CN106222193B - 一种重组载体及无转基因基因编辑植株的筛选方法 - Google Patents
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Abstract
本发明公开了一种重组载体及无转基因基因编辑植株的筛选方法，该重组载体中原始载体为含有sgRNA基因、Cas9基因和筛选标签基因的用于植物基因编辑的CRISPR/Cas9载体，所述重组载体携带有BelRNAi表达元件，所述BelRNAi表达元件转录产生干扰苯达松抗性基因的发夹状RNA干扰片段。本发明在原始载体中引入BelRNAi表达元件，并对后代植株进行苯达松除草剂筛选，既保留了目的基因突变的后代，又确保了该突变后代中不含有转基因片段；筛选方法更为廉价，简单和有效。
Description
技术领域
本发明涉及植物基因编辑技术领域，尤其涉及一种重组载体及无转基因基因编辑植株的筛选方法。
背景技术
基因编辑技术可以准确地对目标基因进行改造(即对DNA序列进行编辑)，不仅是一项先进的生物学技术，同时也是改良农作物品质的有效手段。
目前，可应用于基因编辑的技术主要包括：ZFNs(Zinc finger nucleases，锌指核酸酶)，TALENs(Transcription activator-like effector nucleases，转录起始因子核酸酶)以及CRISPR/Cas9(Clustered regularly interspaced short palir dromicrepeats/CRISPR-associated Cas9,成簇规律间隔的短回文重复序列/Cas9核酸酶)技术(Bogdanove A.J.and Voytas D.F.,2011.TAL effectors:customizable proteins forDNA targeting.Science,333(6051):1843-1846；Carrol D.,2011.Genome engineeringwith zinc-finger nucleases.Genetics,188(4):773-782)。其中，CRISPR/Cas9是新发展起来的一项有着巨大影响力的基因编辑技术，其因简单的操作以及广泛的应用性而受到青睐。在此基础上建立的又一项基因编辑技术CRISPR/Cpf1(CRISPR from Prevotella andFrancisella 1)将基因编辑的重点从引入突变到高效地对基因进行“替换”，“定向编辑”等精准“手术”(Zetsche B.,Gootenberg J.S.,Abudayyeh O.,Slaymaker,I.M.,Makarova,K.S.,Essletzbichler,P.,Volz,S.E.,Joung,J.,Oost J.,Regev,A.,Koonin,E.V.,ZhangF.,2015.Cpf1Is a Single RNA-Guided Endonuclease of a Class 2CRISPR-CasSystem.Cell,163:759-771)。由此可见，基因编辑技术的应用前景是十分广阔的。
CRISPR/Cas9体系中的载体主要由两大元件构成：sgRNA(single guide RNA)以及Cas9。sgRNA是一种非编码小RNA，由U3或者U6启动子启动。Cas9编码核酸酶蛋白，分子量大于1000个氨基酸，可以切割DNA核酸序列。CRISPR/Cas9体系主要借助sgRNA来匹配到基因组的特定位置，之后Cas9核酸酶将DNA切断，形成双链切口。在DNA损伤修复过程中，无论是同源重组修复(Homologous recombination-based repair,HR)还是非同源末端连接修复(Nonhomologous end-joining,NHEJ)，都会在切口处引入突变。此外，在转基因过程中还需要一个筛选基因，例如潮霉素抗性基因(HygR)，草甘膦抗性基因(Bar)。在水稻中，常见的载体有pHun4c12,pRGE等，可以说这些载体的序列结构大同小异。
