CN114107234A - 具有草铵膦抗性的谷氨酰胺合成酶突变体、重组基因、重组载体及其应用 - Google Patents
具有草铵膦抗性的谷氨酰胺合成酶突变体、重组基因、重组载体及其应用 Download PDFInfo
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- CN114107234A CN114107234A CN202111347546.0A CN202111347546A CN114107234A CN 114107234 A CN114107234 A CN 114107234A CN 202111347546 A CN202111347546 A CN 202111347546A CN 114107234 A CN114107234 A CN 114107234A
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- glutamine synthetase
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- ile
- glufosinate
- glu
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Abstract
本发明提供一种具有草铵膦抗性的谷氨酰胺合成酶突变体，将目标植物的谷氨酰胺合成酶中与水稻野生型谷氨酰胺合成酶氨基酸序列的第311位对应的精氨酸(R)突变为丙氨酸(A)、半胱氨酸(C)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、苯丙氨酸(M)、天门冬氨酸(N)、脯氨酸(P)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)中的任一种。本发明还提供了编码该谷氨酰胺合成酶突变体的重组基因、重组载体和重组工程菌及其应用。本发明提供的突变体可以提高多种植物对草铵膦的抗性，同时保持自身的生物酶催化活性，可应用于抗草铵膦植物品种的培育。
Description
技术领域
本发明涉及生物工程技术领域，具体涉及一种具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子及应用。
背景技术
谷氨酰胺合成酶(Glutamine synthetase,GS)是植物氮代谢的关键酶，催化谷氨酸与NH4+合成谷氨酰胺，把无机氮转化为有机氮。谷氨酰胺合成酶一种定位为细胞质内，称为胞质型GS(GS1)；还有一种定位于叶绿体(或质体)内称为质体型GS(GS2)。
草铵膦(Glufosinate ammonium，商品名称保试达Basta)，由赫斯特公司80年代开发成功(后归属于拜耳公司)，属广谱触杀型除草剂，内吸作用不强。其有效成分为phosphinothricin(简称PPT)，化学名称为(RS)-2-氨基-4-(羟基甲基氧膦基)丁酸铵。谷氨酰胺合成酶是草铵膦的靶标蛋白，草铵膦通过与底物谷氨酸的竞争，先与ATP结合，磷酸化的PPT占据谷氨酰胺合成酶反应中心，造成谷氨酰胺合成受到抑制，一方面使得细胞氮代谢紊乱，另一方面还会在细胞内造成NH4 +的积累，质体型谷氨酰胺合成酶参与过量铵的同化会在细胞中产生大量质子，降低pH，导致细胞酸度增加，造成酸胁迫，致使植物死亡，达到除草效果。
现在培育抗草铵膦品种的主要方法是通过转基因方法，把细菌来源的抗草铵膦基因导入农作物中，获得转基因抗草铵膦作物新品种。目前主要应用的抗草铵膦基因来源于细菌的Pat基因和Bar基因，这两个基因都可以编码草铵膦乙酰化酶，该酶可以使PPT乙酰化,乙酰化的产物无抑制谷氨酰胺合成酶的活性。
