CN117511995A - 敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜抗菌核病中的应用 - Google Patents
敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜抗菌核病中的应用 Download PDFInfo
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Abstract
本发明提供了敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜抗菌核病中的应用,涉及基因工程技术领域。甘蓝型油菜BnaC05G0101700WE基因纯合编辑突变体在短日照下相较于野生型westar,对菌核病的抗性显著增强,并且短日照下突变体中茉莉酸(JA)含量以及水杨酸(SA)、茉莉酸(JA)信号传导基因的表达显著上调。
Description
技术领域
本发明涉及基因工程技术领域,特别是涉及敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜抗菌核病中的应用。
背景技术
菌核病是我国油菜生产上第一大病害,由坏死性真菌核盘菌引起,危害油菜的茎、叶、花和菜荚,以茎秆受害损失最大。油菜在开花结荚时,常常一株一株发病枯死。受害茎、叶的菌丝,通过油菜枝叶的相互接触而蔓延邻近健株,进行再次侵染。在我国油菜主产区,其造成的产量损失达10%-30%。菌核病常年发生面积5000多万亩,占播种面积50%以上。油菜植株感染核盘菌后严重影响菜籽中油酸、蛋白质等的积累,降低了菜籽油的品质。长江流域冬春季多雨、潮湿、温暖致使油菜菌核病发生严重。目前生产上主要釆用栽培和化学的方法防治菌核病,但其效果十分有限。因此,选育抗病品种无疑成为油菜抗菌核病育种最根本也是最有效的方法。然而,当前栽培的甘蓝型油菜品种缺少相关抗性基因,严重制约着油菜抗菌核病育种进程。
植物体具有复杂的细胞内和细胞间的信号传导体系,通过产生局部和系统的防御反应,来抵抗病原物的侵染。水杨酸(Salicylic Acid,SA)、茉莉酸(Jasmonic acid,JA)和乙烯(Ethylene,ET)等植物激素在植物抵抗病原物侵染过程中起着非常关键的作用(Bariand Jones,2009;Pieterse et al.,2012)。一般来说JA/ET信号途径与植物对死体型营养型菌的防卫反应相关,而SA信号途径与植物对活体营养型菌的防卫反应相关(Bari andJones,2009)。因此,前期大量研究表明是JA/ET信号途径参与调控植物核盘菌的抗性,比如Wu等(Wu et al.,2016)发现甘蓝型油菜在受到核盘菌侵染后迅速诱导JA、ET的合成及信号转导途径相关基因的表达,而显著抑制SA合成及信号转导途径相关基因的表达。另外发现在接种48hpi时JA和ET合成及信号转导途径在抗病材料中受诱导更剧烈,因此认为甘蓝型油菜中JA/ET信号转导途径正调控菌核病的抗性。以前普遍认为核盘菌为典型的死体营养型菌(Hegedus and Rimmer SR,2005;Amselem et al.,2011),但是越来越多的证据表明,核盘菌也存在活体营养阶段。因此,也有研究指出SA对甘蓝型油菜核盘菌抗性也起到正调控作用(Nováková et al.,2014;Wang et al.,2012);最近在人工合成的RRCC(Raphanusalboglabra)中也发现了这一现象(Alkooranee et al.,2017)。
酪氨酸降解途径在动物中必不可少,若中断将导致严重的代谢疾病。这条途径首先在动物和细菌中被发现,是一条非常重要的代谢途径。FAH是酪氨酸代谢途径的最后一个酶,FAH(SSCD1)基因的突变导致拟南芥在短日照下产生模拟病斑并激活植物防御。模拟病斑突变体是一类在没有明显的逆境、损伤或病原物侵害时,在叶片上能自发地形成类似病原物侵染后的坏死斑的突变体,这类突变体在植物中广泛存在,如:拟南芥、水稻、玉米、高粱、小麦、大麦和花生中等均有报道。这些突变体往往能激活植物体的系统获得性抗性,通常具有与抗病反应相关的细胞学和生物化学特征,对许多病原物表现出局部和系统抗性,是研究植物抗病机理的理想材料。