CN114891833A - Gnaq基因敲除系赤眼鳟模型构建与应用 - Google Patents
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Abstract
本发明属于生物基因技术领域,公开了一种GNAQ基因敲除系赤眼鳟模型构建与应用,获得赤眼鳟的GNAQ基因的基因组信息,确定scGNAQ基因的内含子和外显子的序列;设计scGNAQ基因的敲除靶位点以及靶位点的gRNA引物scGNAQ‑gRNA‑F/R;获得带有T7启动子的scGNAQ基因靶位点的gRNA片段;获得scGNAQ的gRNAmRNA和cas9mRNA;将gRNAmRNA和cas9mRNA共注射进单细胞期的赤眼鳟胚胎,性成熟后通过两代的杂交与自交的方法筛选获得可稳定遗传的GNAQ基因敲除的赤眼鳟模型。本发明为深入研究赤眼鳟GNAQ在性腺发育及性成熟过程中的功能提供了实用的活体模型。
Description
技术领域
本发明属于生物基因技术领域,尤其涉及一种GNAQ基因敲除系赤眼鳟模型构建与应用。
背景技术
目前,赤眼鳟(Squaliobarbus curriculus),与草鱼同属雅罗鱼亚科,又称野草鱼,然而赤眼鳟与草鱼的性成熟年龄相差甚远:赤眼鳟性成熟早,初次性成熟年龄为2冬龄,而草鱼初次性成熟年龄为5~6年。
GNAQ是参与GnRH信号通路中一个关键分子,编码Gaq蛋白,Gαq在神经内分泌系统中的下丘脑-垂体-性腺轴可调节促性腺激素LH和FSH的分泌,GNAQ被认为与性腺发育相关,例如GNAQ敲除系小鼠表现出生殖缺陷,约40%的雄性小鼠不育,而雌性小鼠则不能自发排卵。鱼类性腺分化和性成熟机制十分复杂,鱼类性腺分化和性成熟相关基因的研究有较大进展,但相关分子调控网络机制还不够清晰,通过构建GNAQ基因敲除系的赤眼鳟模型来研究GNAQ基因在鱼类性腺分化及性成熟过程中的功能。
通过上述分析,现有技术存在的问题及缺陷为:现有鱼类性腺分化和性成熟机制十分复杂,但相关分子调控网络机制还不够清晰。
解决以上问题及缺陷的难度为:目前尚未有网站公布赤眼鳟的全基因序列,增大获得scGNAQ的基因组序列的难度;赤眼鳟胚胎小吸水膨胀后体积极大增加并且发育太快,增加注射难度和强度;赤眼鳟胚胎成活率不高,显微注射以后胚胎的成活率更低。
解决以上问题及缺陷的意义为:通过运用赤眼鳟全基因测序技术、RNA提取、cDNA反转录及PCR测序手段获得scGNAQ的基因组序列,包括外显子,内含子等,大大减小了设计scGNAQ基因敲除靶位点的困难,为本发明的实现提供极大的便利;通过将单细胞胚胎放置于冰水浴上,同时通过调节注射房室内温度至20摄氏度,以增加单细胞胚胎的可注射时间,可成功将注射时间延长至50min,为本发明中的后期F0代赤眼鳟的筛选工作提供更多的胚胎供应;通过多年摸索的赤眼鳟繁殖技术,分批次成功繁殖了10对赤眼鳟亲本,并获得可注射用胚胎,通过流水孵化技术增加了胚胎的孵化成活率,最终获得了300尾左右的成活个体,为本发明中突变筛选提供大量的的个体选择。
发明内容
针对现有技术存在的问题,本发明提供了一种GNAQ基因敲除系赤眼鳟模型构建与应用,尤其涉及一种应用CRISPR/cas9技术的构建GNAQ基因敲除系赤眼鳟的方法。
本发明是这样实现的,一种GNAQ基因敲除系赤眼鳟模型构建方法,所述GNAQ基因敲除系赤眼鳟模型构建方法包括以下步骤:
步骤一,在未开始进行敲除工作前,通过全基因组测序技术及PCR技术获得赤眼鳟的GNAQ基因的基因组信息,确定GNAQ基因的内含子和外显子的序列,为后续的靶点设计提供准确的序列依据;
步骤一是最关键的一步,决定本实验的工作开展;
步骤二,得到准确的基因组序列后,利用软件设计scGNAQ基因的敲除靶位点,同时设计好靶位点的gRNA引物scGNAQ-gRNA-F/R,为下一步的靶位点片段扩增提供引物;
