CN106119275A - 基于CRISPR/Cas9技术将非糯性水稻株系改造成糯性株系的打靶载体和方法 - Google Patents
基于CRISPR/Cas9技术将非糯性水稻株系改造成糯性株系的打靶载体和方法 Download PDFInfo
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Abstract
本发明涉及一种基于CRISPR/Cas9技术将非糯性水稻株系改造成糯性株系的打靶载体，其特征在于，包含sgRNA表达框和Cas9表达框，以及筛选标记，其中所述sgRNA表达框包含第一启动子以及由所述第一启动子控制转录的所述sgRNA编码序列，所述Cas9表达框包含第二启动子以及由所述第二启动子控制转录的Cas9编码序列；所述第一启动子和所述第二启动子为在水稻中组成型表达的相同或不同的强启动子；所述sgRNA编码序列由位于上游的位点靶向RNA的编码序列和位于下游的招募Cas9的RNA的编码序列组成，所述位点靶向RNA的编码序列如SEQ ID NO:1所示。本发明还提供了使用所述打靶载体将非糯性水稻株系改造成糯性水稻株系的方法。通过本发明，可简便并且有效实现该技术目的，并且大大节约了时间。
Description
技术领域
本发明涉及植物的遗传改造领域，更特别地，涉及一种将非糯性水稻株系改造成糯性水稻株系的打靶载体，以及改造方法。
背景技术
大米的糯性决定于其中直链淀粉的含量，直链淀粉含量越低，则大米越具有糯性。糯米的直链淀粉含量一般低于5％，而籼米的直链淀粉含量一般高于14％。因此，可通过对水稻的关于直链淀粉合成的基因通路中的一些基因进行遗传操作以部分或完全阻断直链淀粉的合成，从而将产生非糯性大米的籼稻、粳稻和籼粳交株系改造成产生糯性大米的水稻株系。
转录激活样效应因子核酸酶(transcription activator-like effectornuclease,TALEN)技术、锌指核酸酶(Zinc-finger nuclease,ZFN)和成簇规律间隔短回文重复(clustered regulatory interspaced short palindromic repeat,CRISPR)技术是目前基因组编辑领域的三大技术。ZFNs针对目的基因序列设计并合成ZFNs，其特定的DNA结合域特异性识别并结合与其对应的序列，形成有切割活性的Fok I二聚体，在靶位点切割DNA形成DSB。与ZFNs相比，TALENs的构建方便、快捷，但其模块组装过程繁琐。CRISPR-Cas9技术是在植物细胞中进行基因敲除的新方法，该技术对比于以往技术的优点是，具有精准的定位靶点和有效的核酸内切酶活性。是利，
基于CRISPR-Cas9技术用靶点特异性的sgRNA将Cas9核酸酶带到基因组DNA上的具体靶位点，从而对特定基因位点进行切割导致突变，其原理如下：通过基因工程手段将位点靶向序列和Cas9招募序列融合到一起形成表达sgRNA的编码序列，并在该编码序列前添加在宿主中组成型表达的强启动子，形成sgRNA表达盒，然后将sgRNA表达盒和Cas9表达盒导入宿主细胞中，通过sgRNA诱导Cas9特异性地切割基因组的靶位点，从而对靶位点的核酸进行敲除。为了方便操作，通常将sgRNA表达盒与Cas9表达盒集成到一个载体上，得到可同时表达两者的打靶载体，用于转化细胞。
在利用CRISPR-Cas9技术对植物细胞的遗传改造中，Cas9表达盒以及sgRNA表达盒中用于招募Cas9的序列在许多文献中有介绍，因此在本领域是公知的。表达盒中所用的启动子取决于所要求改造的宿主，在本发明中，由于要改造的是水稻，所以这些启动子是能在水稻中组成型表达的强启动子，例如U3启动子、泛素启动子等，可根据操作者的偏好来选择。
