CN107090447A - Make plant that there is paddy rice ALS muteins, gene and its application of Herbicid resistant - Google Patents
Make plant that there is paddy rice ALS muteins, gene and its application of Herbicid resistant Download PDFInfo
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- CN107090447A CN107090447A CN201710486852.XA CN201710486852A CN107090447A CN 107090447 A CN107090447 A CN 107090447A CN 201710486852 A CN201710486852 A CN 201710486852A CN 107090447 A CN107090447 A CN 107090447A
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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Ser627和Gly628位点同时发生突变或者仅在Gly628位点发生突变。植物表达该蛋白序列可以抗（耐）乙酰乳酸合成酶抑制剂类除草剂，特别是咪唑啉酮类除草剂，本发明的Ser627和Gly628位点同时发生突变的突变体抗除草剂效果比只含有Ser627位点或者只含有Gly628位点突变的突变体植株的抗除草剂能力要高30%左右。
The invention discloses a rice ALS mutant protein that makes plants resistant to herbicides. The invention also discloses the nucleic acid encoding the above protein. The protein is derived from rice mutant plants resistant to ALS inhibitor herbicides. Compared with the ALS sequence in the wild-type rice genome, the protein sequence is mutated at both Ser627 and Gly628 or only at Gly628. Plants expressing the protein sequence can resist (resistance) acetolactate synthase inhibitor herbicides, especially imidazolinone herbicides, and the herbicide-resistant effect of the mutants with simultaneous mutations at Ser627 and Gly628 sites of the present invention is better than that of only containing The herbicide resistance of the mutant plants with Ser627 site or only Gly628 site mutation is about 30% higher.
Description
技术领域technical field
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。The invention belongs to the field of plant protein and plant herbicide resistance, and relates to rice ALS mutant protein, gene and application thereof for making plants resistant to herbicides; specifically, the invention relates to rice acetolactate synthase (ALS) mutant protein , the protein can endow plants, especially rice, with the characteristics of resistance to acetolactate synthase inhibitor herbicides. The invention discloses the sequence of the protein and their application in the field of plant herbicide resistance.
背景技术Background technique
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。Weeds are unfavorable factors restricting the stable and high yield of agricultural production. Compared with traditional cultivation methods, artificial weeding and mechanical weeding, the use of chemical herbicides is an efficient, simple and economical method of controlling weeds.
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抑制剂是除草剂通过抑制ALS而破坏植物体内缬氨酸、亮氨酸和异亮氨酸的合成。而亮氨酸、异亮氨酸、缬氨酸是植物体内3种必需的支链氨基酸。ALS是催化缬氨酸和异亮氨酸生物合成过程中第一步反应的一个关键酶，亮氨酸的合成过程则较复杂，其中从丙酮酸到α-酮异戊酸的合成过程与缬氨酸完全一致，然后才形成亮氨酸，所以，这3种氨基酸的生物合成开始阶段均需ALS催化。ALS也是植物支链氨基酸生物合成过程中3个调节酶之一，其活性受产物缬氨酸和异亮氨酸的反馈调节。ALS抑制剂会使植物体内ALS活力降低，导致这3种支链氨基酸合成受阻，影响蛋白质的合成，进一步抑制细胞分裂，导致植物组织失绿、黄化，植株生长受抑，最后逐渐死亡。Acetolactate synthase (ALS) (also known as acetohydroxyacid synthase, AHAS; EC 4.1.3.18) inhibitor herbicides use ALS as a target to cause weed death, mainly including sulfonylureas (Sulfonylureas, SU), Imidazolinones (Imidazolinones, IMI), triazolopyrimidines (TP), pyrimidinylthio(or oxy)–benzoates, PTB; pyrimidinyl-carboxyherbicides; PCs] and sulfonamide carbonyl Triazolones (Sulfonylamino-carbonyltriazolinones, SCT) and other 13 types of compounds. Acetolactate synthase, which exists in the process of plant growth, can catalyze pyruvate into acetolactate with high specificity and high catalytic efficiency, thereby leading to the biosynthesis of branched-chain amino acids. ALS inhibitors are herbicides that inhibit the synthesis of valine, leucine and isoleucine in plants by inhibiting ALS. Leucine, isoleucine, and valine are three essential branched-chain amino acids in plants. ALS is a key enzyme that catalyzes the first step reaction in the biosynthesis process of valine and isoleucine, and the synthesis process of leucine is more complicated. Amino acids are exactly the same, and then leucine is formed. Therefore, the biosynthesis of these three amino acids requires ALS to catalyze the initial stage. ALS is also one of the three regulatory enzymes in the biosynthesis of plant branched-chain amino acids, and its activity is regulated by the feedback of the products valine and isoleucine. ALS inhibitors will reduce the activity of ALS in plants, resulting in the blockage of the synthesis of these three branched chain amino acids, affecting the synthesis of proteins, further inhibiting cell division, causing chlorosis and yellowing of plant tissues, inhibiting plant growth, and finally gradually dying.
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。ALS inhibitor herbicides have the characteristics of strong selectivity, broad herbicidal spectrum, low toxicity and high efficiency, and have been widely used. However, these herbicides also cause phytotoxicity to crops that generally do not have the characteristics of resistance (tolerance) to herbicides, which greatly limits their use time and space. Harmful. Breeding herbicide-resistant (tolerant) crop varieties can reduce crop phytotoxicity and broaden the application range of herbicides.
成熟的ALS蛋白大约由670个氨基酸组成，其序列在不同物种间高度保守。ALS蛋白在Gly 95、Ala 96、Ala 122、Pro 171、Pro 196、Pro 197、Ala 205、Asp 376、Trp 537、Trp548、Trp 552、Trp 557、Trp 563、Trp 574、Ser 621、Ser 627、Ser 638、Ser 653、Gly 654、Val 669等氨基酸位点(以模式植物拟南芥的ALS氨基酸位置计算)发生突变可以产生ALS抑制剂类除草剂抗性，这在多种农作物(包括玉米、水稻、水稻、油菜、向日葵等)、模式植物拟南芥和上百种杂草中均有报道。The mature ALS protein consists of about 670 amino acids, and its sequence is highly conserved among different species. ALS protein in Gly 95, Ala 96, Ala 122, Pro 171, Pro 196, Pro 197, Ala 205, Asp 376, Trp 537, Trp548, Trp 552, Trp 557, Trp 563, Trp 574, Ser 621, Ser 627, Mutations at amino acid sites such as Ser 638, Ser 653, Gly 654, and Val 669 (calculated based on the ALS amino acid positions of the model plant Arabidopsis thaliana) can produce resistance to ALS inhibitor herbicides, which are common in many crops (including corn, Rice, paddy rice, rapeseed, sunflower, etc.), the model plant Arabidopsis thaliana and hundreds of weeds have been reported.
其中，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。Among them, the known anti-ALS inhibitor mutation sites in rice include Gly 95, Ala 96, Ala 122, Trp 548, Ser 627, Ser 653 and Gly 654. The herbicide resistance level of ALS mutants is related to the position of ALS amino acid mutation, and also related to the type of amino acid after mutation and the number of mutated amino acids.
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。At present, the mechanism of action of ALS inhibitor herbicides has not yet been determined, and it is difficult to predict in advance whether mutations at other amino acid sites of the ALS protein will cause herbicide resistance. A new herbicide-resistance locus in the ALS protein may be discovered by luck.
发明内容Contents of the invention
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。Purpose of the invention: The first technical problem to be solved by the present invention is to provide a protein that makes plants resistant to herbicides.
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。The technical problem to be solved by the present invention is to provide the nucleic acid encoding the protein, expression cassette, vector, cell and the like.
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。The technical problem to be solved by the present invention is to provide methods and applications for obtaining plants with herbicide resistance.
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。The technical problem to be solved by the present invention is to provide an identification method for judging whether the plant is obtained by the method of the present invention.