转基因作物在全世界的接受程度仍然比较较低，Pat和Bar转基因作物仍有一定的缺点,另外随着基因编辑技术的快速发展，使得各种除草剂靶标基因的编辑成为可能，且基因编辑作物较转基因作物更易被人们接受，因此，寻找耐受各种除草剂靶标基因的突变体成为当务之急，基于此，提出本发明。
发明内容
针对上述现有技术的缺陷和市场需求，本发明提供一种具有草铵膦抗性的谷氨酰胺合成酶突变体及其应用的方法，该谷氨酰胺合成酶突变体可以赋予植株草铵膦抗性，在农作物转化该谷氨酰胺合成酶突变体，植物可以正常的生长和发育。
一种具有草铵膦抗性的谷氨酰胺合成酶突变体，将目标植物的谷氨酰胺合成酶中与水稻野生型谷氨酰胺合成酶氨基酸序列的第311位对应的精氨酸(R)突变为丙氨酸(A)、半胱氨酸(C)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、苯丙氨酸(M)、天门冬氨酸(N)、脯氨酸(P)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)中的任一种。
在根据本发明的一个实施方案中，所述水稻野生型谷氨酰胺合成酶氨基酸序列为SEQ ID NO:1；
优选地，所述谷氨酰胺合成酶突变体氨基酸序列为SEQ ID NO:7。
在根据本发明的一个实施方案中，所述目标植物选自水稻、玉米、小麦、大麦、燕麦、高粱、谷子、荞麦、棉花、大豆、花生、蚕豆、绿豆、豌豆、菜豆、豇豆、扁豆、苜蓿、向日葵、油菜、萝卜、白菜、芥菜、甘蓝、花椰菜、芥蓝、洋葱、胡萝卜、芹菜、甘薯、马铃薯、甜菜、甜瓜、番茄、茄子、辣椒、韭菜、大葱、菠菜、茼蒿、芝麻、苋菜、莴苣、黄瓜、西葫芦、南瓜、苦瓜、丝瓜、西瓜、木薯、葡萄、草莓、冬瓜、烟草中的任一种。
本发明还提供了一种用于编码上述的谷氨酰胺合成酶突变体的重组基因，目标植物编码谷氨酰胺合成酶的基因中用于编码对应于水稻野生型谷氨酰胺合成酶氨基酸序列第311位的精氨酸(R)的密码子突变为丙氨酸(A)、半胱氨酸(C)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、苯丙氨酸(M)、天门冬氨酸(N)、脯氨酸(P)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)中的任一种氨基酸的密码子。
在根据本发明的一个实施方案中，目标植物编码野生型谷氨酰胺合成酶的基因序列选自SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6中的任一种；
优选地，核苷酸序列突变位点的密码子突变选自表1中的任一种。
表1
本发明进一步提供了一种重组载体或重组工程菌，包含上述的重组基因。所述重组载体的骨架质粒优选为PY16,由YCplac22(GenBank:X75455)改造而来，所述重组工程菌的宿主菌选自酿酒酵母。
本发明进一步提供了上述的谷氨酰胺合成酶突变体、重组基因或重组载体或重组工程菌在培育具有草铵膦抗性的植物突变体中的应用。
本发明的另一方面提供了一种具有草铵膦抗性的植物品种的制备方法，包括：通过同源重组或基因编辑技术使植物株基因组中谷氨酰胺合成酶的编码基因中对应于水稻野生型谷氨酰胺合成酶氨基酸序列第311位的精氨酸(R)的密码子突变为丙氨酸(A)、半胱氨酸(C)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、苯丙氨酸(M)、天门冬氨酸(N)、脯氨酸(P)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)中的任一种氨基酸的密码子。
在根据本发明的一个实施方案中，还进一步包括通过有性杂交或无性培养扩繁获得批量种苗。
本发明最后还提供了一种具有草铵膦抗性的植物突变体，其特征在于，谷氨酰胺合成酶中与水稻野生型谷氨酰胺合成酶氨基酸序列的第311位对应的精氨酸(R)突变为丙氨酸(A)、半胱氨酸(C)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、苯丙氨酸(M)、天门冬氨酸(N)、脯氨酸(P)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)中的任一种；优选地，所述植物突变体是通过上述的制备方法培育得到的。