目前，CRISPR/Cas9系统已经广泛运用于很多物种之中，包括转基因小鼠(CongL.,Ran F.A.,Cox D.,Lin S.,Barretto R.,Habib N.,Hsu P.D.,Wu X.,Jiang W.,Marraffini L.A.,and Zhang F.,2013.Multiplex genome engineering using CRISPR/Cas system.Science,339(6121):819-823；2013；Wang H.,Yang H.,Shivalila C.S.,Dawlaty M.M.,Cheng A.W.,Zhang F.and Jaenisch R.,2013.One-step generation ofmice carrying mutations in multiple genes by CRISPR/Cas9-mediated genomeengineering.Cell,153(4):910-918.)，斑马鱼(Hwang W.Y.,Fu Y.,Reyon D.,MaederM.L.,Tsai S.Q.,Sander J.D.,Peterson R.T.,Yeh J.R.J.and Joung J.K.,2013.Efficient genome editing in zebrafish using a CRISPR-Cassystem.Nat.Biotechnol.,31(3):227-229)等动物；同时在植物中，已经在水稻，拟南芥，烟草等作物之中取得成功(Feng Z.Y.,Mao Y.F.,Xu N.F.,Zhang B.T.,Wei P.L.,Wang Z.,Zhang Z.L.,Yang D.L.,Yang L.,Zeng L.,Liu X.D.,and Zhu J.K.,2014.Muti-generation analysis reveals the inheritance,specificity and patterns ofCRISPR/Cas induced gene modifications in Arabidopsis.Proc.Natl.Acad.Sci.USA,111(12):4632-4637；Miao J.,Guo D.,Zhang J.,Huang Q.,Qin G.,Zhang X.,Wan J.,GuH.,Qu L.J.,2013.Targeted mutagenesis in rice using CRISPR-Cas system.CellRes.,23(10):1233；Nekrasov V.,Staskawicz B.,Weigel D.,Jones J.D.G.and KamounS.,2013.Targeted mutagenesis in the model plant Nicotiana benthamiana usingCas9RNA-guided endonuclease.Nat.Biotechnol.,31:691-693；Shan Q.,Wang Y.,Li J.,Zhang Y.,Chen K.,Liang Z.,Zhang K.,Liu J.,Xi J.J.and Qiu J.L.,2013.Targetedgenome modification of crop plants using a CRISPR/Cas-mediated genomeengineering.Cell,153:910-918)。
以往转基因的经典材料，例如抗除草剂和抗虫的转基因作物，都必须使后代一直保留有转基因成分才能显示出一定的生物学功能。相反，由于CRISPR/Cas9系统在T0代植株中就可以实现靶基因的突变，而在T1代植株中不再需要外源进入的CRISPR/Cas9等T-DNA遗传成分。因此，需要通过自交或者回交使已完成编辑的靶标基因与T-DNA原件分离，获得无T-DNA的基因编辑植株。通过CRISPR/Cas9这样的系统来培育无T-DNA插入植株的优点是显而易见的。第一，从无转基因食品安全的角度出发，是可以被大众广泛接受的。第二，由于T-DNA整合进基因组的随意性，很有可能会破坏植株别的功能基因，造成副作用，因此也需要无转基因的植株进行实验或者生产。