步骤三,利用PCR获得带有T7启动子的scGNAQ基因靶位点的gRNA片段;作为下一步的体外转录的模板;
步骤四,利用酶切及体外转录手段获得scGNAQ的gRNA mRNA和cas9mRNA;
步骤二、三、四是本发明的主要实施步骤,决定了赤眼鳟的敲除效果;
步骤五,设计靶位点前后200bp的基因组筛选引物scGNAQ-screen-F/R,并通过PCR确定引物成功扩增scGNAQ靶位点附近的序列,用于赤眼鳟scGNAQ基因敲除类型的鉴定;
步骤六,繁殖季节时确定可用于繁殖的赤眼鳟亲本,通过人工催产的方式进行赤眼鳟的人工繁殖,获得单细胞期的赤眼鳟胚胎,具体操作如下:采用地欧酮(DOM)(3-5mg/Kg鱼)与促黄体素释放激素A2(LHRH-A2)(5mg/Kg鱼)联合使用注射,约经过12h-16h的效应时间后,开始进行人工授精产卵,获得可用于注射的赤眼鳟胚胎;
这个步骤非常关键,一年只有两个月可以做赤眼鳟的繁殖,错过需再等一年,因此需要由多年的赤眼鳟繁殖经验才可完成;
步骤七,通过改善条件,延长赤眼鳟胚胎的单细胞时间,将gRNAmRNA和cas9 mRNA共注射进单细胞期的赤眼鳟胚胎,性成熟后通过与野生型赤眼鳟杂交的方法筛选获得可稳定遗传的scGNAQ基因敲除的赤眼鳟;这个步骤的显微注射是本发明中最关键的技术之一;注射时间的延长以及注射技术与否决定了赤眼鳟的敲除效率和孵化成活率。
进一步,步骤一中,所述赤眼鳟的GNAQ基因为scGNAQ。
进一步,步骤六中,所述繁殖季节为每年的5月底至~7月初。
进一步,步骤七中,所述赤眼鳟胚胎至少为5000颗胚胎。
本发明的另一目的在于提供一种应用所述的GNAQ基因敲除系赤眼鳟模型构建方法构建得到的赤眼鳟模型。
本发明的另一目的在于提供一种所述的赤眼鳟模型在确定GNAQ基因在鱼类性腺分化及性成熟过程中功能的应用。
结合上述的所有技术方案,本发明所具备的优点及积极效果为:通过草鱼与赤眼鳟的基因组比较分析,在GnRH通路中筛选出了一个功能候选基因——鸟嘌呤核苷酸结合蛋白G(q)α亚基(guanine nucleotide-binding protein G(q)subunit alpha,GNAQ),本发明通过全基因组测序的方法获得了赤眼鳟GNAQ的基因组序列,并结合多年的赤眼鳟繁殖经验及改良的胚胎显微注射方法成功建立了非模式生物——赤眼鳟GNAQ敲除系的构建方法。本发明具备的优点在于团队经过多年的努力获得了赤眼鳟的全基因组序列以及团队多年来从事的赤眼鳟繁殖经验为本发明的实现奠定了最重要的基础,改良的胚胎显微注射技术为基因敲除在赤眼鳟中的应用提供了极大便利。
本发明通过一系列的摸索工作,最终通过应用CRISPR/cas9技术确定了一套构建GNAQ基因敲除的赤眼鳟敲除系的方法。通过一系列操作,本发明可获得scGNAQ基因功能缺失的赤眼鳟活体,为深入研究赤眼鳟GNAQ在性腺发育及性成熟过程中的功能提供了实用的活体模型。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对本发明实施例中所需要使用的附图做简单的介绍,显而易见地,下面所描述的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的GNAQ基因敲除系赤眼鳟模型构建方法流程图。
图2是本发明实施例提供的赤眼鳟胚胎显微注射示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明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发明,并不用于限定本发明。
针对现有技术存在的问题,本发明提供了一种GNAQ基因敲除系赤眼鳟模型构建与应用,下面结合附图对本发明作详细的描述。
如图1所示,本发明实施例提供的GNAQ基因敲除系赤眼鳟模型构建方法包括以下步骤:
S101,在未开始进行敲除工作前,通过赤眼鳟的全基因组测序技术及PCR技术获得赤眼鳟的GNAQ基因的基因组信息,确定GNAQ基因的内含子和外显子的序列;
S102,得到准确的基因组序列后,利用软件设计scGNAQ基因的敲除靶位点,同时设计好靶位点的gRNA引物scGNAQ-gRNA-F/R;
S103,利用PCR获得带有T7启动子的scGNAQ基因靶位点的gRNA片段;
S104,利用酶切及体外转录手段获得scGNAQ的gRNAmRNA和cas9 mRNA;
S105,设计靶位点前后200bp的基因组筛选引物scGNAQ-screen-F/R,并通过PCR确定引物成功扩增scGNAQ靶位点附近的序列;
S106,繁殖季节时确定可用于繁殖的赤眼鳟亲本,通过人工催产的方式进行赤眼鳟的人工繁殖,获得单细胞期的赤眼鳟胚胎;
S107,将gRNAmRNA和cas9 mRNA共注射进单细胞期的赤眼鳟胚胎,性成熟后通过与野生型赤眼鳟杂交的方法筛选获得可稳定遗传的scGNAQ基因敲除的赤眼鳟。
下面结合具体实施例对本发明的技术方案作进一步描述。
本发明利用基因编辑方法构建GNAQ基因敲除的赤眼鳟模型,并利用此模型研究草鱼与赤眼鳟性成熟差异现象的分子机制,探讨鱼类性腺发育的机理。
本发明通过一系列的摸索工作,最终通过应用CRISPR/cas9技术确定了一套构建GNAQ基因敲除的赤眼鳟敲除系的方法。
工作过程如下:
1.在未开始进行敲除工作前,本发明首先通过赤眼鳟的全基因组测序技术及PCR技术获得赤眼鳟的GNAQ(scGNAQ)的基因组信息,确定GNAQ基因的内含子和外显子的序列;
2.得到准确的基因组序列后,利用软件设计scGNAQ基因的敲除靶位点,同时设计好靶位点的gRNA引物scGNAQ-gRNA-F/R;
3.利用PCR获得带有T7启动子的scGNAQ基因靶位点的gRNA片段;
4.利用酶切及体外转录手段获得scGNAQ的gRNAmRNA和cas9 mRNA;
5.设计靶位点前后约200bp的基因组筛选引物scGNAQ-screen-F/R,并通过PCR确定引物可成功扩增scGNAQ靶位点附近的序列,以确保可用于后续敲除系的筛选;
6.繁殖季节(每年的5月底至~7月初)时确定可用于繁殖的赤眼鳟亲本,通过人工催产的方式进行赤眼鳟的人工繁殖,获得单细胞期的赤眼鳟胚胎;
7.将gRNAmRNA和cas9 mRNA共注射进单细胞期的赤眼鳟胚胎(至少5000颗胚胎),性成熟后通过与野生型赤眼鳟杂交的方法筛选获得可稳定遗传的scGNAQ基因敲除的赤眼鳟。
通过以上操作,可获得scGNAQ基因功能缺失的赤眼鳟活体,为深入研究赤眼鳟GNAQ在性腺发育及性成熟过程中的功能提供了实用的活体模型。
下面结合实验对本发明的技术效果作详细的描述。
为证实本发明中的显微注射的改良实用性,附上显微注射的操作图(图2)及赤眼鳟scGNAQ赤眼鳟F0代的敲除突变情况(表1)
表1赤眼鳟GNAQ敲除系F0代突变类型
注:黄底表示pam(NGG)序列;绿底表示靶位点序列;虚线代表缺失碱基;红色代表替换碱基。
以上所述,仅为本发明的具体实施方式,但本发明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发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
<110> 湖南农业大学
<120>GNAQ基因敲除系赤眼鳟模型构建与应用
<160>9
<210>1
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400>1
GGCGTGCTGCCTCAGCGAGGAGGCGAAAGAAGCTCGGCGGATCAAG
<210>2
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400>2
