CN111778274A - 一种通过基因编辑提高番茄耐贮藏性的方法 - Google Patents
一种通过基因编辑提高番茄耐贮藏性的方法 Download PDFInfo
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Abstract
本发明提供一种通过基因编辑提高番茄耐贮藏性的方法,利用CRISPR/Cas9***对番茄材料基因组中的催化合成乙烯前体调控基因SlACS2进行编辑,进而使催化合成乙烯前体调控基因SlACS2性能丧失,从而提高番茄耐贮藏性能;该***包括两个sgRNA靶位点,分别为sgRNA1和sgRNA2;sgRNA1和sgRNA2识别的靶序列均为所述番茄材料基因组中编码SlACS2蛋白的DNA片段。利用上述编辑位点,可以在核酸内切酶Cas9的介导下对番茄自身的SlACS2基因进行编辑,形成SlACS2基因的定点突变。本发明对促进内源基因敲除或外源基因定点整合技术在番茄基因育种中的应用具有十分重要的作用。
Description
技术领域
本发明属于植物基因编辑技术领域,具体涉及一种通过基因编辑提高番茄耐贮藏性的方法,具体涉及使用CRISPR-Cas9***对番茄进行SlACS2基因的双位点(197-215和275-257)进行编辑的方法。
背景技术
番茄果实容易成熟过度而***,致使耐压性能变差、贮藏期缩短、抗病原菌能力降低,严重影响到生产、贮运及加工品质。
杂交育种是常用的番茄品质改良途径,但需要的周期长,易受不良基因连锁和种间生殖隔离的限制。将外源基因导入番茄改善耐贮性取得了一定的进展,比常规育种缩短了育种周期,拓宽了基因来源,但由于番茄基因组上整合的是外源基因,易使民众产生安全性疑虑,影响了基因工程育种效率和推广应用。
近年来,以多种新型高效的DNA靶向内切酶为基础建立的基因组编辑技术,是对植物自身的基因进行定点修饰,提高了育种效率,规避了常规转基因可能产生的不良效应。基因编辑可分为锌指核酸酶(Zinc-finger nuclease,ZFN)、类转录激活因子效应物核酸酶(transcription activator-like effector nuclease,TALEN)和CRISPR/Cas(clusteredregulatory interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas))技术。ZFN和TALEN是利用蛋白质识别特定的基因组靶位点,针对每一个突变位点需要构建两个相应的核酸酶,操作繁琐;CRISPR/Cas是利用更简单的核苷酸互补配对方式识别特定的基因组靶位点,比ZFN和TALEN编辑效率高,构建更简单。
番茄为呼吸跃变型果实,伴随呼吸跃变产生大量乙烯,即***Ⅱ乙烯,催熟果实。***II乙烯具有自催化机制,使得番茄成熟过程难以控制,造成果实过熟软化、腐烂变质。番茄1-氨基环丙烷1-羧酸ACC(1-aminocyclopropane-1-carboxylic acid)是合成乙烯的直接前体,由ACC合酶(ACC synthase)SlACS催化生成。番茄SlACS2催化***Ⅱ乙烯合成前体ACC的生成,是***II乙烯合成的重要限速酶。利用CRISPR/Cas9技术敲除SlACS2基因,不仅有利于研究SlACS2基因对番茄果实成熟及贮藏性能等方面的影响,进而培育耐贮藏品种,也为改良番茄的其它性状建立新途径。
发明内容
为了解决现有技术中存在的问题,本发明提供了一种通过基因编辑提高番茄耐贮藏性的方法,是使用CRISPR-Cas9***对番茄进行SlACS2基因编辑,并提供可高效敲除SlACS2基因的双编辑位点(197-215和275-257)。该编辑位点可用于Cas9介导的番茄SlACS2基因打靶,使其功能失活,从而提高番茄的耐贮藏性能。本发明针对SlACS2基因设计、筛选可高效切割的基因编辑位点,为构建SlACS2基因内源性敲除或定点敲入外源基因的番茄提供新途径。