本发明人通过长期、艰苦的研究，对籼稻扬州恢复系的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。Through long-term and arduous research, the inventors have continuously screened the EMS mutant plants of the indica rice Yangzhou restorer line, and found a series of ALS mutant proteins, including some known ALS mutant proteins and new ALS mutants mentioned above. A protein that is insensitive to ALS inhibitor herbicides, thereby conferring resistance to ALS inhibitor herbicides in plants. The application of the present invention in plant breeding can be used to breed plants with herbicide resistance, especially crops, and the application of these proteins and their coding genes in transgenic or non-transgenic crops such as rice has also been developed.
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627位氨基酸由丝氨酸突变为天冬酰胺，和/或在氨基酸序列的第628位氨基酸由甘氨酸突变为谷氨酸。目前还没有报道表明，在来自水稻品种扬州恢复系ALS蛋白的Ser627和Gly628位点同时突变的突变株和在Gly628位点突变的突变株。Technical solution: In order to solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: the present invention provides an ALS protein that makes plants resistant to herbicides, wherein the 627th amino acid in the amino acid sequence is mutated from serine to asparagine, and /or the 628th amino acid in the amino acid sequence is mutated from glycine to glutamic acid. At present, there is no report showing that the mutant strains mutated at the Ser627 and Gly628 sites of the ALS protein from the Yangzhou restorer line of the rice variety and the mutant strains mutated at the Gly628 site.
优选本发明的第一方面的蛋白带有Ser627和Gly628位点同时突变的突变株和在Gly628位点突变的突变株，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或SEQ ID NO:6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。Preferably, the protein of the first aspect of the present invention has mutations at both Ser627 and Gly628 sites and mutants at the Gly628 site, except for the mutant plant screening or amplification methods described in the embodiments of the present invention , when the protein sequence is known, those skilled in the art can prepare a protein with substitution, addition or deletion of amino acid residues by changing the coding gene sequence of the known protein and introducing it into a vector. for conventional means. In a specific embodiment of the present invention, the amino acid sequence of the herbicide-resistant ALS protein of the present invention is shown in SEQ ID NO:2 or SEQ ID NO:6. This protein is an acetolactate synthase that has been shown to be insensitive to imidazolinone herbicides.
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或SEQ IDNO:5所示。具体的，本发明的DNA序列，是在野生型水稻品种扬州恢复系的ALS基因序列的第1880位核苷酸由G变成核苷酸A，和/或第1883位核苷酸由G变成核苷酸A。A second aspect of the invention provides a nucleic acid encoding the protein of the first aspect of the invention. In the present invention, nucleic acid may be DNA or RNA, among which DNA is preferred. On the premise of knowing the encoded protein sequence or nucleic acid sequence, through conventional codon correspondence and host expression frequency, using PCR method, DNA recombination method or artificial synthesis method, those skilled in the art have the ability to obtain and optimize the coding method of the present invention. The nucleic acid of the protein of the first aspect. Once the nucleic acid is obtained, it can be cloned into a vector, transformed or transfected into a corresponding cell, and then propagated through a conventional host cell, from which a large amount of nucleic acid is isolated. Preferably, the nucleotide sequence of the nucleic acid of the second aspect of the present invention is shown in SEQ ID NO: 1 or SEQ ID NO: 5. Specifically, the DNA sequence of the present invention is that the 1880th nucleotide of the ALS gene sequence of the wild-type rice variety Yangzhou restorer line is changed from G to A, and/or the 1883rd nucleotide is changed from G to A. into nucleotide A.
此外，本发明还获得了仅包含有Ser627位点突变的抗除草剂突变体2和仅包含有Gly628位点突变的抗除草剂突变体3，通过实验比较发现，含有Ser627和Gly628位点突变的突变体比仅含有Ser627位点的突变体2或仅含有Gly628位点突变的抗除草剂突变体3的抗除草剂抗性要强。其中，仅含有Ser627位点突变的抗除草剂突变体2的ALS核苷酸序列如SEQID NO.3所示，氨基酸如SEQ ID NO.4所示。仅含有Gly628位点突变的抗除草剂突变体3的ALS核苷酸序列如SEQ ID NO.5所示，氨基酸如SEQ ID NO.6所示。In addition, the present invention also obtained the herbicide-resistant mutant 2 containing only the Ser627 site mutation and the herbicide-resistant mutant 3 containing only the Gly628 site mutation. The mutants were more resistant to herbicides than mutant 2 containing only Ser627 site or herbicide-resistant mutant 3 containing only Gly628 site mutation. Wherein, the ALS nucleotide sequence of the herbicide-resistant mutant 2 containing only the Ser627 site mutation is shown in SEQ ID NO.3, and the amino acid is shown in SEQ ID NO.4. The ALS nucleotide sequence of the herbicide-resistant mutant 3 containing only the Gly628 site mutation is shown in SEQ ID NO.5, and the amino acid is shown in SEQ ID NO.6.
此外，本发明突变前的野生型水稻为籼型常规水稻种子扬州恢复系，该ALS抑制剂类除草剂敏感的野生型扬州恢复系水稻ALS的核苷酸序列如SEQ ID NO.7所示，氨基酸如SEQ ID NO.8所示。In addition, the wild-type rice before the mutation of the present invention is an indica-type conventional rice seed Yangzhou restorer line, and the nucleotide sequence of the wild-type Yangzhou restorer rice ALS sensitive to ALS inhibitor herbicides is shown in SEQ ID NO.7, The amino acid is shown in SEQ ID NO.8.
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。The content of the third aspect of the present invention includes expression cassettes, recombinant vectors or cells, which contain the above-mentioned nucleic acid of the second aspect.
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。The fourth aspect of the present invention also includes the application of the above-mentioned herbicide-resistant ALS protein, nucleic acid, expression cassette, recombinant vector or cell in green plant resistance to herbicides.
其中，上述绿色植物为水稻、烟草、拟南芥、棉花等。Wherein, the above-mentioned green plants are rice, tobacco, Arabidopsis, cotton and the like.
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：The content of the fifth aspect of the present invention also includes a method for obtaining a herbicide-resistant plant, comprising the steps of:
1)使植物包含本发明所述的核酸；或1) causing plants to contain a nucleic acid according to the invention; or
2)使植物表达所述的蛋白。2) Allowing plants to express the protein.
其中，上述的方法，包括转基因、杂交、回交或无性繁殖步骤。Wherein, the above method includes the steps of transgenic, hybrid, backcross or asexual reproduction.
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：The content of the sixth aspect of the present invention includes a method for identifying a plant, wherein the plant is a plant containing the nucleic acid, a plant expressing the protein or a plant obtained by the method, specifically comprising the following steps:
1)测定所述植物是否包含所述的核酸；或，1) determining whether said plant comprises said nucleic acid; or,
2)测定所述植物是否表达所述的蛋白。2) Determining whether the plant expresses the protein.
有益效果：Beneficial effect:
1)本发明通过对突变株的ALS酶活测定结果来看，扬州恢复系抗除草剂突变体1的ALS活性比扬州恢复系抗除草剂突变体2的ALS活性高30％，比扬州恢复系抗除草剂突变体3的ALS活性高31％。1) According to the ALS enzyme activity determination results of the mutant strains in the present invention, the ALS activity of the Yangzhou restorer line herbicide-resistant mutant 1 is 30% higher than the ALS activity of the Yangzhou restorer line herbicide-resistant mutant 2, which is higher than that of the Yangzhou restorer line The ALS activity of the herbicide-resistant mutant 3 was 31% higher.
