸,类型:氨基酸,链型:单链,特性:预测分子量为33.0kDa,等电点为6.30,具有保守的DM结构域和Pfam结构域。
实施例3Dmrt1重组蛋白体外活性验证
1.Dmrt1重组蛋白的组织孵育
将实施例2中重组纯化的牙鲆Dmrt1蛋白在前述PBS中稀释至1000μg/mL。
取健康的野生型牙鲆雌雄成鱼各4尾的性腺组织,用含有1%青霉素(10kU/mL)-链霉素(10mg/mL)混合抗生素的前述PBS反复清洗后,切成约1.0mm3的小块并置于24孔板中,用含有1%抗生素的L15培养基在25℃下预孵育6h,随后用含有实施例2中重组纯化的Dmrt1重组蛋白(100μg)和LipofectamineTM3000转染试剂的新鲜培养基在25℃下孵育24h,孵育后的样品储存于-80℃备用。
2.WB及qPCR分析
一部分样品使用放射免疫沉淀法(RIPA)提取总蛋白,加入5×上样缓冲液后,沸水浴8-10min,用于WB检测。剩余的样品使用Trizol法提取总RNA,用NanoDrop 2000分光光度计和1.5%琼脂糖凝胶电泳检测RNA的浓度和纯度。利用反转录了试剂盒合成第一链cDNA,用于qPCR分析基因的表达量。
结果显示,重组蛋白借助体外转染试剂可以进入性腺组织,见图2A。Dmrt1重组蛋白孵育后,卵巢和精巢中cyp19a基因的表达量均显著降低(p<0.05),如图2B所示。结果表明,纯化的Dmrt1重组蛋白能够显著抑制性腺中cyp19a的表达(p<0.05),具有生物活性。
实施例4Dmrt1重组蛋白对牙鲆成体卵巢发育的作用
1.Dmrt1重组蛋白的雌性成体腹腔注射
选取20尾健康的野生型牙鲆雌鱼成体,按照每g体重注射100μg实施例2中重组纯化蛋白,同时加入in vivo-jetPEI转染试剂,每3d注射一次,连续注射3次后,养殖21d。
2.组织学观察
取出卵巢,放置于Davidsions固定液,利用组织切片观察组织学变化。实验组注射Dmrt1重组蛋白,对照组注射EGFP蛋白。
结果显示,与对照组相比,Dmrt1重组蛋白注射后,有50%个体的卵巢出现异常,如图3所示,卵巢的卵母细胞细胞核染色质凝缩,说明Dmrt1对成体卵巢发育有一定抑制作用。
实施例5Dmrt1重组蛋白对牙鲆性别表型形成的作用
1.Dmrt1重组蛋白的遗传雌性幼鱼腹腔注射
将全长1.2-1.5cm的健康遗传雌性(雌核发育)牙鲆幼鱼随机分为3组:Dmrt1重组蛋白注射组、EGFP蛋白注射组和未注射组。每7d用in vivo-jetPEI转染试剂转染Dmrt1重组蛋白和EGFP蛋白。在全长1.2-1.5cm时注射浓度为10μg/尾,后随幼鱼的生长成比例增加,至全长7.0cm时,浓度增加至100μg/尾,并停止注射,继续培育。
2.性激素测定及组织学观察
收集全长10.0cm的样品,去除其头部、躯干部及内脏团,剩余的性腺部位用于性激素测定,使用酶联免疫吸附剂测定(ELISA)试剂盒测定17β-雌二醇(17β-E2)、睾酮(T)和11-酮基睾酮(11-KT)水平。随机选取20尾,利用组织切片观察其性别比例及组织学特征。
结果显示,与EGFP组和未注射组相比,注射Dmrt1重组蛋白后,性腺中17β-E2处于较低水平(p<0.05),而T和11-KT处于较高水平(p<0.05),如图4所示。EGFP蛋白处理组和未注射组的雌性率均为100%,而Dmrt1重组蛋白处理组的雌性率为18%(雄性率为82%),如图5A所示。EGFP蛋白处理组和未注射组的卵巢发育正常,Dmrt1重组蛋白处理组的精巢发育正常,卵巢出现异常,卵巢中卵母细胞的细胞核染色质高度凝缩,如图5B所示。
综上述所述牙鲆性腺组织孵育表明Dmrt1重组蛋白能够抑制cyp19a的表达,具有生物活性。雌性成鱼的腹腔注射实验结果显示,Dmrt1重组蛋白可引起卵巢中卵母细胞的细胞核染色质凝缩,对成体卵巢发育有一定抑制作用。进一步的遗传雌性牙鲆幼鱼腹腔注射实验表明,Dmrt1重组蛋白注射后会影响幼鱼体内雌、雄激素水平的变化,进而改变其性别表型,这为水产养殖中的单性生产提供新的生物学见解和方法。
以上记载的仅为本发明的优选实施例,不能以此来限定本发明之权利范围,因此,依据本发明权利要求所作的等同变化,仍属于本发明所涵盖的范围。
序列表
<110> 中国科学院海洋研究所
<120> 一种牙鲆雄性相关Dmrt1重组蛋白及应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 302
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
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Gln His Ala Gln Glu Glu Glu Leu Gly Ile Cys Ser Pro Val Thr Leu
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Pro Gly Pro Glu Val Leu Val Lys Asn Glu Ala Gly Ala Asp Cys Leu
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Phe Ser Val Asp Gly Arg Ser Pro Thr Pro Thr Gly Ser Ala Ser Ala
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Ser Ser Leu Ala Phe Thr Gly Ser Arg Ser Ala Ser Ser Ser Ser Pro
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Ser Ala Gly Val Arg Ala His Ala Glu Gly Ala Ser Asp Leu Leu Met
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Asn Asn Tyr Ser Asp Thr Met Ala Ala Ser Phe Ser Pro Ser Asn Val
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Claims (5)
1.一种牙鲆雄性相关Dmrt1重组蛋白,其特征在于:重组蛋白为在pET-30a-EGFP载体上插入dmrt1编码区,由此获得牙鲆Dmrt1重组蛋白。
2.按权利要求1所述的牙鲆雄性相关Dmrt1重组蛋白,其特征在于:所述dmrt1基因核苷酸序列XM_020107068.1的编码区。
3.按权利要求1或2所述的牙鲆雄性相关Dmrt1重组蛋白,其特征在于:
(1)牙鲆精巢cDNA为模板进行PCR扩增;
(2)PCR产物经纯化后与大肠杆菌表达载体pET-30a-EGFP进行连接,连接产物转化至大肠杆菌,测序鉴定重组子,得到重组质粒pET-30a-EGFP-Dmrt1;
(3)将重组质粒转入E.coli Transetta(DE3)化学感受态细胞,并接种于含0.1%卡那霉素的自动诱导复合培养基中,随后进行诱导培养、纯化、复性,即得到Dmrt1重组蛋白。
4.按权利要求3所述的牙鲆雄性相关Dmrt1重组蛋白,其特征在于:所述步骤(1)中PCR扩增采用的引物为
Dmrt1-F:5’-AAGGCCATGGCTGATATGAACAAGGACAAGCAGAG-3’;
Dmrt1-R:5’-TGAATCGATACGCATATTGATGCCATCCACGTCGA-3’。
5.一种权利要求1所述的牙鲆雄性相关Dmrt1重组蛋白的应用,其特征在于:所述Dmrt1重组蛋白在牙鲆性别控制或单性养殖中的应用。
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