目前,通过对植物模拟病斑突变体的研究,发现许多模拟病斑突变体与植物抗病信号中的水杨酸(Salicylic Acid,SA)、茉莉素(Jasmonic acid,JA)、乙烯(Ethylene,ET)等信号分子均有相关,它们不同程度地参与了抗病过程中的不同环节,起着非常重要的作用。前期研究证实sscd1突变体产生模拟病斑的同时积累JA,同时激活控JA/ET信号途径的部分病程相关基因PDF1.2,VSP1和SA病程相关基因PR1。尽管sscd1中的模拟病斑的产生伴随着SA诱导基因PR1的上调,但与SA信号转导无关;而JA信号通路的中断显著抑制sscd1模拟病斑的产生,因此JA信号传导受体COI1正向调控sscd1模拟病斑。这些结果说明FAH基因突变激活拟南芥抗病信号途径,并且这些信号参与调控突变体模拟病斑的形成。
发明内容
为了解决上述问题,本发明提供了敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜抗菌核病中的应用,敲除甘蓝型油菜BnaC05G0101700WE基因后明显提高油菜对盘核菌的抗性,为甘蓝型油菜抗病育种提供宝贵的基因资源和种质资源。
为了实现上述目的,本发明提供如下技术方案:
本发明提供了敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜抗菌核病中的应用。
本发明还提供了敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜茉莉酸含量中的应用。
优选的,敲除甘蓝型油菜BnaC05G0101700WE基因的油菜在短日照下提高油菜抗菌核病和菜茉莉酸含量。
本发明还提供了敲除甘蓝型油菜BnaC05G0101700WE基因在油菜抗菌核病育种中的应用。
本发明提供了一种获取抗菌核病油菜的方法,包括以下步骤后:
1)将CRISPR/Cas9质粒酶切,得到酶切质粒;
2)将所述步骤1)得到的酶切质粒分别与sgRNA1、sgRNA2、sgRNA3连接,得到连接产物1、连接产物2和连接产物3;
所述sgRNA1由引物对1退火得到,所述引物对1的上游引物的核苷酸序列如SEQ IDNo.1所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.2所示;
所述sgRNA2由引物对2退火得到,所述引物对2的上游引物的核苷酸序列如SEQ IDNo.3所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.4所示;
所述sgRNA3由引物对3退火得到,所述引物对3的上游引物的核苷酸序列如SEQ IDNo.5所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.6所示;
3)将所述步骤2)得到的连接产物1、连接产物2和连接产物3分别转化大肠杆菌,提取质粒,将得到的3个质粒构建多靶标载体,得到zmpl-BnaC05FAH载体;
4)将所述步骤3)得到的zmpl-BnaC05FAH载体转化农杆菌,得到转化菌;
5)将所述步骤4)得到的转化菌浸染油菜的下胚轴,再进行植株再生培养,得到抗菌核病油菜。
优选的,所述步骤1)使用BsaI/Eco31I酶切CRISPR/Cas9质粒。
优选的,所述步骤2)退火的程序为:95℃10min,55℃10min,14℃5min。
优选的,采用CaCl2冻融法将zmpl-BnaC05FAH载体转化农杆菌中。
优选的,所述步骤5)转化菌以菌液形式浸染下胚轴,所述菌液的OD600值为0.5~0.6;所述浸染的时间为8~10min。
本发明的有益效果为:
甘蓝型油菜BnaC05G0101700WE基因纯合编辑突变体在短日照下相较于野生型westar,对菌核病的抗性显著增强,并且短日照下突变体中茉莉酸(JA)含量以及水杨酸(SA)、茉莉酸(JA)信号传导基因的表达显著上调。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍。
图1为BnaC05G0101700WE(BnC05FAH)基因结构和CRISPR/Cas9基因编辑靶标位点示意图,注释:BnaC05FAH的基因结构及靶标位点;标靶1和标靶2(Target 1/2)位于BnaC05FAH的外显子4上、标靶3(Target 3)位BnaC05FAH的外显子6上的序列分别用下划线表示。PAM位点(5’-NGG-3’)用蓝色突出显示;
图2为BnaC05G0101700WE(BnC05FAH)基因纯合突变体的编辑位点;
