CN105505945A - 一种脊尾白虾蜕皮激素基因EcEH及其应用 - Google Patents
一种脊尾白虾蜕皮激素基因EcEH及其应用 Download PDFInfo
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Abstract
本发明涉及一种脊尾白虾蜕皮激素基因EcEH及其在蜕皮调控中的应用。本发明所涉及的脊尾白虾蜕皮激素基因具有序列列表中SEQ？ID？No.1所示的核苷酸序列；基于该序列设计合成的双链RNA(dsRNA)注射蜕皮前期的脊尾白虾，明显抑制了其蜕皮的进度，可用于调控脊尾白虾的蜕皮过程。
Description
技术领域
本发明涉及脊尾白虾蜕皮，具体地说是一种脊尾白虾蜕皮激素基因及其在蜕皮调控中的应用。
背景技术
虾类(甲壳动物)的蜕皮与其生长发育、变态和繁殖关系密切。虾类(甲壳动物)的蜕皮过程包括新甲壳的逐渐形成，旧甲壳的蜕去，个体吸水增大以及新甲壳的硬化等。依据不同蜕皮阶段的特征变化把蜕皮过程分为蜕皮前期、蜕皮期、蜕皮后期和蜕皮间期。虾类的蜕皮过程受到内分泌激素的精准调控，Y-器官分泌的固醇类激素ecdysteroids及其衍生物20-羟基蜕皮酮(20-hydroxyecdysone，20E)诱发并调控着蜕皮过程的进行(Gilbert,L.I.,Rewitz,K.F.,2009.ThefunctionandevolutionoftheHalloweengenes:thepathwaytothearthropodmoltinghormone,in:G.Smagghe(Ed.),Ecdysone:StructuresandFunction.SpringerNetherlands.)。眼柄X-器官/窦腺复合体内分泌的蛋白类激素蜕皮抑制激素(molt-inhibitinghormone，MIH)和甲壳动物高血糖激素(crustaceanhyperglycemichormone，CHH)共同调控着Y器官内固醇类激素的分泌合成能力，进而对蜕皮过程起到调节作用(Chung,J.S.,Webster,S.G.,2003.Moultcycle-relatedchangesinbiologicalactivityofmoult-inhibitinghormone(MIH)andcrustaceanhyperglycaemichormone(CHH)inthecrab,Carcinusmaenas.EurJBiochem270,3280-3288.)。
蜕皮激素(eclosionhormone，EH)是一类蛋白类激素，在昆虫中的研究发现EH的表达受到20E的调控，并在蜕皮前期诱发其蜕皮程序，在昆虫蜕皮过程中发挥重要作用(Clark,A.C.,delCampo,M.L.,Ewer,J.,2004.NeuroendocrinecontroloflarvalecdysisbehaviorinDrosophila:complexregulationbypartiallyredundantneuropeptides.JNeurosci24,4283-4292.)。本发明在脊尾白虾中获得一条蜕皮激素基因EcEH，利用体外转录方法获得了基于该基因的双链RNA(dsRNA)，通过对脊尾白虾蜕皮前期的个体注射该dsRNA，可以有效控制脊尾白虾的蜕皮进程。
发明内容
本发明描述了一种脊尾白虾蜕皮激素基因及其在蜕皮调控中的应用。
本发明的技术方案如下：
利用转录组分析方法获得了脊尾白虾蜕皮激素基因EcEH，具有SEQIDNo.1所示的核苷酸序列，其序列特征为：
SEQIDNo.1：
CCCCTTTCAACATCGGCGCACCGCTCCTCAACTCTAAAGGTCAACATTTGCGGTCCATTTCTATCTAAGCTCTACCCATATTGAAAACAGAATGTTTGTTTCCAGAAAGGCTGTATCTTCATCCCTGCTGGTCTTAAGCATTTTGCTCGCACTGTTTCTGGTCAGGGCGCATGCGCTTTCCATTGACGTCACGAGGAAGGTCGGAATCTGCATTGACAACTGCGGTCACTGCAAGGAAATGTACCACGATTACTTCAAAGGGGGCCTCTGCGCCGAATTCTGCCAGAAGCTCCGAGGACGCCTCATCATCGACTGCGGCGACCCCCATACGCTTCTGCCCTTCTTCCTTGAGAGGCTCGAGTGATCGTTGAAGAATGAATCACGTTATTTTCAAAGTGGGAAAAACATATCAGAAGATGGCATAACCAAATGTTGACCAGGCCCTAAGTT
所述的脊尾白虾蜕皮激素基因EcEH的应用，利用基于该基因核苷酸序列进行体外合成的双链RNA注射蜕皮前期的脊尾白虾，达到控制脊尾白虾蜕皮过程的目的。