本发明提供一种通过基因编辑提高番茄耐贮藏性的方法,利用CRISPR/Cas9***对番茄材料基因组中的催化合成乙烯前体调控基因SlACS2进行编辑,进而使催化合成乙烯前体调控基因SlACS2性能丧失,从而提高番茄耐贮藏性能;
所述CRISPR/Cas9***包括两个sgRNA靶位点,分别命名为sgRNA1和sgRNA2;
所述sgRNA1和所述sgRNA2识别的靶序列均为所述番茄材料基因组中编码SlACS2蛋白的DNA片段。
作为优选,所述sgRNA1靶位点序列为:5'-GGATTAAGAGAAACCCAAAAGG-3';
所述sgRNA2靶位点序列为:5'-TAATCTTGAAAGTTGGCAATGG-3'。
作为优选,所述编辑的方法为向所述番茄材料中导入番茄基因组编辑的载体;
所述番茄基因组编辑的载体含有所述sgRNA1靶位点序列、所述sgRNA2靶位点序列和Cas9蛋白的编码基因。
作为优选,所述方法还包括筛选SlACS2纯合突变体的步骤。
本发明提供一种耐贮藏番茄材料的获得方法,包括以下步骤:将上述方法获得的番茄材料自交,得到自交子代,选择SlACS2纯合突变且不携带外源DNA片段的自交子代,即为耐贮藏番茄材料。
本发明提供上述方法在培育耐贮藏番茄材料中的应用。
本发明提供如下(1)-(3)中任一种生物材料:
(1)上述的靶位点序列;
(2)上述的番茄基因组编辑的载体;
(3)含有上述的番茄基因组编辑的载体的微生物转化体。
本发明提供上述的载体或微生物转化体或靶位点序列在提高番茄耐储藏性能中的应用;
或,上述的载体或微生物转化体或靶位点序列在培育耐贮藏番茄中的应用;
或,上述的载体或微生物转化体或靶位点序列在番茄育种中的应用。
本发明提供一种鉴定待测番茄是否为上述方法获得的耐贮藏番茄或其子代的方法,包括以下步骤:
分别提取番茄野生株和待测番茄转化株基因组DNA,以上游引物和下游引物对待测番茄的基因组DNA进行PCR扩增,分别得到PCR扩增产物,根据所述PCR扩增产物测序结果,判断待测番茄是否为上述方法获得的耐贮藏番茄或其子代,判断方法为将转化体植株测序结果与野生型植株测序结果进行比对,如果在sgRNA1、sgRNA2附近或二者之间出现碱基的缺失或***,即可判断其为上述方法获得的耐贮藏番茄或其子代;
所述上游引物:CTCTTACACCATAACACAAC;
所述下游引物:CCAGCCATAACAACTCTTTC。
本发明提供一种鉴定或鉴定待测番茄是否为上述方法获得的耐贮藏番茄或其子代的产品,为如下(1)-(3)中的任一种:
(1)上述的上游引物和下游引物;
(2)含有(1)所述的上游引物和下游引物的PCR试剂;
(3)含有(1)所述的上游引物和下游引物或(2)所述的PCR试剂的试剂盒。
在确定上游引物和下游引物的情况下,构成PCR试剂及构成PCR试剂盒的方法为本领域公知,在此不再一一赘述。
与现有技术相比,本发明的有益效果为:
利用本发明提供的编辑位点(197-215和275-257),可以在核酸内切酶Cas9的介导下以较高效率对番茄自身的SlACS2基因进行编辑,形成SlACS2基因的定点突变。本发明对促进内源基因敲除或外源基因定点整合技术在番茄基因育种研究和生产中的应用具有十分重要的作用。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1为植物表达载体pSlACS2-DsgRNA结构示意图。
图2为番茄突变体的基因编辑形式。
图3为T0代植株ACS2基因的PCR检测电泳结果。其中,-为阴性空白对照,WT为未转化的野生型植株,M为maker(DL2000),1-8为T0代番茄植株。
图4为基因编辑植株T0代。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为市售。
