CN116836996A - 基因duyao和jieyao在建立细胞筛选系统中的应用 - Google Patents
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Abstract
本发明公开一种基因DUYAO和JIEYAO在建立细胞筛选系统中的应用，本发明提供的生殖隔离基因座及组成基因，培育RHS12位点亲和性水稻品系的策略，以及利用DUYAO和JIEYAO基因使转基因事件优势传递，快速纯合的方法对提高水稻杂种育性，加快水稻品种改良进程具有重要的应用价值。此外，通过对DUYAO和JIEYAO作用机制的阐明，以及两者对植物(例如水稻)细胞，真菌(例如酵母)细胞，动物(例如果蝇S2细胞)细胞同样具有毒性，解毒作用，拓宽了两基因的应用范围，对其它物种利用该基因进行遗传改良，医疗，生物制药等具有极高的科研和应用价值。
Description
本申请为申请日为2022年8月30日，申请号为202211059979.0，发明名称为水稻生殖隔离基因座RHS12基因DUYAO和JIEYAO及其育种利用的中国发明专利的分案申请。
技术领域
本发明属于植物基因工程技术领域，具体涉及RHS12基因座的鉴定，RHS12基因座组成基因DUYAO和JIEYAO的克隆，DUYAO和JIEYAO两个基因在植物(水稻)，真菌(酵母)和动物(果蝇)中的功能验证，以及在水稻育种中的利用。
技术背景
水稻是全世界一半人口，中国60％人口的主粮，因此提高水稻产量对确保粮食安全具有重要的意义。水稻种间，亚种间存在强大的杂种优势，籼粳杂交具有能提高水稻约30％产量的潜力(Qian,2016)。此外，野生稻一些优良的农艺性状，如耐逆性、抗虫性、抗病性等导入到栽培稻中能维持栽培稻产量的稳定(徐鹏,2005)。然而，种间，亚种间存在的生殖隔离严重影响了水稻杂种优势的利用，即存在的杂种不育基因一方面严重影响了水稻的产量提高，另一方面严重限制了种间或亚种间有利基因的交流和聚合(Ouyang and Zhang,2013)。
为了克隆杂种不育基因，解析杂种不育机理，以期更好得利用杂种优势，几十个控制杂种不育的位点已经被鉴定。根据导致的性状不同，这些基因被分为杂种雄配子败育基因，例如Sa,Sb,Sc,Sd,Se,S24(Zhao et al.,2011；Li et al.,2006,2008；Long et al.,2008；Shen et al.,2017；朱文银et al.,2008)等。还有一些是雌配子败育基因，例如S1,S5,S7,S29,S31(Zhao et al.,2007；Xie et al.,2019；Yang et al.,2012；Yu et al.,2016；Zhu et al.,2005)等。然而，目前仅有Sa,S5,S1,Sc,qHMS7(Long et al.,2008；Shenet al.,2017；Xie et al.,2019；Yang et al.,2012；Yu et al.,2018)等少数几个座位的基因被克隆，尚不能解决杂种不育问题，因此需要鉴定和克隆更多的杂种不育基因。在克隆杂种不育基因后，就可以去筛选或创造广亲和材料。所谓的广亲和材料是指粳稻携带S-j型基因，籼稻携带S-i型等位基因，而携带中性基因S-n的即为广亲和材料。广亲和材料和粳稻或者籼稻杂交育性都正常。此外，目前已克隆的水稻杂种不育基因仅限于对水稻生殖细胞(配子)起作用，其是否对体细胞甚至水稻以外的物种起作用值得深入的研究和具有重要的意义。
发明内容
本发明目的之一在于提供水稻种间、籼粳亚种间、粳稻和籼稻品种间均广泛存在的生殖隔离位点RHS12。
