CN113862272A - 沉默中华绒螯蟹Dsx基因的dsRNA以及其应用 - Google Patents
沉默中华绒螯蟹Dsx基因的dsRNA以及其应用 Download PDFInfo
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Abstract
本申请涉及中华绒螯蟹Dsx基因相关技术领域,尤其涉及沉默中华绒螯蟹Dsx基因的dsRNA以及其应用。制备该dsRNA的模板cDNA的核苷酸序列如SEQ ID NO.1所示。用于制备该dsRNA的引物对的核苷酸序列如SEQ ID NO.2~3所示。该dsRNA不仅可实现对中华绒螯蟹精巢特化Dsx基因的沉默,还能够调控中华绒螯蟹性别决定基因IAG的基因表达;该dsRNA对Dsx基因的沉默准确可靠、沉默效率高,为中华绒螯蟹单性化养殖及遗传改良提供了研究基础。
Description
技术领域
本申请涉及中华绒螯蟹Dsx基因相关技术领域,尤其涉及沉默中华绒螯蟹Dsx基因的dsRNA以及其应用。
背景技术
中华绒螯蟹(Eriocheir sinensis),俗称河蟹,隶属于节肢动物门(Arthropoda)、甲壳纲(Crustacea)、十足目(Decapoda)。因其肉质鲜美、营养丰富而成为中国重要的水产养殖蟹类之一。在其生产过程中,根据不同需要,通过单性化养殖,实现河蟹全雄或全雌养殖可以节约养殖成本,提高生产效率,进而在满足消费者需求的同时创造出更大的经济效益。因此,中华绒螯蟹的性别决定与性腺分化机制越来越受到人们的关注。
Doublesex(Dsx)基因最早是在果蝇中被发现的,作为昆虫性别级联系统中的关键基因,Dsx基因的结构和功能在不同昆虫中具有保守性,可调节下游基因的表达来参与雌雄性别的决定和分化。Dsx基因编码的DSX蛋白主要包括两部分,其N端包含一个能与DNA结合的保守基序DM(doublesex and mab-3)结构域,此结构域是DSX蛋白结合下游靶标基因的重要位点;而C端的OD2结构域激活或者抑制下游靶标基因的表达,进而起到调控个体性腺分化的作用。研究发现,河蟹的Dsx基因与昆虫Dsx基因高度同源(见刘志强,中华绒螯蟹雌雄个体早期形态分化及雄性个体中性别相关基因的研究[D].上海海洋大学,2018)。虽然Dsx基因的功能在昆虫性别决定的研究中已阐明,但河蟹关于Dsx基因的报道还未见。因此,有必要在河蟹中开展Dsx基因的功能研究,并应用于河蟹的性别决定与性腺分化中。
发明内容
有鉴于此,本申请的目的在于针对Dsx基因的表达进行研究,以利于将其应用于中华绒螯蟹的性别决定和性腺分化研究中。
第一方面,本申请实施例公开了制备dsRNA的模板cDNA,所述模板cDNA的核苷酸序列如SEQ ID NO.1所示,其中,所述dsRNA用于沉默中华绒螯蟹Dsx基因。
第二方面,本申请实施例公开了所述引物对的核苷酸序列如SEQ ID NO.2~3所示。
第三方面,本申请实施例公开了连接有所述的模板cDNA的重组表达载体。
第四方面,本申请实施例公开了携带有所述的重组表达载体的宿主。
第五方面,本申请实施例公开了制备所述的dsRNA的方法,包括以下步骤:
获得所述模板cDNA;
PCR扩增,利用所述引物对对所述模板cDNA进行扩增,得到长度为571bp的靶序列;
构建重组表达载体,所述重组表达载体连接有所述靶序列片段;
获得转化有所述重组表达载体的大肠杆菌菌体;
诱导表达所述大肠杆菌菌体;以及
得到所述dsRNA,由所述大肠杆菌菌体诱导表达后,经过菌体裂解、RNA提取、变性、退火和纯化得到所述dsRNA。
在本申请实施例中,所述PCR扩增体系为:10×Buffer 5μL,正向引物0.5μL,反向引物0.5μL,cDNA模板1μL,Taq聚合酶1.0μL,dd H2O补足50μL;所述PCR扩增反应条件为:98℃预变性30s;95℃变性30s,60℃退火30s,72℃延伸1min,共循环30次后;72℃延伸10min。
在本申请实施例中,大肠杆菌菌体诱导表达后的步骤包括:
收集菌体,利用Trizol Reagent提取上述细胞总RNA后,于72℃水浴条件下对上述总RNA变性处理10min,自然冷却至室温后,退火即可形成靶向Dsx基因的dsRNA。
