CN116926117A - 一种通过基因编辑培育早熟水稻品种的方法 - Google Patents
一种通过基因编辑培育早熟水稻品种的方法 Download PDFInfo
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Abstract
本发明涉及植物基因工程和生物育种领域,具体涉及基因编辑结合分子轮回选择技术提早水稻品种熟期的方法。通过对qDeh3候选基因28Kb编码区内含子区域引入基因组变异,以提早水稻熟期,且对其它农艺性状没有显著影响。本发明的方法创制的早熟材料可以作为供体,结合分子轮回选择等技术在水稻育种中加以利用。
Description
技术领域
本发明涉及植物基因工程和生物育种领域,具体涉及基因编辑结合分子轮回选择技术提早水稻品种熟期的方法。
背景技术
随着疫情、贸易战乃至局部战争等国际形势变化的影响,粮食安全战略意义日益凸显。早稻是重要的战略储备粮来源,也提供了食品加工的重要原料,2019年以来我国早稻产量连续三年稳步增长[1]。采用早熟高产品种能够有效缩短种植周期,降低生产成本并提高生产者的抗风险能力。因此,培育早熟高产的水稻品种已成为水稻分子育种工作者关注的热点[2]。值得注意的是,当前生产上大面积应用的早稻品种仍以常规稻品种为主[3],杂种优势未能充分利用。究其原因,与现有杂交组合在实现高产过程中,恢复系生育期较长而导致杂交组合熟期偏晚有密切关系。因此培育早熟高产特别是显性早熟高产品系,对水稻育种意义重大[4]。
水稻早熟的分子基础研究有利于了解早熟机理。早熟与早花(水稻中也为早穗)直接相关。开花分子机制在长日照模式植物拟南芥中研究非常详细,其中包括几个重要基因:GI(GIGANTEA)基因与昼夜节律相关,CO(CONSTANS)基因通过激活FT(FLOWERING LOCUS T)基因促进开花。SOC1(SUPPRESSOR OF OVEREXPRESSION OF CO 1)位于CO下游,是整合光照、赤霉素和春化等多种开花信号的关键调控因子[5],在豆科作物中也起着早熟的重要作用[6]。水稻是短日照模式作物,存在相似的调控机制,包括:Hd1与CO[7],Hd3a与FT[8],OsGI与GI[9]等分别属于同源基因。同时有两条主要调控通路:途径1:OsGI-Hd1-Hd3a和途径2:Ghd7-Ehd1-RFT(图1)。
短日照条件下,水稻通过途径1中的OsGI感受短日昼夜节律,再借助Hd1基因来调控Hd3a(编码成花素)表达,将信号从叶片中传递到茎尖分生组织来促进抽穗[10,11]。同时,途径2中的Ehd1(B型响应调节因子[11])也可以诱导Hd3a表达而促进抽穗。除了这两条途径,与Hd1同源的OsCO3基因在短日照条件下通过负调控Hd3a、FTL的表达则会延迟抽穗[12]。
与短日照条件下相反,途径1的OsGI在长日照条件下通过调控Hd1基因来延迟抽穗[10,13]。同时,途径2的Ghd7、DTH8等通过抑制Ehd1来延迟抽穗[14,15]并增加株高和穗粒数。
值得注意的是,生物学现象往往比理论模型复杂,水稻对熟期的调控亦然。除了这两条途径,长日照下还存在促进抽穗的调控通路。例如,OsMADS50/OsSOC1/DTH3基因通过促进Ehd1基因的表达而提早抽穗[16,17];而OsID1/Ehd2/RID1在长日照和短日照下均能上调Ehd1的表达[18,19];OsDof12基因编码DOF转录因子,长日照下通过上调Hd3a的表达量促进抽穗[20]。
产量是水稻最重要的育种目标性状,适宜的熟期是品种生态适应性的重要指标。从目前分子育种关注的熟期基因(图1)看,与产量相关的熟期基因,其增加产量的同时往往与熟期延迟有关。因此,早熟与丰产存在一定的矛盾[21]。
