CN107217044A - 使植物具有除草剂抗性的水稻als突变型蛋白及其应用 - Google Patents
使植物具有除草剂抗性的水稻als突变型蛋白及其应用 Download PDFInfo
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Abstract
本发明公开了使植物具有除草剂抗性的水稻ALS突变型蛋白。本发明还公开了编码上述蛋白的核酸。该蛋白来源于抗ALS抑制剂类除草剂的水稻突变体植株，与野生型水稻基因组中的ALS序列相比，其蛋白序列在Pro171和Asp350位点同时发生突变。本发明通过田间喷施ALS抑制剂类除草剂“百垄通”的实验结果表明，含有本发明的具有除草剂抗性的ALS蛋白的水稻3‑4叶幼苗在施用3.3mL百垄通/L水（10倍推荐使用浓度）后，植株仍然正常生长发育和结实，而野生型水稻3‑4叶幼苗在施用1 mL百垄通/3L水（1倍推荐使用浓度）30天后表现为整株死亡。
Description
技术领域
本发明属于植物蛋白和植物抗除草剂领域，涉及使植物具有除草剂抗性的水稻ALS 突变型蛋白、基因及其应用；具体而言，本发明涉及水稻的乙酰乳酸合成酶(ALS)突变蛋白，该蛋白能赋予植物尤其水稻抗乙酰乳酸合成酶抑制剂类除草剂的特性。本发明公开了该蛋白的序列，以及它们在植物抗除草剂领域中的应用。
背景技术
杂草是制约农业生产稳产高产的不利因素。与传统依靠栽培措施、人工除草和机械除草等方法相比，化学除草剂的使用是一种高效的、简便的和经济的治理杂草方法。
植物和微生物支链氨基酸(缬氨酸、亮氨酸和异亮氨酸)的生物合成需要4种酶共同催化作用，乙酰乳酸合成酶(acetolactate synthase，ALS)、酮醇还原异构酶(ketol-acid reductoisomerase)、二羟基酸脱水酶(dihydroxyavid dehydratase)、支链氨基酸转氨酶 (branched-chain amino acidtransaminase)。乙酰乳酸合成酶是生物合成过程中第一阶段的关键酶，在缬氨酸和亮氨酸的合成中催化2分子丙酮酸生成乙酰乳酸和二氧化碳，在异亮氨酸的合成中催化1分子丙酮酸与1分子α丁酮酸生成2-乙醛基-2-羟基丁酸和二氧化碳。ALS抑制剂类除草剂通过抑制植物体内的ALS酶活性，从而阻止支链氨基酸的合成，导致蛋白质的合成受到破坏，阻碍细胞分裂期的DNA合成，从而使植物细胞的有丝分裂停止在Gl阶段的S期(DNA合成期)和G2阶段的M期，干扰了DNA的合成，细胞因此不能完成有丝分裂，进而使植物停止生长，最终导致植物个体死亡。
乙酰乳酸合成酶(ALS)(也称乙酰羟酸合成酶，AHAS；EC 4.1.3.18)抑制剂类除草剂以ALS作为靶标而导致杂草死亡，主要包括磺酰脲类(Sulfonylureas,SU)、咪唑啉酮类(Imidazolinones,IMI)、三唑嘧啶类(Triazolopyrimidines,TP)、嘧啶氧(硫)苯甲酸类[Pyrimidinylthio(or oxy)–benzoates,PTB；pyrimidinyl-carboxyherbicides；PCs]和磺酰胺基羰基三唑啉酮类(Sulfonylamino-carbonyltriazolinones,SCT)等13类化合物。乙酰乳酸合成酶，存在于植物生长过程中，它能以高度专一性和极高的催化效率催化丙酮酸为乙酰乳酸，从而导致支链氨基酸的生物合成。
ALS抑制剂类除草剂具有选择性强、杀草谱广、低毒高效等特点，目前已大面积推广使用。但这些除草剂对一般不具有抗(耐)除草剂特性的农作物本身也产生药害，极大限制了其使用时间和使用空间，如需要在农作物播种前一段时间使用除草剂才能避免农作物遭受药害。培育抗(耐)除草剂作物品种可减少作物药害、拓宽除草剂的使用范围。
目前，水稻已知的抗ALS抑制剂突变位点包括Gly 95、Ala 96、Ala 122、Trp 548、Ser 627、Ser 653和Gly 654。ALS突变体抗除草剂水平与ALS氨基酸突变的位置有关，还与突变后的氨基酸种类及突变氨基酸的数目有关。
目前，ALS抑制剂类除草剂的作用机理尚未确定，很难提前预测ALS蛋白其它氨基酸位点的突变是否会产生除草剂抗性，只能依赖于科研人员长期、艰苦的实践探索，并凭借一些运气才可能发现ALS蛋白新的除草剂抗性位点。
发明内容
发明目的：本发明所要解决的第一个技术问题是提供使植物具有除草剂抗性的蛋白。
本发明还要解决的技术问题是提供了编码该蛋白的核酸以及表达盒、载体和细胞等。
本发明还要解决的技术问题是提供了获得具有除草剂抗性的植物的方法和应用。
本发明还要解决的技术问题是提供了判断植物是否采用本发明方法获得的鉴定方法。
本发明人通过长期、艰苦的研究，对野生型水稻为籼型常规水稻种子合肥不育系的 EMS突变植株进行长期、不断地筛选，发现了一系列ALS突变蛋白，包括前文所述的某些已知ALS突变蛋白和新ALS突变蛋白，其对ALS抑制剂类除草剂不敏感，从而使得植物具有ALS抑制剂类除草剂抗性。本发明在植物育种中的应用，可用于培育具有除草剂抗性的植物，尤其是农作物，还开发了这些蛋白及其编码基因在转基因或者非转基因如水稻等作物中的应用。
技术方案：为解决上述技术问题，本发明采用的技术方案如下：本发明提供了使得植物具有除草剂抗性的ALS蛋白，其在氨基酸序列的第171位氨基酸由脯氨酸变为组氨酸，第350位氨基酸由天冬氨酸突变为谷氨酸。目前还没有报道表明，在来自水稻品种合肥不育系ALS蛋白的Pro171和Asp350位点同时突变的突变株。
优选本发明的第一方面的蛋白带有Pro171和Asp350位点同时突变的突变株，除了本发明具有的实施方式所述的突变植株筛选或扩增方法之外，在蛋白质序列已知的情形下，本领域技术人员有能力通过改变已知蛋白质的编码基因序列并将其导入载体，就可以制备出取代、添加或缺失了氨基酸残基的蛋白质，这些方法均为常规手段。在本发明的具体实施方式中，本发明的具有除草剂抗性的ALS蛋白的氨基酸序列如SEQ ID NO:2 所示。该蛋白经过证实对咪唑啉酮类除草剂不敏感的乙酰乳酸合成酶。
本发明的第二方面提供了核酸，其编码本发明第一方面的蛋白质。在本发明中，核酸可以是DNA、RNA，其中优选为DNA。在知晓所编码蛋白序列或核酸序列的前提下，通过常规的密码子对应关系和宿主表达频率，运用PCR方法、DNA重组法或人工合成的方法，本领域技术人员有能力获得并优化编码本发明第一方面的蛋白质的核酸。一旦获得该核酸，就可以将其克隆入载体，再转化或转染入相应的细胞，然后通过常规的宿主细胞进行增殖，从中分离得到大量的核酸。优选本发明第二方面的核酸的核苷酸序列如SEQ ID NO：1所示。具体的，本发明的DNA序列，是在野生型水稻为籼型常规水稻种子合肥不育系的ALS基因序列的第512位核苷酸由C变成核苷酸A、第1050位核苷酸由T变成核苷酸A。
此外，本发明突变前的野生型水稻为籼型常规水稻种子合肥不育系，该ALS抑制剂类除草剂敏感的野生型合肥不育系水稻ALS的核苷酸序列如SEQ ID NO.3所示，氨基酸如SEQ ID NO.4所示。
本发明的第三方面的内容包括表达盒、重组载体或细胞，其含有上述的第二方面的核酸。
本发明第四方面内容还包括上述的具有除草剂抗性的ALS蛋白、核酸、表达盒、重组载体或细胞在绿色植物抗除草剂方面的应用。
其中，上述绿色植物为水稻、烟草、拟南芥、棉花等。
本发明第五方面的内容还包括获得具有除草剂抗性的植物的方法，包括如下步骤：
1)使植物包含本发明所述的核酸；或
2)使植物表达所述的蛋白。
其中，上述的方法，包括转基因、杂交、回交或无性繁殖步骤。
本发明第六方面的内容包括鉴定植物的方法，其中植物是包含所述的核酸的植物、表达所述的蛋白的植物或所述的方法获得的植物，具体包括以下步骤：
1)测定所述植物是否包含所述的核酸；或，
2)测定所述植物是否表达所述的蛋白。
有益效果：与现有技术相比，本发明具有以下优点：
1)本发明通过田间喷施ALS抑制剂类除草剂“百垄通”的实验结果表明，含有本发明的具有除草剂抗性的ALS蛋白的水稻3-4叶幼苗在施用3.3mL百垄通/L水(10倍推荐使用浓度)后，植株仍然正常生长发育和结实，而野生型水稻3-4叶幼苗在施用1mL 百垄通/3L水(1倍推荐使用浓度)30天后表现为整株死亡。