目前，从利用CRISPR/Cas 9体系获得的后代群体中鉴定无转基因植株仍依赖于传统方法，即：通过PCR技术对后代植株进行分析，需要通过扩增转基因元件中携带的标记基因，例如报告基因(Gus)，筛选基因(潮霉素抗性基因,HygR)来确定，如果扩增结果是阳性就表示是转基因植株。上述方法不仅需要提取DNA、进行PCR和凝胶电泳等繁琐的实验步骤，而且若上述步骤中任何一个环节出现错误，都会造成特异性条带无法扩增出来，而误以为某植株是无转基因片段的植株，进而引物假阴性结论。
因此，有必要针对CRISPR/Cas 9体系工作原理的特殊性及优势所在，探究一种更为简便的鉴定方法，以解决上述传统方法带来的缺陷。
发明内容
本发明提供了一种应用于基因编辑(如CRISPR/Cas9系统)研发中培育无转基因植株的载体及筛选方法，为通过基因编辑培育无转基因植株提供了一种更为廉价，简单和有效的筛选手段。
一种重组载体，原始载体为含有sgRNA基因、Cas9基因和筛选标签基因的用于植物基因编辑的CRISPR/Cas9载体，所述重组载体携带有BelRNAi表达元件，所述BelRNAi表达元件转录产生干扰苯达松抗性基因的发夹状RNA干扰片段。
除草剂苯达松的作用靶点是光合系统，而正常水稻的基因组中含有可以解除毒性的苯达松抗性基因CYP81A6/Bel(Os03g0760200)，使得苯达松处理后的水稻依旧能够存活，表现出抗性；但是，若该抗性基因突变或者RNA水平沉默，则会表现出苯达松敏感。
上述特点在本发明中被巧妙地运用，通过在原始载体中引入BelRNAi单元(即BelRNAi表达元件)，使得含有转基因片段(即该重组载体的片段，T-DNA)与BelRNAi绑定。在进行苯达松除草剂筛选时，若植株表现出敏感致死，则为携带转基因成分的植株，也是不需要保留的；而存活下来的则为不含有转基因片段的后代，既保留了目的基因的突变，又确保了该突变后代中不含有T-DNA。
作为优选，所述原始载体为pHun4c12，其已经在水稻中成熟运用。
筛选标签基因主要用于转基因过程中的抗性筛选以及T0代植株中是否已经转入重组载体的筛选；选取筛选标签基因阳性的T0代植株进行后续实验。T0代的筛选过程中，不需要进行苯达松除草剂的喷施，因为成功转入重组载体的转基因后代均对苯达松除草剂敏感。
作为优选，所述筛选标签基因为潮霉素抗性基因。
作为优选，所述BelRNAi表达元件中启动子为d35S，终止子为NOS Terminal。这两个元件应用广泛而且被成熟地运用于载体改造中。
进一步优选，所述BelRNAi基因的碱基序列如SEQ ID NO.1所示。
更优选，所述重组载体的碱基序列如SEQ ID NO.2所示。
本发明还提供了一种CRISPR/Cas9系统中无转基因片段植株的筛选方法，包括：
(1)将所述的重组载体转入水稻植株中，培养获得转基因水稻植株；
(2)从转基因水稻植株中筛选得到含重组载体同时目标基因已完成编辑的T0代植株；
(3)继续培养T0代植株直至获得T1代种子；
(4)种植T1代种子，在T1代幼苗期间，喷施除草剂苯达松；若水稻幼苗死亡，则为含有外源重组载体插入的水稻植株；反之，若幼苗正常生长，则为无外源重组载体插入的水稻植株，判定为无转基因基因编辑材料。
具体地，步骤(2)中，所述筛选的过程包括：
(a)选取存在所述筛选标签基因的植株，得到含重组载体的转基因植株；
(b)提取步骤(a)中含重组载体的转基因植株DNA，经测序，确定转基因植株中sgRNA锚定的靶片段区域的序列，从中挑取已发生基因突变的序列所对应的植株，获得含重组载体的转基因突变植株T0代。
与现有技术相比，本发明具有以下有益效果：
本发明在原始载体中引入BelRNAi表达元件，并对后代植株进行苯达松除草剂筛选，既保留了目的基因突变的后代，又确保了该突变后代中不含有转基因片段；筛选方法更为廉价，简单和有效。
附图说明
图1为实施例1中重组载体的图谱示意图；
其中，sgRNA受U3启动子控制；壮观霉素抗性基因通过Bsa I酶切来替换成与靶基因匹配的20bp；Cas9核酸酶由Ubi启动子启动转录并在水稻细胞中翻译成蛋白，与sgRNA配合切开靶点；HygR是潮霉素抗性基因，主要是在转基因过程中用于筛选阳性植株；在RB附近，引入Bel RNAi单元，其转录产物会形成发夹结构，干扰水稻中的Bel基因RNA，阻碍其翻译。