Ghd7对很多农艺性状都有影响,包括株高,熟期和穗粒数。长日照条件下,增加Ghd7的表达,熟期延迟,株高和穗粒数增加[14]。最新研究表明,Ghd7作为一个转录抑制因子,Ghd7能直接与ARE1(ABC1 REPRESSOR1)基因位于启动子和第一内含子的2个EveningElement-like基序结合,从而抑制ARE1基因表达并提高氮素利用效率和籽粒产量[22]。Ghd7.1编码伪应答调控类蛋白,长日照条件下有功能性的Ghd7.1使水稻抽穗延迟、产量增加[23]。长日照条件下,DTH8/Ghd8/EF8通过负调控Ehd1、RFT1和Hd3a的表达延迟水稻抽穗并增加产量。MOC1基因负责控制水稻分蘖和侧枝,DTH8/Ghd8能促进MOC1的表达增加蘖数、一次枝梗和二次枝梗数,从而增加产量[24]。
DTH8可以负调控水稻中叶绿素生物合成基因的表达量以降低叶绿素含量,在光周期开花通路、产量潜力以及叶绿素合成中发挥重要作用[25]。Hd1和Ehd1能够降低穗部一次枝梗的数目,导致穗粒数的减少,独立于抽穗期的调控;Hd1Ehd1株系在开花转换时期的叶片中,Hd3a和RFT1这两个成花素基因的表达上调。Hd1和(或)Ehd1导致穗发育时顶端分生组织中类Terminal Flower 1上调、穗形成相关基因的表达提前。因此,Hd1和Ehd1这两个重要的开花基因具有调控水稻穗发育的功能,可能是通过影响叶片中成花素基因的表达进而影响作物产量[26]。
真核生物基因的非编码序列主要包括启动子(promoter)上下游非翻译区(UTR)和编码区内含子(intron)。其中编码区内含子可以转录,在mRNA成熟过程中被加工剪切。近来研究发现,编码区内含子在基因的表达调控中有着重要的作用,其起作用的方式除了可变剪接之外,主要表现为内含子保留(intron retention,IR)[27]。内含子保留对于压力条件下的酿酒酵母细胞生长调节起重要作用[28]。在饥饿细胞中,内含子还能够通过抑制核糖体蛋白基因表达,来实现细胞代谢减缓、减少能量消耗,最终延长细胞寿命的目的[29]。
此外,内含子本身还可能包含调控结合位点,影响基因的表达。例如,拟南芥中,非编码区的SNP差异会导致FLC基因表观遗传记忆的不稳定[30]。保留的内含子中包含重要的顺式元件,可能在小麦抗白粉病基因表达调控中起重要作用[31]。
一般认为水稻的早熟性由非感光基因、感光抑制基因、早熟基本营养生长期基因及相关的修饰基因所决定[32],属于主基因控制的隐性性状或多基因控制的数量性状,显性早熟性状的报道较少。除了突变体,育种工作者更加关注来自育种材料的显性早熟基因。目前报导的显性早熟基因大多来自不育系/保持系(表1),且F2群体大多符合3:1的分离比例。目前仅有1例被克隆的显性早熟基因Ef-cd,研究推测其分子机理是通过lncRNA编码基因的启动子变异导致表达水平上升,来调控下游抽穗期基因表达促进开花[33]。
基因组编辑技术是指可以在基因组水平上对DNA序列进行定点改造的遗传操作技术,其在基因功能研究和改造、生物医学和植物遗传改良等方面都具有重大应用价值。科学家自20世纪90年代末就开始探索基因组定点编辑技术,但直到2002年,也仅在小鼠[34]和果蝇[35]等少数模式生物中实现了同源重组介导的基因组定点编辑,且因同源重组的效率很低,限制了其应用前景。进入21世纪后,随着蛋白质结构与功能研究的新突破和人工核酸内切酶(engineered endonuclease,EEN)技术的出现,将特异识别并结合DNA的蛋白结构域和EEN融合,创造出了能够特异切割DNA序列的核酸酶(sequence-specific nucleases,SSNs),从而可以对基因组特定位点进行高效和精确的靶向编辑[36]。