本发明还进一步提供了具有草铵膦抗性的植物细胞、组织或外植体，其谷氨酰胺合成酶中与水稻野生型谷氨酰胺合成酶氨基酸序列的第311位对应的精氨酸(R)突变为丙氨酸(A)、半胱氨酸(C)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、苯丙氨酸(M)、天门冬氨酸(N)、脯氨酸(P)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)中的任一种。
本发明的上述技术方案的有益效果如下：
本发明提供的突变体可以提高多种植物对草铵膦的抗性，同时保持自身的生物酶催化活性。转化了本发明提供的植物谷氨酰胺合成酶突变体的植株或重组菌均能够在草铵膦存在的条件下正常生长，该谷氨酰胺合成酶突变体不仅用于转基因作物培育外，也可应用于培育抗草铵膦非转基因植物，应用前景广泛。
附图说明
图1为水稻野生型GS与突变体的氨基酸序列比对图；
图2为水稻、玉米和油菜野生型GS氨基酸序列与对应突变体的氨基酸序列比对图；
图3为PY16酵母表达载体图；
图4为GS突变体酵母抗性验证图；
图5为水稻、玉米和油菜GS突变体酵母抗性验证图；
图6为水稻和油菜GS突变体转化酵母的生长曲线图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。
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材料、试剂、仪器等，如无特殊说明，均来自常规生化试剂供应商购买得到。
实施例1：
本实施例提供了来源于水稻(Oryza sativa)的多个谷氨酰胺合成酶(glutaminesynthetase，GS)突变体，这些GS突变体命名为：OS_WT、OS_RA、OS_RC、OS_RH、OS_RI、OS_RK、OS_RM、OS_RN、OS_RP、OS_RQ、OS_RT、OS_RV；这些GS突变体与水稻野生型GS氨基酸序列(SEQID NO.1)相比较，对应于水稻野生型GS的氨基酸序列(OS_WT)的第311位发生突变，具体的突变类型见图1，灰色底纹处为311位点，这些GS突变体的长度和其余位点的氨基酸残基类型与水稻野生型GS相同。
实施例2：
本实施例提供了玉米谷氨酰胺合成酶突变体ZmGS1_RK，与水稻野生型GS氨基酸序列(SEQ ID NO.1)相比较，如图2所示的水稻、玉米和油菜野生型GS氨基酸序列与对应突变体的氨基酸序列比对图。对应于水稻野生型GS氨基酸序列的第311位发生突变，具体的突变R311K；ZmGS1_RK的长度和其余位点的氨基酸残基类型与玉米野生型GS(SEQ ID NO.2)相同。
实施例3：
本实施例提供了油菜谷氨酰胺合成酶突变体BnGS1_RK，与水稻野生型GS(SEQ IDNO.1)相比较，对应于水稻野生型GS氨基酸序列的第311位发生突变，具体的突变R311K；BnRK的长度和其余位点的氨基酸残基类型与油菜野生型GS氨基酸序列(SEQ ID NO.3)相同。
实施例4：
本实施例提供了编码上述实施例中GS突变体的核酸分子。
编码水稻野生型GS(SEQ ID NO.1)的核酸序列如SEQ ID NO.4；编码玉米野生型GS(SEQ ID NO.2)的核酸序列如SEQ ID NO.5；编码油菜野生型GS(SEQ ID NO.3)的核酸序列如SEQ ID NO.6。
水稻野生型GS氨基酸序列(SEQ ID NO.1)、玉米野生型GS氨基酸序列(SEQ IDNO.2)和油菜野生型GS氨基酸序列(SEQ ID NO.3)三者的序列一致性(identity)和相似性(similarity)如表2：