2)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3mL百垄通/L水(9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) The experimental results of the present invention spraying the ALS inhibitor herbicide "Bailongtong" in the field show that rice seedlings with 3-4 leaves containing the herbicide-resistant ALS protein of the present invention are treated with 3mL Bailongtong/ After L water (9 times the recommended use concentration), the plants still grow and develop normally and bear fruit, while wild-type rice seedlings with 3-4 leaves show the whole plant after 30 days of application of 1mL Bailongtong/3L water (1 times the recommended use concentration) die.
附图说明Description of drawings
图1百垄通除草剂筛选获得的抗性水稻突变体1；Fig. 1 Resistant rice mutant 1 obtained by screening with Bailongtong herbicide;
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3kb、2kb、1kb、750bp、500bp、250bp，第2泳道为扬州恢复系野生型水稻DNA，第3泳道为抗除草剂突变体1的DNA，第4泳道为抗除草剂突变体2的DNA，第5泳道为抗除草剂突变体3的DNA；目的片段长度2091bp；Figure 2 PCR amplification results of ALS gene; the first lane is Marker; the molecular weight of Marker from top to bottom is 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, the second lane is the wild-type rice DNA of Yangzhou restorer line, and the third lane The DNA of the herbicide-resistant mutant 1, the fourth lane is the DNA of the herbicide-resistant mutant 2, and the fifth lane is the DNA of the herbicide-resistant mutant 3; the length of the target fragment is 2091bp;
图3百垄通除草剂对野生型和抗除草剂水稻抗除草剂突变体1、抗除草剂突变体2和抗除草剂突变体3的ALS酶活性抑制的吸光值测定；Fig. 3 Determination of the absorbance value of Bailongtong herbicide on the inhibition of ALS enzyme activity of wild-type and herbicide-resistant rice herbicide-resistant mutant 1, herbicide-resistant mutant 2 and herbicide-resistant mutant 3;
图4抗除草剂突变体1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3kb、2kb、1kb、750bp、500bp、250bp；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；Fig. 4 ALS gene overexpression carrier BamHI/SacI double enzyme digestion verification diagram of herbicide-resistant mutant 1; the first swimming lane is Marker; Marker molecular weight is 3kb, 2kb, 1kb, 750bp, 500bp, 250bp from top to bottom; The second lane is the undigested recombinant vector, and the third lane is the gene fragment and plasmid fragment DNA produced by the recombinant vector after BamHI/SacI double digestion. The size of the gene fragment meets the expectation, which proves that the vector is successfully constructed;
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泳道为质粒DNA，作为阳性对照，第4泳道为转化抗除草剂突变体1ALS基因的转基因植株DNA，第5泳道为转化抗除草剂突变体2ALS基因的转基因植株DNA，第6泳道为转化抗除草剂突变体3ALS基因的转基因植株DNA。Figure 5 PCR detection diagram of mutant ALS gene rice; there are a total of 8 lanes, the first lane is Marker; the molecular weight of Marker is 3kb, 2kb, 1kb, 750bp, 500bp, 250bp from top to bottom; the second lane is non-transgenic wild Type DNA, as a negative control, the third lane is plasmid DNA, as a positive control, the fourth lane is the DNA of the transgenic plant transformed with the herbicide-resistant mutant 1 ALS gene, and the fifth lane is the transgenic plant transformed with the herbicide-resistant mutant 2 ALS gene DNA, lane 6 is the DNA of the transgenic plant transformed with the herbicide-resistant mutant 3ALS gene.
图6百垄通除草剂对野生型水稻和含有抗除草剂突变体1ALS基因的转基因水稻、含有抗除草剂突变体2ALS基因的转基因水稻和含有抗除草剂突变体3ALS基因的转基因水稻的ALS酶活性抑制的吸光值测定；Fig. 6 ALS enzyme of Bailongtong herbicide on wild-type rice and transgenic rice containing herbicide-resistant mutant 1 ALS gene, transgenic rice containing herbicide-resistant mutant 2 ALS gene and transgenic rice containing herbicide-resistant mutant 3 ALS gene Absorbance determination of activity inhibition;
图7百垄通除草剂对野生型烟草和含有抗除草剂突变体1ALS基因的转基因烟草、含有抗除草剂突变体2ALS基因的转基因烟草和含有抗除草剂突变体3ALS基因的转基因水稻的ALS酶活性抑制的吸光值测定；Fig. 7 The ALS enzyme of Bailongtong herbicide on wild-type tobacco and transgenic tobacco containing herbicide-resistant mutant 1 ALS gene, transgenic tobacco containing herbicide-resistant mutant 2 ALS gene and transgenic rice containing herbicide-resistant mutant 3 ALS gene Absorbance determination of activity inhibition;
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2和抗除草剂突变体3的ALS酶活性抑制的吸光值测定。Fig. 8 Absorbance determination of the inhibition of the ALS enzyme activity of the hybrid plants, the herbicide-resistant mutant 2 and the herbicide-resistant mutant 3 by Bailongtong herbicide.
具体实施方式detailed description
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。Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
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)Example 1: The process of obtaining rice mutants resistant to imidazolinone herbicides (Bailongtong)
将籼型常规水稻种子扬州恢复系(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211株，这些抗性单株进行常规肥水管理，有198个M2单株可正常结实，种子成熟后，单株收种、晾干，过冬保存。150 kg of indica conventional rice seed Yangzhou restorer line (purchased from Jiangsu Provincial Agricultural Germplasm Resources Protection and Utilization Platform) (this is M0, soaked in water for 2 hours) was divided into 6 times with 0.5-1.0% (w/w) methanesulfonic acid Soak in ethyl acetate (EMS) at room temperature for 6-9 hours, shake the seeds every 1 hour during this period; discard the EMS solution, soak the seeds 5 times with tap water, 5 minutes each time, then rinse the seeds with tap water overnight, and carry out field work the next day Sow seeds and perform conventional fertilizer and water management (this is M1). After the plants are mature, the seeds are mixed, dried, and stored for the winter. Sow in the field the following year. When the rice (this is M2) seedlings grow to the 3-4 leaf stage, spray 3mL Bailongtong/L water ("Bailongtong" is a water-based imidazolinone herbicide produced by BASF in Germany, it is recommended The minimum concentration used is 1mL Bailongtong/1.5-3L water), and after 30 days, the normal green plants are rice mutants resistant to imidazolinone herbicides (Figure 1). A total of 211 herbicide-resistant M2 individual plants were obtained. These resistant individual plants underwent conventional fertilizer and water management, and 198 M2 individual plants could bear fruit normally. After the seeds matured, the individual plants were harvested, dried, and stored for winter.