图3为BnaC05G0101700WE(BnC05FAH)基因纯合突变体对菌核病的抗性检测;
图4为CRISPR/Cas9编辑BnaC05FAH突变体短日照下积累JA激活JA抗病途径;
图5为BnaC05G0101700WE基因的核苷酸和氨基酸序列图。
具体实施方式
本发明提供了敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜抗菌核病中的应用。
本发明还提供了敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜茉莉酸含量中的应用。
在本发明中,敲除甘蓝型油菜BnaC05G0101700WE基因的油菜优选在短日照下提高油菜抗菌核病和菜茉莉酸含量。
本发明还提供了敲除甘蓝型油菜BnaC05G0101700WE基因在油菜抗菌核病育种中的应用。
本发明还提供了一种获取抗菌核病油菜的方法,包括以下步骤后:
1)将CRISPR/Cas9质粒酶切,得到酶切质粒;
2)将所述步骤1)得到的酶切质粒分别与sgRNA1、sgRNA2、sgRNA3连接,得到连接产物1、连接产物2和连接产物3;
所述sgRNA1由引物对1退火得到,所述引物对1的上游引物的核苷酸序列如SEQ IDNo.1所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.2所示;
所述sgRNA2由引物对2退火得到,所述引物对2的上游引物的核苷酸序列如SEQ IDNo.3所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.4所示;
所述sgRNA3由引物对3退火得到,所述引物对3的上游引物的核苷酸序列如SEQ IDNo.5所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.6所示;
3)将所述步骤2)得到的连接产物1、连接产物2和连接产物3分别转化大肠杆菌,提取质粒,将得到的3个质粒构建多靶标载体,得到zmpl-BnaC05FAH载体;
4)将所述步骤3)得到的zmpl-BnaC05FAH载体转化农杆菌,得到转化菌;
5)将所述步骤4)得到的转化菌浸染油菜的下胚轴,再进行植株再生培养,得到抗菌核病油菜。
本发明将CRISPR/Cas9质粒酶切,得到酶切质粒。本发明优选使用BsaI/Eco31I酶切CRISPR/Cas9质粒,本发明对所述酶切的体系和条件没有特殊限定,本领域技术人员按照常规即可。
本发明将得到的酶切质粒分别与sgRNA1、sgRNA2、sgRNA3连接,得到连接产物1、连接产物2和连接产物3。
在本发明中,所述sgRNA靶位点的选择:
针对甘蓝型油菜BnaC05G0101700WE序列信息及同源序列比对结果,结合靶位点设计网站(http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR),设计出3个sgRNA靶位点,2个位于BnaC05G0101700WE基因编码区的第四个外显子和1个位于第六个外显子,靶标序列如下:
靶标1SEQ ID No.7:gcagttcttggcgtgatgcatgg;
靶标2SEQ ID No.8:gtgtagtccccaatcaccatagg;
靶标3SEQ ID No.9:agaggtcaaggccatccacaagg。
在本发明中,所述sgRNA1由引物对1退火得到,所述引物对1的上游引物的核苷酸序列如SEQ ID No.1所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.2所示,具体如下:
SEQ ID No.1:cagtGGTCTCagtca gcagttcttggcgtgatgca;
SEQ ID No.2:cagtGGTCTCaaaac tgcatcacgccaagaactgc。
在本发明中,所述sgRNA2由引物对2退火得到,所述引物对2的上游引物的核苷酸序列如SEQ ID No.3所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.4所示,具体如下:
SEQ ID No.3:cagtGGTCTCagtca gtgtagtccccaatcaccat;
SEQ ID No.4:cagtGGTCTCaaaac atggtgattggggactacac。