2)本发明的含有合肥不育系抗除草剂突变体的ALS基因的转基因水稻ALS酶活性比野生型的ALS酶活高4.7倍，本发明的含有合肥不育系抗除草剂突变体的ALS基因的转基因烟草后代植株的ALS酶活性比野生型的ALS酶活高5.2倍。
3)本发明的合肥不育系突变体与专利号201610226003.6中CGMCC No.12265的抗除草剂突变体植株进行ALS酶活进行测定，发现本专利申请中的合肥不育系突变体 ALS酶活性比CGMCC No.12265高18％。
附图说明
图1百垄通除草剂筛选获得的抗性水稻突变体；
图2PCR扩增ALS基因结果图；第1泳道为Marker；Marker分子量从上到下依次为5kb、3kb、2kb、1kb、750bp、500bp、250bp、100bp，第2泳道为合肥不育系野生型水稻DNA，第3泳道为抗除草剂突变体的DNA，目的片段长度2091bp；
图3百垄通除草剂对野生型和抗除草剂突变体的ALS酶活性抑制的吸光值测定；
图4抗除草剂突变体的ALS基因的过量表达载体BamHI/SacI双酶切验证图；第1 泳道为Marker；Marker分子量从上到下依次为5kb、3kb、2kb、1kb、750bp、500bp、 250bp、100bp；第2泳道为未经酶切的重组载体，第3泳道为重组载体经BamHI/SacI 双酶切后产生的基因片段和质粒片段DNA，基因片段大小符合预期，证明载体构建成功；
图5突变型ALS基因水稻PCR检测图；总共有8个泳道，第1泳道为Marker； Marker分子量从上到下依次为3kb、2kb、1kb、750bp、500bp、250bp；第2泳道为非转基因野生型DNA，作为阴性对照，第3泳道为含有ALS突变序列的质粒DNA为模板，作为阳性对照，第4～6泳道为转化抗除草剂突变体ALS基因的转基因植株DNA。
图6百垄通除草剂对野生型水稻和含有抗除草剂突变体ALS基因的转基因水稻的ALS酶活性抑制的吸光值测定；
图7百垄通除草剂对野生型烟草和含有抗除草剂突变体ALS基因的转基因烟草的ALS酶活性抑制的吸光值测定。
图8百垄通除草剂对杂交后代中的杂合植株、抗除草剂突变体和CGMCC No. 12265的ALS酶活性抑制的吸光值测定。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：水稻抗咪唑啉酮类除草剂突变体获取过程(百垄通)
将籼型常规水稻种子合肥不育系(购自江苏省农业种质资源保护与利用平台)(此为M0，用清水浸泡2小时)150kg分6次用0.5-1.0％(w/w)甲磺酸乙酯(EMS)室温下浸泡6-9小时，期间每1小时摇动一次种子；弃去EMS溶液，自来水翻动浸泡种子5次，每次5分钟，然后用自来水冲洗种子过夜，次日进行田间播种，并进行常规肥水管理(此为M1)。植株成熟后，种子混收、晾干，过冬保存。次年播种田间。待水稻(此为M2) 幼苗长至3-4叶期时，喷施为3.3mL百垄通/L水(“百垄通”是德国巴斯夫公司生产一种水剂型咪唑啉酮类除草剂，推荐最低使用浓度为1mL百垄通/1.5～3L水)，30天后还呈正常绿色植株即为抗咪唑啉酮类除草剂的水稻突变体(图1)。共计获得抗除草剂的 M2单株128株，这些抗性单株进行常规肥水管理，有101个M2单株可正常结实，种子成熟后，单株收种、晾干，过冬保存。
实施例2：抗咪唑啉酮类除草剂水稻突变体突变位点分析
取上述实施例1获得的抗除草剂水稻突变植株选取突变体植株叶片，提取基因组DNA，送南京一道生物科技有限公司进行基因组测序。测序结果与合肥不育系野生型 ALS基因比较，发现抗除草剂水稻突变体在ALS基因上发生了2个位点的突变，第512、 1050位点碱基发生突变，分别由C变成A、T变成A，导致其相应编码的氨基酸序列的第171、350位点由脯氨酸变为组氨酸、天冬酰胺变为谷氨酸，即抗除草剂突变体的ALS 基因的核苷酸序列如SEQID NO.1所示，其编码的ALS蛋白的氨基酸序列如SEQ ID NO. 2所示。
本发明的抗除草剂水稻突变体其分类命名为水稻种子HF-407(Oryza sativaIndica Group HF-407)，该菌株已于2017年6月2日保藏于中国典型培养物保藏中心(CCTCC)，地址：湖北省武汉市武昌区武汉大学保藏中心(武汉大学第一附属小学对面)，邮编： 430072，保藏编号为CCTCC No：P201714。
实施例3：抗咪唑啉酮类除草剂水稻突变体ALS基因克隆
取上述抗除草剂水稻突变体提取基因组DNA。根据合肥不育系野生型ALS基因序列设计扩增ALS基因全长的特异引物为：正向引物
F5’-TCGCCCAAACCCAGAAACCC-3’、反向引物R 5’-
CTCTTTATGGGTCATTCAGGTC-3’。
采用Takara PrimerSTAR Max DNA Polymerase聚合酶(购自Takara公司)扩增ALS基因，其反应体系如下：
PCR扩增反应程序采用两步法，退火和延伸合为一起，采用68度。程序如下：预变性：98℃3min；35个循环：变性98℃10sec；延伸68℃1min；保温： 72℃10min。
取2μl PCR产物经1％琼脂糖凝胶电泳检测，发现有预期大小的片段后(图2)，剩余的PCR产物经PCR清洁试剂盒(购自Axygen公司)清洁回收后，克隆至pMD19-T 载体(购自Takara公司)，然后转化大肠杆菌。每个转化随机挑取12个大肠杆菌单克隆进行PCR检测，取PCR结果呈阳性的6个单克隆，送南京一道生物科技有限公司测序，获得突变ALS基因序列，该基因片段长度为2091bp，抗除草剂水稻突变体1ALS 基因片段的测序结果如下：
CTGCATCGCGTGTCGCCCTTTCGCCCAAACCCAGAAACCCTCGCCGCCGCCGCCGCCGCCATCACCCACCATGGCTACGACCGCCGCGGCCGCGGCCGCCACCTTGTCCGCCGCCGCGACGGCCAAGACCGGCCGTAAGAACCACCAGCGACACCACGTCCTTCCCGCTCGAGGCCGGGTGGGGGCGGCGGCGGTCAGGTGCTCGGCGGTGTCCCCGGTCACCCCGCCGTCCCCGGCGCCGCCGGCCACGCCGCTCCGGCCGTGGGGGACGGCCGAGCCCCGCAAGGGCGCGGACATCCTCGTGGAGGCGCTGGAGCGGTGCGGCGTCAGCGACGTGTTCGCCTACCCGGGCGGCGCGTCCATGGAGATCCACCAGGCGCTGACGCGCTCCCCGGTCATCACCAACCACCTCTTCCGCCACGAGCAGGGCGAGGCGTTCGCGGCGTCCGGGTACGCGCGCGCGTCCGGCCGCGTCGGGGTCTGCGTCGCCACCTCCGGCCCCGGGGCAACCAACCTCGTGTCCGCGCTCGCCGACGCGCTGCTCGACTCCGTCCCGATGGTCGCCATCACGGGCCAGGTCACCCGCCGCATGATCGGCACCGACGCCTTCCAGGAGACGCCCATAGTCGAGGTCACCCGCTCCATCACCAAGCACAATTACCTTGTCCTTGATGTGGAGGACATCCCCCGCGTCATACAGGAAGCCTTCTTCCTCGCGTCCTCGGGCCGTCCTGGCCCGGTGCTGGTCGACATCCCCAAGGACATCCAGCAGCAGATGGCTGTGCCAGTCTGGGACACCTCGATGAATCTACCGGGGTACATTGCACGCCTGCCCAAGCCACCCGCGACAGAATTGCTTGAGCAGGTCTTGCGTCTGGTTGGCGAGTCACGGCGCCCGATTCTCTATGTCGGTGGTGGCTGCTCTGCATCTGGTGATGAATTGCGCCGGTTTGTTGAGCTGACCGGCATCCCAGTTACAACCACTCTGATGGGCCTCGGCAATTTCCCCAGTGATGATCCGTTGTCCCTGCGCATGCTTGGGATGCATGGCACGGTGTACGCAAATTATGCGGTGGATAAGGCTGACCTGTTGCTTGCATTTGGCGTGCGGTTTGATGAACGTGTGACAGGGAAAATTGAGGCTTTTGCAAGCAGGGCCAAGATTGTGCACATTGACATTGATCCAGCGGAGATTGGAAAGAACAAGCAACCACATGTGTCAATTTGCGCAGATGTTAAGCTTGCTTTACAGGGCTTGAATGCTCTGCTAGACCAGAGCACAACAAAGACAAGTTCTGATTTTAGTGCATGGCACAATGAGTTGGACCAGCAGAAGAGGGAGTTTCCTCTGGGGTACAAGACTTTTGGTGAAGAGATCCCACCGCAATATGCTATTCAGGTGCTGGATGAGCTGACGAAAGGGGAGGCAATCATCGCTACTGGTGTTGGACAGCACCAGATGTGGGCGGCACAATATTACACCTACAAGCGGCCACGGCAGTGGCTGTCTTCGGCTGGTCTGGGCGCAATGGGATTTGGGCTGCCTGCTGCAGCTGGTGCTTCTGTGGCTAACCCAGGTGTCACAGTTGTTGATATTGATGGGGATGGTAGCTTCCTCATGAACATTCAGGAGTTGGCATTGATCCGCATTGAGAACCTCCCGGTGAAGGTGATGGTGTTGAACAACCAACATTTGGGTATGGTTGTGCAATGGGAGGATAGGTTTTACAAGGCAAATAGGGCGCATACATACTTGGGCAACCCAGAATGTGAGAGCGAGATATATCCAGATTTTGTGACTATTGCTAAAGGGTTCAATATTCCTGCAGTCCGTGTAACAAAGAAGAGTGAAGTCCGTGCCGCCATCAAGAAGATGCTCGAGACCCCAGGGCCATACTTGTTGGATATCATCGTCCCACACCAGGAGCATGTGCTGCCTATGATCCCAAGTGGGGGCGCATTCAAGGACATGATCCTGGATGGTGATGGCAGGACTATGTATTAATCTATAATCTGTATGTTGGCAAAGCACCAGCCCGGCCTATGTTTGACCTGAATGACCCATAAAGAGAAGGGCGACACGCGATGCAG
实施例4：水稻M3突变体抗咪唑啉酮类除草剂(百垄通)
将合肥不育系抗除草剂突变体收获的种子播种出苗(此为M3)，待M3水稻幼苗长至3-4叶期时，喷施3.3mL百垄通/L水(推荐最低使用浓度为1mL百垄通/3L水，相当于10倍浓度)。喷施除草剂15天后，抗性苗M3呈正常绿色、能继续长高至20-30cm，而非抗性苗叶片失去绿色甚至部分枯黄，植株不长高，只有5-9cm。30天后，M3抗性株均呈正常绿色植株，而喷施同样浓度除草剂的野生型水稻都已全部枯死，表明水稻突变体至少抗10倍浓度的百垄通除草剂。
实施例5：水稻M3抗除草剂突变体的ALS酶活测定
为了验证水稻突变体的除草剂抗性是否由ALS突变所致，本发明人进行了ALS酶活性测定。测定方法参照Singh等的方法(Singh B.K.,Stidham M.A.,Shaner D.L.Assay ofacetohydroxyacid synthase.Analytical Biochemistry,1988,171:173-179.)。具体的，分别取野生型、合肥不育系抗除草剂突变体的M3植株叶片0.2g，在研钵中用液氮研磨粉碎，加入2mL提取液(100mM K2HPO4，pH 7.5、10mM丙酮酸钠、5mM EDTA、1mM缬氨酸、1mM亮氨酸、10mM半胱氨酸、0.1mM黄素腺嘌呤二核苷酸、5mM氯化镁、 10％(V/V)甘油、1％(w/v)聚乙烯吡咯烷酮)，待提取液解冻后继续研磨1min左右。 12000rpm、4℃离心30min，吸取上清液，加入硫酸铵使之达到50％饱和度，于冰上放置半小时，12000rpm、4℃离心30min，弃上清，将沉淀溶解于0.2mL反应缓冲液(100 mM K2HPO4,pH 7.0、1mM EDTA、10mM氯化镁、100mM丙酮酸钠、1mM焦磷酸硫胺素、0.1mM黄素腺嘌呤二核苷酸)，分别得各植株的ALS提取液。
在获得ALS提取液中分别加入10μL除草剂“百垄通”(水剂，有效成分240g/L)，混匀，37℃孵育1h，加0.1ml 3M硫酸终止反应，反应混合物在60℃反应孵育30分钟便于脱羧。然后加0.4mL显色液(0.09g/L 1-萘酚和0.009g/L肌酸，用2.5M NaOH溶解)。混合液在37℃孵育30分钟进行显色(ALS催化2个丙酮酸形成乙酰乳酸，乙酰乳酸脱羧形成3-羟基丁酮，再与肌酸和1-萘酚形成粉红色复合物，该复合物在530nm 处有最大吸收值)，随后测定其530nm的吸光度，ALS活性用A530吸光值表示，A530 吸光值的高低反映ALS活性的高低。实验以水为对照，野生型、合肥不育系抗除草剂突变体各测5个单株。
A530吸光值测定结果发现，当野生型、合肥不育系抗除草剂突变体的ALS提取液中没有ALS抑制剂百垄通时，它们的A530吸光值均在1.2-1.4间，表明野生型和突变体的ALS酶活性无显著性差异(图3)；而加入ALS抑制剂百垄通后，野生型的A530 吸光值仅为0.25，合肥不育系抗除草剂突变体的A530吸光值为1.21，即野生型的ALS 酶活性仅为对照的18.9％，而合肥不育系抗除草剂突变体的ALS酶活性仍有89％以上 (图3)，表明合肥不育系抗除草剂突变体的ALS对百垄通不敏感，从而赋予了抗性。
实施例6：转基因ALS水稻抗百垄通
设计特异引物5’-CGCGGATCCATCCGAGCCACACATCGCCTC-3’和 5’-TCCCCGCGGCCTACGGAAAACAACACAC-3’，其5’分别加入BamHI和SacI酶切修饰位点。参考实施例3的方法，通过PCR从上述水稻突变体合肥不育系抗除草剂突变体的基因组DNA中扩增出突变ALS基因，测序正确后，用BamHI和SacI分别双酶切突变ALS基因片段和植物表达载体pCAMBIA1301质粒(购自pcambia公司)，酶切产物用T4-DNA酶(购自TaKaRa公司)连接，连接产物转化大肠杆菌。重组质粒提取 DNA，用BamHI和SacI双酶切验证，可以产生大的质粒片段和小的基因片段(图4)，证明已将核苷酸序列如SEQ ID NO.1所示的ALS基因克隆至植物表达载体 pCAMBIA1301质粒(购自pcambia公司)中。将构建好的质粒载体转化农杆菌EHA105，培养菌体。采用常规的农杆菌介导法转化粳型水稻日本晴(购自江苏省农业种质资源保护与利用平台)，获得转基因植株收种后，后代植株长至3-4叶期时，PCR检测转基因植株(图5)。PCR检测引物为正向引物35SF 5’-ATGGTTAGAGAGGCTTACGC-3’，反向引物5R 5’-AGCAACAGGTCAGCCTTATCCAC-3’，扩增片段涵括CaMV35S启动子和ALS基因的5’端序列，大小约2kb。PCR扩增反应体系参考实施例3，PCR扩增反应程序采用两步法，退火和延伸合为一起，采用68度。扩增程序如下：预变性：98℃ 3min；30个循环：变性98℃10sec；延伸68℃2min；保温：72℃10min。经PCR鉴定呈阳性后，喷施3.3mL百垄通/L水(10倍推荐使用浓度)，7天后，参照实施例6所述方法测定ALS酶活性，发现转基因水稻的ALS酶活性显著高于非转基因水稻；30天后发现转基因水稻生长状态良好，而非转基因日本晴水稻则全部枯死。比较转化合肥不育系抗除草剂突变体的ALS基因的转基因水稻后代植株的ALS酶活性，发现含有合肥不育系抗除草剂突变体的ALS基因的转基因植株ALS酶活性比野生型的ALS酶活高4.7 倍(图6)。
实施例7：转基因ALS烟草抗百垄通