目前,SSNs主要包括锌指核酸酶(Zinc finger nucleases,ZFNs)[37]、类转录激活因子效应物核酸酶(transcription activator-like effector nucleases,TALENs)[38]、成簇的规律间隔的短回文重复序列及其相关系统(clustered regularlyinterspaced short palindromic repeats/CRISPR-associated Cas9,CRISPR/Cas9system)[39]和CRISPR/Cpf1系统[40]。这些SSNs的共同特点是都能在基因组特定部位精确切割DNA双链,造成DNA双链断裂(DNA double-strand breaks,DSBs);而DSBs能够极大地提高染色体重组事件发生的概率[41]。DSBs的修复机制在真核生物细胞中高度保守,主要包括同源重组(homology-directed repair,HDR)和非同源末端连接(non-homologous endjoining,NHEJ)[42]两种修复途径。当没有供体DNA时,细胞则通过NHEJ途径修复[42]。由于NHEJ方式的修复往往不够精确,在DNA链断裂位置常会产生少量核酸碱基的插入或缺失(insertion-deletion,InDel),从而导致基因突变;而存在同源序列供体DNA时,以HDR方式的修复能够产生精确的定点替换或插入[42]。
2010年出现TALENs和2013年出现CRISPR/Cas9技术后,世界上掀起了基因组定点编辑研究热潮[43]。尤其是CRISPR/Cas9技术,因其相对简单、精确、高效,很快被广泛应用于医学、农业、基础研究等领域[44,45]。
基因编辑领域历经多年的技术积累和发展,从早期的Zinc Finger核酸酶技术、TALEN技术发展至更具可编程性的CRISPR技术、碱基编辑技术(Base Editing)和引导编辑技术(Prime Editing)等,在生物育种、生物制药、合成生物学等多个应用方向迎来快速发展的风口。国际顶尖学术期刊Nature亦将精准基因组编辑技术列为2022年值得关注的7大技术。
基因序列根据其是否在编码蛋白中起主要作用,通常分为编码区和非编码区,后者包括启动子(promoter)、上游(5’)和下游(3’)非翻译区(untranslated region,UTR),它们的主要功能是调控编码序列的表达。编码区和UTR在转录的过程中会出现在前体mRNA中,在从前体mRNA到成熟mRNA的加工过程中,被切除的部分称为内含子(intron);被保留下来的部分称为外显子(exon),经过拼接用于作为后续翻译蛋白质的模板,因此在UTR和编码区中均存在内含子和外显子。当前的基因编辑技术主要靶点是编码区外显子、启动子和UTR,而编码区内含子未有关注。
在水稻中,通过编辑Waxy基因5’UTR内含子来改变目标基因的表达水平,能够实现对稻米品质性状的数量调控[46];通过编辑前文提到的与产量相关的抽穗期基因Ghd7.1(Hd2)上游开放阅读框(Upstream Open Reading Frame,uORF),即5’UTR外显子能够改变水稻品种熟期[47],但是由于这些抽穗期基因与产量之间存在显著关联即一因多效,采用这种方法提前熟期将导致产量的降低。因此如何找到能够提早熟期而对产量影响不大的编辑靶点对于生物育种应用至关重要。
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发明内容