实施例2：抗咪唑啉酮类除草剂水稻突变体突变位点分析Example 2: Analysis of mutation sites in rice mutants resistant to imidazolinone herbicides
取上述实施例1获得的抗除草剂水稻突变植株选取多个突变体叶片，分别提取基因组DNA，送南京一道生物科技有限公司进行基因组测序。测序结果与扬州恢复系野生型ALS基因比较，发现上述水稻突变植株中有三组突变植株，即抗除草剂水稻突变体1、抗除草剂水稻突变体2和抗除草剂水稻突变体3，其中，抗除草剂水稻突变体1在ALS基因上发生了2个位点的突变，第1880、1883位点碱基发生突变，分别由G变成A、G变成A，导致其相应编码的氨基酸序列的第627、628位点由丝氨酸变为天冬酰胺、甘氨酸变为谷氨酸，即抗除草剂突变体1的ALS基因的核苷酸序列如SEQ ID NO.1所示，其编码的ALS蛋白的氨基酸序列如SEQ IDNO.2所示；抗除草剂突变体2在ALS基因的仅在第1880位点碱基发生突变，分别由G变成A，导致其相应编码的氨基酸序列的第627位点分别由丝氨酸变为天冬酰胺，即抗除草剂突变体2的ALS基因的核苷酸序列如SEQ ID NO.3所示，其编码的ALS蛋白的氨基酸序列如SEQ IDNO.4所示；抗除草剂突变体3在ALS基因的仅在第1883位点碱基发生突变，由G变成A，导致其相应编码的氨基酸序列的第628位点分别由甘氨酸变为谷氨酸，即抗除草剂突变体3的ALS基因的核苷酸序列如SEQ ID NO.5所示，其编码的ALS蛋白的氨基酸序列如SEQ ID NO.6所示。From the herbicide-resistant mutant rice plants obtained in Example 1 above, a plurality of mutant leaves were selected, and genomic DNA was extracted respectively, and sent to Nanjing Yidao Biotechnology Co., Ltd. for genome sequencing. Comparing the sequencing results with the wild-type ALS gene of the Yangzhou restorer line, it was found that there were three groups of mutant plants in the above-mentioned mutant rice plants, namely, herbicide-resistant rice mutant 1, herbicide-resistant rice mutant 2 and herbicide-resistant rice mutant 3, among which, The herbicide-resistant rice mutant 1 has 2 mutations on the ALS gene, the 1880th and 1883th bases are mutated, respectively changing from G to A, and G to A, resulting in the corresponding encoded amino acid sequence The 627th and 628th positions of the ALS gene are changed from serine to asparagine, and glycine to glutamic acid, that is, the nucleotide sequence of the ALS gene of the herbicide-resistant mutant 1 is shown in SEQ ID NO.1, and the ALS gene encoded by it is shown in SEQ ID NO.1. The amino acid sequence of the protein is shown in SEQ ID NO.2; the herbicide-resistant mutant 2 is only mutated at the 1880th base of the ALS gene, changing from G to A, resulting in the 627th base of its correspondingly encoded amino acid sequence The positions are respectively changed from serine to asparagine, that is, the nucleotide sequence of the ALS gene of the herbicide-resistant mutant 2 is shown in SEQ ID NO.3, and the amino acid sequence of the ALS protein encoded by it is shown in SEQ ID NO.4 ; The herbicide-resistant mutant 3 is only mutated at the 1883rd position of the ALS gene, changing from G to A, resulting in the 628th position of the corresponding encoded amino acid sequence being changed from glycine to glutamic acid, namely The nucleotide sequence of the ALS gene of the herbicide-resistant mutant 3 is shown in SEQ ID NO.5, and the amino acid sequence of the ALS protein encoded by it is shown in SEQ ID NO.6.
本发明的抗除草剂水稻突变体1其分类命名为水稻种子YXL-25(Oryza sativaIndica Group YXL-25)，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：P201713。The herbicide-resistant rice mutant 1 of the present invention is classified as rice seed YXL-25 (Oryza sativaIndica Group YXL-25), and the strain has been preserved in the China Center for Type Culture Collection (CCTCC) on June 2, 2017. Address: Wuhan University Preservation Center (opposite to the First Affiliated Primary School of Wuhan University), Wuchang District, Wuhan City, Hubei Province, Zip Code: 430072, and the deposit number is CCTCC No: P201713.
本发明的抗除草剂水稻突变体3其分类命名为水稻种子YXL-63(Oryza sativaIndica Group YXL-63)，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：P201712。The herbicide-resistant rice mutant 3 of the present invention is classified as rice seed YXL-63 (Oryza sativaIndica Group YXL-63), and the strain has been preserved in the China Center for Type Culture Collection (CCTCC) on June 2, 2017. Address: Wuhan University Preservation Center (opposite to the First Affiliated Primary School of Wuhan University), Wuchang District, Wuhan City, Hubei Province, Zip Code: 430072, and the deposit number is CCTCC No: P201712.
实施例3：抗咪唑啉酮类除草剂水稻突变体ALS基因克隆Embodiment 3: ALS gene cloning of rice mutant resistant to imidazolinone herbicides
取上述抗除草剂水稻突变体1、抗除草剂水稻突变体2和抗除草剂水稻突变体3的叶片，分别提取基因组DNA。根据扬州恢复系野生型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’。The leaves of the above herbicide-resistant rice mutant 1, herbicide-resistant rice mutant 2 and herbicide-resistant rice mutant 3 were taken, and genomic DNA was extracted respectively. The specific primers for amplifying the full-length ALS gene were designed according to the wild-type ALS gene sequence of Yangzhou restorer line: forward primer F5'-TCGCCCAAACCCAGAAACCC-3', reverse primer 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：Adopt Takara PrimerSTAR Max DNA Polymerase polymerase (purchased from Takara company) to amplify ALS gene, its reaction system is as follows:
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。The PCR amplification reaction program adopts a two-step method, annealing and extension are combined together, and 68 degrees are used.
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。The program is as follows: pre-denaturation: 98°C for 3min; 35 cycles: denaturation at 98°C for 10sec; extension at 68°C for 1min; incubation: 72°C for 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：Take 2 μl of the PCR product and detect it by 1% agarose gel electrophoresis. After a fragment of the expected size is found (Figure 2), the remaining PCR product is cleaned and recovered by a PCR cleaning kit (purchased from Axygen), and cloned into pMD19-T Vector (purchased from Takara Company), and then transformed into Escherichia coli. For each transformation, 12 single clones of Escherichia coli were randomly selected for PCR detection, and 6 single clones with positive PCR results were taken and sent to Nanjing Yidao Biotechnology Co., Ltd. for sequencing to obtain the mutant ALS gene sequence. The sequencing results of the ALS gene fragment of the herbicide rice mutant 1 are as follows:
CTGCATCGCGTGTCGCCCTTTCGCCCAAACCCAGAAACCCTCGCCGCCGCCGCCGCCGCCATCACCCACCATGGCTACGACCGCCGCGGCCGCGGCCGCCACCTTGTCCGCCGCCGCGACGGCCAAGACCGGCCGTAAGAACCACCAGCGACACCACGTCCTTCCCGCTCGAGGCCGGGTGGGGGCGGCGGCGGTCAGGTGCTCGGCGGTGTCCCCGGTCACCCCGCCGTCCCCGGCGCCGCCGGCCACGCCGCTCCGGCCGTGGGGGACGGCCGAGCCCCGCAAGGGCGCGGACATCCTCGTGGAGGCGCTGGAGCGGTGCGGCGTCAGCGACGTGTTCGCCTACCCGGGCGGCGCGTCCATGGAGATCCACCAGGCGCTGACGCGCTCCCCGGTCATCACCAACCACCTCTTCCGCCACGAGCAGGGCGAGGCGTTCGCGGCGTCCGGGTACGCGCGCGCGTCCGGCCGCGTCGGGGTCTGCGTCGCCACCTCCGGCCCCGGGGCAACCAACCTCGTGTCCGCGCTCGCCGACGCGCTGCTCGACTCCGTCCCGATGGTCGCCATCACGGGCCAGGTCCCCCGCCGCATGATCGGCACCGACGCCTTCCAGGAGACGCCCATAGTCGAGGTCACCCGCTCCATCACCAAGCACAATTACCTTGTCCTTGATGTGGAGGACATCCCCCGCGTCATACAGGAAGCCTTCTTCCTCGCGTCCTCGGGCCGTCCTGGCCCGGTGCTGGTCGACATCCCCAAGGACATCCAGCAGCAGATGGCTGTGCCAGTCTGGGACACCTCGATGAATCTACCGGGGTACATTGCACGCCTGCCCAAGCCACCCGCGACAGAATTGCTTGAGCAGGTCTTGCGTCTGGTTGGCGAGTCACGGCGCCCGATTCTCTATGTCGGTGGTGGCTGCTCTGCATCTGGTGATGAATTGCGCCGGTTTGTTGAGCTGACCGGCATCCCAGTTACAACCACTCTGATGGGCCTCGGCAATTTCCCCAGTGATGATCCGTTGTCCCTGCGCATGCTTGGGATGCATGGCACGGTGTACGCAAATTATGCGGTGGATAAGGCTGACCTGTTGCTTGCATTTGGCGTGCGGTTTGATGATCGTGTGACAGGGAAAATTGAGGCTTTTGCAAGCAGGGCCAAGATTGTGCACATTGACATTGATCCAGCGGAGATTGGAAAGAACAAGCAACCACATGTGTCAATTTGCGCAGATGTTAAGCTTGCTTTACAGGGCTTGAATGCTCTGCTAGACCAGAGCACAACAAAGACAAGTTCTGATTTTAGTGCATGGCACAATGAGTTGGACCAGCAGAAGAGGGAGTTTCCTCTGGGGTACAAGACTTTTGGTGAAGAGATCCCACCGCAATATGCTATTCAGGTGCTGGATGAGCTGACGAAAGGGGAGGCAATCATCGCTACTGGTGTTGGACAGCACCAGATGTGGGCGGCACAATATTACACCTACAAGCGGCCACGGCAGTGGCTGTCTTCGGCTGGTCTGGGCGCAATGGGATTTGGGCTGCCTGCTGCAGCTGGTGCTTCTGTGGCTAACCCAGGTGTCACAGTTGTTGATATTGATGGGGATGGTAGCTTCCTCATGAACATTCAGGAGTTGGCATTGATCCGCATTGAGAACCTCCCGGTGAAGGTGATGGTGTTGAACAACCAACATTTGGGTATGGTTGTGCAATGGGAGGATAGGTTTTACAAGGCAAATAGGGCGCATACATACTTGGGCAACCCAGAATGTGAGAGCGAGATATATCCAGATTTTGTGACTATTGCTAAAGGGTTCAATATTCCTGCAGTCCGTGTAACAAAGAAGAGTGAAGTCCGTGCCGCCATCAAGAAGATGCTCGAGACCCCAGGGCCATACTTGTTGGATATCATCGTCCCACACCAGGAGCATGTGCTGCCTATGATCCCAAATGAGGGCGCATTCAAGGACATGATCCTGGATGGTGATGGCAGGACTATGTATTAATCTATAATCTGTATGTTGGCAAAGCACCAGCCCGGCCTATGTTTGACCTGAATGACCCATAAAGAGAAGGGCGACACGCGATGCAGCTGCATCGCGTGTCGCCCTTTCGCCCAAACCCAGAAACCCTCGCCGCCGCCGCCGCCGCCATCACCCACCATGGCTACGACCGCCGCGGCCGCGGCCGCCACCTTGTCCGCCGCCGCGACGGCCAAGACCGGCCGTAAGAACCACCAGCGACACCACGTCCTTCCCGCTCGAGGCCGGGTGGGGGCGGCGGCGGTCAGGTGCTCGGCGGTGTCCCCGGTCACCCCGCCGTCCCCGGCGCCGCCGGCCACGCCGCTCCGGCCGTGGGGGACGGCCGAGCCCCGCAAGGGCGCGGACATCCTCGTGGAGGCGCTGGAGCGGTGCGGCGTCAGCGACGTGTTCGCCTACCCGGGCGGCGCGTCCATGGAGATCCACCAGGCGCTGACGCGCTCCCCGGTCATCACCAACCACCTCTTCCGCCACGAGCAGGGCGAGGCGTTCGCGGCGTCCGGGTACGCGCGCGCGTCCGGCCGCGTCGGGGTCTGCGTCGCCACCTCCGGCCCCGGGGCAACCAACCTCGTGTCCGCGCTCGCCGACGCGCTGCTCGACTCCGTCCCGATGGTCGCCATCACGGGCCAGGTCCCCCGCCGCATGATCGGCACCGACGCCTTCCAGGAGACGCCCATAGTCGAGGTCACCCGCTCCATCACCAAGCACAATTACCTTGTCCTTGATGTGGAGGACATCCCCCGCGTCATACAGGAAGCCTTCTTCCTCGCGTCCTCGGGCCGTCCTGGCCCGGTGCTGGTCGACATCCCCAAGGACATCCAGCAGCAGATGGCTGTGCCAGTCTGGGACACCTCGATGAATCTACCGGGGTACATTGCACGCCTGCCCAAGCCACCCGCGACAGAATTGCTTGAGCAGGTCTTGCGTCTGGTTGGCGAGTCACGGCGCCCGATTCTCTATGTCGGTGGTGGCTGCTCTGCATCTGGTGATGAATTGCGCCGGTTTGTTGAGCTGACCGGCATCCCAGTTACAACCACTCTGATGGGCCTCGGC AATTTCCCCAGTGATGATCCGTTGTCCCTGCGCATGCTTGGGATGCATGGCACGGTGTACGCAAATTATGCGGTGGATAAGGCTGACCTGTTGCTTGCATTTGGCGTGCGGTTTGATGATCGTGTGACAGGGAAAATTGAGGCTTTTGCAAGCAGGGCCAAGATTGTGCACATTGACATTGATCCAGCGGAGATTGGAAAGAACAAGCAACCACATGTGTCAATTTGCGCAGATGTTAAGCTTGCTTTACAGGGCTTGAATGCTCTGCTAGACCAGAGCACAACAAAGACAAGTTCTGATTTTAGTGCATGGCACAATGAGTTGGACCAGCAGAAGAGGGAGTTTCCTCTGGGGTACAAGACTTTTGGTGAAGAGATCCCACCGCAATATGCTATTCAGGTGCTGGATGAGCTGACGAAAGGGGAGGCAATCATCGCTACTGGTGTTGGACAGCACCAGATGTGGGCGGCACAATATTACACCTACAAGCGGCCACGGCAGTGGCTGTCTTCGGCTGGTCTGGGCGCAATGGGATTTGGGCTGCCTGCTGCAGCTGGTGCTTCTGTGGCTAACCCAGGTGTCACAGTTGTTGATATTGATGGGGATGGTAGCTTCCTCATGAACATTCAGGAGTTGGCATTGATCCGCATTGAGAACCTCCCGGTGAAGGTGATGGTGTTGAACAACCAACATTTGGGTATGGTTGTGCAATGGGAGGATAGGTTTTACAAGGCAAATAGGGCGCATACATACTTGGGCAACCCAGAATGTGAGAGCGAGATATATCCAGATTTTGTGACTATTGCTAAAGGGTTCAATATTCCTGCAGTCCGTGTAACAAAGAAGAGTGAAGTCCGTGCCGCCATCAAGAAGATGCTCGAGACCCCAGGGCCATACTTGTTGGATATCATCGTCCCACACCAGGAGCATGTGCTGCCTATGATCCCAAATGAGGGCGCATTCAAGGACATGATCCTGGATGGTGATGGCAGGACTATGT ATTAATCTATAATCTGTATGTTGGCAAAGCACCAGCCCGGCCTATGTTTGACCTGAATGACCCATAAAGAGAAGGGCGACACGCGATGCAG
实施例4：水稻M3突变体抗咪唑啉酮类除草剂(百垄通)Example 4: Rice M3 mutants are resistant to imidazolinone herbicides (Bailongtong)
将扬州恢复系抗除草剂突变体1和扬州恢复系抗除草剂突变体2和扬州恢复系抗除草剂突变体3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3mL百垄通/L水(推荐最低使用浓度为1mL百垄通/3L水，相当于9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，表明突变水稻至少抗9倍浓度的百垄通除草剂。我们又对扬州恢复系抗除草剂突变体1和扬州恢复系抗除草剂突变体2和扬州恢复系抗除草剂突变体3喷施4mL百垄通/L水(相当于12倍浓度)进行鉴定，发现抗除草剂突变体1还是呈现正常绿色，而抗除草剂突变体2和抗除草剂突变体3部分植株已经枯黄。表明含有同时含有Ser627位点和Gly628位点突变的抗除草剂突变体1的抗除草剂能力比抗除草剂突变体2和抗除草剂突变体3都要好。The seeds of Yangzhou restorer herbicide-resistant mutant 1, Yangzhou restorer herbicide-resistant mutant 2 and Yangzhou restorer herbicide-resistant mutant 3 are sown and emerge (this is M3), and the rice seedlings of M3 grow to 3-4 At the leaf stage, spray 3mL Bailongtong/L water (the recommended minimum concentration is 1mL Bailongtong/3L water, which is equivalent to 9 times the concentration). 15 days after spraying the herbicide, the resistant seedling M3 was normally green and could continue to grow up to 20-30cm in height, while the leaves of non-resistant seedlings lost their green color or even partially withered yellow, and the plant did not grow taller, only 5-9cm in height. After 30 days, the M3-resistant plants were all normal green plants, while the wild-type rice sprayed with the same concentration of herbicide all died, indicating that the mutant rice was at least resistant to 9 times the concentration of Bailongtong herbicide. We also identified the herbicide-resistant mutant 1 of the Yangzhou restorer line, the herbicide-resistant mutant 2 of the Yangzhou restorer line, and the herbicide-resistant mutant 3 of the Yangzhou restorer line by spraying 4mL Bailongtong/L water (equivalent to 12 times the concentration) , it was found that herbicide-resistant mutant 1 still showed normal green color, while some plants of herbicide-resistant mutant 2 and herbicide-resistant mutant 3 had withered and yellowed. It shows that the herbicide-resistant mutant 1 containing both Ser627 and Gly628 mutations has better herbicide resistance than herbicide-resistant mutant 2 and herbicide-resistant mutant 3.