第六方面,本申请实施例公开了沉默中华绒螯蟹Dsx基因表达的方法,包括对中华绒螯蟹注射包含所述dsRNA的试剂的步骤。
第七方面,本申请实施例公开了所述的dsRNA或所述的模板cDNA的应用,所述应用包括以下任一项:
(1)在中华绒螯蟹杂交育种中的应用;
(2)在中华绒螯蟹单性化养殖中的应用;
(3)在中华绒螯蟹遗传改良中的应用。
与现有技术相比,本申请至少具有以下有益效果:
本申请中涉及一种靶向中华绒螯蟹精巢特化表达的Dsx基因的dsRNA,该dsRNA不仅可实现对中华绒螯蟹精巢特化Dsx基因的沉默,还能够调控中华绒螯蟹性别决定基因IAG的基因表达;该dsRNA对Dsx基因的沉默准确可靠、沉默效率高,为中华绒螯蟹单性化养殖及遗传改良提供了研究基础。
附图说明
图1为本申请实施例提供的中华绒螯蟹总RNA的电泳检测图;左泳道为Marker,右泳道为总RNA样本。
图2为本申请实施例提供的中华绒螯蟹Dsx基因的RNAi靶序列的电泳检测图;左泳道为Marker,右泳道为RNAi靶序列PCR结果。
图3为本申请实施例提供的重组表达载体的双酶切电泳图;左泳道为Marker,右泳道为重组表达载体的双酶切产物。
图4为本申请实施例对目的片段的测序鉴定结果。
图5为本申请实施例提供的原核表达制备靶向Dsx基因的dsRNA(经RNase A处理过)的电泳检测图;M泳道为Marker;1泳道为IPTG诱导后退火形成的dsRNA样本;2泳道为未经IPTG诱导的样本;3泳道为IPTG诱导L4440空载体样本。
图6为本申请实施例提供的RNA干扰中华绒螯蟹24h后性别相关Dsx和IAG基因mRNA的定量检测结果。
具体实施方式
为了使本申请的目的、技术方案及优点更加清楚明白,以下结合实施例对本申请进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本申请,并不用于限定本申请。
本申请发明人长期实践和研究中创造性发现了一种靶向中华绒螯蟹精巢特化表达的Dsx基因的dsRNA,该dsRNA不仅可实现对中华绒螯蟹精巢特化Dsx基因的沉默,还能够调控中华绒螯蟹性别决定基因IAG的基因表达;该dsRNA对Dsx基因的沉默准确可靠、沉默效率高,为中华绒螯蟹单性化养殖及遗传改良提供了研究基础。
为了进一步说明本发明,下面结合实施例对本发明提供的沉默中华绒螯蟹精巢特化表达的Dsx基因的dsRNA制备过程以及沉默效果进行详细说明,但不能将它们理解为对本发明保护范围的限定。下方未特别说明的试剂或仪器均可从常规商业途径获得,其中未特别说明的实验方法均为本领域常规实验方法。
一、试验材料及来源
Trizol Reagent总RNA提取试剂盒、Taq聚合酶、RNase-Free DNase I、RNase A、PrimeScript RT Kit反转录试剂盒和荧光定量PCR试剂盒,均购自(TaKaRa)宝生物工程有限公司;PCR产物纯化试剂盒和DNA割胶纯化试剂盒均购自天根生化科技(北京)有限公司;T4 DNA连接酶和限制性核酸内切酶购自普洛麦格(北京)生物技术有限公司;L4440载体购自上海柯雷生物科技有限公司;HT115菌株购自北京华越洋生物;各种PCR引物由上海生工制备。
二、实验步骤
1、提取中华绒螯蟹总RNA
取中华绒螯蟹精巢,按照Trizol Reagent总RNA提取试剂盒的说明书提取总RNA,使用Nanodrop 2000c检测RNA浓度和纯度。同时制备2.0%的琼脂糖凝胶,于120V电压电泳20min检测,如图1所示,图中可明显观察到28S rRNA、18S rRNA和5.8S rRNA的条带,且前两者亮度远高于5.8S rRNA,说明得到了完整的中华绒螯蟹总RNA。
2、反转录制备cDNA
取0.5μg中华绒螯蟹总RNA,用DNase I除去RNA中混有的少量DNA污染后,按照PrimeScript RT Kit反转录试剂盒的说明书制备cDNA。
3、制备中华绒螯蟹Dsx基因的RNAi靶序列