在本发明中,所述sgRNA3由引物对3退火得到,所述引物对3的上游引物的核苷酸序列如SEQ ID No.5所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.6所示,具体如下:
SEQ ID No.5:cagtGGTCTCagtca agaggtcaaggccatccaca;
SEQ ID No.6:cagtGGTCTCaaaac tgtggatggccttgacctct。
在本发明中,所述退火的程序优选为:95℃10min,55℃10min,14℃5min。
本发明将得到的连接产物1、连接产物2和连接产物3分别转化大肠杆菌,提取质粒,将得到的3个质粒构建多靶标载体,得到zmpl-BnaC05FAH载体。本发明对所述连接产物转化到大肠杆菌中以及构建多靶标载体的方法没有特殊限定,本领域技术人员采用常规即可。
本发明将得到的zmpl-BnaC05FAH载体转化农杆菌,得到转化菌。本发明优选采用CaCl2冻融法将zmpl-BnaC05FAH载体转化农杆菌中。
本发明将得到的转化菌浸染油菜的下胚轴,再进行植株再生培养,得到抗菌核病油菜。在本发明中,所述转化菌优选以菌液形式浸染下胚轴,所述菌液的OD600值优选为0.5~0.6;所述浸染的时间优选为8~10min。
为了进一步说明本发明,下面结合实施例对本发明进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
实施例1
1、甘蓝型油菜BnaC05G0101700WE基因CRISPR/Cas9表达载体的构建
1.1甘蓝型油菜westar中BnaC05G0101700WE基因全长编码区核酸序列的克隆:
待甘蓝型油菜westar长到四叶期,取适量的新鲜的叶片用液氮研磨,用Trizol(RNAiso Plus,宝生物工程有限公司)提取总RNA,进一步用DNase I(RNase Free,ThermoFisher Scientific)去除基因组DNA利用反转录试剂盒ReverTraAce qPCR RT试剂盒(perfect real time,Toyobo),将RNA反转录成cDNA,以cDNA为模板,设计引物BnaC05FAH-ty-F(SEQ ID No.105’-AACCTACCACTGCCCTTT-3’)和BnaC05FAH-ty-R(SEQ ID No.115’-TTTCCACCAACTTCCTGC-3’),通过TaKaRa高保真DNA聚合酶进行PCR扩增,对BnaC05G0101700WE基因进行克隆。
将克隆得到的PCR产物进行1%的琼脂糖凝胶电泳,PCR扩增目的片段大小预期为1266bp,PCR产物经1.0%琼脂糖凝胶电泳检测后切胶回收并纯化后,送上海生工测序,得到甘蓝型油菜westar中BnaC05G0101700WE基因的全长编码区。
BnaC05G0101700WE基因的核酸序列和氨基酸序列如图5所示。
1.2sgRNA靶位点的选择和引物设计
针对甘蓝型油菜BnaC05G0101700WE序列信息及同源序列比对结果,结合靶位点设计网站(http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR),设计出3个sgRNA靶位点,2个位于BnaC05G0101700WE基因的第四个外显子和1个位于第六个外显子,设计sgRNA序列如图1所示。靶标序列如下:
靶标1SEQ ID No.7:gcagttcttggcgtgatgcatgg;
靶标2SEQ ID No.8:gtgtagtccccaatcaccatagg;
靶标3SEQ ID No.9:agaggtcaaggccatccacaagg;
根据实验需求,为得到最终载体,需要构建3个中间载体,分别命名为:zmpl-BnaC05FAH-Y1、zmpl-BnaC05FAH-B1、zmpl-BnaC05FAH-A1。以下是中间载体引物:
zmpl-BnaC05FAH-Y1中间载体引物:
zmpl-BnaC05FAH-Y1-FP SEQ ID No.1:
cagtGGTCTCagtca gcagttcttggcgtgatgca;
zmpl-BnaC05FAH-Y1-RP SEQ ID No.2:
cagtGGTCTCaaaac tgcatcacgccaagaactgc。
zmpl-BnaC05FAH-B1中间载体引物:
zmpl-BnaC05FAH-B1-FP SEQ ID No.3:
cagtGGTCTCagtca gtgtagtccccaatcaccat;