本发明目的之二在于提供组成RHS12位点的基因：DUYAO和JIEYAO。
本发明目的之三在于提供DUYAO和JIEYAO基因所编码的蛋白。
本发明目的之四在于提供DUYAO和JIEYAO基因在水稻育种中培育育性亲和性水稻品系的应用。
本发明所述目的的具体技术方案如下：
本发明所述的生殖隔离位点RHS12位于水稻第12号染色体短臂，具体位置为标记DK24和TGR4之间30.8kb的区间范围内。
其中分子标记DK24的序列为：
DK24-F TGTAATTGCAAGAATGGGCC
DK24-R CTCCGTTCGTCCGTAAAGTA。
分子标记TGR4的序列为：
TGR4-F ATTCGAATAGCCGTGCCCGT
TGR4-R CCAACCAACCCACCTGAACT。
本发明所发现的RHS12基因座能决定种间，籼粳亚种间，籼稻或粳稻品种间杂交是否会产生生殖隔离，具体表现为发生花粉或雌配子败育。
本发明克隆了组成RHS12基因座的两个基因DUYAO和JIEYAO，其中基因DUYAO核苷酸序列如SEQ ID NO.1所示，蛋白序列如SEQ ID NO.2所示；基因JIEYAO核苷酸序列如SEQID NO.3所示，蛋白序列如SEQ ID NO.4所示。
本发明还阐明了DUYAO和JIEYAO两个基因引起RHS12位点生殖隔离的遗传和分子机制。DUYAO蛋白通过与OsCOX11互作产生细胞毒性，引起细胞及配子死亡；而JIEYAO蛋白通过与DUYAO互作，改变DUYAO蛋白的线粒体定位，并使其降解，进而解除毒性，细胞及配子存活。
本发明还提供了上述培育的育性亲和性品系在水稻育种的利用，具体应用方法为将DUYAO基因无功能而JIEYAO基因有功能的育性亲和性品系与其它任何品种杂交，培育没有RHS12位点生殖隔离效应的杂交材料，用于水稻育种。本发明所述的DUYAO基因无功能是指DUYAO基因没有正常表达的情况，例如基因DUYAO被敲除或被沉默。培育的没有RHS12位点生殖隔离效应的杂交材料为育性正常的杂交材料，也就是RHS12位点不引起花粉或胚囊育性降低的材料，用于水稻育种。
本发明还提供了培育水稻育性亲和性品系的策略，具体为破坏目标水稻品种中的DUYAO基因或者将JIEYAO基因转入目标水稻品种中。破坏基因DUYAO的方法可以采用本领域常用的方法，例如通过但不局限于CRISPR/Cas9技术，对DUYAO基因进行突变，使其丧失功能。
本发明还提供培养杂种不育生物品系的策略，其特征在于，具体为将权利要求2所述的基因DUYAO和基因JIEYAO一起转入目标生物中。
本发明还提供一种打破水稻生殖隔离的方法，破坏目标生物中的基因DUYAO或者将基因JIEYAO转入目标水稻品种中。
本发明还提供一种建立生物生殖隔离的方法，其特征在于，将基因DUYAO和基因JIEYAO一起转入目标生物中。
本发明所述的生物为含有基因座RHS12的植物，和不含有RHS12位点的真菌或动物；更优选的为水稻、酵母或果蝇。
本发明还提供了一种通过转基因将目的基因进行优势传递，快速纯合的策略。具体应用方法为将DUYAO和JIEYAO两个基因组成一个元件，需要转基因的基因与该元件串联组成一个新的元件，该单拷贝新元件能接近100％传递到T1代，而且接近50％的T1代为转基因纯合型。
本发明还提供了DUYAO和JIEYAO两个基因在体细胞(以水稻为例)中的应用。具体方法为将DUYAO基因在水稻愈伤细胞表达时，其能杀死细胞，而同时表达DUYAO和JIEYAO时，JIEYAO能消除DUYAO的毒性，使细胞正常生长。
本发明还提供了DUYAO和JIEYAO两个蛋白在酵母中的应用。其具体方法为将DUYAO蛋白在酵母中表达时，酵母不能正常生长，而将DUYAO与JIEYAO同时在酵母中表达时，JIEYAO能解除DUYAO的抑制作用，使酵母正常生长。