以上述反转录获得的cDNA为模板;以正向引物:5'-ataagaatgcggccgccctgaacctcatgacgacct-3',如SEQ ID NO.2所示;以反向引物,具体为:5'-cccaagcttggcttgactgggcttactgt-3',如SEQ ID NO.3所示,对Dsx基因的RNAi靶序列进行PCR扩增。
PCR反应体系为:10×Buffer 5μL,正向引物0.5μL,反向引物0.5μL,cDNA模板1μL,Taq聚合酶1.0μL,dd H2O补足50μL。扩增反应条件为:98℃预变性30s;95℃变性30s,60℃退火30s,72℃延伸1min,共循环30次;72℃延伸10min。如图2所示,经过琼脂糖凝胶电泳检测的获得的RNAi靶序列片段大小为571bp。根据天根生化科技(北京)有限公司PCR产物纯化试剂盒的说明书进行PCR产物的纯化。
4、构建重组表达载体
用限制性核酸内切酶Not I和Hind III对上述PCR纯化片段和L4440质粒分别进行双酶切。酶切反应体系均为100μL:目的片段/L4440质粒2μg,10×酶切Buffer 10μL,限制性内切酶Not I和Hind III各3μL,dd H2O补足100μL。37℃消化5h后分别按照DNA割胶纯化试剂盒的说明书进行割胶纯化。
连接反应16℃过夜,其反应体系为:双酶切线性化的L4440质粒1μL,双酶切的PCR纯化片段3μL,T4 DNA连接酶0.5μL,10×连接反应Buffer 1μL,dd H2O补足10μL。如图3所示,酶切得到了约2790bp的载体片段和571bp的目的片段。
5、重组表达载体的转化
将上述重组表达载体转化大肠杆菌HT115感受态。操作步骤如下:
(1)将10μL的重组表达载体加入刚刚融化的HT115感受态细胞悬浮液中,混匀,冰上放置30min;
(2)42℃热激90秒,迅速转至冰上静置3分钟;
(3)加900μL的LB液体培养基,37℃,在180rpm条件下摇菌1h;
(4)4℃,4500rpm条件下离心3min;
(5)吸去900μL的上清液,用微量移液器吹打混匀细胞沉淀后,涂布至已加入氨苄青霉素的LB固体培养基上,37℃条件下过夜培养;
(6)挑选LB固体培养基上长出的单个菌落进行扩大培养并进行双酶切和测序验证。双酶切的产物经琼脂糖凝胶电泳检测,对同样能够得到571bp长度条带的阳性克隆进行测序鉴定,测序结果如图4所示,由此得到其核苷酸序列如SEQ ID NO.1所示。
6、双链RNA(dsRNA)的制备
挑取上述已鉴定的阳性克隆,获得携带重组表达载体的大肠杆菌HT115,对其进行诱导表达,以制备靶向Dsx基因的dsRNA。具体步骤如下:
(1)将测序正确的、含重组表达载体的HT115菌液100μL,接种到4mL含氨苄青霉素的LB液体培养基上过夜扩大培养;
(2)取250μL扩大培养的菌液接种到30mL的2×YT液体培养基,37℃条件下震荡培养。当OD595=0.5时,加入50μL(50mg/mL)的IPTG诱导5h;
(3)4℃,5000rpm条件下离心5min,收集细胞沉淀;
(4)利用Trizol Reagent提取上述细胞总RNA后,用RNase-Free DNase I处理总RNA中可能混有的少量DNA污染;
(5)72℃水浴条件下对上述总RNA变性10min。
(6)自然冷却至室温后,退火形成靶向Dsx基因的dsRNA。
(7)加1μL的RNase A去除没有形成dsRNA的单链RNA。如图5可见IPTG诱导后退火形成了dsRNA。
7、dsRNA的纯化
使用苯酚氯仿抽提法纯化dsRNA。在25μL上述dsRNA产物的体系中迅速加入125μLDEPC水、20μL(3M,pH=5.2)醋酸钠和等体积的酚/氯仿(1:1)混合液,混匀。在4℃,12,500rpm条件下离心10min后,取出上清,并加入等体积的氯仿混匀。在4℃,12,500rpm条件下离心10min,取出上清,向其中加入十分之一体积的醋酸钠(3M,pH=5.2)和2.5倍体积的预冷无水乙醇,于-80℃放置120min。放置结束以后,离心回收沉淀。将得到的沉淀于超净工作台干燥后,用适量的DEPC水溶解,并测定dsRNA的浓度。
8、RNA干扰试验
将中华绒螯蟹I期仔蟹放入玻璃养殖箱中,养殖条件为正常的光照周期(12h光照、12h黑暗),且溶解氧充足。向实验组注射100μL(2.5μg/μL)/仔蟹的dsRNA,共注射60只。同时设置对照组,对照组注射100μL/仔蟹的PBS。