图2为实施例2的T0代中重组载体的工作原理示意图；
其中，sgRNA会匹配到基因OsLCT上，在Cas9核酸酶的作用下切开DNA双链，修复过程中引入突变；尽管Bel RNAi也会转录出来并干扰基因Bel，但是在没有苯达松的环境下，植株可以正常生长。
图3为实施例2的T1代植株中的原理示意图；
其中，在T1代植株中，CRISPR/Cas9元件已经履行完生物学功能；若植株是带有T-DNA的植株，那么就会携带有T-DNA，那么Bel RNAi单元会干扰水稻细胞中的Bel转录本。此时，外源的苯达松除草剂处理便会引起植株致死，从而达到清除T-DNA携带植株的目的。
图4为实施例2中T1代在苯达松处理之后的表型；
其中，(a)野生型水稻品种嘉浙B，表现出苯达松抗性；(b)苯达松敏感植株，通过γ射线诱变Bel突变产生的；(c)未改造过的原始载体pHun4c12进行基因敲除获得植株，并不表现出苯达松敏感；(d)上述三种材料的单株；(e)筛选出来的目的基因OsLCT突变的16个转基因系(T1代植株)，苯达松处理前的生长状况；(f)16个转基因系经苯达松处理之后的表型。
图5为实施例2中针对16个突变系中的第6和第30号，对每个单株进行分子标记水平验证是否携带T-DNA片段。
图6为实施例3中T1代植株经苯达松处理之后的表型。
具体实施方式
实施例1
1、BelRNAi基因转录单元的制备
这里用到的RNA干扰主要是通过引入300bp长度的发夹(Hairpin)结构来干扰细胞内的Bel转录本。
用引物Beli-F1(gagctcAGCTTAGCCATGGATAACGCCTAC,小写下划线为SacⅠ酶切位点)和Beli-R1(ctgcagAAGGTCACGTCGTGCTCGGTGAAGCACTC,小写下划线为PstⅠ酶切位点)，经PCR扩增获得正向序列。
另一方面，用引物Beli-F2(ggtaccAGCTTAGCCATGGATAACGCCTAC,小写下划线为KpnⅠ酶切位点)和Beli-R2(ctcgagAAGGTCACGTCGTGCTCGGTGAAGCACTC,小写下划线为XhoⅠ酶切位点)。将这两段序列连接T载体之后进行测序，测序结果为SEQ ID NO.1。
测序正确之后，将序列装进PCAMBIA-1301，与其载体上的d35S和NOS terminal拼装，最终形成完整功能单元d35S-BelRNAi-NOS terminal。
2、重组载体的构建
将这个完整单元用EcoRI和HindⅢ酶切之后，装进已经被HpaI切开的原始载体pHun4c12，形成最终的载体。整个载体序列为SEQ ID NO.2。
3、重组载体的转入和植株细胞的培养
携带有目的基因敲除靶片段的pHun4c12-Beli主要是通过农杆菌介导的方法进行转基因。主要方法参照Li et al.(2014).(Li W.X.,Huang J.Z.,Zhao H.J.,Tan Y.Y.,CuiH.R.,Poirier Y.,Shu Q.Y.,2014.Production of low phytic acid rice by hairpinRNA-and artificial microRNA-mediated silencing of OsMIK in seeds.Plant CellTiss.Organ Cult.,119:15-25)。
实施例2以镉(Cd)转运相关的基因OsLCT1(Os06g0579200)作为靶基因，进行无转基因片段植株的筛选
以镉(Cd)转运相关的基因OsLCT1(Os06g0579200)作为靶基因，引入20bp的片段5'-TACTATCCCGCGTGCCAATG-3'作为sgRNA来产生OsLCT1突变体。
如图2所示，在T0代中，一方面，sgRNA在启动子U3的启动下会转录出来，并匹配到基因OsLCT1上；Cas9基因也在水稻细胞中转录翻译出来核酸酶。两者协同作用，切割靶点，之后水稻细胞DNA修复过程中引入突变。另一方面，Bel RNAi发夹结构转录出来形成RNA，与水稻细胞内存在的Bel RNA匹配，打断其片段，影响翻译。
通过实施例1的上述步骤，获得58株水稻，选取前30株进行后续实验。