设计特异引物5’-CGCGGATCCATCCGAGCCACACATCGCCTC-3’和 5’-TCCCCGCGGCCTACGGAAAACAACACAC-3’，其5’分别加入BamHI和SacI酶切修饰位点。通过PCR从合肥不育系抗除草剂突变体的基因组DNA中扩增出突变ALS 基因，测序正确后，参照实施例7方法将核苷酸序列如SEQ ID NO.1所示的ALS基因克隆至植物表达载体pCAMBIA2301质粒(购自pcambia公司)中。挑选阳性克隆转化农杆菌EHA105，采用常规的农杆菌介导法转化本氏烟叶盘，获得转基因植株收种后，后代植株长至3-4叶期时，经PCR鉴定呈阳性后，喷施3.3mL百垄通/L水(10倍推荐使用浓度)，7天后，参照实施例6所述方法测定ALS酶活性，发现转基因烟草的ALS 酶活性显著高于非转基因烟草；30天后发现转基因烟草生长状态良好，而非转基因烟草则全部枯死。比较转化合肥不育系抗除草剂突变体的ALS基因的转基因烟草后代植株的ALS酶活性，发现含有合肥不育系抗除草剂突变体的ALS基因的转基因植株ALS酶活性比野生型的ALS酶活高5.2倍(图7)。
实施例8：抗性材料的比较和鉴定
为了检测是否具有本专利中提到的2个突变位点的抗性植株比之前专利申请(201610226003.6)中提到的抗性植株的除草耐受能力更强，我们将实施例2中的合肥不育系突变体与专利号201610226003.6中CGMCC No.12265的抗除草剂突变体植株进行ALS酶活进行测定，发现本发明中的合肥不育系突变体ALS酶活性比CGMCC No. 12265高18％(图8)。
尽管本发明的具体实施方式已经得到详细描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些均在本发明的保护范围内。本发明的全部范围由所附权利要求及其任何等同物给出。
SEQUENCE LISTING
<110> 江苏省农业科学院
<120> 使植物具有除草剂抗性的水稻ALS突变型蛋白及其应用
<130> SG20170801001
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 1935
<212> DNA
<213> 水稻种子合肥不育系抗除草剂突变体ALS基因
<220>
<221> CDS
<222> (1)..(1935)
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atg gct acg acc gcc gcg gcc gcg gcc gcc acc ttg tcc gcc gcc gcg 48
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
acg gcc aag acc ggc cgt aag aac cac cag cga cac cac gtc ctt ccc 96
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
gct cga ggc cgg gtg ggg gcg gcg gcg gtc agg tgc tcg gcg gtg tcc 144
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
ccg gtc acc ccg ccg tcc ccg gcg ccg ccg gcc acg ccg ctc cgg ccg 192
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
tgg ggg acg gcc gag ccc cgc aag ggc gcg gac atc ctc gtg gag gcg 240
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
ctg gag cgg tgc ggc gtc agc gac gtg ttc gcc tac ccg ggc ggc gcg 288
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
tcc atg gag atc cac cag gcg ctg acg cgc tcc ccg gtc atc acc aac 336
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
cac ctc ttc cgc cac gag cag ggc gag gcg ttc gcg gcg tcc ggg tac 384
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
gcg cgc gcg tcc ggc cgc gtc ggg gtc tgc gtc gcc acc tcc ggc ccc 432
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
ggg gca acc aac ctc gtg tcc gcg ctc gcc gac gcg ctg ctc gac tcc 480
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
gtc ccg atg gtc gcc atc acg ggc cag gtc cac cgc cgc atg atc ggc 528
Val Pro Met Val Ala Ile Thr Gly Gln Val His Arg Arg Met Ile Gly
165 170 175
acc gac gcc ttc cag gag acg ccc ata gtc gag gtc acc cgc tcc atc 576
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
acc aag cac aat tac ctt gtc ctt gat gtg gag gac atc ccc cgc gtc 624
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
ata cag gaa gcc ttc ttc ctc gcg tcc tcg ggc cgt cct ggc ccg gtg 672
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
ctg gtc gac atc ccc aag gac atc cag cag cag atg gct gtg cca gtc 720
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
tgg gac acc tcg atg aat cta ccg ggg tac att gca cgc ctg ccc aag 768
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
cca ccc gcg aca gaa ttg ctt gag cag gtc ttg cgt ctg gtt ggc gag 816
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
tca cgg cgc ccg att ctc tat gtc ggt ggt ggc tgc tct gca tct ggt 864
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
gat gaa ttg cgc cgg ttt gtt gag ctg acc ggc atc cca gtt aca acc 912
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
act ctg atg ggc ctc ggc aat ttc ccc agt gat gat ccg ttg tcc ctg 960
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
cgc atg ctt ggg atg cat ggc acg gtg tac gca aat tat gcg gtg gat 1008
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