zmpl-BnaC05FAH-B1-RP SEQ ID No.4:
cagtGGTCTCaaaac atggtgattggggactacac。
zmpl-BnaC05FAH-A1中间载体引物:
zmpl-BnaC05FAH-A1-FP SEQ ID No.5:
cagtGGTCTCagtca agaggtcaaggccatccaca;
zmpl-BnaC05FAH-A1-RP SEQ ID No.6:
cagtGGTCTCaaaac tgtggatggccttgacctct。
1.3双链gDNA的获得
将上述引物送至上海生工生物工程股份有限公司进行引物合成,合成好的引物通过PCR反应经过变性退火合成双链gDNA用于后续载体构建。PCR反应和程序如表1-1至表1-3和表2所示。
表1-1双链gDNA的获得PCR反应
成分 | 体积 |
zmpl-BnaC05FAH-Y1-FP | 5uL |
zmpl-BnaC05FAH-Y1-RP | 5uL |
Nuclease-free water | 40uL |
50uL(Total) |
表1-2双链gDNA的获得PCR反应
成分 | 体积 |
zmpl-BnaC05FAH-B1-FP | 5uL |
zmpl-BnaC05FAH-B1-RP | 5uL |
Nuclease-free water | 40uL |
50uL(Total) |
表1-3双链gDNA的获得PCR反应
成分 | 体积 |
zmpl-BnaC05FAH-A1-FP | 5uL |
zmpl-BnaC05FAH-A1-RP | 5uL |
Nuclease-free water | 40uL |
50uL(Total) |
表2双链gDNA的获得PCR程序
将变性退火得到的PCR产物从PCR仪中取出,放入4℃保存,以方便与载体进行连接。
1.4双链gDNA与CRISPR/Cas9载体的连接:
3个中间载体zmpl-BnaC05FAH-Y1、zmpl-BnaC05FAH-B1、zmpl-BnaC05FAH-A1的构建:分别用BsaI/Eco31I限制性内切酶将CRISPR/Cas9质粒在37℃恒温培养箱中酶切和连接2小时,酶切连接反应体系如表3所示。
表3连接双链gDNA与CRISPR/Cas9载体反应体系
试剂 | 体积 |
T4 Buffer | 1uL |
T4-ligase | 0.5uL |
BsaI/Eco31I | 0.5uL |
CRISPR/Cas9载体 | 1.5uL |
双链gDNA | 2uL |
Nuclease-free water | 4.5uL |
10uL(Total) |
1.5重组质粒转化和鉴定
分别将5uL连接产物转化大肠杆菌DH5a感受态,中间载体zmpl-BnaC05FAH-Y1转化涂(卡纳霉素)抗性平皿,中间载体zmpl-BnaC05FAH-B1转化涂(氨苄霉素)抗性平皿,中间载体zmpl-BnaC05FAH-A1转化涂(氯霉素)抗性平皿,37℃培养12小时,进行菌斑PCR鉴定。验证正确的送上海生工进行测序,将测序验证正确的阳性克隆进行摇菌和质粒提取,菌落PCR反应体系如表4所示,PCR反应条件如表5所示。
表4菌落PCR反应体系
表5菌落PCR扩增程序
温度 | 时间 |
94℃预变性 | 2min |
94℃ | 30sec |
67℃ | 30sec |
72℃ | 1min 15sec |
1.6多靶标载体构建
1.6.1质粒抽提和酶切连接
将构建好的中间载体zmpl-BnaC05FAH-Y1、zmpl-BnaC05FAH-B1、zmpl-BnaC05FAH-A1的质粒进行提取,根据表6进行酶切连接反应,以进行多靶标载体的构建。
表6酶切连接反应体系
试剂 | 体积 |
T4Buffer | 1uL |
T4-ligase | 0.5uL |
LguI | 0.5uL |
zmpl-BnaC05FAH-Y1 | 1uL |
zmpl-BnaC05FAH-B1 | 1.5uL |
Nuclease-free water | 5.5uL |
10uL(Total) |
把配置好的体系置于37℃培养箱2h,得到中间载体zmpl-油菜FAH(1),将该载体转化转化大肠杆菌DH5a感受态,转化涂(卡纳霉素)抗性平皿,37℃培养12小时,进行菌斑PCR鉴定。挑取10个菌斑同时进行1.5mLEP管接菌和PCR鉴定