本发明还提供了DUYAO和JIEYAO两个蛋白在果蝇S2细胞中的应用。其具体方法为将DUYAO蛋白单独在果蝇S2细胞中表达时，果蝇S2细胞的增殖速率降低，而当单独表达JIEYAO时，果蝇S2细胞增殖速率提高；而同时表达DUYAO和JIEYAO时，JIEYAO能消除DUYAO对果蝇S2细胞的抑制作用，使其增殖速率得到恢复。
与现有技术相比，本发明具有如下有益效果：
本发明提供的生殖隔离基因座及组成基因，培育RHS12位点亲和性水稻品系的策略，以及利用DUYAO和JIEYAO基因使转基因事件优势传递，快速纯合的方法对提高水稻杂种育性，加快水稻品种改良进程具有重要的应用价值。此外，通过对DUYAO和JIEYAO作用机制的阐明，以及两者对植物(例如水稻)细胞，真菌(例如酵母)细胞，动物(例如果蝇S2细胞)细胞同样具有毒性，解毒作用，拓宽了两基因的应用范围，对其它物种利用该基因进行遗传改良，医疗，生物制药等具有极高的科研和应用价值。
附图说明
图1两个籼粳交F2群体的花粉育性QTL检测；
图2RHS12位点引起花粉败育表型观察和精细定位；
图3转基因敲除和互补确认DUYAO和JIEYAO的遗传作用；
图4转基因互补确认DUYAO和JIEYAO的遗传作用；
图5DUYAO通过与OsCOX11互作来产生毒性；
图6JIEYAO通过与DUYAO互作来解除其毒性；
图7RHS12位点在野生稻间、种间、亚种间以及籼稻品种间的效应；
图8DUYAO-JIEYAO系统的转基因应用及RHS12位点在粳稻中的扩散；
图9DUYAO和JIEYAO在水稻愈伤，酵母和果蝇S2细胞中的体细胞效应。
具体实施方式
实施例1RHS12位点的鉴定，表型观察和精细定位
两个籼粳交F1植株(台中65(T65)/广陆矮4号(G4),滇粳优1号(DJY1)/RD23)具有强大的杂种优势，但是存在严重的生殖隔离，表现为F1花粉育性的严重降低(图1A-1B)。通过对上述两个F2群体的全基因组QTL鉴定，我们在12号染色体都发现了一个控制花粉败育的主效位点，并将其命名为RHS12(图1A-1B)。
随后我们通过回交构建了以DJY1为背景，RD23为插入片段的NIL，命名为DJYRD23。当DJY1与DJYRD23杂交时，其F1表现为花粉半不育，败育类型为染败，而且败育花粉不能萌发(图2A)。对F1花粉进行扫描和透射电镜观察时，发现败育花粉形状偏小，淀粉粒积累不全，花粉粒壁发育也不完全(图2B)。进一步的细胞学观察发现F1花粉败育的原因在于小孢子单核靠边期的发育停滞导致其发育一直落后正常的花粉(图2C)。最后，我们通过图位克隆的方法将该位点进行了精细定位。在粳稻基因组中，该位点为30.7kb，包括两个基因(jORF1和jORF5)和一个转座子；而在籼稻基因组中，除了iORF1和iORF5之外，还多了三个基因，分别命名为iORF2，iORF3(DUYAO)，iORF4(JIEYAO)。其中基因DUYAO核苷酸序列如SEQ ID NO.1所示，蛋白序列如SEQ ID NO.2所示；基因JIEYAO核苷酸序列如SEQ ID NO.3所示，蛋白序列如SEQ ID NO.4所示。
实施例2：RHS12位点的基因克隆
为了克隆组成RHS12位点的基因，确定其遗传模式，我们进行了以下的转基因敲除和互补实验。首先，当我们将F1中的DUYAO敲除时，发现其恢复正常育性(图3A)，而且分离比恢复为1:2:1(图3B)，说明DUYAO是个“毒性”基因。然后将JIEYAO转入F1时，单拷贝的转基因植株恢复到75％的育性(图3C)，且分离比变成1:3:2(图3D)，说明JIEYAO是个“解毒”基因。接下来，我们将DUYAO和JIEYAO串联转入粳稻亲本和F1，发现单拷贝转基因引起了亲本的半不育(图3E)和1:1的偏分离(图3F),而且转基因的效果和RHS12位点效应是等价的(图4)。