aag gct gac ctg ttg ctt gca ttt ggc gtg cgg ttt gat gaa cgt gtg 1056
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Glu Arg Val
340 345 350
aca ggg aaa att gag gct ttt gca agc agg gcc aag att gtg cac att 1104
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
gac att gat cca gcg gag att gga aag aac aag caa cca cat gtg tca 1152
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
att tgc gca gat gtt aag ctt gct tta cag ggc ttg aat gct ctg cta 1200
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
gac cag agc aca aca aag aca agt tct gat ttt agt gca tgg cac aat 1248
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
gag ttg gac cag cag aag agg gag ttt cct ctg ggg tac aag act ttt 1296
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
ggt gaa gag atc cca ccg caa tat gct att cag gtg ctg gat gag ctg 1344
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
acg aaa ggg gag gca atc atc gct act ggt gtt gga cag cac cag atg 1392
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
tgg gcg gca caa tat tac acc tac aag cgg cca cgg cag tgg ctg tct 1440
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
tcg gct ggt ctg ggc gca atg gga ttt ggg ctg cct gct gca gct ggt 1488
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
gct tct gtg gct aac cca ggt gtc aca gtt gtt gat att gat ggg gat 1536
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
ggt agc ttc ctc atg aac att cag gag ttg gca ttg atc cgc att gag 1584
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
aac ctc ccg gtg aag gtg atg gtg ttg aac aac caa cat ttg ggt atg 1632
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
gtt gtg caa tgg gag gat agg ttt tac aag gca aat agg gcg cat aca 1680
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
tac ttg ggc aac cca gaa tgt gag agc gag ata tat cca gat ttt gtg 1728
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
act att gct aaa ggg ttc aat att cct gca gtc cgt gta aca aag aag 1776
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
agt gaa gtc cgt gcc gcc atc aag aag atg ctc gag acc cca ggg cca 1824
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
tac ttg ttg gat atc atc gtc cca cac cag gag cat gtg ctg cct atg 1872
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
atc cca agt ggg ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
agg act atg tat taa 1935
Arg Thr Met Tyr
<210> 2
<211> 644
<212> PRT
<213> 水稻种子合肥不育系抗除草剂突变体ALS基因
<400> 2
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val His Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Glu Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 3
<211> 1935
<212> DNA
<213> 水稻种子合肥不育系野生型 ALS基因
<220>
<221> CDS
<222> (1)..(1935)
<400> 3
atg gct acg acc gcc gcg gcc gcg gcc gcc acc ttg tcc gcc gcc gcg 48
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
acg gcc aag acc ggc cgt aag aac cac cag cga cac cac gtc ctt ccc 96
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
gct cga ggc cgg gtg ggg gcg gcg gcg gtc agg tgc tcg gcg gtg tcc 144
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
ccg gtc acc ccg ccg tcc ccg gcg ccg ccg gcc acg ccg ctc cgg ccg 192
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
tgg ggg acg gcc gag ccc cgc aag ggc gcg gac atc ctc gtg gag gcg 240
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
ctg gag cgg tgc ggc gtc agc gac gtg ttc gcc tac ccg ggc ggc gcg 288
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
tcc atg gag atc cac cag gcg ctg acg cgc tcc ccg gtc atc acc aac 336