表2
编码实施例1-3中GS突变体的核酸序列均可以在编码相同植物来源的野生型GS的核酸序列上对应于第311位的碱基进行突变获得，得到的突变核酸序列编码出对应的GS突变体。本领域技术人员可以根据密码子的简并性可以理解到，在编码第311位突变位点碱基的密码子可存在多种情况，见表1，无论哪种密码子，只要是编码上述GS氨基酸序列的第311位点处突变体的氨基酸，其均属于本发明的保护范围。
实验例1：
本发明研究过程中首先发现了突变体Os_RK(R311K)具有草铵膦抗性，进而对311位点氨基酸进行饱和突变，从而检测实施例1提供的水稻GS突变体OS_RA、OS_RC、OS_RH、OS_RI、OS_RK、OS_RM、OS_RN、OS_RP、OS_RQ、OS_RT、OS_RV的草铵膦抗性。方法如下：根据实施例1和实施例4提供的核酸分子的序列，采用化学合成的方法合成编码水稻GS突变体OsGSRn(第311为R变为n,R311n,n＝A、C、D、E、F、G、H、I、K、L、M、N、P、Q、S、T、V、W、Y)的核酸序列，PCR扩增后通过Gibson组装连接到表达载体(PY16载体，由YCplac22 GenBank:X75455改造而来，如图3所示)上，然后转化大肠杆菌，提取阳性克隆，测序分析后把含有OsGSRn突变体编码基因的PY16-OsGSRn载体，转化到谷氨酰胺合成酶缺陷型BY4741酵母菌中，挑取阳性菌，接种至含不同浓度草铵膦的SC-W培养基上，观察酵母菌生长情况。以含有野生型水稻GS氨基酸序列为负对照，检测GS突变体的草铵膦抗性。结果如图4所示。
在0mM草铵膦的SC-W培养基上，转化编码野生型水稻GS1(OS_WT)及水稻GS突变体R311n的编码基因的缺陷型菌株均能正常生长，表明由R311n编码的19种GS突变体都具有正常GS酶活力；
在30mM草铵膦的SC-W培养基上，转化野生型水稻GS1的缺陷型BY4741已经不能生长，但转化了水稻突变体OS_RA、OS_RC、OS_RH、OS_RI、OS_RK、OS_RM、OS_RN、OS_RP、OS_RQ、OS_RT、OS_RV的缺陷型BY4741生长明显优于负对照，说明这些突变体抗草铵膦的能力明显优于野生型；
在50mM-75mM草铵膦的SC-W培养基上，转化水稻GS1突变体OS_RA、OS_RC、OS_RH、OS_RI、OS_RK、OS_RM、OS_RN、OS_RP、OS_RQ、OS_RT、OS_RV的缺陷型BY4741都还可以生长。这些结果说明OS_RA、OS_RC、OS_RH、OS_RI、OS_RK、OS_RM、OS_RN、OS_RP、OS_RQ、OS_RT、OS_RV这些突变体具有抗草铵膦的能力。
实验例2：
本试验例检测把水稻OsRK(R311K)突变位点在玉米和油菜GS氨基酸序列中的对应位置进行突变后草铵膦抗性。实施例2和实施例3中的玉米GS突变体ZmRK和油菜GS突变体BnRK，可采用化学合成法合成玉米GS突变体ZmRK和油菜GS突变体BnRK的核酸序列，PCR扩增后通过Gibson组装连接到表达载体(PY16载体，如图3所示)上，然后转化大肠杆菌，阳性克隆测序后提取质粒，转化到谷氨酰胺合成酶缺陷型BY4741酵母菌中，挑取阳性菌，接种至含不同浓度草铵膦的SC-W培养基上，观察酵母菌生长情况。以野生型水稻GS(OsWT,SEQ IDNO.1)，野生型玉米GS(ZmWT，SEQ ID NO.2)和野生型油菜GS(BnWT,SEQ ID NO.3)为负对照，检测OsRK、ZmRK和BnRK突变体的草铵膦抗性。结果如图5所示。
在0mM草铵膦的SC-W培养基上，转化编码野生型水稻GS，野生型玉米GS氨基酸序列和野生型油菜GS氨基酸序列，以及水稻OsRK，玉米ZmRK和油菜BnRK的突变体的缺陷型BY4741菌株均能正常生长，表明由OsRK，ZmRK和BnRK编码的GS突变体都具有正常GS酶活力；