所述的脊尾白虾蜕皮激素基因EcEH双链RNA的获取，提取脊尾白虾总RNA，反转录获得cDNA，基于EcEH基因核苷酸序列设计PCR引物EcEH-dsF(TAATACGACTCACTATAGGGCACCGCTCCTCAACTCTAAA)和EcEH-dsR(TAATACGACTCACTATAGGGGGGCCTGGTCAACATTTGGT)，用脊尾白虾cDNA做模版进行扩增获得PCR产物，利用回收的PCR产物做模版进行体外转录获得EcEH的双链RNA(dsEcEH)。
本发明有如下优点
1、本发明确定了一种脊尾白虾蜕皮激素基因及其在蜕皮调控中的作用。
2、通过本发明可获得有效的调控脊尾白虾蜕皮过程的双链RNA。
附图说明
图1脊尾白虾蜕皮激素基因EcEH的双链RNA电泳检测；
图2注射双链RNA后对脊尾白虾蜕皮过程的影响；
图3注射双链RNA后对脊尾白虾蜕皮率的影响。
具体实施方式
下面结合实施例对本发明作进一步详细说明。
一种脊尾白虾蜕皮激素基因EcEH，其序列及来源序列信息如下：
SEQIDNo.1的信息
(a)序列特征
*长度：450碱基对
*类型：核苷酸
*链型：双链
*拓扑结构：线性
(b)分子类型：核酸
序列描述：SEQIDNo.1
CCCCTTTCAACATCGGCGCACCGCTCCTCAACTCTAAAGGTCAACATTTGCGGTCCATTTCTATCTAAGCTCTACCCATATTGAAAACAGAATGTTTGTTTCCAGAAAGGCTGTATCTTCATCCCTGCTGGTCTTAAGCATTTTGCTCGCACTGTTTCTGGTCAGGGCGCATGCGCTTTCCATTGACGTCACGAGGAAGGTCGGAATCTGCATTGACAACTGCGGTCACTGCAAGGAAATGTACCACGATTACTTCAAAGGGGGCCTCTGCGCCGAATTCTGCCAGAAGCTCCGAGGACGCCTCATCATCGACTGCGGCGACCCCCATACGCTTCTGCCCTTCTTCCTTGAGAGGCTCGAGTGATCGTTGAAGAATGAATCACGTTATTTTCAAAGTGGGAAAAACATATCAGAAGATGGCATAACCAAATGTTGACCAGGCCCTAAGTT
所述脊尾白虾总RNA提取、蜕皮激素基因EcEH的获得、cDNA反转录、双链RNA的合成和作用分析：
1、脊尾白虾总RNA提取
利用Unizol试剂(上海博星公司)提取脊尾白虾的总RNA。步骤如下：
1)将脊尾白虾头胸部组织在液氮中研碎后加入匀浆器中，100mg组织中加入1mLUnizol，匀浆5-10s，使组织彻底裂解；
2)把匀浆液转移到1.5mL离心管中，4℃，10000rpm离心10min，除去不溶性细胞碎片等；
3)将上清转移到新的1.5mL离心管中，室温放置5min，充分裂解核蛋白复合体，变性蛋白；
4)每1mLUnizol加入0.2mL氯仿，剧烈振荡15s，室温静置2-3min，4℃，12000rpm离心15min；
5)小心转移上层水相至新的1.5mL离心管中，加入等体积的异丙醇，缓慢颠倒混匀溶液后，室温放置15min，4℃，12000rpm离心15min；
6)弃上清，将离心管倒置，流尽液体后，加入1mL75％乙醇，将沉淀悬起进行洗涤，之后4℃，7500rpm离心10min；
7)弃上清，将离心管倒置于吸水纸上，自然干燥RNA沉淀，加入适量的无RNase水溶解RNA，-80℃存放。
8)总RNA的定量检测：取1μL总RNA，加入99μLDEPC水，将其稀释100倍，利用紫外分光光度计测定RNA的OD值；
9)总RNA完整性检测：取1μL总RNA，加入4μLDEPC水及0.5μLloadingbuffer，1.2％琼脂糖凝胶电泳，100V，约30min，检测RNA的电泳条带。
2、EcEH基因的获得
1)提取的总RNA用带有Oligo(dT)的磁珠富集mRNA，富集的mRNA加入fragmentationbuffer打断成短片段；
2)以mRNA为模板，用六碱基随机引物(randomhexamers)合成一链cDNA，然后加入缓冲液、dNTPs和DNApolymeraseI和RNaseH合成二链cDNA，再用AMPureXPbeads纯化双链cDNA；
3)纯化的双链cDNA进行末端修复、加A尾并连接测序接头，再用AMPureXPbeads进行片段大小选择，最后进行PCR扩增，并用AMPureXPbeads纯化PCR产物，得到最终的文库。
4)库检合格后，把不同文库按照有效浓度及目标下机数据量的需求pooling后进行IlluminaHiSeq/MiSeq测序；
5)测序得到的原始序列去除接头序列和低质量序列(质量值Qphred<＝5的碱基数占整个reads的50％以上的测序序列)；