注射24小时候后分别取实验组和对照组的中华绒螯蟹各3只,按照TrizolReagent总RNA提取试剂盒的说明书提取总RNA,并按照PrimeScript RT Kit反转录试剂盒的说明书制备cDNA备用。
9、qRT-PCR方法检测Dsx、IAG基因表达
Dsx基因检测用的正向引物的核苷酸序列为ccgacgaaggcaagagaga,如SEQ IDNO.4所示;Dsx基因检测用的反向引物的核苷酸序列为ctggcagtagcgtttgtgg,如SEQ IDNO.5所示。
IAG基因检测用的正向引物的核苷酸序列为cggttacaggaggactcgag,如SEQ IDNO.6所示;IAG基因检测用的反向引物的核苷酸序列为tgagtgtaggcgtcaacagt,如SEQ IDNO.7所示。
以β-actin为内参基因,正向引物的核苷酸序列为cgacggtcaggtcatcacca,如SEQID NO.8所示;反向引物的核苷酸序列为acgtcgcacttcatgatgga,如SEQ ID NO.9所示。
反应体系为:2×SYBR Green 10μL,正向和反向引物各0.2μL,cDNA模板1μL,ddH2O补足至20μL。PCR循环程序为:初始95℃30s,之后进行95℃5s,60℃30s的40个循环。每个qRT-PCR检测试验设三个独立的生物学重复,每个生物学重复再设3个技术性重复。最终结果使用2-ΔΔCt法计算。基因沉默处理后,目标Dsx基因的表达量的降低程度视为该基因的沉默效率。如图6所示,与β-actin相比,对照组Dsx、IAG基因的相对表达量分别为12.2%和43.4%;而实验组个体干扰24h后,二者的相对表达量分别为1.05%和6.25%,仅为对照组Dsx、IAG基因表达量的8.6%和14.4%,由此说明本申请实施例提供的dsRNA不仅能够沉默Dsx基因表达,还能抑制IAG表达。
上述实施例的结果表明,本申请实施例提供的dsRNA不仅可实现对中华绒螯蟹精巢特化Dsx基因的沉默,还能够调控中华绒螯蟹性别决定基因IAG的基因表达;该dsRNA对Dsx基因的沉默准确可靠、沉默效率高,为中华绒螯蟹单性化养殖及遗传改良提供了研究基础。
以上所述,仅为本申请较佳的具体实施方式,但本申请的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本申请揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本申请的保护范围之内。
序列表
<110> 上海海洋大学
<120> 沉默中华绒螯蟹Dsx基因的dsRNA以及其应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 571
<212> DNA
<213> Artificial Sequence
<400> 1
cctgaacctc atgacgacct cctccactac cgatgaaggc aagagagagg ccatgtcccc 60
gcccttgcca taccacggac tgccccttca tctccagcag cagcagcaac aggcggagtg 120
cagcagccga gttcccaaat gcgcccgctg taggaaccat aacaagcaca cgccattaag 180
aggccacaaa cgctactgcc agttcaggaa gtgccaatgt tcgcgatgtc agcccaccgt 240
ggacaggcag cgcatcatgg cccgtcaggt ggctctccgt cgggcgcagg aacaggacga 300
ggcacgcaga gttctccttc aggaaacgct gcaaatggag acttcgccgg cgcttacatc 360
ctcagacttg gacgggtatc gaagtgaggg gtcgatgtcg cctggaagct gcccacctgc 420