本发明发现qDeh3候选基因28Kb编码区内含子的变异控制显性早熟材料DEH229长短日照下的熟期,进而通过基因编辑手段,在qDeh3候选基因28Kb编码区内含子区域引入基因组变异,能够提早水稻熟期,对其它农艺性状没有显著影响。该方法创制的早熟材料可以作为供体,结合分子轮回选择等技术在水稻等作物育种中加以利用。由此完成本发明。
本发明提供一种通过基因编辑培育早熟作为品种的方法,其特征在于,通过对禾本科作物中与水稻第3染色体的QTL编号为qDeh3的候选基因的同源的基因作为靶点进行基因编辑突变以使作物获得早熟特性,优选地所述作物是禾本科或豆科作物,更多具体的例如是水稻、玉米、大豆。经调研,在禾本科植物和豆科植物中均存在上述的同源基因且均与熟期相关,因此本发明的方法能够适用于禾本科或豆科作物。
优选地,所述基因编辑的靶点位于编号为qDeh3的候选基因或其同源基因的内含子区域。
进一步优选地,对于水稻作物而言,其靶点是第3染色体的QTL编号为qDeh3的候选基因的编码区内含子的28Kb区域内缺失、插入或替换突变或其组合突变,例如大片段的缺失插入和小片段的缺失插入,或单个碱基替换,以获得早熟特性。
更具体地,所述基因编辑的靶点的核苷酸序列是:靶点1-AGCCGTACGTCTAAGCAGCC,靶点2-TCGGGCGGAGACGTGCGGTT。
在具体操作中,在靶点两端分别设计引物;再将靶点引物接头与启动子连接,再通过Overlapping PCR方法构建包含靶点的sgRNA表达盒;把载体和sgRNA表达盒连接到一起,完成载体构建。
进一步,将构建完成的载体遗传转化水稻。
更进一步优选地,通过基因编辑获得T0代转基因苗,收获自交种子,进一步种植T1代并鉴定靶点编辑的纯合体,进一步获得早熟特性的稳定株系。
在具体实施方式中,水稻起始品种是各生态区的偏中熟品种或偏晚熟品种,例如籼稻区的明恢63(MH63)和粳稻区的中农粳11等。
本发明也提供所述的方法获得的早熟作物品种的应用,其特征在于,将其作为供体,结合分子轮回选择技术,即选择熟期偏晚的材料作为轮回亲本,编辑早熟材料作为供体亲本,根据编辑早熟材料的靶点信息设计引物,开发分子标记,每一轮回交,都使用这对标记辅助,在杂交后代分离世代中,选择早熟单株作为杂交对象,与轮回亲本继续杂交,再通过杂交或花培等快速稳定方法,最终培育目标早熟水稻品种;
所述作物是禾本科和豆科作物,优选地所述作物是水稻、玉米或大豆。
本发明基于研究发现qDeh3候选基因28Kb编码区内含子的变异控制显性早熟材料的熟期,进而通过基因编辑手段在此处引入基因组变异,能够提早作物尤其是禾本科作物的熟期,而对其它农艺性状没有显著影响。因而,本发明方法可以用于创制的作物早熟材料,进而可以作为供体运用于作物育种中。
附图说明
图1长/短日照下的水稻熟期调控网络。
图2A基于MH63/DEH229组合的F2群体BSA-seq穗期定位结果。
图2B基于MH63/DEH229组合的F2群体ES-RIL群体海南短日穗期定位结果。
图2C基于MH63/DEH229组合的F2群体ES-RIL群体北京长日穗期定位结果。
图3基于三代基因组测序的DEH229与MH63的qDeh3候选基因变异分析。
图4接头引物与启动子连接(A)以及表达盒构建(B)效果检测。其中,Ladder:2000+的marker,U6aT1:包括靶点1的序列,gRT1:包括靶点1的反向序列,U6bT2:包括靶点2的序列,gRT2:包括靶点2的反向序列,sgRT1:加上靶点1之后的sgRNA,sgRT2:加上靶点2之后的sgRNA。
图5qDeh3候选基因的28Kb编码区内含子基因编辑两个株系(Line1和Line2)及其受体亲本(MH63)在海南(短日照)和北京(长日照)条件下的熟期表型。