实施例5：水稻M3抗除草剂突变体的ALS酶活测定Example 5: ALS Enzyme Activity Determination of Rice M3 Herbicide-resistant Mutants
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、扬州恢复系抗除草剂突变体1、扬州恢复系抗除草剂突变体2和扬州恢复系抗除草剂突变体3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(100mMK2HPO4，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(100mM 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。In order to verify whether the herbicide resistance of the rice mutants is caused by the ALS mutation, the present inventors performed an ALS enzyme activity assay. The determination method refers to the method of Singh et al. (Singh B.K., Stidham M.A., Shaner D.L. Assay of facetohydroxyacid synthase. Analytical Biochemistry, 1988, 171:173-179.). Specifically, 0.2 g of M3 plant leaves of wild type, Yangzhou restorer herbicide-resistant mutant 1, Yangzhou restorer herbicide-resistant mutant 2 and Yangzhou restorer herbicide-resistant mutant 3 were taken respectively, and mixed with liquid Nitrogen grinding, adding 2mL extract (100mM K2HPO4, pH 7.5, 10mM sodium pyruvate, 5mM EDTA, 1mM valine, 1mM leucine, 10mM cysteine, 0.1mM flavin adenine dinucleotide, 5mM Magnesium chloride, 10% (V/V) glycerin, 1% (w/v) polyvinylpyrrolidone), and continue grinding for about 1 min after the extract is thawed. Centrifuge at 12000rpm, 4°C for 30min, absorb the supernatant, add ammonium sulfate to make it reach 50% saturation, place on ice for half an hour, centrifuge at 12000rpm, 4°C for 30min, discard the supernatant, dissolve the precipitate in 0.2mL reaction buffer (100mM K2HPO4, pH 7.0, 1mM EDTA, 10mM magnesium chloride, 100mM sodium pyruvate, 1mM thiamine pyrophosphate, 0.1mM flavin adenine dinucleotide), and obtain the ALS extracts of each plant.
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、扬州恢复系抗除草剂突变体1和扬州抗除草剂突变体2和扬州恢复系抗除草剂突变体3都各测5个单株。Add 10 μL of the herbicide “Bailongtong” (aqua, active ingredient 240g/L) to the obtained ALS extract, mix well, incubate at 37°C for 1 hour, add 0.1ml of 3M sulfuric acid to terminate the reaction, and incubate the reaction mixture at 60°C 30 minutes facilitates decarboxylation. Then add 0.4mL color developing solution (0.09g/L 1-naphthol and 0.009g/L creatine, dissolved with 2.5M NaOH). The mixture was incubated at 37°C for 30 minutes for color development (ALS catalyzed 2 pyruvic acids to form acetolactate, decarboxylated acetolactate to form 3-hydroxybutanone, and then formed a pink complex with creatine and 1-naphthol, the complex There is a maximum absorbance value at 530nm), and then its absorbance at 530nm is measured, and the ALS activity is represented by the A530 absorbance value, and the level of the A530 absorbance value reflects the level of ALS activity. Water was used as the control in the experiment, and 5 individual plants were tested for each of wild type, Yangzhou restorer herbicide-resistant mutant 1, Yangzhou herbicide-resistant mutant 2 and Yangzhou restorer herbicide-resistant mutant 3.
A530吸光值测定结果发现，当野生型、扬州恢复系抗除草剂突变体1、扬州恢复系抗除草剂突变体2和扬州恢复系抗除草剂突变体3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0.2，扬州恢复系抗除草剂突变体1的A530吸光值为1.28，扬州恢复系抗除草剂突变体2的A530吸光值为0.98，扬州恢复系抗除草剂突变体3的A530吸光值为0.97，即野生型的ALS酶活性仅为对照的16.4％，而扬州恢复系抗除草剂突变体1、抗除草剂突变体2和抗除草剂突变体3的ALS酶活性仍有75％以上(图3)，表明扬州恢复系抗除草剂突变体1、扬州恢复系抗除草剂突变体2和扬州恢复系抗除草剂突变体3的ALS对百垄通不敏感，从而赋予了抗性。从抗除草剂的效果来看，扬州恢复系抗除草剂突变体1的ALS活性比扬州恢复系抗除草剂突变体2高30％，比扬州恢复系抗除草剂突变体3高31％。因此扬州恢复系抗除草剂突变体1的抗除草剂效果比扬州恢复系抗除草剂突变体2和扬州恢复系抗除草剂突变体3都要好。The results of A530 absorbance measurement showed that there was no ALS inhibitor in the ALS extracts of wild type, Yangzhou restorer herbicide-resistant mutant 1, Yangzhou restorer herbicide-resistant mutant 2 and Yangzhou restorer herbicide-resistant mutant 3. The A530 absorbance values of the wild-type and mutants were all between 1.2 and 1.4 when the ALS inhibitor was added (Fig. 3); however, the A530 absorbance of the wild-type The value is only 0.2, the A530 absorbance value of the Yangzhou restorer line herbicide-resistant mutant 1 is 1.28, the A530 absorbance value of the Yangzhou restorer line herbicide-resistant mutant 2 is 0.98, and the A530 absorbance value of the Yangzhou restorer line herbicide-resistant mutant 3 is 0.97, that is, the ALS enzyme activity of the wild type is only 16.4% of that of the control, while the ALS enzyme activity of the Yangzhou restorer line herbicide-resistant mutant 1, herbicide-resistant mutant 2 and herbicide-resistant mutant 3 is still more than 75%. (Fig. 3), indicating that ALS of Yangzhou restorer herbicide-resistant mutant 1, Yangzhou restorer herbicide-resistant mutant 2 and Yangzhou restorer herbicide-resistant mutant 3 are not sensitive to Bailongtong, thus conferring resistance. From the effect of herbicide resistance, the ALS activity of the Yangzhou restorer mutant 1 was 30% higher than that of the Yangzhou restorer mutant 2, and 31% higher than that of the Yangzhou restorer mutant 3. Therefore, the herbicide-resistant mutant 1 of the Yangzhou restorer line is better than the herbicide-resistant mutant 2 of the Yangzhou restorer line and the herbicide-resistant mutant 3 of the Yangzhou restorer line.