在30mM草铵膦的SC-W培养基上，转化编码野生型水稻GS氨基酸序列、野生型玉米GS氨基酸序列和野生型油菜GS氨基酸序列的缺陷型BY4741菌株已经不能生长，但转化了水稻OsRK，玉米ZmRK和油菜BnRK的缺陷型BY4741菌株生长明显优于负对照，说明这些突变体抗草铵膦的能力明显优于野生型；
在50和75mM草铵膦的SC-W培养基上，转化水稻OsRK，玉米ZmRK和油菜BnRK的缺陷型BY4741菌都还可以生长。这说明水稻OsRK，玉米ZmRK和油菜BnRK的突变体具有抗草铵膦的能力。
由水稻GS发现的具有草铵膦抗性的第311位点在其他作物中进行突变也具有草铵膦抗性，这可进一步以扩展到以下植物的GS基因，扩展植物包含水稻、玉米、小麦、大麦、燕麦、高粱、谷子、荞麦、棉花、大豆、花生、蚕豆、绿豆、豌豆、菜豆、豇豆、扁豆、苜蓿、向日葵、油菜、萝卜、白菜、芥菜、甘蓝、花椰菜、芥蓝、洋葱、胡萝卜、芹菜、甘薯、马铃薯、甜菜、甜瓜、番茄、茄子、辣椒、韭菜、大葱、菠菜、茼蒿、芝麻、苋菜、莴苣、黄瓜、西葫芦、南瓜、苦瓜、丝瓜、西瓜、木薯、葡萄、草莓、冬瓜、烟草等。
实验例3：
本试验例通过测量转化谷氨酰胺合成酶突变体酵母生长曲线来验证水稻OsRK和油菜BnRK谷氨酰胺合成酶突变体酶活力。方法如下，把构建好的含有GS突变体基因的质粒PY16-OsRK，PY16-BnRK，水稻野生型PY16-OsWT、油菜PY16-BnWT转化到谷氨酰胺合成酶缺陷型BY4741酵母菌中，挑取阳性菌落接菌扩繁后稀释到一定浓度，通过定量接菌培养，30℃震动摇床培养14小时后开始测OD值，每2小时测量一次OD值并记录，测量到34小时，收集数据做图分析，数据分析和作图由Excel软件完成，见图6。
从含0mM草铵膦的SC-W的培养基生长曲线可以看出，在14-34小时时间段中，含有水稻突变体PY16-OsRK的BY4741酵母菌生长速度高于含有水稻野生型PY16-OsWT的BY4741酵母菌，可以看出OsRK酶活力不低于野生型OsWT；含有油菜突变体PY16-BnRK的BY4741酵母菌生长速度也高于含有油菜野生型PY16-BnWT的BY4741酵母菌,可以看出BnRK酶活力不低于野生型BnWT；
从含100mM草铵膦的SC-W的培养基生长曲线可以看出，在14-34小时时间段中，含有水稻突变体PY16-OsRK的BY4741酵母菌可以生长，含有突变体PY16-OsWT的BY4741酵母菌基本没有生长；含有油菜突变体PY16-BnRK的BY4741酵母菌可以生长，含有突变体PY16-BnWT的BY4741酵母菌基本没有生长；这些结果说明OsRK，BnRK对草铵膦有耐受性，并具有酶活，且酶活不低于野生型，可以正常生长。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 科稷达隆（北京）生物技术有限公司
<120> 具有草铵膦抗性的谷氨酰胺合成酶突变体、重组基因、重组载体及其应用
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<210> 7
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<213> 人工序列(Artificial Sequence)
<220>
<221> MUTAGEN
<222> (311)..(311)
<223> 选自A、C、H、I、K、M、N、P、Q、T或V中任一种
<220>
<221> UNSURE
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<220>
<221> UNSURE
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<223> The 'Xaa' at location 311 stands for Gln, Arg, Pro, or Leu.