6)采用Trinity(GrabherrMG,HaasBJ,YassourM,etal.,2011.Full-lengthtranscriptomeassemblyfromRNA-Seqdatawithoutareferencegenome.NatureBiotechnology29,644-652.)对处理后的序列进行拼接组装，获得组装序列；
7)获得的拼接序列通过与NCBI官方的蛋白序列数据库进行比对注释，获得基因序列的功能注释信息；
8)根据脊尾白虾转录组序列的功能注释信息，获得一条注释为蜕皮激素的核苷酸序列EcEH。
3、cDNA反转录
应用六聚体随机引物反转录合成第一链cDNA。每50μL反应体系加入mRNA1μg，反应体系如下：
轻轻混匀，离心，37℃孵育1.5h，95℃变性5min，冰上冷却3min，将合成的cDNA模板-20℃保存用于双链RNA体外合成实验。
4、双链RNA的合成
1)根据脊尾白虾蜕皮激素EcEH的核苷酸序列(SEQIDNo.1)设计扩增引物EcEH-dsF(TAATACGACTCACTATAGGGCACCGCTCCTCAACTCTAAA)和EcEH-dsR(TAATACGACTCACTATAGGGGGGCCTGGTCAACATTTGGT)；
2)以合成的cDNA为模版，用引物EcEH-dsF和EcEH-dsR按以下体系进行PCR扩增：
PCR程序如下：
3)按照胶回收试剂盒(货号：DP209；生产厂家：天根生化科技(北京)有限公司)的步骤回收PCR产物；
4)以回收的PCR产物为模版，按照体外转录试剂盒(货号：K0441；生产厂家：ThermoFisherScientific)的步骤体外合成EcEH基因的双链RNA；
5)合成的双链RNA经琼脂糖检测后(图1)存于-20℃，用于后续实验。
5、注射双链RNA对脊尾白虾蜕皮的作用分析
1)对处于蜕皮前早期的5尾脊尾白虾(体长：3.9±0.5cm)按每尾1μg双链RNA的剂量进行注射，同时用处于蜕皮前早期的5尾脊尾白虾做对照。注射两天后用显微镜观察脊尾白虾的尾扇，发现注射双链RNA后的脊尾白虾蜕皮进程仍处于蜕皮前早期，而未注射双链RNA的对照组脊尾白虾已发育至蜕皮前中期(图2)，结果显示EcEH的双链RNA明显抑制了脊尾白虾的蜕皮进程。
2)对处于蜕皮前早期的60尾脊尾白虾(体长：3.9±0.5cm)按每尾1μg双链RNA的剂量进行注射，同时用处于蜕皮前早期的60尾脊尾白虾做对照。注射后1-6天，统计每天脊尾白虾蜕皮率。结果显示，至注射后第6天，双链RNA注射组的蜕皮率为4.9％，而未注射双链RNA的对照组脊尾白虾蜕皮率达到20.7％(图3)。
Claims (5)
1.一种脊尾白虾蜕皮激素基因EcEH，具有序列列表中SEQIDNo.1所示的核苷酸序列。
2.按照权利要求1所述的脊尾白虾蜕皮激素基因EcEH，其特征在于：
SEQIDNo.1：
CCCCTTTCAACATCGGCGCACCGCTCCTCAACTCTAAAGGTCAACATTTGCGGTCCATTTCTATCTAAGCTCTACCCATATTGAAAACAGAATGTTTGTTTCCAGAAAGGCTGTATCTTCATCCCTGCTGGTCTTAAGCATTTTGCTCGCACTGTTTCTGGTCAGGGCGCATGCGCTTTCCATTGACGTCACGAGGAAGGTCGGAATCTGCATTGACAACTGCGGTCACTGCAAGGAAATGTACCACGATTACTTCAAAGGGGGCCTCTGCGCCGAATTCTGCCAGAAGCTCCGAGGACGCCTCATCATCGACTGCGGCGACCCCCATACGCTTCTGCCCTTCTTCCTTGAGAGGCTCGAGTGATCGTTGAAGAATGAATCACGTTATTTTCAAAGTGGGAAAAACATATCAGAAGATGGCATAACCAAATGTTGACCAGGCCCTAAGTT。
3.一种权利要求1所述的脊尾白虾蜕皮激素基因EcEH的应用，其特征在于：
基于脊尾白虾蜕皮激素基因EcEH序列获得脊尾白虾体外转录的双链RNA，可作为脊尾白虾蜕皮抑制剂，抑制其蜕皮进程，到达控制脊尾白虾蜕皮的作用。
4.按照权利要求3所述的应用，其特征在于：
基于该基因序列获得其体外转录的双链RNA，注射蜕皮前期的脊尾白虾，可明显抑制其蜕皮进程，到达控制脊尾白虾蜕皮的作用。
5.按照权利要求3或4所述的应用，其特征在于：所述的双链RNA的获取过程为，提取脊尾白虾总RNA，反转录获得cDNA，基于EcEH基因核苷酸序列设计PCR引物EcEH-dsF(TAATACGACTCACTATAGGGCACCGCTCCTCAACTCTAAA)和EcEH-dsR(TAATACGACTCACTATAGGGGGGCCTGGTCAACATTTGGT)，用脊尾白虾cDNA做模版进行扩增获得PCR产物，利用回收的PCR产物做模版进行体外转录获得EcEH的双链RNA(dsEcEH)。
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