cgtctccacc accagcacgc caccgcctcc gccgctgcac ttcccgcccc tcctgcagcc 480
tcatcacacc cgctcagagc accctccaca ccgccccgac ctcgcccccc tcgccttcac 540
gccctcgcca cacagtaagc ccagtcaagc c 571
<210> 2
<211> 36
<212> DNA
<213> Artificial Sequence
<400> 2
ataagaatgc ggccgccctg aacctcatga cgacct 36
<210> 3
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 3
cccaagcttg gcttgactgg gcttactgt 29
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 4
ccgacgaagg caagagaga 19
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 5
ctggcagtag cgtttgtgg 19
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 6
cggttacagg aggactcgag 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 7
tgagtgtagg cgtcaacagt 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 9
cgacggtcag gtcatcacca 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 9
acgtcgcact tcatgatgga 20
Claims (9)
1.制备dsRNA的模板cDNA,所述模板cDNA的核苷酸序列如SEQ ID NO.1所示,其中,所述dsRNA用于沉默中华绒螯蟹Dsx基因。
2.用于制备权利要求1所述的dsRNA的引物对,其特征在于,所述引物对的核苷酸序列如SEQ ID NO.2~3所示。
3.连接有权利要求2所述的模板cDNA的重组表达载体。
4.携带有权利要求3所述的重组表达载体的宿主。
5.制备权利要求1所述的dsRNA的方法,其特征在于,包括以下步骤:
获得如权利要求1所述的模板cDNA;
PCR扩增,利用权利要求2所述的引物对对所述模板cDNA进行扩增,得到长度为571bp的靶序列片段;
构建重组表达载体,所述重组表达载体连接有所述靶序列片段;
获得转化有所述重组表达载体的大肠杆菌菌体;
诱导表达所述大肠杆菌菌体;以及
得到所述dsRNA,由所述大肠杆菌菌体诱导表达后,经过收集菌体、提取RNA、变性、退火和纯化得到所述dsRNA。
6.根据权利要求5所述的方法,其特征在于,所述PCR扩增体系为:10×Buffer 5μL,正向引物0.5μL,反向引物0.5μL,cDNA模板1μL,Taq聚合酶1.0μL,dd H2O补足50μL;所述PCR扩增反应条件为:98℃预变性30s;95℃变性30s,60℃退火30s,72℃延伸1min,共循环30次;72℃延伸10min。
7.根据权利要求5所述的方法,其特征在于,大肠杆菌菌体诱导表达后的步骤包括:
收集菌体,利用Trizol Reagent提取上述细胞总RNA后,于72℃水浴条件下对上述总RNA变性处理10min,自然冷却至室温后,退火即可形成靶向Dsx基因的dsRNA。
8.沉默中华绒螯蟹Dsx基因表达的方法,包括对中华绒螯蟹注射包含如权利要求1所述的dsRNA的试剂的步骤。
9.权利要求1所述的dsRNA或权利要求2所述的模板cDNA的应用,其特征在于,所述应用包括以下任一项:
(1)在中华绒螯蟹杂交育种中的应用;
(2)在中华绒螯蟹单性化养殖中的应用;
(3)在中华绒螯蟹遗传改良中的应用。
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