<400> 7
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Ser Ala Gly Asp Gln Val Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile
210 215 220
Thr Glu Ile Ala Gly Val Val Val Ser Phe Asp Pro Lys Pro Ile Pro
225 230 235 240
Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser
245 250 255
Met Arg Asn Asp Gly Gly Tyr Glu Ile Ile Lys Ser Ala Ile Glu Lys
260 265 270
Leu Lys Leu Arg His Lys Glu His Ile Ser Ala Tyr Gly Glu Gly Asn
275 280 285
Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300
Ser Trp Gly Val Ala Asn Xaa Gly Ala Ser Val Arg Val Gly Arg Glu
305 310 315 320
Thr Glu Gln Asn Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser
325 330 335
Asn Met Asp Pro Tyr Ile Val Thr Ser Met Ile Ala Glu Thr Thr Ile
340 345 350
Ile Trp Lys Pro
355
Claims (9)
1.一种具有草铵膦抗性的谷氨酰胺合成酶突变体，其特征在于，将目标植物的谷氨酰胺合成酶中与水稻野生型谷氨酰胺合成酶氨基酸序列的第311位对应的精氨酸(R)突变为丙氨酸(A)、半胱氨酸(C)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、苯丙氨酸(M)、天门冬氨酸(N)、脯氨酸(P)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)中的任一种。
2.如权利要求1所述的谷氨酰胺合成酶突变体，其特征在于，所述水稻野生型谷氨酰胺合成酶氨基酸序列为SEQ ID NO:1；
优选地，所述谷氨酰胺合成酶突变体氨基酸序列为SEQ ID NO:7。
3.如权利要求1所述的谷氨酰胺合成酶突变体，其特征在于，所述目标植物选自水稻、玉米、小麦、大麦、燕麦、高粱、谷子、荞麦、棉花、大豆、花生、蚕豆、绿豆、豌豆、菜豆、豇豆、扁豆、苜蓿、向日葵、油菜、萝卜、白菜、芥菜、甘蓝、花椰菜、芥蓝、洋葱、胡萝卜、芹菜、甘薯、马铃薯、甜菜、甜瓜、番茄、茄子、辣椒、韭菜、大葱、菠菜、茼蒿、芝麻、苋菜、莴苣、黄瓜、西葫芦、南瓜、苦瓜、丝瓜、西瓜、木薯、葡萄、草莓、冬瓜、烟草中的任一种。
4.一种用于编码如权利要求1-3中任一项所述的谷氨酰胺合成酶突变体的重组基因，其特征在于，目标植物编码谷氨酰胺合成酶的基因中用于编码对应于水稻野生型谷氨酰胺合成酶氨基酸序列第311位的精氨酸(R)的密码子突变为丙氨酸(A)、半胱氨酸(C)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、苯丙氨酸(M)、天门冬氨酸(N)、脯氨酸(P)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)中的任一种氨基酸的密码子。
5.如权利要求4所述的重组基因，其特征在于，目标植物编码野生型谷氨酰胺合成酶的基因序列选自SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6中的任一种；
优选地，核苷酸序列突变位点的密码子突变选自表1中的任一种
表1
6.一种重组载体或重组工程菌，其特征在于，包含如权利要求4或5所述的重组基因。
7.如权利要求1-3中任一项所述的谷氨酰胺合成酶突变体、如权利要求4或5所述的重组基因或权利要求6所述的重组载体或重组工程菌在培育具有草铵膦抗性的植物突变体中的应用。
8.一种具有草铵膦抗性的植物品种的制备方法，其特征在于，包括：通过同源重组或基因剪接技术使植株基因组中谷氨酰胺合成酶的编码基因中对应于水稻野生型谷氨酰胺合成酶氨基酸序列第311位的精氨酸(R)的密码子突变为丙氨酸(A)、半胱氨酸(C)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、苯丙氨酸(M)、天门冬氨酸(N)、脯氨酸(P)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)中的任一种氨基酸的密码子。
9.如权利要求8所述的制备方法，其特征在于，还进一步包括通过有性杂交或无性培养扩繁获得批量种苗。
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