CN112522299A - 一种利用OsTNC1基因突变获得分蘖增加的水稻的方法 - Google Patents
一种利用OsTNC1基因突变获得分蘖增加的水稻的方法 Download PDFInfo
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Abstract
本发明公开了一种利用OsTNC1基因突变获得分蘖增加的水稻的方法,实质是通过基因编辑控制了水稻分蘖数量的蛋白质,该蛋白来源于稻属水稻石狩白毛(Oryza sativa var.Ishikari‑shiroke),名称为OsTNC1。具体是利用CRISPR/Cas9基因编辑技术对OsTNC1进行编辑从而得到不同基因突变的植株,结果表明基因突变植株相比于对照野生型植株分蘖数量增多。另外本发明阐释了不同突变点与植株分蘖数量之间的关系,实验提供的sgRNA编辑效率较高,有良好的应用前景。
Description
技术领域
本发明属于生物技术领域,具体涉及一种利用OsTNC1基因突变获得分蘖增加的水稻的方法。
背景技术
水稻(Oryza sativa L.)系禾本科、稻属中一种一年生禾本植物,是世界上超过三十亿人口的主要粮食作物,也是研究单子叶植物的模式植物。有研究表明,水稻的株高与分蘖数负相关,而株高与分蘖数量在一定程度上决定了水稻的产量。在对水稻分蘖机制的研究中,已有多个与水稻分蘖相关的基因被克隆。
近年来,随着基因编辑技术的快速发展,各类编辑方法如ZFN、TALEN、CRISPR和BE在水稻基因功能、遗传多样性研究中已有了较多应用。目前CRISPR/Cas***因其操作技术要求简单、专一性好、基因定点突变效率高,已获得广泛研究,并被成功运用于如拟南芥、烟草、番茄、小麦和玉米等多种物种的基因编辑中。CRISPR/Cas***是由CRISPR序列元件和Cas家族组成的,可分为I型、II型和III型。三种类型的***都具有识别和切割与crRNA互补的核苷酸序列的特性,目前的CRISPR/Cas***由II型***改造而来。其中CRISPR/Cas9是目前报道最强大的基因编辑工具,是唯一被优先应用于基因编辑的II型***。CRISPR/Cas9及其优化版本在动物、植物、微生物中均有相关应用,同时在农作物的生产改良方面成果显著
基因编辑技术在水稻抗病研究及新品种选育方面有着巨大的发展前景。关于利用基因编辑技术培育高抗高产水稻是关乎全球粮食安全的战略性议题,具有重大的社会和经济学意义。其中CRISPR/Cas9***在培育高产抗病水稻品种目标,完成到2050年可能达到97亿人口的庞大养活任务中发挥着巨大的作用。未来CRISPR/Cas9技术的抗病研究结合水稻传统育种方式的新型抗病育种形式,可能得到大量的应用与推广,为此可以更加有效、精准、快速地培育优良水稻抗病高产新品种。
发明内容
本发明的目的在于解决现有技术中存在的问题,提供一种利用OsTNC1基因突变获得分蘖增加的水稻的方法。具体提出了一种以CRISPR/Cas9基因编辑技术打靶OsTNC1基因获得分蘖数量改变水稻品系的方法。
本发明的目的通过下述技术方案实现:
本发明所编辑的与植物分蘖数量相关的基因,名称为OsTNC1(Tiller numbercontrol 1)。OsTNC1是一个在水稻育种实践中被广泛应用的株型功能基因。利用CRISPR/Cas9基因编辑技术分别将多分蘖基因各个外显子及内含子处能被识别的序列打靶,然后对基因组序列切割后引发修复,从而产生缺失突变,获得功能缺失的OsTNC1基因;
本发明选用稻属水稻石狩白毛(Oryza sativa var.Ishikari-shiroke)为研究品种,利用CRISPR/Cas9技术对Ishikari-shiroke基因组上的OsTNC1基因进行编辑。在OsTNC1基因序列第一个外显子处设计3个sgRNA位点,分别记为sgRNA-exon1-1、sgRNA-exon1-2和sgRNA-exon1-3(序列如下)(所述序列均是发明人通过创造性劳动筛选获得的,非通过常规软件简单设计获得)。通过CRISPR/Cas9蛋白的定点切割与随机性修复,产生了不同的编辑类型。
下划线所示序列为20bp的sgRNA,加粗碱基为PAM位点
本发明的另一个目的是提供一种使植株分蘖数量增加、株高变矮的转基因方法。
