CN109402169A - 一种spo11基因的敲除方法 - Google Patents
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Abstract
本发明公开了一种spo11基因的敲除方法,利用CRISPR/Cas9技术对减数***相关基因spo11进行敲除。通过本发明的方法,通过敲除spo11基因,继而可以特异性获得雄性不育个体,可以通过建立敲除家系。
Description
技术领域
本发明涉及一种spo11基因的敲除方法,属于分子生物学领域。
背景技术
spo11是Ⅱ型拓扑异构酶的同源蛋白,参与DNA双链断裂复合物(DSBs)的形成,在减数***同源重组的发生中发挥重要功能。在大多数有性生殖的生物的减数***中,重组扮演着至关重要的角色,它是遗传多样性的重要来源,能够产生单倍体的配子,同时姐妹染色单体交叉互换也增加了配子的多样性。发明人在多年的实验中,发现敲除一个减数***相关基因spo11,导致斑马鱼雄性特异性不育,雌性几乎不受影响,而目前还没有人报道基因spo11具有这种功能。
发明内容
针对上述现有技术存在的不足,本发明的目的是提供一种spo11基因的敲除方法。
为实现上述目的,本发明采用的技术方案是:spo11基因的敲除方法,其特征在于,利用CRISPR/Cas9技术对减数***相关基因spo11进行敲除,具体操作如下:
(1)通过ensembl在线数据库查找斑马鱼spo11基因组序列no:ENSDART00000005373.9,在该基因第一个外显子上设计敲除靶位点,靶位点序列为SEQ ID NO:1:TGGAAACGGTCGACAGATGCCAGG;
(2)按常规方法检测靶位点是否存在单核苷酸多态性;
(3)gRNA的获得:
以gRNA-plasmid为模板,以gRNA-F/gRNA-R为引物,gRNA-F的序列为SEQ ID NO:2:TAATACGACTCACTATAGGGTGGAAACGGTCGACAGATGCCGTTTTAGAGCTAGAAATAAG;gRNA-R的序列为SEQID NO:3:AGCACCGACTCGGTGCCACT,利用KOD Plus(TAKARA)高保真酶进行PCR扩增,扩增产物割胶回收,回收产物连接pMD18-T载体(TAKARA)后转化感受态大肠杆菌,挑取5个大肠杆菌单克隆扩大培养后提取质粒DNA,然后质粒DNA测序进行序列验证,序列正确克隆扩大培养后提取质粒DNA作为下一步转录gRNA的模板;再利用MEGAscript® T7 Transcription Kit(InvitrogenTM)将构建好的含有spo11靶序列的质粒体外逆转录20ul反应体系如下:
10x Reaction Buffer 2ul;
Plasmid DNA 4ul (约1ug);
T7 Enzyme 2ul;
10mmol/L NTP 1ul;
DEPC water 11ul;
以上体系37℃反应一个小时后纯化备用;
(4)体外转录获得Cas9 mRNA :
Cas9质粒来自AddgeneTM(#63154),使用MAXIscript®T7/T3 Transcription Kit(InvitrogenTM),合成Cas9 mRNA,体系及反应条件同步骤3;
(5)Cas9 mRNA和gRNA显微共注射:首先配置显微注射体系如下:
Cas9 mRNA 300 ng/ul;
gRNA 30ng/ul;
Phenol-red 0.2ul;
DEPC Water up to 2ul;
将上述溶液配好混匀后,显微注射单细胞时期的斑马鱼受精卵中,养殖24小时后检测spo11基因的突变效率;
(6)spo11 F1代杂合突变个体的筛选及突变类型确定:
经过步骤(5)显微注射后斑马鱼受精卵,养殖3个月至性成熟后,与野生型个体杂交获得杂交F1代,F1代个体养殖2个月后,剪取部分尾鳍组织,提取基因组DNA,以F-spo11/R-spo11为引物,F-spo11:5’-ATGGCTTACCAGTTTACTG-3’; R-spo11: 5’-CGTAGCTCTTTCAGTAACTC-3’,利用KOD PLUS高保真酶(TAKARA)进行PCR扩增,PCR产物连接pMD18-T载体(TAKARA)后转化感受态大肠杆菌,分别挑取10个大肠杆菌单克隆扩大培养后提取质粒DNA,然后质粒DNA测序比对后筛选获得10条F1代个体突变效率和突变类型;
(7)spo11 F2代纯合突变个体的获得:
待spo11 F1代杂合突变个体性成熟后,选取突变类型一致的雌雄F1代个体各一条进行人工受精,获得spo11 F2代纯合突变个体;
(8)雄性不育雄性个体的选取:
spo11 F2代纯合突变群体中所有的雄性个体均为雄性不育个体,但是雌性个体中有卵子,且与野生雄鱼能够正常受精。
与现有技术相比,本发明的有益效果:通过本发明的方法,可以特异性获得雄性不育个体,可以通过建立敲除家系,源源不断提供雄性不育个体,操作简单,节省时间。
附图说明