图6载体(A和B分别是靶点1和靶点2)和qDeh3编码区28Kb内含子编辑获得早熟株系(C)的靶点变异。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。其中对于没有特别描述的具体实验操作是常规操作。
实施例中采用的实验材料:亲本明恢63(MH63)、DEH229及其杂交组合MH63/DEH229的F2群体1个、该组合所衍生的极端选择重组自交系(Extreme-Select RecombinantInbred Lines,ES-RIL)1套。
本发明所使用的材料DEH229来源于MH63的高代回交后代,同MH63、等中晚熟材料杂交的F1代表现显性早熟。与其它已报道的显性早熟育种材料不同,MH63/DEH229的F2群体穗期呈双峰分布但严重偏离3:1;对定位到的主效位点进行候选基因分析发现,双亲在编码区没有变异[4]。
表1.目前育种材料中已定位/克隆的显性早熟基因
而根据日本晴参考基因组,在qDeh3候选基因(一个MADS-box家族基因)有着长达28Kb的编码区内含子,DEH229与MH63的变异集中在内含子区域,这很可能是导致qDeh3与目前已知的编码区变异存在不同的显性早熟调控机制。
实施例一 基因定位
2021年在海南(短日照)下种植MH63/DEH229的F2群体,再海南和北京(长日照)两个环境条件下种植ES-RIL群体,设置两个生物学重复。对群体进行全基因组分子标记基因型鉴定,其中F2群体采用BSA分池测序结合双亲重测序,测序深度超过100X;ES-RIL采用芯片进行SNP标记基因型鉴定。定位也是采用通用常规方法,前者利用QTG-Seq[50],后者采用QTL定位软件进行单标记分析(SMA)和区间作图[51],为减少假阳性,LOD阈值采用5.0。选取两种方法都检测到的作为可靠的位点。
使用BSA-seq方法对MH63/DEH229的F2群体的早熟池和晚熟池的进行分池测序,再QTG-Seq进行定位。当window size=50时,在第3染色体短臂上定位到一个QTL,依据日本晴参考基因组,区间是269,914-3,266,858bp,大小为2.99Mb,我们把这个QTL位点命名为qDeh3(图2A)。再结合ES-RIL群体在长/短日照下连锁定位验证,其中qDeh3在长短日照下均能检测到。短日照下3号染色体的qDeh3的贡献率是63.2%(图2B),长日照条件下qDeh3的贡献率是55.0%(图2C)。促进抽穗期的有利等位基因来源于DEH229。
实施例二28Kb编码区内含子变异分析
对亲本MH63和DEH229分别取样进行长片段三代测序。
具体流程如下:
1.基因组DNA样品检测
(1)利用安捷伦4200系统,检测提取DNA的片段大小,查看样品是否存在降解;
(2)采用Nanodrop系统,检测DNA纯度,OD260/280在1.8-2.0之间,OD260/230在2.0-2.2之间;
(3)采用Qubit系统,精确定量DNA。
2.文库构建
DNA质检合格后,采用使用g-TUBE进行片段打断,之后进行片段分选获得~20kb的插入片段。进一步利用磁珠富集、纯化大片段DNA,对片段末端进行修复、在DNA片段两端连接茎环状测序接头,再次经过片段分选之后,进行退火绑定DNA聚合酶,经过Agilent 2100检测文库质量合格后进行上机测序。
3.上机测序:
DNA文库经定量后,将一定浓度和体积的文库模板和酶复合物转移到PacBioSequel测序仪的纳米孔里面,上机进行测序。
4.下机数据处理及质控