本发明提供的培育分蘖数量增加、株高变矮转基因植物的方法,具体是将上述的编码基因利用CRISPR/Cas9基因编辑技术进行敲除从而得到转基因植物,所述转基因植物的分蘖数量相比于对照野生型植株变多,株高相比于对照野生型植株变矮。
本发明的实验证明,将提供OsTAC1的编码基因在Ishikari-shiroke中敲除后获得的转基因植株,与未敲除该基因的野生型植株相比,株高变矮,分蘖数量大于野生型植株,因此,该基因可应用于植物株型遗传改良等工作。
本研究所设计的sgRNA位点、CRISPR/Cas9编辑产生的不同编辑类型以及突变体分蘖数量的表型特征均属于本发明的保护范围。
本研究阐述的功能弱化型突变和功能丧失型突变这两种突变类型所属含义以及其与分蘖数量之间的对应关系属于本发明的保护范围。
含有所述编码基因的重组载体、重组菌、转基因细胞系或表达盒均属于本发明的保护范围。
附图说明
此处所说明的附图用来提供对本申请的进一步理解,构成本申请的一部分,本申请的示意性实施例及其说明用于解释本申请,并不构成对本申请的不当限定。在附图中:
图1为水稻D3基因结构及CRISPR/Cas9靶位点示意图。
图2为编辑类型和植株表型图。
图3为分蘖个数对比图。
具体实施方式
下述实施例中所使用的方法如无特殊说明均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
举例说明本发明的具体实施过程,使本领域技术人员按照其不需要创造性劳动就能完成该发明即可,实施例的限定不能作为限定发明人保护范围的局限。
实施例1表达载体的构建
以筛选到的靶向核苷酸序列5’-GN19NGG-3’中NGG前的20bp为基础,按照5’-GN19GTTTT-3’,3’-ACACACN19-5’的模板编辑正向和反向引物,送交公司合成(序列如下)。
sgRNA-exon1-1-F GGAGTAGACGTCGCCCATGGGTTTT
sgRNA-exon1-1-R CCATGGGCGACGTCTACTCCACACA
sgRNA-exon1-2-F GTGCCATTTGCAACACTGCAGTTTT
sgRNA-exon1-2-R TGCAGTGTTGCAAATGGCACACACA
sgRNA-exon1-3-F GCGGGACATGCAGTTGCGGGGTTTT
sgRNA-exon1-3-R CCCGCAACTGCATGTCCCGCACACA
(下划线所示序列为20bp的sgRNA)
分别溶解引物F及R引物,并稀释到10μM,各取10μL混合,在PCR仪上退火生成接头,退火条件如下:95℃,3min;22℃,1min;降温速率,0.1℃/s;22℃,∞。
将粘性接头产物与CRISPR/Cas9基因编辑载体Bae I酶切产物用T4连接酶置于25℃反应2h,获得相应CRISPR/Cas9表达载体。随后将连接产物通过热激转化到大肠杆菌热激感受态细胞DH5 α中,均匀地涂布在LB/K+固体培养基上(含卡那霉素50μg/mL),37℃倒置培养过夜。挑取阳性单克隆,提取质粒测序验证,将测序正确含有sgRNA序列的重组载体分别命名为sgRNA-exon1-1、sgRNA-exon1-2、sgRNA-exon1-3。
将sgRNA-exon1-1、sgRNA-exon1-2、sgRNA-exon1-3即构建得到的CRISPR/Cas9表达载体的质粒DNA用电击转入农杆菌EHA105中得到重组体,提取质粒进行测序验证。将测序验证正确的重组菌株分别命名为EHA-sgRNA-exon1-1、EHA-sgRNA-exon1-2、EHA-sgRNA-exon1-3。
实施例2农杆菌介导转化
以EHA-sgRNA-exon1-1为例。将构建得到的CRISPR/Cas9表达载体的农杆菌转入日本晴的愈伤组织中,经遗传转化获得相应的转基因植株。具体方法为:
1)诱导愈伤颗粒:选取饱满的水稻种子,去除颖壳后,用75%酒精消毒2min,30%次氯酸钠消毒30min,并用无菌水清洗3-5次,置于滤纸上,晾干后转到诱导培养基上,光照培养箱28℃培养4-5周,经继代培养获得获得颗粒;
2)农杆菌介导的侵染:将活化的含目的载体的农杆菌菌液悬于AAM培养液中,挑选生长良好的愈伤颗粒置于其中,侵染15min,弃掉菌悬液,置于滤纸上,晾干后转到共培养培养基上,20℃暗培养3天;