位点靶向序列设计十分重要，靶序列一般要求满足以下条件：由20-21个核苷酸构成，靶序列前面必须是碱基G作为转录的起始信号，如果没有需人工添加一个G，靶序列末端的PAM序列必须是NGG，进行靶位点序列预测以保证靶序列具有唯一性。然而，并非只要满足这些条件就能达到目的，脱靶时有发生。因此，需要针对水稻选择合适的启动子、靶位点，设计出合适的靶向该靶位点的RNA的编码序列，从而构建用于将非糯性水稻株系改造成糯性的水稻株系的打靶载体。
发明内容
发明人在对水稻的直链淀粉合成的基因调控通路的研究中，发现直链淀粉的含量与Wx基因的表达密切相关。Wx基因的敲除可将水稻籽粒中的淀粉含量降低至接近于0，但是对水稻的遗传操作并非易事。
为了更加方便、简易并且精确地敲除非糯性水稻中的Wx基因，发明人设计了一种基于CRISPR-Cas9技术将非糯性水稻株系改造成糯性株系的打靶载体，其包含sgRNA表达框和Cas9表达框，以及筛选标记，
其中所述sgRNA表达框包含第一启动子以及由所述第一启动子控制转录的所述sgRNA编码序列，所述Cas9表达框包含第二启动子以及由所述第二启动子控制转录的Cas9编码序列；
所述第一启动子和所述第二启动子可为在水稻中组成型表达的相同或不同的强启动子，例如U3启动子、泛素启动子pOsUBQ、花椰菜花叶病毒(CaMV)的35S启动子、根癌农杆菌Ti质粒的胭脂碱合成酶基因NOS启动子、水稻Actl启动子；
所述sgRNA编码序列由位于上游的位点靶向RNA的编码序列和位于下游的招募Cas9的RNA的编码序列组成，所述位点靶向RNA的编码序列如SEQ ID NO:1所示。
优选地，所述sg RNA表达框的序列如SEQ ID NO:2所示。
优选地，所述Cas9表达框的序列如SEQ ID NO:3所示。
优选地，所述所述筛选标记为抗潮霉素基因。
优选地，所述打靶载体通过将所述sg RNA表达框和所述Cas9表达框插入于水稻的表达质粒载体上而得到，例如，插入农杆菌质粒改造的载体，例如pCAMBIA1301质粒。
本发明还公开了一种基于CRISPR/Cas9技术将非糯性水稻株系改造成糯性株系的方法，其特征在于，包括以下步骤：
1)诱导所述非糯性水稻株系的组织获得愈伤组织；
2)将权利要求1-5中任一项所述的打靶载体导入所述愈伤组织中；
3)筛选阳性转化子；
4)将得到的阳性转化子培养成成熟植株，即得到糯性水稻株系。
优选地，步骤2)中将所述打靶载体导入所述愈伤组织中通过基因枪法或农杆菌转染法来进行。
优选地，其特征在于，所述愈伤组织由成熟胚、幼穗、子房、花药、花粉或未受精的子房培育而成。
当所述愈伤组织由花药、花粉或未受精的子房培育而成时，在将所述阳性转化子培养成成熟植株之前，还包括使所述阳性转化子染色体加倍的步骤，例如用秋水仙素处理。
附图说明
图1为集成了sgRNA表达框和Cas9表达框的片段的构建示意图；
图2为本发明的打靶载体的示意图。
具体实施方式
以下结合实施例对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
实施例1构建打靶载体
1.靶位点的选择
发明人通过预测和实验，选择Wx基因上的5’-GCGAGGGAATTGGAACTAATCGG-3’作为靶位点，由此合成两条寡核苷酸：
olig1：5’-TGGC GCGAGGGAATTGGAACTAAT-3’
olig2：5’-AAAC ATTAGTTCCAATTCCCTCGC-3’
将这两条寡核苷酸以1:1的比率混合，并退火，得到具有突出5’端的双链分子：
2.产生集成了sgRNA表达框和Cas9表达框的片段
将其与用BbsI完全酶切的质粒pSGR-Cas9-Os混合，使用T4DNA连接酶连接成环状质粒(图1)，然后通过HindIII和EcoRI双酶切，回收集成了sgRNA表达框和Cas9表达框的片段，其中sgRNA表达框的序列如SEQ ID NO:2所示，并且Cas9表达框如SEQ ID NO:3所示。