对下机的原始数据Polymerase reads进行测序接头去除和拆分处理,获得Subreads。然后,基于测序仪本身,对Subreads进行自动化质量控制,包括去除仍然含有测序接头序列的Subreads以及过滤掉平均碱基质量小于0.8的Subreads。使用Smrtlink中的CCS工具,将Subreads转化为HiFi reads。最后,对Polymerase reads、Subreads、HiFireads进行描述性统计分析。
获得的数据质量如下:在单碱基准确率99%的前提下,Reads N50和平均长度均>14kb(表2)。将Reads拼接成fasta格式文件,获得的Contig N50均大于30Mb(表3)。在此基础利用BLAST建立本地序列检索库,用于后续分析。
表2.三代测序原始数据统计
表3.三代测序数据拼装结果
利用候选基因序列对MH63和DEH229的本地序列检索库分别进行BLAST,将调取的FASTA格式序列文件用于序列比对。分析结果发现,MH63和DEH229的基因组差异集中在qDeh3候选基因的28Kb编码区内含子区域。与MH63相比,DEH229的qDeh3候选基因28Kb编码区内含子有两个3-4Kb长度的大片段插入,另有15个小的InDel(图5)。
实施例三靶点设计及遗传转化
先以日本晴作为参考基因组,利用在target Design网站,针对定位候选基因的编码区内含子进行靶点设计;再根据脱靶率、特异性和靶点+sgRNA的二级结构等特征,筛选两个合适靶点。然后,在靶点两端分别设计引物,检测MH63和DEH229基因组中是否也存在该靶点且序列一致。再将靶点引物接头与U6a/U6b启动子连接,通过Overlapping PCR方法构建包含靶点的sgRNA表达盒。使用Golden Gate cloning的方法,把pYLCRISPR/Cas9载体和sgRNA表达盒连接到一起,完成载体构建。
根据上述qDeh3候选基因28Kb编码区内含子的靶点设计流程,选择的靶点及检测用的引物如表5所示,PCR反应体系如表6所示。接头引物与U6a启动子连接一共四个PCR反应,分别命名为U6aT1、gRT1、U4bT2和gRT2(表4)。通过琼脂糖电泳,检测启动子连接和表达盒构建的PCR产物。U6aT1和U4bT2的产物大小在140-600bp;表达盒构建gRT1和gRT2获得的目标片段长度分别为629bp和515bp(图4),构建载体涉及的引物也已在表5中列出。
表4.SgRNA与启动子连接以及表达盒构建使用的4个PCR反应
试剂 | U6aT1 | gRT1 | U4bT2 | gRT2 |
buffer | 5ul | 5ul | 5ul | 5ul |
dNTP mix | 2ul | 2ul | 2ul | 2ul |
GXL | 0.5ul | 0.5ul | 0.5ul | 0.5ul |
ddH2O | 16ul | 16ul | 16ul | 16ul |
U6a/U6b | U6a 0.5 | U6a 0.5 | U6b 0.5 | U6b 0.5 |
U-F/gRT | U-F 0.5ul | gRT1 0.5ul | U-F 0.5ul | gRT2 0.5ul |
U-T/gR-R | Ua-T1 0.5ul | gR-R 0.5ul | Ub-T1 0.5ul | gR-R 0.5ul |
表5.实验使用的靶点/引物序列
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表6.实验所用的PCR反应体系
PCR反应参数:98℃变性5分钟,然后进入PCR循环,即98℃10秒,55℃15秒,68℃1分钟,共进行35个循环,最后68℃延伸10分钟。
遵循常规转化方法,将载体转化到农杆菌中,测序鉴定,确认靶点已连接到载体,即可送遗传转化,受体材料为明恢63,植物抗性为潮霉素,具体操作步骤如下。
具体操作步骤
1、愈伤诱导与继代