ZMPL-CHEN SEQ ID No.12:CCAGAAATTGAACGCCGAAG;
M13F-CHEN SEQ ID No.13:GTAAAACGACGGCCAGT。
表7菌落PCR反应体系
试剂 | 体积 |
5×buffer | 4uL |
2.5mM dNTPs | 1.6uL |
Taq | 0.2uL |
菌体悬浮液 | 5uL |
ZMPL-CHEN | 1uL |
M13F-CHEN | 1uL |
Nuclease-free water | 7.2uL |
20uL(Total) |
表8菌落PCR扩增程序
温度 | 时间 |
94℃预变性 | 2min |
94℃ | 30sec |
67℃ | 30sec |
72℃ | 1min 15sec |
1.6.2最终载体构建
total:10uL
H2O:5.5uL
T4 Buffer:1uL
LguI:0.5uL
zmpl-油菜FAH(1)(质粒):1uL
zmpl-油菜FAH-A1(质粒):1.5uL
T4-ligase:0.5uL。
把配置好的体系置于37℃培养箱2h,得到最终载体zmpl-油菜FAH。
1.7农杆菌的转化
用CaCl2冻融法将已验证的重组质粒转入农杆菌GV3101,挑选菌落PCR验证正确的农杆菌单菌落进行培养,28℃、220rpm振荡培养18~24h。6000g离心大量活化菌液2min,弃上清,用农杆菌悬浮液(5%蔗糖+1/2MS+0.02%Silwet-L77+0.01%6-BA)将菌体重悬至OD600在0.6-1.0之间。
1.8CRISPR/Cas9基因编辑载体的甘蓝型油菜遗传转化
1)无菌苗的制备
外植体的制备:在超净工作台上,选取颗粒饱满、种皮完好、外观无病斑的油菜种子在75%酒精中浸泡1min,无菌水清洗一遍后,用0.15%HgCl2(加1uLTritonX-100)浸泡10-15min,再用灭菌的蒸馏水清洗4-5次,每次5min。灭菌后播种于MS固体培养基(MS+30g/L葡萄糖+0.6%植物凝胶,pH 5.6-5.8)的三角瓶中,每瓶20-30粒,于25℃黑暗条件下培养4-5d(新种子与旧种子萌发有差异)。种子发芽后,然后转光照培养箱25±2℃、16h光照/8h黑暗,光强为60μmol/m2/s的条件下培养2-4d,使子叶展开,至子叶变绿。下胚轴首先要在培养基中预培养2-3d。组培条件:光周期为16h/d,温度为25±2℃。
2)农杆菌活化和浸染
从-80℃取出含有已验证的重组质粒农杆菌,操作台内用接种环挑取菌液后,在含有卡那霉素、庆大霉素、利福平三种抗生素的固体LB培养基划线涂布,倒置放于28℃培养箱培养1-2天,挑取单菌落摇菌,菌液OD600值为0.5~0.6左右活性最高,4000rmp离心6-8min收集菌体,将预培养的下胚轴置于农杆菌悬浮液中,侵染8-10min后,用无菌滤纸将下胚轴表面的农杆菌菌液吸干。
3)外植体与农杆菌的共培养
将表面晾干的下胚轴置于共培培养基中,25℃,暗培养2-3d。侵染后的下胚轴用无菌滤纸吸干多余的菌液。
4)愈伤的诱导
共培后将下胚轴转移至愈伤诱导培养基中,光周期16h光照/8h黑暗,培养5~7天。在该阶段没有加入相应的抗生素筛选剂,相当于延迟筛选,转化后的下胚轴受到一定的胁迫和损伤,如果直接加入抗生素可能加剧外界胁迫并导致下胚轴死亡,延迟筛选使转化后的细胞有一个恢复的阶段,延迟的时间并不影响后期的筛选和分化。
5)胚性细胞的诱导
将产生愈伤的下胚轴转移至诱导诱导胚性愈伤生成的培养基上,在此培养基中同样没有添加筛选剂(原因同上述(4)),下胚轴诱导出的愈伤慢慢变绿。诱导时间为5-7d。
6)分化与筛选
油菜遗传转化幼苗的分化与筛选同时进行,在分化培养基中加入潮霉素,分为初筛和二筛,分化前期为初筛阶段,抗生素浓度较低,待分化的幼苗伸长以后提高抗生素浓度,该阶段为二筛,这样可以在检测前筛掉假阳性苗,提高阳性率并且减少后期工作量。
7)幼苗的生根
将苗子从愈伤上切下来,转入生根培养基,光照培养。一个月后幼苗生根,洗去底部培养基成分,移栽到温室进行培育生长。
2、甘蓝型油菜BnaC05G0101700WE基因CRISPR/Cas9基因编辑突变体的获得与鉴定