图1为本发明涉及到的相关靶位点、突变类型及靶位点突变基因序列所对应的氨基酸变化,其中A为CRISPR/Cas9技术作用靶位点;B为靶位点序列突变检测(红色虚线所框为建立家系所选突变类型);C为靶位点突变基因序列所对应的氨基酸变化(红色虚线所框为建立家系所选氨基酸突变型);
图2为雌鱼性腺组织显微镜放大图,其中A为对照性成熟雌鱼性腺组织中***,B为spo11性成熟雌鱼性腺组织中***;
图3为雄鱼性腺组织显微镜放大图,其中A为对照性成熟雄鱼性腺组织(存在***),B为spo11性成熟雄鱼性腺组织(无***)。
具体实施方式
现结合具体实施例来说明本发明的技术方案和技术效果,但并不是限制本发明保护范围的依据。
实施例一
鱼类雄性不育模型的建立方法,是利用CRISPR/Cas9技术对减数***相关基因spo11进行敲除,spo11的序列号为no:ENSDART00000005373.9;具体步骤如下:
(1)通过ensembl在线数据库查找斑马鱼spo11基因组序列no:ENSDART00000005373.9,在该基因第一个外显子上设计敲除靶位点,靶位点序列为:TGGAAACGGTCGACAGATGCC[AGG],中括号内为PAM序列。
(2)按常规方法检测靶位点是否存在单核苷酸多态性(SNP):
随机选取6条斑马鱼分别提取尾鳍组织的基因组DNA,以此为PCR模板,以F-spo11/R-spo11为引物,F-spo11:5’-ATGGCTTACCAGTTTACTG-3’; R-spo11: 5’-CGTAGCTCTTTCAGTAACTC-3’,利用KOD PLUS高保真酶(TAKARA)进行PCR扩增,PCR产物连接pMD18-T载体(TAKARA)后转化感受态大肠杆菌,分别挑取5个大肠杆菌单克隆扩大培养后提取质粒DNA,然后质粒DNA测序比对后发现所选靶位点中无SNP位点。
(3)gRNA的获得:以gRNA-plasmid为模板,以gRNA-F/gRNA-R为引物,gRNA-F:TAATACGACTCACTATAGGGTGGAAACGGTCGACAGATGCCGTTTTAGAGCTAGAAATAAG;gRNA-R:AGCACCGACTCGGTGCCACT,利用KOD Plus(TAKARA)高保真酶进行PCR扩增,扩增产物割胶回收,回收产物连接pMD18-T载体(TAKARA)后转化感受态大肠杆菌,挑取5个大肠杆菌单克隆扩大培养后提取质粒DNA,然后质粒DNA测序进行序列验证,序列正确克隆扩大培养后提取质粒DNA作为下一步转录gRNA的模板;再利用MEGAscript® T7 Transcription Kit(InvitrogenTM)将构建好的含有spo11靶序列的质粒体外逆转录20ul反应体系如下:
10x Reaction Buffer 2ul;
Plasmid DNA 4ul (约1ug);
T7 Enzyme 2ul;
10mmol/L NTP 1ul;
DEPC water 11ul;
以上体系37℃反应一个小时后纯化备用。
(4)体外转录获得Cas9 mRNA :
Cas9质粒来自AddgeneTM(#63154),使用MAXIscript®T7/T3 Transcription Kit(InvitrogenTM),合成Cas9 mRNA,体系及反应条件同步骤3。
(5)Cas9 mRNA和gRNA显微共注射:首先配置显微注射体系如下:
Cas9 mRNA 300 ng/ul;
gRNA 30ng/ul;
Phenol-red 0.2ul;
DEPC Water up to 2ul;
将上述溶液配好混匀后,显微注射单细胞时期的斑马鱼受精卵中,养殖24小时后检测spo11基因的突变效率。方法如下:随机选取8个胚胎提取基因组DNA,以F-spo11/R-spo11为引物,F-spo11:5’-ATGGCTTACCAGTTTACTG-3’; R-spo11: 5’-CGTAGCTCTTTCAGTAACTC-3’,利用KOD PLUS高保真酶(TAKARA)进行PCR扩增,PCR产物连接pMD18-T载体(TAKARA)后转化感受态大肠杆菌,分别挑取20个大肠杆菌单克隆扩大培养后提取质粒DNA,然后质粒DNA测序比对后获得突变效率。
(6)spo11 F1代杂合突变个体的筛选及突变类型确定:
经过步骤(5)显微注射后斑马鱼受精卵,养殖3个月至性成熟后,与野生型个体杂交获得杂交F1代,F1代个体养殖2个月后,剪取部分尾鳍组织,提取基因组DNA,以F-spo11/R-spo11为引物,F-spo11:5’-ATGGCTTACCAGTTTACTG-3’; R-spo11: 5’-CGTAGCTCTTTCAGTAACTC-3’,利用KOD PLUS高保真酶(TAKARA)进行PCR扩增,PCR产物连接pMD18-T载体(TAKARA)后转化感受态大肠杆菌,分别挑取10个大肠杆菌单克隆扩大培养后提取质粒DNA,然后质粒DNA测序比对后筛选获得10条F1代个体突变效率和突变类型。
(7)spo11 F2代纯合突变个体的获得:
待spo11 F1代杂合突变个体性成熟后,选取突变类型一致的雌雄F1代个体各一条进行人工受精,获得spo11 F2代纯合突变个体。选择附图1B中标出的突变类型,该突变类型在突变位点附近产生了终止密码,即突变个体中只能产生spo11突变位点之前序列对应的氨基酸。