3)抗性愈伤的选择:用无菌水将愈伤清洗4-5遍,置于滤纸上晾干,将其整齐摆放在选择培养基上(含潮霉素抗性),光照培养箱28℃,培养2-3周,更换至新的选择培养基,继续培养至其长出颗粒抗性愈伤;
4)抗性愈伤的分化:将颗粒抗性愈伤转至分化培养基,光照培养箱28℃培养,直至分化得到转基因植株,共获得31株T0代tac1-exon1-1转基因水稻植株。
实施例3转基因水稻分子鉴定
在靶序列上下游200bp左右处设计正向引物与反向引物,作为鉴定是否发生突变的特异性引物,用于扩增包含靶序列在内的目的片段。以粗提转基因植株的总DNA为模板,用相应的特异性引物PCR扩增目的片段,将扩增产物送到铂尚生物技术有限公司进行测序,最终通过序列比对鉴定获得的突变植株。
依上所述方法,得到转基因植株数目如表1:
从表1的结果可以看出,3个sgRNA均可以实现tacl的特异性突变效果,总体突变率为29.23%,这也充分说明,在前期申请人付出创造性劳动筛选得到的这3种sgRNA具有本领域常规方法所无以比拟的优势,具有较强的编辑效率。
实施例3转基因水稻表型鉴定
将从阳性T0代转基因植株收获的T1代种子种于大田进行表型分析,以Nip为对照。表型结果如图2所示,3种转基因植物与野生型对照相比,tac1-sgRNA-exon1-1和tac1-sgRNA-exon1-3分蘖数明显上升,株高较对照组明显变低。
本发明中利用CRISPR/Cas9基因编辑技术获得OsTNC1基因,与TALEN和ZFN技术相比,操作过程简单方便,节约成本,一般实验室都可操作,而且用农杆菌介导法将多分蘖基因打靶载体分别导入到二倍体和单倍体细胞组织中,与常规的杂交育种相比,大大节约了时间成本,是一种新的获得多分蘖水稻品系的分子育种方法。
以上所述,仅为本发明较佳的具体实施方式,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,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。此外,虽然本说明书按照实施方式描述,但并非实施方式仅包括一个技术方案,本领域技术人员应将说明书作为一个整体,各实施例中的技术方案可以通过适当重新组合,形成本领域技术人员可以实施方式的方案。
序列表
<110> 浙江万里学院
宁波市农业科学研究院
浙江省农业科学院
<120> 一种利用OsTNC1基因突变获得分蘖增加的水稻的方法
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggagtagacg tcgcccatgg 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gtgccatttg caacactgca 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcgggacatg cagttgcggg 20
Claims (2)
1.一种利用OsTNC1基因突变获得分蘖增加的水稻的方法,其特征在于,以石狩白毛的OsTNC1基因为靶点,OsTNC1基因有4个外显子,在OsTNC1基因第1个外显子处设计3个sgRNA位点,即以序列12bp、1445bp、2068bp处的NGG前20bp作为3个靶向序列,分别记为sgRNA-exon1、sgRNA-exon2和sgRNA-exon3,将sgRNA与cas9一起导入到水稻中,通过CRISPR/Cas9蛋白的定点切割与随机性修复,制备得到基因编辑水稻;其中,
sgRNA-exon1-1:GGAGTAGACGTCGCCCATGGCGC
sgRNA-exon1-2:GTGCCATTTGCAACACTGCAGGG
sgRNA-exon1-3:GCGGGACATGCAGTTGCGGGAGG。
2.权利要求1所述的一种利用OsTNC1基因突变获得分蘖增加的水稻的方法,其中敲除采用CRISPR技术,其中sgRNA序列分别为:
sgRNA-exon1-1:GGAGTAGACGTCGCCCATGGCGC
sgRNA-exon1-2:GTGCCATTTGCAACACTGCAGGG
sgRNA-exon1-3:GCGGGACATGCAGTTGCGGGAGG。
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