实施例1 CRISPR/Cas9特异性敲除番茄SlACS2基因中用于特异性靶向SlACS2基因的sgRNA的设计和合成
1、靶向番茄SlACS2基因的sgRNA的设计
利用在线工具设计SlACS2-sgRNA,具体步骤为:
①登陆GenBank网站,搜索SlACS2基因序列并下载,其核苷酸序列如SEQ ID NO:4所示。
②打开靶点预测网站(https://crispr.dbcls.jp/),利用靶位点在线设计工具“CRISPRdirect”,在文本框中输入基因序列,选择番茄物种,即可得出该基因序列所有的靶位点,综合考虑评分结果、GC含量、靶位点的特异性及两个靶位点之间的距离最终选取一组靶位点进行打靶。
③长19nt的寡核苷酸sgRNA核心序列按GG(N)19NGG序列进行设计。
得到的sgRNA靶位点序列1为:5'-GGATTAAGAGAAACCCAAAAGG-3'
sgRNA靶位点序列2为:5'-TAATCTTGAAAGTTGGCAATGG-3'。
sgRNA靶位点序列1和2在番茄基因组1号染色体,SlACS2编码区的第2外显子上,染色体坐标为78221289-78221271和78221211-78221229,编码区坐标为(197-215和275-257)。
2、构建sgRNA的寡聚核苷酸
根据选择的sgRNA核心序列,设计正义链引物和反义链引物,两条引物的5'端添加含有核酸内切酶AarI识别位点的序列,正义链添加的序列为ATATCACCTGCACACTTTGG,以与载体质粒的黏性末端互补。正义链引物在添加序列的3'端为编辑位点197-215的序列GGATTAAGAGAAACCCAAA,编辑位点的3'端添加GTTTCAGAGCTATGCTGGAA;反义链5'端添加序列为ATATCACCTGCACACAAAC,在添加序列的3'端为编辑位点275-257的反向互补序列TTGCCAACTTTCAAGATTA,编辑位点的3'端添加CCAAACTACACTGTTAGATTC。
最终得到的正义链引物和反义链引物分别为:
正义链引物:5'-ATATCACCTGCACACTTTGGGGATTAAGAGAAACCCAAAGTTTCAGAGCTATGCTGGAA-3'(SEQ ID NO:5);
反义链引物:5'-ATATCACCTGCACACAAACTTGCCAACTTTCAAGATTACAAACTACACTGTTAGATTC-3'(SEQ ID NO:6)。
实施例2植物表达载体的构建
CP185载体和CP178载体由中国农业科学院蔬菜花卉研究所提供,此载体为公开载体。以CP185载体为模板,用正义链引物(SEQ ID NO:5)和反义链引物(SEQ ID NO:6)进行PCR扩增,得到含有双sgRNA的DNA片段并连接至pEasy-blunt,测序无误后,用AarI酶切连接至CP178载体上,形成植物表达载体pSlACS2-DsgRNA(结构见图1,核苷酸序列为SEQ ID NO:3)。植物表达载体上的Cas9基因受35S驱动,两条sgRNA基因均受番茄U6启动子驱动。构建好的植物表达载体通过热激法转化到大肠杆菌DH5α菌株中扩增,通过测序验证载体序列正确,再通过冻融法转化农杆菌LBA4404菌株,扩增以备侵染利用。
实施例3番茄遗传转化及T0代SlACS2基因编辑植株的获得
1、无菌苗的获得
将准备好的番茄种子用75%的酒精处理30s,用无菌水冲洗2遍,加入10%的漂白水,放置在摇床上晃动1小时后,取出种子用双蒸水冲洗5遍,放置在4℃冰箱中冷藏12h,接种至1/2MS培养基中(不含蔗糖)培养6d,便可获得无菌苗。
2、菌液制备