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
cac ctc ttc cgc cac gag cag ggc gag gcg ttc gcg gcg tcc ggg tac 384
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
gcg cgc gcg tcc ggc cgc gtc ggg gtc tgc gtc gcc acc tcc ggc ccc 432
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
ggg gca acc aac ctc gtg tcc gcg ctc gcc gac gcg ctg ctc gac tcc 480
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
gtc ccg atg gtc gcc atc acg ggc cag gtc ccc cgc cgc atg atc ggc 528
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
acc gac gcc ttc cag gag acg ccc ata gtc gag gtc acc cgc tcc atc 576
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
acc aag cac aat tac ctt gtc ctt gat gtg gag gac atc ccc cgc gtc 624
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
ata cag gaa gcc ttc ttc ctc gcg tcc tcg ggc cgt cct ggc ccg gtg 672
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
ctg gtc gac atc ccc aag gac atc cag cag cag atg gct gtg cca gtc 720
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
tgg gac acc tcg atg aat cta ccg ggg tac att gca cgc ctg ccc aag 768
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
cca ccc gcg aca gaa ttg ctt gag cag gtc ttg cgt ctg gtt ggc gag 816
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
tca cgg cgc ccg att ctc tat gtc ggt ggt ggc tgc tct gca tct ggt 864
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
gat gaa ttg cgc cgg ttt gtt gag ctg acc ggc atc cca gtt aca acc 912
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
act ctg atg ggc ctc ggc aat ttc ccc agt gat gat ccg ttg tcc ctg 960
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
cgc atg ctt ggg atg cat ggc acg gtg tac gca aat tat gcg gtg gat 1008
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
aag gct gac ctg ttg ctt gca ttt ggc gtg cgg ttt gat gat cgt gtg 1056
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
aca ggg aaa att gag gct ttt gca agc agg gcc aag att gtg cac att 1104
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
gac att gat cca gcg gag att gga aag aac aag caa cca cat gtg tca 1152
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
att tgc gca gat gtt aag ctt gct tta cag ggc ttg aat gct ctg cta 1200
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
gac cag agc aca aca aag aca agt tct gat ttt agt gca tgg cac aat 1248
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
gag ttg gac cag cag aag agg gag ttt cct ctg ggg tac aag act ttt 1296
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
ggt gaa gag atc cca ccg caa tat gct att cag gtg ctg gat gag ctg 1344
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
acg aaa ggg gag gca atc atc gct act ggt gtt gga cag cac cag atg 1392
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
tgg gcg gca caa tat tac acc tac aag cgg cca cgg cag tgg ctg tct 1440
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
tcg gct ggt ctg ggc gca atg gga ttt ggg ctg cct gct gca gct ggt 1488
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
gct tct gtg gct aac cca ggt gtc aca gtt gtt gat att gat ggg gat 1536
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
ggt agc ttc ctc atg aac att cag gag ttg gca ttg atc cgc att gag 1584
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
aac ctc ccg gtg aag gtg atg gtg ttg aac aac caa cat ttg ggt atg 1632
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
gtt gtg caa tgg gag gat agg ttt tac aag gca aat agg gcg cat aca 1680
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