实施例3：DUYAO通过与OsCOX11互作来产生“毒性”
亚细胞定位结果显示DUYAO定位于线粒体(图5A)，暗示DUYAO是通过线粒体来发挥“毒性”功能的。OsCOX11是线粒体电子呼吸传递链的重要组成亚基，为细胞的正常生长发育提供能量。我们发现DUYAO与OsCOX11在线粒体上共定位(图5A)。此外，酵母双杂、Pull-down和Co-IP实验证实了DUYAO与OsCOX11的互作(图5B-5E)。虽然OsCOX11形成二聚体，DUYAO形成寡聚体，但是当两者在一起时，OsCOX11并不能破坏DUYAO的寡聚体状态(图5F-5H)。然而，随着互作体系中DUYAO蛋白的增加，OsCOX11自身互作的能力减弱(图5I)，说明DUYAO的存在破坏了OsCOX11蛋白二聚体的形成。随后我们设计了两个靶点去对OsCOX11进行敲除，发现得不到OsCOX11的敲除植株，而且敲除杂合植株符合1:1分离，暗示OsCOX11是细胞(配子)生长发育的必须因子(图5J和5K)。综上，我们认为DUYAO通过与OsCOX11互作产生“毒性”，影响细胞(配子)的生长和发育。
实施例4：JIEYAO通过与DUYAO互作来进行“解毒”
在解析了DUYAO是如何产生“毒性”后，我们接下来对JIEYAO如何“解毒”的分子机制进行了阐明。通过酵母双杂，LCI，Pull-down和Co-IP实验，我们证实DUYAO和JIEYAO两者能够互作(图6A-6D)。前面的研究结果证实DUYAO能形成寡聚体，而当JIEYAO与DUYAO一起表达时，JIEYAO破坏了DUYAO的寡聚状态(图6E和6F)。此外，JIEYAO的存在还破坏了DUYAO与OsCOX11之间的互作(图6G)。接下来我们在亚细胞层面探究了JIEYAO是如何影响DUYAO的。结果显示与DUYAO定位于线粒体不同的是，JIEYAO定位于MVB和自噬体(图6H)。进一步得，当将JIEYAO与DUYAO一起表达时，结果显示JIEYAO改变了DUYAO的线粒体定位，而使其定位于MVB和自噬体(图6I)。由此，这也解释了为什么JIEYAO能完全破坏DUYAO和OsCOX11的互作，因为其改变了DUYAO的线粒体定位。那么，JIEYAO将DUYAO定位于自噬体对DUYAO有什么影响呢？通过体外降解实验，我们发现JIEYAO能促进DUYAO的降解，而自噬途径抑制剂BFA1的加入能明显抑制其降解(图6J)。综上，我们在分子层面解析了JIEYAO解除DUYAO“毒性”的机理，即JIEYAO通过与DUYAO互作，破坏DUYAO的寡聚状态，改变其线粒体定位，最后将其经自噬途径降解。
实施例5：RHS12位点普遍存在于种间、亚种间、品种间
为了研究RHS12位点在水稻中的普遍性与重要性，我们构建了以粳稻DJY1为背景，不同类型的野生稻和栽培籼稻为插入片段来源的NILs。通过不同NILs间双列杂交的花粉表型结果可以看出，RHS12在野生稻种间，野生稻与栽培稻种间，籼粳亚种间以及籼籼品种间均存在(图7A)。随后，通过更多类型的以DJY1为背景，不同的野生稻和栽培稻来源片段插入的NILs证实了上述结果，而且我们发现JIEYAO蛋白115位的氨基酸由K变为E或Q时就能使其丧失功能(图7B)。更多的不同类型的籼粳，籼籼组合的NILs也证实了RHS12位点广泛存在于籼粳亚种和籼籼品种间(图7C)。
实施例6：DUYAO-JIEYAO系统在水稻育种改良中的应用