CRISPR/Cas9基因编辑载体进行油菜遗传转化后获得多个潮霉素抗性的CRISPR/Cas9基因编辑突变体油菜株系。提取每个突变体油菜株系的基因组DNA,然后以基因组DNA为模板,以FAH-M3-F SEQ ID No.14:gaaccagaactggaggatagt、FAH-M3-257R SEQ IDNo.15:aaaGGTTTAAGTTAGTTGCGG、FAH-M12-F SEQ ID No.16:ATTTGAATTGTGGGGATATTGG、FAH-M12-381Rcx SEQ ID No.17:CACCAAGAAACACCTCAGC为扩增引物,对BnaC05G0101700WE基因进行PCR扩增,将扩增产物切胶纯化,检测其质量和浓度,送至上海生工生物工程股份有限公司进行测序,以检测每个CRISPR/Cas9基因编辑突变体中BnaC05G0101700WE基因的编辑情况。
3、验证甘蓝型油菜BnaC05G0101700WE基因编辑纯合突变体对菌核病抗性的影响
通过对CRISPR/Cas9基因编辑突变体油菜进行测序分析,如图2所示目前获得4株不同编辑位点的油菜BnaC05G0101700WE基因纯合编辑突变体,其中1个纯合突变体(T55-4-1)编辑位点同时发生在靶位点2(Target 2,删除1个碱基)和靶位点3(Target 3,删除5个碱基),另外3个纯合突变体只有1个靶位点发生编辑:T34-3-1突变体(删除4个碱基)和T35-1突变体编辑位点都在靶位点2(Target 2,增加1个碱基),T22-7突变体编辑位点位于靶点3(Target3,删除2个碱基)。
抗菌实验结果表明4株纯合油菜BnaC05G0101700WE基因纯合编辑突变体在短日照下相较于野生型westar,对菌核病的抗性显著增强(如图3),并且短日照下突变体中茉莉酸(JA)含量以及水杨酸(SA)、茉莉酸(JA)信号传导基因的表达显著上调(图4)。
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例,而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (9)
1.敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜抗菌核病中的应用。
2.敲除甘蓝型油菜BnaC05G0101700WE基因在提高油菜茉莉酸含量中的应用。
3.根据权利要求1或2所述的应用,其特征在于,敲除甘蓝型油菜BnaC05G0101700WE基因的油菜在短日照下提高油菜抗菌核病和菜茉莉酸含量。
4.敲除甘蓝型油菜BnaC05G0101700WE基因在油菜抗菌核病育种中的应用。
5.一种获取抗菌核病油菜的方法,其特征在于,包括以下步骤后:
1)将CRISPR/Cas9质粒酶切,得到酶切质粒;
2)将所述步骤1)得到的酶切质粒分别与sgRNA1、sgRNA2、sgRNA3连接,得到连接产物1、连接产物2和连接产物3;
所述sgRNA1由引物对1退火得到,所述引物对1的上游引物的核苷酸序列如SEQ IDNo.1所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.2所示;
所述sgRNA2由引物对2退火得到,所述引物对2的上游引物的核苷酸序列如SEQ IDNo.3所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.4所示;
所述sgRNA3由引物对3退火得到,所述引物对3的上游引物的核苷酸序列如SEQ IDNo.5所示,所述引物对1的下游引物的核苷酸序列如SEQ ID No.6所示;
3)将所述步骤2)得到的连接产物1、连接产物2和连接产物3分别转化大肠杆菌,提取质粒,将得到的3个质粒构建多靶标载体,得到zmpl-BnaC05FAH载体;
4)将所述步骤3)得到的zmpl-BnaC05FAH载体转化农杆菌,得到转化菌;
5)将所述步骤4)得到的转化菌浸染油菜的下胚轴,再进行植株再生培养,得到抗菌核病油菜。
6.根据权利要求1所述的方法,其特征在于,所述步骤1)使用BsaI/Eco31I酶切CRISPR/Cas9质粒。
7.根据权利要求1所述的方法,其特征在于,所述步骤2)退火的程序为:95℃10min,55℃10min,14℃5min。
8.根据权利要求1所述的方法,其特征在于,采用CaCl2冻融法将zmpl-BnaC05FAH载体转化农杆菌中。
9.根据权利要求1所述的方法,其特征在于,所述步骤5)转化菌以菌液形式浸染下胚轴,所述菌液的OD600值为0.5~0.6;所述浸染的时间为8~10min。
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