集成了sgRNA表达框和Cas9表达框的片段的获得不限于以上方法，还可通过用其他科研常用的质粒来构建，也可通过人工合成直接得到。
3.将集成了sgRNA表达框和Cas9表达框片段插入表达载体
将上述片段插入到表达载体pCAMBIA1301质粒的HindIII与EcoRI之间，由此得到打靶载体(图2)。
实施例2农杆菌介导的遗传转化。
将上述打靶载体转入农杆菌中，通过潮霉素筛选阳性转化子。将水稻株系鄂早17的成熟胚诱导成愈伤组织，然后用所得到的农杆菌侵染该愈伤组织细胞，用潮霉素筛选阳性的愈伤组织细胞，将愈伤组织细胞培养成成熟植株，即，产生糯米的水稻株系。通过检测，鄂早17所产生的籽粒的直链淀粉的含量大于13.5％，而通过本方法得到的水稻株系产生的籽粒的直链淀粉含量小于3％。
实施例3基因枪法介导的转化。
基因枪转化转化前4h，将胚性愈伤组织转移到到0.6mol/L高渗透压培养基(MS培养基附加46.67g/L的甘露醇和46.67g/L的山梨醇)上，每培养皿50-70块愈伤组织。用质粒上述打靶载体包裹直径1.0μm金粉。采用基因枪，选择可裂膜压力为1100Psi,靶材料至载样膜距离为8cm，使用基因枪对靶材料进行轰击，每皿材料轰击2次。用潮霉素筛选阳性的愈伤组织细胞，将愈伤组织细胞培养成成熟植株，即，产生糯米的水稻株系而通过本方法得到的水稻株系产生的籽粒的直链淀粉含量小于3％。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种基于CRISPR/Cas9技术将非糯性水稻株系改造成糯性株系的打靶载体，其特征在于，包含sgRNA表达框和Cas9表达框，以及筛选标记，
其中所述sgRNA表达框包含第一启动子以及由所述第一启动子控制转录的sgRNA编码序列，所述Cas9表达框包含第二启动子以及由所述第二启动子控制转录的Cas9编码序列；
所述第一启动子和所述第二启动子为在水稻中组成型表达的相同或不同的强启动子；
所述sgRNA编码序列由位于上游的位点靶向RNA的编码序列和位于下游的招募Cas9的RNA的编码序列组成，所述位点靶向RNA的编码序列如SEQ ID NO:1所示。
2.根据权利要求1所述的打靶载体，其特征在于，所述sg RNA表达框的序列如SEQ IDNO:2所示。
3.根据权利要求1所述的打靶载体，其特征在于，所述Cas9表达框的序列如SEQ ID NO:3所示。
4.根据权利要求1所述的打靶载体，其特征在于，所述筛选标记为抗潮霉素基因。
5.根据权利要求1所述的打靶载体，其特征在于，所述打靶载体通过将所述sg RNA表达框和所述Cas9表达框插入pCAMBIA1301质粒载体上而得到。
6.一种基于CRISPR/Cas9技术将非糯性水稻株系改造成糯性株系的方法，其特征在于，包括以下步骤：
1)诱导所述非糯性水稻株系的组织获得愈伤组织；
2)将权利要求1-5中任一项所述的打靶载体导入所述愈伤组织中；
3)根据所述打靶载体中的筛选标记筛选阳性转化子；
4)将得到的阳性转化子培养成成熟植株，即得到糯性水稻株系。
7.根据权利要求6所述的方法，其特征在于，步骤2)中将所述打靶载体导入所述愈伤组织中通过基因枪法或农杆菌转染法来进行。
8.根据权利要求6所述的方法，其特征在于，所述愈伤组织由成熟胚、幼穗、子房、花药、花粉或未受精的子房培育而成。
9.根据权利要求8所述的方法，其特征在于，当所述愈伤组织由花药、花粉或未受精的子房培育而成时，在将所述阳性转化子培养成成熟植株之前，还包括使所述阳性转化子染色体加倍的步骤。
10.根据权利要求9所述的方法，其特征在于，所述染色体加倍通过用秋水仙素处理来实现。
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