由于DUYAO-JIEYAO系统独特的配子传递特性，我们尝试利用该系统来研究其在水稻品种改良中的作用。首先我们在DUYAO-JIEYAO基因组合的中间插入一个Ubi驱动的GFP，然后分析其单拷贝转基因的效果。结果发现与单独Ubi驱动的GFP单拷贝转基因事件相比，串联了DUYAO-JIEYAO系统的转基因花粉为半不育，而且只有携带GFP的花粉才正常(图8A-B)，说明通过该系统能使约50％的后代为转基因纯合型，约50％为转基因杂合型，远高于常规转基因的25％为纯合型，25％为杂合型，25％不携带转基因。由此可见，当将Ubi驱动的GFP替换为任意元件时，该元件能优势传递到下一代，并伴以极高的纯合比例。这一特性使利用DUYAO-JIEYAO元件进行转基因育种改良时，大大加快了育种进程。
前面的结果显示DUYAO-JIEYAO系统特异存在于籼稻中的，而粳稻中并没有。通过对1970年代之前的粳稻品种分析结果显示确实如此，但是1970年代之后，DUYAO-JIEYAO系统开始入侵入粳稻并逐步扩散。到2020年时，DUYAO-JIEYAO系统在粳稻中的占比已经达到了接近90％(图8C)，这一结果也同该系统的遗传特性相一致，即DUYAO-JIEYAO系统会优势传递到下一代。那么，粳稻品种中本来是没有生殖隔离位点的，新入侵的DUYAO-JIEYAO系统会导致粳稻品种间产生生殖隔离吗？我们通过将不同类型的粳稻品种进行杂交，结果显示DUYAO-JIEYAO系统使粳稻品种中产生了新的生殖隔离(图8D)。
实施例7：DUYAO-JIEYAO系统的跨物种体细胞效果
前面的细胞学观察和遗传学结果证实DUYAO对雄配子有毒性，而JIEYAO能解除这种毒性，那么除了生殖细胞，DUYAO-JIEYAO系统对体细胞或者植物以外的物种是否有类似的作用值得我们进一步的研究。通过将35S启动子驱动的DUYAO与Ubi驱动的GFP相连进行水稻愈伤转化时，我们发现细胞不表达GFP光；而将Actin驱动的JIEYAO与之共转时，水稻愈伤恢复GFP光。当然，换成水稻自身启动子驱动DUYAO和JIEYAO也有同样的效果(图9A-B)。说明DUYAO对水稻体细胞也有“毒性”，而JIEYAO能解除DUYAO产生的“毒性”。接下来我们将DUYAO-JIEYAO系统转入酵母中也得到了类似的效果，在酵母中表达DUYAO使酵母不能生长，而同时表达的JIEYAO能解毒DUYAO的毒性，使酵母恢复正常生长(图9C)。在果蝇S2细胞中也是如此，当表达DUYAO时，细胞增殖速率下降，而当共表达JIEYAO时，细胞的增殖速率得到了恢复(图9D)。综上，我们认为DUYAO-JIEYAO系统不仅在植物中，在其它物种(比如酵母和果蝇)中也有类似的“毒性-解毒”效果。正是DUYAO-JIEYAO系统作用的广泛性，我们认为该系统不光能被利用来进行植物的育种改良，也能在其它物种中的遗传改良，制药医疗中进行进一步的应用。
Claims (2)
1.SEQ ID NO.1所示基因DUYAO或SEQ ID NO.2所示基因JIEYAO在建立细胞筛选系统中的应用，其特征在于，具体为将SEQ ID NO.1所示基因DUYAO转入需淘汰的细胞，获得不能生长或增殖缓慢的细胞；或者将SEQ ID NO.2所示基因JIEYAO转入需保留的具有基因DUYAO的细胞，获得正常生长或增殖的细胞，所述细胞为酵母或果蝇细胞。
2.一种将目的基因进行优势传递并快速纯合的方法，其特征在于，将SEQ ID NO.1所示的基因DUYAO，SEQ ID NO.2所示的基因JIEYAO和目的基因三者串联一起转入目标生物中，所述目标生物为水稻。
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