将pSlACS2-DsgRNA甘油菌在含有50mg/L利福平和100mg/L的卡那霉素YEB固体培养基(酵母提取物5g/L,蛋白胨5g/L,牛肉浸膏5g/L,七水硫酸镁0.5g/L,蔗糖1g/L)上划线,28℃培养两天后,挑取单克隆接种到5ml含有50mg/L利福平和100mg/L卡那霉素的液体YEB培养基中,28℃,220rpm恒温震荡培养过夜,次日取120μL摇好的菌液至50mL液体YEB培养基(含有50mg/L利福平和100mg/L卡那霉素)中摇菌至菌液浓度为OD600=0.7,5000rpm离心10min收集菌体,将收集的菌体用MSO液体培养基重新悬浮,即可使用。
3、T0代再生植株的获得
取无菌培养的番茄子叶剪切后于预培养培养基上预培养1d(黑暗条件),再置于步骤2制备的OD600=0.7的pSlACS2-DsgRNA农杆菌悬浮液中15min,之后,用滤纸吸干多余菌液,置于预培养培养基中28℃共培养2d(黑暗条件)。
其中,预培养培养基的配方为:MS+玉米素(1mg/L)+吲哚乙酸(1mg/L)。
共培养的子叶置于分化培养基上进行不定芽的分化筛选,待筛选出的抗性再生芽长到2cm高时,转移到生根培养基,培养条件为:温度25±1℃、光周期16h/d、光照强度12000lx,培养至生根得到T0代再生植株。
分化培养基的配方为:MS+玉米素(2mg/L)+吲哚乙酸(1mg/L)+卡那霉素(100mg/L)+特美汀(300mg/L)。
生根培养基的配方为:MS+吲哚乙酸(1mg/L)+卡那霉素(100mg/L)+特美汀(3001mg/L)。
3、T0代SlACS2基因编辑植株的获得
将获得的T0代再生植株编号,每株取嫩叶0.2g提取基因组DNA,利用靶位点上游引物(SEQ ID NO:7)和靶位点下游引物(SEQ ID NO:8)进行PCR扩增。扩增结束后,于1%琼脂糖凝胶中分别点样进行电泳,然后将对应的PCR产物回收转化大肠杆菌DH5α,进行测序(图2)。测序所得结果中,SlACS2基因编辑位点197-215处缺失2个核苷酸(突变类型A),编辑位点276-254处缺失7个核苷酸(突变类型B),基因编辑成功,得到T0代两种类型的SlACS2基因编辑植株。
其中,
靶位点上游引物:CTCTTACACCATAACACAAC;
靶位点下游引物:CCAGCCATAACAACTCTTTC。
PCR扩增体系为:基因组DNA1uL,Premix Taq DNA聚合酶Mix 10uL,前后引物各0.8uL,加双蒸水至20uL;PCR扩增条件分别为:94℃5min;94℃30s;58℃30s;72℃30s;35个循环;72℃5min,PCR产物的大小通过1.0%的琼脂糖凝胶电泳得到。
实施例4基因编辑番茄的耐贮藏性能检测
番茄果实呼吸跃变是果实进入完全成熟的一种特征,其果实耐压力是与耐储运性密切相关的重要品质性状。对于加工番茄来说,由于成熟过于集中,如何能够提高番茄果实的耐压力与延长挂枝时间,在成熟采收储运中,重要的目的是延迟其成熟。本发明通过CRISPR-Cas9基因组编辑***修饰该基因,调控***Ⅱ乙烯过量表达,以期迟滞番茄过熟腐烂。下述为新番72号番茄突变株系的耐压力测定试验。
4.1试验设计
将应用实施例1-3的方法得到的新番72号番茄T0代种子A与B与对照野生型亲本同时播种育苗后移载至田间,进行T1加工番茄主要农艺性状观察研究。田间试验采取宽窄双行滴灌栽培模式种植,宽行100cm,窄行50cm,株距30cm,每小区种植20株,随机三次生物学重复试验,5月3日定植,整个生育期田间管理同大田。
4.2试验结果
2019年7月10日对野生型亲本和两个T1突变体植株的转色期番茄进行选择标记,每个试验材料标记60个果实,至试验小区达到成熟果50%时,记为该小区该材料的始熟期,并于始熟期后每隔5天取样一次,选取形状、大小均匀一致的果实10个,测定其耐压力。耐压力测定用电子台秤改置测定,具体测定方法为:将番茄放在待测压板上,旋转把手使上部压板逐渐加压,直至番茄破裂,同时读取电子台秤上显示的压力公斤数。结果发现经基因编辑后的两种突变体类型的耐压力均高于野生型植株,尤其是田间挂枝在20天以后,其对照的耐压力下降明显快于突变株系。见下表:
表1基因编辑对新番72号番茄突变株系果实耐压力的影响测试数据
实施例5获得基因编辑耐贮藏性能得到提高的番茄种质
将实施例4获得的T1代SlACS2基因编辑植株自花授粉后,收获自交种子进行种植,应用实施例3步骤3中的方法,在T2代植株中筛选出不含T-DNA序列的,从而获得在197-215处缺失2个碱基的突变体(A)和197-215处缺失7个碱基的突变体(B)的两种SlACS2突变植株,通过自交获得耐贮藏性能得到提高的无cas9番茄新种质材料。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。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)
1.一种通过基因编辑提高番茄耐贮藏性的方法,其特征在于:利用CRISPR/Cas9***对番茄材料基因组中的催化合成乙烯前体调控基因SlACS2进行编辑,进而使催化合成乙烯前体调控基因SlACS2性能丧失,从而提高番茄耐贮藏性能;
所述CRISPR/Cas9***包括两个sgRNA靶位点,分别命名为sgRNA1和sgRNA2;
所述sgRNA1和所述sgRNA2识别的靶序列均为所述番茄材料基因组中编码SlACS2蛋白的DNA片段。
2.根据权利要求1所述的方法,其特征在于:
所述sgRNA1靶位点序列为:5'-GGATTAAGAGAAACCCAAAAGG-3';
所述sgRNA2靶位点序列为:5'-TAATCTTGAAAGTTGGCAATGG-3'。
3.根据权利要求1或2所述的方法,其特征在于:所述编辑的方法为向所述番茄材料中导入番茄基因组编辑的载体;
所述番茄基因组编辑的载体含有所述sgRNA1靶位点序列、所述sgRNA2靶位点序列和Cas9蛋白的编码基因。
4.根据权利要求1或2所述的方法,其特征在于:所述方法还包括筛选SlACS2纯合突变体的步骤。
5.一种耐贮藏番茄材料的获得方法,其特征在于:包括以下步骤:将权利要求1-4任一所述方法获得的番茄材料自交,得到自交子代,选择SlACS2纯合突变且不携带外源DNA片段的自交子代,即为耐贮藏番茄材料。
6.权利要求5所述的方法在培育耐贮藏番茄材料中的应用。
7.如下(1)-(3)中任一种生物材料:
(1)权利要求1中所述的靶位点序列;
(2)权利要求3中所述的番茄基因组编辑的载体;
(3)含有权利要求3中所述的番茄基因组编辑的载体的微生物转化体。
8.权利要求7所述的载体或微生物转化体或靶位点序列在提高番茄耐储藏性能中的应用;
或,权利要求7所述的载体或微生物转化体或靶位点序列在培育耐贮藏番茄中的应用;
或,权利要求7所述的载体或微生物转化体或靶位点序列在番茄育种中的应用。
9.一种鉴定待测番茄是否为权利要求1-4任一所述方法获得的耐贮藏番茄或其子代的方法,其特征在于:包括以下步骤:
分别提取番茄野生株和待测番茄转化株基因组DNA,以上游引物和下游引物对待测番茄的基因组DNA进行PCR扩增,分别得到PCR扩增产物,根据所述PCR扩增产物测序结果,判断待测番茄是否为上述方法获得的耐贮藏番茄或其子代,判断方法为将转化体植株测序结果与野生型植株测序结果进行比对,如果在sgRNA1、sgRNA2附近或二者之间出现碱基的缺失或***,即可判断其为上述方法获得的耐贮藏番茄或其子代;
所述上游引物:CTCTTACACCATAACACAAC;
所述下游引物:CCAGCCATAACAACTCTTTC。
10.一种鉴定或鉴定待测番茄是否为权利要求1-4任一所述方法获得的耐贮藏番茄或其子代的产品,为如下(1)-(3)中的任一种:
(1)权利要求9中所述的上游引物和下游引物;
(2)含有(1)所述的上游引物和下游引物的PCR试剂;
(3)含有(1)所述的上游引物和下游引物或(2)所述的PCR试剂的试剂盒。
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