GGCGTGCTGCCTCAGGAGGCGAAAGAAGCGGCGGATCAAG
<210>3
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400>3
GGCGTGCTGCCTCAGCGAAAGAAGGATCAAG
<210>4
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400>4
GGCGTGCTGCCTCAGCGAGGAGGGGAAAGAAGCTTGGCGGATTAAG
<210>5
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400>5
GGGGGAAAGCATCGGATAGGAGGCGAAATAAGCTAGGAGATTAACG
<210>6
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400>6
GGCGGAGGACGTCAAAGAAGGTGGGGAAATAACG
<210>7
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400>7
GGCGTGCTGCCTCAGCGAGGAGGCGAAAGAAGAGA
<210>8
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400>8
GGCGTGGAGGAGGCGAAAGAAGGCGGATCAACG
<210>9
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400>9
GTCCATAATGGTCAGCGACGTGGGGAGGGGAAATAGGGGTATCAGGG
Claims (6)
1.一种GNAQ基因敲除系赤眼鳟模型构建方法,其特征在于,所述GNAQ基因敲除系赤眼鳟模型构建方法包括以下步骤:
步骤一,在未开始进行敲除工作前,通过赤眼鳟的全基因组测序技术及PCR技术获得赤眼鳟的GNAQ基因的基因组信息,确定GNAQ基因的内含子和外显子的序列;
步骤二,得到准确的基因组序列后,利用软件设计scGNAQ基因的敲除靶位点,同时设计好靶位点的gRNA引物scGNAQ-gRNA-F/R;
步骤三,利用PCR获得带有T7启动子的scGNAQ基因靶位点的gRNA片段;
步骤四,利用酶切及体外转录手段获得scGNAQ的gRNA mRNA和cas9mRNA;
步骤五,设计靶位点前后200bp的基因组筛选引物scGNAQ-screen-F/R,并通过PCR确定引物成功扩增scGNAQ靶位点附近的序列;
步骤六,繁殖季节时确定可用于繁殖的赤眼鳟亲本,通过人工催产的方式进行赤眼鳟的人工繁殖,获得单细胞期的赤眼鳟胚胎;
步骤七,将gRNA mRNA和cas9 mRNA共注射进单细胞期的赤眼鳟胚胎,性成熟后通过与野生型赤眼鳟杂交的方法筛选具有效突变的敲除系杂合子,再经过一代的自交便可获得可稳定遗传的scGNAQ基因敲除的赤眼鳟。
2.如权利要求1所述的GNAQ基因敲除系赤眼鳟模型构建方法,其特征在于,步骤一中,所述赤眼鳟的GNAQ基因为scGNAQ。
3.如权利要求1所述的GNAQ基因敲除系赤眼鳟模型构建方法,其特征在于,步骤六中,所述繁殖季节为每年的5月底至~7月初。
4.如权利要求1所述的GNAQ基因敲除系赤眼鳟模型构建方法,其特征在于,步骤七中,所述赤眼鳟胚胎至少为5000颗胚胎。
5.一种应用如权利要求1~4任意一项所述的GNAQ基因敲除系赤眼鳟模型构建方法构建得到的赤眼鳟模型。
6.一种如权利要求5所述的赤眼鳟模型在确定GNAQ基因在鱼类性腺分化及性成熟过程中功能的应用。
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