挑选成熟水稻种子(最好是当年新收种),剥离颖壳,倒入50ml离心管中,加入75%乙醇消毒1min,倒掉乙醇,无菌水冲洗一遍,倒掉,再加入30%次氯酸钠消毒20min,倒掉次氯酸钠后用无菌水冲洗5-6遍。移液枪吸去多余的水份(可用灭菌过的滤纸吸干),将种子转移到诱导培养基上,每皿20-25颗种子。
愈伤长出后可用原胚直接做转化,原胚旁边长出的小颗粒可挑取到新的诱导培养基上进行继代培养,长到适宜的大小时同样可以进行转化。
2、农杆菌培养
将含有目的基因载体的农杆菌EHA105在含有相应抗生素的平板上划线,28℃黑暗培养2天至出现单菌落。
3、农杆菌侵染
准备AAM侵染液,加入AS(1000倍稀释),用移液器吸取AAM将平板上的农杆菌冲洗下来,调整菌体浓度至OD600为0.3-0.5,即为共培养转化水稻用的农杆菌悬浮液。
挑选足够数量的愈伤组织(愈伤状态良好,颜色鲜黄,质地圆润坚硬,颗粒直径在3mm左右为宜,)放入100ml无菌三角瓶中,加入适量农杆菌悬浮液(保证有足够的菌液与材料接触即可),室温放置侵染20分钟,并不时晃动。倒掉菌液,将愈伤组织放在无菌滤纸上吸去多余菌液,随即转移到铺有一层无菌滤纸的固体共培养基上,26℃黑暗培养3天。
4、筛选培养
共培养3天后的愈伤组织要进行清洗步骤,用1ml的蓝枪头将共培养基上的愈伤播到已灭菌的三角瓶中,加入无菌水冲洗两边,第三遍用含有500ul/L羧苄青霉素的无菌水冲洗一遍,移液枪吸掉多余水分后将愈伤转移到无菌滤纸上利用超净台的风吹干愈伤上的水,吹风时间控制在30min左右,待愈伤吹干后转移到筛选培养基上进行筛选培养,培养条件28-30度,暗培养。筛选时长3-4周。
5、分化再生
筛选一个月后,可见颜色鲜黄,直径1-2mm的阳性愈伤长出,此时可将阳性愈伤挑取到分化培养基上进行分化再生。每个分化皿上放16颗阳性愈伤,置于28-30度温室中光照培养。一般10天左右可将愈伤冒出绿点,在经过10天左右会有幼苗分化出。
6、幼苗生根
待分化出的幼苗长到2-3cm左右,有明显根系的时候就可以将幼苗转移到生根培养基上让幼苗长大,生根培养基要倒在比较高的瓶子或管子里,生根后的苗子才有足够的空间长高,生根培养条件28-30度,无菌光照培养。
实施例四基因编辑效果
对获得的转基因植株,通过靶点特异引物,扩增编辑后代和受体亲本(MH63)的基因组DNA,PCR产物测序,获得序列进行比对分析。
水稻总DNA的提取:
TPS配方:1M Tris-HCL(PH8.0)10mL,0.5M EDTA(PH8.0)2mL,KCl 7.45g,ddH2O定容到100mL。
1)取约4cm左右的水稻叶片放置于2mL离心管里,加入两个钢珠。根据自封袋上的编号在离心管上做标记。
2)按照6*8的方式放置在100孔板中,盖上盖子。
3)将离心管转移到固定的板子上,上下盖住,放入液氮中冷冻1分钟左右取出,放入打样机中粉碎样品。
4)开盖,加TPS缓冲液1mL,盖上盖子,65℃烘箱放置40分钟以上(每5分钟摇晃一次),离心12000rpm,10min。
5)取上清放入加500μL异丙醇(等体积)的1.5mL的离心管中,异丙醇需要放置在4℃冰箱里。(多余的加异丙醇的孔板可以先放置在4℃冰箱)
6)将混有上清和异丙醇的离心管放置在﹣20℃冰箱里,1小时以上。
7)12000rpm离心6min,去上清。
8)加200μL的75%的酒精洗涤,去上清,30℃烘箱20-30min。
9)加200μL dd H2O溶解,4℃冰箱保存。
表型观察:
拿到T0代转基因苗之后,缓苗、移栽,收获足量自交种子。2021年在北京继续种植T1代,同时种植亲本作为对照。秧苗期每个株系混合取样,鉴定靶点是否纯合。调查抽穗期表型。2022年海南种植T2代,同样取样鉴定靶点和调查表型。通过对qDeh3候选基因28Kb编码区内含子的基因编辑,获得了Line1和Line2这样的稳定株系,与受体MH63相比,熟期提早而其它农艺性状变化不大。如图5所示,qDeh3候选基因的28Kb编码区内含子基因编辑两个株系(Line1和Line2)及其受体亲本(MH63)在海南(短日照)和北京(长日照)条件下的熟期表型,可知通过编辑qDeh3候选基因的内含子,使内含子DNA产生缺失之后,水稻的抽穗显著提前。与野生型受体MH63相比,编辑株系在长日照下可提前14天左右,短日照下可提前22天左右(表7)。
表7.编辑株系的抽穗期表现
编辑靶点验证:
在同源左臂的左边和同源右臂的右边的基因组序列上分别设计正向引物C2-F和反向引物C2-R(表3),通过PCR反应扩增发生重组后的目标区段(表4),再通过Sanger测序,将获得编辑株系序列和野生型序列信息,进行比对查看靶点编辑情况。根据对载体和编辑株系的靶点变异比较发现,Line1和Line2的主要在靶点1附近qDeh3候选基因28Kb编码区内含子引入了InDel变异。如图6所示,A和B分别是包含靶点1和靶点2的载体部分序列,C为qDeh3编码区28Kb内含子编辑获得早熟株系的靶点变异的序列比较,由此可知在第一个靶点附近缺失一段序列,包括Line1和Line2类型的缺失,都会促进水稻抽穗。
Claims (10)
1.一种通过基因编辑培育早熟作为品种的方法,其特征在于,通过对禾本科作物中与水稻第3染色体的QTL编号为qDeh3的候选基因的同源基因作为靶点进行基因编辑实现基因组变异以使作物获得早熟特性,优选地所述作物是禾本科或豆科作物,更多具体的例如是水稻、玉米、大豆。
2.如权利要求1所述的方法,其特征在于,所述基因编辑的靶点位于编号为qDeh3的候选基因的同源基因的编码区内含子区域。
3.如权利要求2所述的方法,其特征在于,对于水稻作物而言,其靶点是第3染色体的QTL编号为qDeh3的候选基因的编码区内含子的28Kb区域内缺失、插入或替换突变或其组合突变,例如大片段的缺失插入和小片段的缺失插入,或单个碱基替换,以引起早熟特性。
4.如权利要求3所述的方法,其特征在于,所述基因编辑的靶点的核苷酸序列是:靶点1-AGCCGTACGTCTAAGCAGCC,靶点2-TCGGGCGGAGACGTGCGGTT。
5.如权利要求3所述的方法,其特征在于,在靶点两端分别设计引物;再将靶点引物接头与启动子连接,将再通过Overlapping PCR方法构建包含靶点的sgRNA表达盒;把载体和sgRNA表达盒连接到一起,完成载体构建。
6.如权利要求5所述的方法,其特征在于,将构建完成的载体遗传转化水稻。
7.如权利要求6所述的方法,其特征在于,通过基因编辑获得T0代转基因苗,收获自交种子,进一步种植T1代并鉴定靶点编辑的纯合体,进一步获得早熟特性的稳定株系。
8.如权利要求1至7任一项所述的方法,其特征在于,水稻起始品种是各生态区的偏中熟品种或偏晚熟品种,例如籼稻区的明恢63(MH63)或粳稻区的中农粳11。
9.如权利要求1-8任一项所述的方法获得的早熟作物品种的应用,其特征在于,将其作为供体,结合分子轮回选择技术,即选择熟期偏晚的材料作为轮回亲本,编辑早熟材料作为供体亲本,根据编辑早熟材料的靶点信息设计引物,开发分子标记,每一轮回交,都使用这对标记辅助,在杂交后代分离世代中,选择早熟单株作为杂交对象,与轮回亲本继续杂交,再通过杂交或花培快速稳定方法,最终培育目标早熟水稻品种;
所述作物是禾本科或豆科作物。
10.如权利要求9所述的应用,其特征在于,所述作物是水稻、玉米或大豆。
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