tac ttg ggc aac cca gaa tgt gag agc gag ata tat cca gat ttt gtg 1728
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
act att gct aaa ggg ttc aat att cct gca gtc cgt gta aca aag aag 1776
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
agt gaa gtc cgt gcc gcc atc aag aag atg ctc gag acc cca ggg cca 1824
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
tac ttg ttg gat atc atc gtc cca cac cag gag cat gtg ctg cct atg 1872
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
atc cca agt ggg ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
agg act atg tat taa 1935
Arg Thr Met Tyr
<210> 4
<211> 644
<212> PRT
<213> 水稻种子合肥不育系野生型 ALS基因
<400> 4
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 5
<211> 30
<212> DNA
<213> 上游引物
<400> 5
cgcggatcca tccgagccac acatcgcctc 30
<210> 6
<211> 28
<212> DNA
<213> 下游引物
<400> 6
tccccgcggc ctacggaaaa caacacac 28
<210> 7
<211> 2091
<212> DNA
<213> 抗除草剂水稻突变体ALS基因片段
<400> 7
ctgcatcgcg tgtcgccctt tcgcccaaac ccagaaaccc tcgccgccgc cgccgccgcc 60
atcacccacc atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac 120
ggccaagacc ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt 180
gggggcggcg gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc 240
gccggccacg ccgctccggc cgtgggggac ggccgagccc cgcaagggcg cggacatcct 300
cgtggaggcg ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc 360
catggagatc caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca 420
cgagcagggc gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt 480
ctgcgtcgcc acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct 540
gctcgactcc gtcccgatgg tcgccatcac gggccaggtc caccgccgca tgatcggcac 600
cgacgccttc caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta 660
ccttgtcctt gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc 720
ctcgggccgt cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc 780
tgtgccagtc tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc 840
acccgcgaca gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat 900
tctctatgtc ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct 960
gaccggcatc ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc 1020
gttgtccctg cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa 1080
ggctgacctg ttgcttgcat ttggcgtgcg gtttgatgaa cgtgtgacag ggaaaattga 1140
ggcttttgca agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa 1200
gaacaagcaa ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa 1260
tgctctgcta gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga 1320
gttggaccag cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc 1380
accgcaatat gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac 1440
tggtgttgga cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca 1500
gtggctgtct tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc 1560
ttctgtggct aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat 1620
gaacattcag gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt 1680
gaacaaccaa catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag 1740
ggcgcataca tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac 1800
tattgctaaa gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc 1860
cgccatcaag aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca 1920
ccaggagcat gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga 1980
tggtgatggc aggactatgt attaatctat aatctgtatg ttggcaaagc accagcccgg 2040
cctatgtttg acctgaatga cccataaaga gaagggcgac acgcgatgca g 2091
Claims (11)
1.使植物具有除草剂抗性的水稻ALS突变型蛋白，其氨基酸序列如SEQ ID NO：2所示。