提取这30株T0代植株的DNA，对其进行PCR验证，以HgyR作为标记基因进行筛选。引物如下：
HygR F:AGAAGAAGATGTTGGCGACCT；
HygR R:GTCCTGCGGGTAAATAGCT。
PCR体系如下：
20μl体系包括：1μl DNA,10μl反应缓冲液,0.4μl上下引物(10μM),双蒸水补足20μl。
PCR反应条件如下：94℃2min；之后进行35个三步循环94℃10s,60℃30s,72℃60s。
再利用引物OsLCT1F:CTCGATGTTAAGCATGCTCC，
和Os LCT1R:AGAGTCAGGAACGCGGCTAC进行扩增，方法及程序如上所述。再通过测序找出突变体以及确定突变位点(如表1所示)。
表1T0代30株植株靶点测序
注：加下环线表示插入，*表示缺失。
将aa和aa'类型植株的T1代种子进行种植，共16个品系，待其长至幼苗进行1000mg/L的苯达松处理。
结果如图4中的(e)、(f)所示，在每个突变系中都表现出一定植株的致死。这个致死的比例并不相同(如表2所示)，取决于T-DNA整合进水稻基因组的拷贝数及位置。存活率最大的是第16个系，33％；而第17号突变系表现出全致死，这很有可能是插入的拷贝数过多引起的，这样的情况需要种植更多的植株来筛选，以便获得理想的植株。另外一个角度，正是因为需要大群体来筛选，因此更能体现出此方法的优势所在。
表1 16个突变系经苯达松处理之后的存活植株统计结果
实施例3以控制水稻稻米香味的基因甜菜碱醛脱氢酶2(Betaine aldehydedehydrogenase 2，OsBADH2)作为靶基因，进行无转基因片段植株的筛选
为了进一步验证新构建载体pHun4c12-Beli的适用普遍性。同时，以基因甜菜碱醛脱氢酶2(Betaine aldehyde dehydrogenase 2，OsBADH2)作为靶基因进行实验，OsBADH2突变的植株会表现出稻米芳香，提高稻米品质。
实验过程及步骤同实施例2一致。
选取其中的两个突变系，对其T1代植株进行苯达松处理。
结果如图6所示，同一个系中表现出苯达松敏感和抗性的植株。其中，存活的植株正是基因OsBADH2已经突变，同时不带有T-DNA插入的理想植株，可以用于生产及水稻育种。
Claims (3)
1.一种无转基因基因编辑植株的筛选方法，其特征在于，包括：
(1)将重组载体转入水稻植株中，培养获得转基因水稻植株；
所述重组载体的原始载体为pHun4c12，含有sgRNA基因、Cas9基因和潮霉素抗性基因；BelRNAi表达元件中启动子为d35S，终止子为NOS Terminal；
还携带有BelRNAi表达元件，所述BelRNAi表达元件转录产生干扰苯达松抗性基因的发夹状RNA干扰片段；
(2)从转基因水稻植株中筛选得到含重组载体同时目标基因已完成编辑的T0代植株；
所述筛选的过程包括：
(a)选取存在所述筛选标签基因的植株，得到含重组载体的转基因植株；
(b)提取步骤(a)中含重组载体的转基因植株DNA，经测序，确定转基因植株中sgRNA锚定的靶片段区域的序列，从中挑取已发生基因突变的序列所对应的植株，获得含重组载体的转基因突变植株T0代；
(3)继续培养T0代植株直至获得T1代种子；
(4)种植T1代种子，在T1代幼苗期间，喷施除草剂苯达松；若水稻幼苗死亡，则为含有外源重组载体插入的水稻植株；反之，若幼苗正常生长，则为无外源重组载体插入的水稻植株，判定为无转基因基因编辑材料。
2.如权利要求1所述的无转基因基因编辑植株的筛选方法，其特征在于，所述BelRNAi基因的碱基序列如SEQ ID NO.1所示。
3.如权利要求1所述的无转基因基因编辑植株的筛选方法，其特征在于，所述重组载体的碱基序列如SEQ ID NO.2所示。
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| Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice;Xu等;《Rice》;20141231;第7卷(第1期);5-8 * |
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