实施例6：转基因ALS水稻抗百垄通Example 6: Transgenic ALS rice resistant to Bailongtong
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从上述水稻突变体扬州恢复系抗除草剂突变体1、扬州恢复系抗除草剂突变体2和扬州恢复系抗除草剂突变体3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或3或5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3mL百垄通/L水(9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。比较转化扬州恢复系抗除草剂突变体1、扬州恢复系抗除草剂突变体2和扬州恢复系抗除草剂突变体3的ALS基因的转基因水稻后代植株的ALS酶活性，发现含有扬州恢复系抗除草剂突变体1的ALS基因的转基因植株ALS酶活性比含有扬州恢复系抗除草剂突变体2的ALS基因的转基因植株的ALS酶活高32％，比含有扬州恢复系抗除草剂突变体3的ALS基因的转基因植株的ALS酶活高34％(图6)。Specific primers 5'-CGCGGATCCATCCGAGCCACACATCGCCTC-3' and 5'-TCCCCGCGGCCTACGGAAAACAACACAC-3' were designed, and BamHI and SacI restriction modification sites were added to the 5' respectively. Referring to the method of Example 3, amplified by PCR from the genomic DNA of the above-mentioned rice mutant Yangzhou restorer herbicide-resistant mutant 1, Yangzhou restorer herbicide-resistant mutant 2 and Yangzhou restorer herbicide-resistant mutant 3 Mutate the ALS gene, after the sequencing is correct, use BamHI and SacI to double-digest the mutant ALS gene fragment and the plant expression vector pCAMBIA1301 plasmid (purchased from pcambia company), respectively, and connect the digested product with T4-DNase (purchased from TaKaRa company). The product was transformed into Escherichia coli. The DNA extracted from the recombinant plasmid is verified by double digestion with BamHI and SacI, and large plasmid fragments and small gene fragments (Fig. 4) can be produced, which proves that the nucleotide sequence has been shown in SEQ ID NO.1 or 3 or 5 The ALS gene was cloned into the plant expression vector pCAMBIA1301 plasmid (purchased from pcambia company). The constructed plasmid vector was transformed into Agrobacterium EHA105, and the cells were cultured. Transform the japonica rice Nipponbare (purchased from the Agricultural Germplasm Resources Protection and Utilization Platform of Jiangsu Province) using the conventional Agrobacterium-mediated method, and obtain the transgenic plants. (Figure 5). The PCR detection primers were forward primer 35SF 5'-ATGGTTAGAGAGGCTTACGC-3', reverse primer 5R 5'-AGCAACAGGTCAGCCTTATCCAC-3', and the amplified fragment included the CaMV35S promoter and the 5' end sequence of the ALS gene, with a size of about 2 kb. Referring to Example 3 for the PCR amplification reaction system, the PCR amplification reaction procedure adopts a two-step method, combining annealing and extension at 68°C. The amplification program is as follows: pre-denaturation: 98°C for 3min; 30 cycles: denaturation at 98°C for 10sec; extension at 68°C for 2min; incubation: 72°C for 10min. After positive identification by PCR, spray 3mL Bailongtong/L water (9 times the recommended concentration), after 7 days, measure the ALS enzyme activity with reference to the method described in Example 6, and find that the ALS enzyme activity of transgenic rice is significantly higher than that of non-transgenic rice. Genetically modified rice: After 30 days, it was found that the genetically modified rice was growing well, while the non-transgenic Nipponbare rice all died. Comparing the ALS enzyme activity of transgenic rice progeny plants transformed with the ALS gene of Yangzhou restorer line mutant 1, Yangzhou restorer line mutant 2 and Yangzhou restorer mutant 3, it was found that the plants containing Yangzhou restorer line resistant The ALS enzyme activity of transgenic plants with the ALS gene of the herbicide mutant 1 was 32% higher than that of the transgenic plants containing the ALS gene of the Yangzhou restorer herbicide-resistant mutant 2, and was 32% higher than that of the transgenic plants containing the Yangzhou restorer herbicide-resistant mutant 3 The ALS enzyme activity of the transgenic plants with the ALS gene was 34% higher ( FIG. 6 ).
实施例7：转基因ALS烟草抗百垄通Example 7: Transgenic ALS Tobacco Resistant to Bailongtong
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从扬州恢复系抗除草剂突变体1、扬州恢复系抗除草剂突变体2和扬州恢复系抗除草剂突变体3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SEQID NO.1或3或5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3mL百垄通/L水(9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。比较转化扬州恢复系抗除草剂突变体1、抗除草剂突变体2和抗除草剂突变体3的ALS基因的转基因烟草后代植株的ALS酶活性，发现含有扬州恢复系抗除草剂突变体1的ALS基因的转基因植株ALS酶活性比含有扬州恢复系抗除草剂突变体2的ALS基因的转基因植株的高31％，比含有扬州恢复系抗除草剂突变体3的ALS基因的转基因植株的高27％(图7)。Specific primers 5'-CGCGGATCCATCCGAGCCACACATCGCCTC-3' and 5'-TCCCCGCGGCCTACGGAAAACAACACAC-3' were designed, and BamHI and SacI restriction modification sites were added to the 5' respectively. Mutant ALS genes were amplified by PCR from the genomic DNA of Yangzhou restorer herbicide-resistant mutant 1, Yangzhou restorer herbicide-resistant mutant 2, and Yangzhou restorer herbicide-resistant mutant 3. After the sequence was correct, refer to the examples Method 7 Cloning the ALS gene whose nucleotide sequence is shown in SEQ ID NO.1 or 3 or 5 into the plant expression vector pCAMBIA2301 plasmid (purchased from pcambia company). Select positive clones to transform Agrobacterium EHA105, and use the conventional Agrobacterium-mediated method to transform tobacco leaf discs to obtain transgenic plants. After harvesting, when the progeny plants grow to the 3-4 leaf stage and are positive by PCR, spray 3mL Bailongtong/L water (9 times the recommended use concentration), after 7 days, the ALS enzyme activity was measured with reference to the method described in Example 6, and it was found that the ALS enzyme activity of transgenic tobacco was significantly higher than that of non-transgenic tobacco; after 30 days, the growth state of transgenic tobacco was found Good, while the non-GM tobaccos were all dead. Comparing the ALS enzyme activity of transgenic tobacco progeny plants transformed with the ALS gene of the Yangzhou restorer line herbicide-resistant mutant 1, herbicide-resistant mutant 2 and herbicide-resistant mutant 3, it was found that the ALS gene containing the Yangzhou restorer line herbicide-resistant mutant 1 The ALS enzyme activity of the transgenic plants with ALS gene was 31% higher than that of the transgenic plants containing the ALS gene of the Yangzhou restorer herbicide-resistant mutant 2, and 27% higher than that of the transgenic plants containing the ALS gene of the Yangzhou restorer herbicide-resistant mutant 3 % (Figure 7).