2.权利要求1所述的蛋白，其中除草剂是咪唑啉酮类除草剂。
3.权利要求2所述的蛋白，其中咪唑啉酮类除草剂是百垄通。
4.核酸，其编码权利要求1~3任一项所述的蛋白。
5.根据权利要求4所述的核酸，其核苷酸序列如SEQ ID NO:1所示。
6.表达盒、重组载体或细胞，其含有权利要求4或5所述的核酸。
7.权利要求1~3任一项所述的蛋白，权利要求4或5所述的核酸，权利要求6所述的表达盒、重组载体或细胞在植物抗除草剂方面的应用。
8.根据权利要求7所述的应用，其特征在于，所述植物为水稻、烟草、拟南芥或棉花。
9.获得具有除草剂抗性的植物的方法，其特征在于，包括如下步骤：
1）使植物包含权利要求4或5所述的核酸；或
2）使植物表达权利要求1~3之任一所述的蛋白。
10.根据权利要求9所述的方法，其特征在于，其包括转基因、杂交、回交或无性繁殖步骤。
11.鉴定植物的方法，其中植物是包含权利要求4或5所述的核酸的植物、表达权利要求1~3之任一所述的蛋白的植物或权利要求9~10之任一所述的方法获得的植物，其特征在于，包括以下步骤：
1) 测定所述植物是否包含权利要求4或5所述的核酸；或，
2) 测定所述植物是否表达权利要求1~3之任一所述的蛋白。
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710655418.XA CN107217044A (zh) | 2017-08-03 | 2017-08-03 | 使植物具有除草剂抗性的水稻als突变型蛋白及其应用 |
| CN201711477560.6A CN108004224B (zh) | 2017-08-03 | 2017-12-29 | 使植物具有除草剂抗性的水稻als突变型蛋白及其应用 |
| PCT/CN2018/082758 WO2019024534A1 (zh) | 2017-08-03 | 2018-04-12 | 使植物具有除草剂抗性的水稻als突变型蛋白及其应用 |
Applications Claiming Priority (1)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019024534A1 (zh) * | 2017-08-03 | 2019-02-07 | 江苏省农业科学院 | 使植物具有除草剂抗性的水稻als突变型蛋白及其应用 |
| CN111100852A (zh) * | 2019-12-16 | 2020-05-05 | 中国农业科学院植物保护研究所 | OsALS1的定向突变及农作物内源基因定向进化的方法 |
| CN112029742B (zh) * | 2020-08-28 | 2022-03-15 | 天津市农业科学院 | 植物als突变型蛋白及其应用 |
| CN115873088A (zh) * | 2022-11-24 | 2023-03-31 | 北京大学现代农业研究院 | Als抗性突变蛋白及其应用 |
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| CN109097346B (zh) * | 2018-09-06 | 2021-07-13 | 江苏省农业科学院 | 基于基因编辑技术的als突变型蛋白及其基因在植物育种中的应用 |
| CN113005105B (zh) * | 2019-12-21 | 2022-07-29 | 科稷达隆(北京)生物技术有限公司 | 一组抗除草剂蛋白及其在植物育种中的应用 |
| CN111593031B (zh) * | 2020-05-07 | 2022-04-08 | 海南波莲水稻基因科技有限公司 | 水稻ALS突变基因、含有该基因的植物转基因筛选载体pCALSm3及其应用 |
| CN112410308B (zh) * | 2020-11-20 | 2023-11-10 | 江苏省农业科学院 | 水稻ACCase突变型基因及其蛋白在植物抗除草剂中的应用 |
| CN112813045B (zh) * | 2021-02-09 | 2023-02-28 | 江苏省农业科学院 | 棉花GhALS突变型蛋白、基因及其分子标记和应用 |
| CN115851650B (zh) * | 2022-09-06 | 2024-11-01 | 海南波莲科技有限公司 | 一种als突变蛋白及其编码基因和应用 |
| CN115851651B (zh) * | 2022-09-06 | 2024-10-18 | 海南波莲科技有限公司 | 水稻als突变蛋白及其编码基因和应用 |
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| AU6135801A (en) * | 2000-05-10 | 2001-11-20 | Univ Louisiana State | Resistance to acetohydroxyacid synthase-inhibiting herbicides |
| CN102212537B (zh) * | 2002-03-29 | 2013-08-14 | 组合化学工业株式会社 | 编码乙酰乳酸合酶的基因 |
| CN101297040A (zh) * | 2005-08-24 | 2008-10-29 | 先锋高级育种国际公司 | 提供对多种除草剂的耐受性的组合物和其使用方法 |
| CN102559646B (zh) * | 2011-11-24 | 2013-01-23 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | 小麦抗除草剂蛋白及其在植物育种中的应用 |
| CN105695493A (zh) * | 2016-04-12 | 2016-06-22 | 江苏省农业科学院 | 一种als突变型基因在抗除草剂方面的应用 |
| CN105779479B (zh) * | 2016-04-12 | 2017-08-25 | 江苏省农业科学院 | 一种als突变型基因及其在抗除草剂方面的应用 |
| CN106867977B (zh) * | 2017-01-20 | 2018-06-05 | 湖南杂交水稻研究中心 | 一种水稻抗除草剂蛋白与基因及其应用 |
| CN107217044A (zh) * | 2017-08-03 | 2017-09-29 | 江苏省农业科学院 | 使植物具有除草剂抗性的水稻als突变型蛋白及其应用 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019024534A1 (zh) * | 2017-08-03 | 2019-02-07 | 江苏省农业科学院 | 使植物具有除草剂抗性的水稻als突变型蛋白及其应用 |
| CN111100852A (zh) * | 2019-12-16 | 2020-05-05 | 中国农业科学院植物保护研究所 | OsALS1的定向突变及农作物内源基因定向进化的方法 |
| CN111100852B (zh) * | 2019-12-16 | 2021-04-13 | 中国农业科学院植物保护研究所 | OsALS1的定向突变及农作物内源基因定向进化的方法 |
| CN112029742B (zh) * | 2020-08-28 | 2022-03-15 | 天津市农业科学院 | 植物als突变型蛋白及其应用 |
| CN115873088A (zh) * | 2022-11-24 | 2023-03-31 | 北京大学现代农业研究院 | Als抗性突变蛋白及其应用 |
| CN115873088B (zh) * | 2022-11-24 | 2023-06-27 | 北京大学现代农业研究院 | Als抗性突变蛋白及其应用 |
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| CN108004224B (zh) | 2019-01-18 |
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