(8)雄性不育雄性个体的选取:
spo11 F2代纯合突变群体中所有的雄性个体均为雄性不育个体,如附图2,spo11 F2代纯合突变雄性个体***中无***,但是雌性个体中有卵子,且与野生雄鱼能够正常受精。
序列1
<110>湖南文理学院
<120>一种spo11基因的敲除方法
<160>3
<210> 1
<211>24
<212>DNA
<213>人工合成
<400>1
TGGAAACGGT CGACAGATGC CAGG 24
序列2
<110>湖南文理学院
<120>一种spo11基因的敲除方法
<160>3
<210> 2
<211>61
<212>RNA
<213>人工合成
<400>2
TAATACGACT CACTATAGGG TGGAAACGGT CGACAGATGC CGTTTTAGAG CTAGAAATAA 60
G 61
序列3
<110>湖南文理学院
<120>一种spo11基因的敲除方法
<160>3
<210> 3
<211>20
<212>RNA
<213>人工合成
<400>3
AGCACCGACT CGGTGCCACT 20
Claims (1)
1.一种spo11基因的敲除方法,其特征在于,利用CRISPR/Cas9技术对减数***相关基因spo11进行敲除,具体操作如下:
(1)通过ensembl在线数据库查找斑马鱼spo11基因组序列no:ENSDART00000005373.9,在该基因第一个外显子上设计敲除靶位点,靶位点序列为:TGGAAACGGTCGACAGATGCCAGG;
(2)按常规方法检测靶位点是否存在单核苷酸多态性;
(3)gRNA的获得:
以gRNA-plasmid为模板,以gRNA-F/gRNA-R为引物,gRNA-F:TAATACGACTCACTATAGGGTGGAAACGGTCGACAGATGCCGTTTTAGAGCTAGAAATAAG;gRNA-R:AGCACCGACTCGGTGCCACT,利用KODPlus(TAKARA)高保真酶进行PCR扩增,扩增产物割胶回收,回收产物连接pMD18-T载体(TAKARA)后转化感受态大肠杆菌,挑取5个大肠杆菌单克隆扩大培养后提取质粒DNA,然后质粒DNA测序进行序列验证,序列正确克隆扩大培养后提取质粒DNA作为下一步转录gRNA的模板;再利用MEGAscript® T7 Transcription Kit (InvitrogenTM)将构建好的含有spo11靶序列的质粒体外逆转录20ul反应体系如下:
10x Reaction Buffer 2ul;
Plasmid DNA 4ul (约1ug);
T7 Enzyme 2ul;
10mmol/L NTP 1ul;
DEPC water 11ul;
以上体系37℃反应一个小时后纯化备用;
(4)体外转录获得Cas9 mRNA :
Cas9质粒来自AddgeneTM(#63154),使用MAXIscript®T7/T3 Transcription Kit(InvitrogenTM),合成Cas9 mRNA,体系及反应条件同步骤3;
(5)Cas9 mRNA和gRNA显微共注射:首先配置显微注射体系如下:
Cas9 mRNA 300 ng/ul;
gRNA 30ng/ul;
Phenol-red 0.2ul;
DEPC Water up to 2ul;
将上述溶液配好混匀后,显微注射单细胞时期的斑马鱼受精卵中,养殖24小时后检测spo11基因的突变效率;
(6)spo11 F1代杂合突变个体的筛选及突变类型确定:
经过步骤(5)显微注射后斑马鱼受精卵,养殖3个月至性成熟后,与野生型个体杂交获得杂交F1代,F1代个体养殖2个月后,剪取部分尾鳍组织,提取基因组DNA,以F-spo11/R-spo11为引物,F-spo11:5’-ATGGCTTACCAGTTTACTG-3’; R-spo11: 5’-CGTAGCTCTTTCAGTAACTC-3’,利用KOD PLUS高保真酶(TAKARA)进行PCR扩增,PCR产物连接pMD18-T载体(TAKARA)后转化感受态大肠杆菌,分别挑取10个大肠杆菌单克隆扩大培养后提取质粒DNA,然后质粒DNA测序比对后筛选获得10条F1代个体突变效率和突变类型。
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CN118127040A (zh) * | 2024-05-06 | 2024-06-04 | 中国农业科学院生物技术研究所 | 从蒺藜苜蓿中分离的spo11-1基因及其编码蛋白和应用 |
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CN118127040A (zh) * | 2024-05-06 | 2024-06-04 | 中国农业科学院生物技术研究所 | 从蒺藜苜蓿中分离的spo11-1基因及其编码蛋白和应用 |
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