实施例8：抗性材料的杂交和鉴定Example 8: Hybridization and Identification of Resistant Materials
为了检测是否具有2个突变位点的抗性植株比具有单个位点突变点的抗性植株的除草耐受能力更强，我们将实施例2中的单个位点突变进行杂交，选择仅含有Ser627单个位点突变的扬州恢复系抗除草剂突变体2和仅含有Gly628单个位点突变的的扬州恢复系抗除草剂突变体3在温室种植，开花期将扬州恢复系抗除草剂突变体2和扬州恢复系抗除草剂突变体3进行杂交。具体方法是将扬州恢复系突变体2人工去雄并套袋，扬州恢复系突变体3植株开花的时候，将颖壳剪掉，光下放置10分钟，将父本花粉抖到扬州恢复系突变体2的柱头上，重复2次，待种子成熟后收种，即为F1代杂交植株。收获的F1代杂交中繁殖一代后，收获F2代群体，在该群体中鉴定同时携带有Ser627和Gly628位点突变的个体。用除草剂对杂合植株以及抗除草剂突变体2和抗除草剂突变体3亲本同时进行喷施实验，结果表明，同时含有2个位点突变的植株相对于只含有单个位点突变的植株，对除草剂的抗性明显提高。我们又对杂合植株、抗除草剂突变体2和抗除草剂突变体3的ALS酶活进行测定，发现杂合植株的ALS酶活性比抗除草剂突变体2高31％，比抗除草剂突变体3高25％(图8)。In order to detect whether the resistant plants with two mutation sites have stronger herbicide tolerance than the resistant plants with a single mutation site, we crossed the single site mutation in Example 2, and selected only those containing Ser627 The Yangzhou restorer line herbicide-resistant mutant 2 with a single site mutation and the Yangzhou restorer line herbicide-resistant mutant 3 containing only a single site mutation of Gly628 were planted in the greenhouse, and the Yangzhou restorer line herbicide-resistant mutant 2 and Yangzhou restorer line herbicide-resistant mutant 3 was crossed. The specific method is to artificially detassell the Yangzhou restorer mutant 2 and bag it. When the plants of the Yangzhou restorer mutant 3 bloom, cut off the chaff, place it under the light for 10 minutes, and shake the pollen of the male parent to the Yangzhou restorer mutant. On the stigma of body 2, repeat twice, and harvest the seeds after the seeds are mature, which is the F1 generation hybrid plant. After breeding one generation in the harvested F1 generation cross, the F2 generation population is harvested, and individuals carrying both Ser627 and Gly628 site mutations are identified in this population. The heterozygous plants and the parents of herbicide-resistant mutant 2 and herbicide-resistant mutant 3 were sprayed with herbicide at the same time. The results showed that the plants containing two site mutations at the same time were more effective than the plants containing only a single site mutation. , significantly increased resistance to herbicides. We also measured the ALS enzyme activity of heterozygous plants, herbicide-resistant mutant 2 and herbicide-resistant mutant 3, and found that the ALS enzyme activity of heterozygous plants was 31% higher than that of herbicide-resistant mutant 2, and higher than that of herbicide-resistant mutant 2. Mutant 3 was 25% taller (Figure 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专利要求极其任何等同物给出。While specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, which are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
SEQUENCE LISTINGSEQUENCE LISTING
<110> 江苏省农业科学院<110> Jiangsu Academy of Agricultural Sciences
<120> 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<120> Rice ALS mutant protein, gene and application thereof for making plants resistant to herbicides
<130> SG20170620001<130> SG20170620001
<160> 10<160> 10
<170> PatentIn version 3.3<170> PatentIn version 3.3
<210> 1<210> 1
<211> 1935<211> 1935
<212> DNA<212>DNA
<213> 水稻种子扬州恢复系抗除草剂突变体1 ALS基因<213> Herbicide-resistant mutant 1 ALS gene of rice seed Yangzhou restorer line
<220><220>
<221> CDS<221> CDS
<222> (1)..(1935)<222> (1)..(1935)
<400> 1<400> 1
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Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala AlaMet Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
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acg gcc aag acc ggc cgt aag aac cac cag cga cac cac gtc ctt ccc 96acg 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 ProThr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
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gct cga ggc cgg gtg ggg gcg gcg gcg gtc agg tgc tcg gcg gtg tcc 144gct 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 SerAla Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
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ccg gtc acc ccg ccg tcc ccg gcg ccg ccg gcc acg ccg ctc cgg ccg 192ccg 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 ProPro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
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tgg ggg acg gcc gag ccc cgc aag ggc gcg gac atc ctc gtg gag gcg 240tgg 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 AlaTrp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
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ctg gag cgg tgc ggc gtc agc gac gtg ttc gcc tac ccg ggc ggc gcg 288ctg 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 AlaLeu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
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Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr AsnSer Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
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His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly TyrHis Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
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gcg cgc gcg tcc ggc cgc gtc ggg gtc tgc gtc gcc acc tcc ggc ccc 432gcg 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 ProAla Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
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Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp SerGly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
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Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile GlyVal Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
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Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser IleThr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
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acc aag cac aat tac ctt gtc ctt gat gtg gag gac atc ccc cgc gtc 624acc 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 ValThr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
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Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro ValIle Gln Glu Ala Phe Phe Leu Ala Ser Ser Ser Gly Arg Pro Gly Pro Val
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ctg gtc gac atc ccc aag gac atc cag cag cag atg gct gtg cca gtc 720ctg 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 ValLeu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
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tgg gac acc tcg atg aat cta ccg ggg tac att gca cgc ctg ccc aag 768tgg 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 LysTrp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
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cca ccc gcg aca gaa ttg ctt gag cag gtc ttg cgt ctg gtt ggc gag 816cca 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 GluPro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
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Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser GlySer Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
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Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr ThrAsp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
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Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser LeuThr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
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cgc atg ctt ggg atg cat ggc acg gtg tac gca aat tat gcg gtg gat 1008cgc 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 AspArg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
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Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg ValLys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
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aca ggg aaa att gag gct ttt gca agc agg gcc aag att gtg cac att 1104aca 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 IleThr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
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gac att gat cca gcg gag att gga aag aac aag caa cca cat gtg tca 1152gac 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 SerAsp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
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att tgc gca gat gtt aag ctt gct tta cag ggc ttg aat gct ctg cta 1200att 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 LeuIle Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
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gac cag agc aca aca aag aca agt tct gat ttt agt gca tgg cac aat 1248gac 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 AsnAsp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415 405 410 415
gag ttg gac cag cag aag agg gag ttt cct ctg ggg tac aag act ttt 1296gag 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 PheGlu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430 420 425 430
ggt gaa gag atc cca ccg caa tat gct att cag gtg ctg gat gag ctg 1344ggt 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 LeuGly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
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acg aaa ggg gag gca atc atc gct act ggt gtt gga cag cac cag atg 1392acg 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 MetThr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460 450 455 460
tgg gcg gca caa tat tac acc tac aag cgg cca cgg cag tgg ctg tct 1440tgg 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 SerTrp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480465 470 475 480
tcg gct ggt ctg ggc gca atg gga ttt ggg ctg cct gct gca gct ggt 1488tcg 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 GlySer Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495 485 490 495
gct tct gtg gct aac cca ggt gtc aca gtt gtt gat att gat ggg gat 1536gct 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 AspAla Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510 500 505 510
ggt agc ttc ctc atg aac att cag gag ttg gca ttg atc cgc att gag 1584ggt 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 GluGly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525 515 520 525
aac ctc ccg gtg aag gtg atg gtg ttg aac aac caa cat ttg ggt atg 1632aac 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 MetAsn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540 530 535 540
gtt gtg caa tgg gag gat agg ttt tac aag gca aat agg gcg cat aca 1680gtt 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 ThrVal Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560545 550 555 560
tac ttg ggc aac cca gaa tgt gag agc gag ata tat cca gat ttt gtg 1728tac 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 ValTyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575 565 570 575
act att gct aaa ggg ttc aat att cct gca gtc cgt gta aca aag aag 1776act 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 LysThr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590 580 585 590
agt gaa gtc cgt gcc gcc atc aag aag atg ctc gag acc cca ggg cca 1824agt 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 ProSer Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605 595 600 605
tac ttg ttg gat atc atc gtc cca cac cag gag cat gtg ctg cct atg 1872tac 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 MetTyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620 610 615 620
atc cca aat gag ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920atc cca aat gag ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920
Ile Pro Asn Glu Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp GlyIle Pro Asn Glu Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640625 630 635 640
agg act atg tat taa 1935agg act atg tat taa 1935
Arg Thr Met TyrArg Thr Met Tyr
<210> 2<210> 2
<211> 644<211> 644
<212> PRT<212> PRT
<213> 水稻种子扬州恢复系抗除草剂突变体1 ALS基因<213> Herbicide-resistant mutant 1 ALS gene of rice seed Yangzhou restorer line
<400> 2<400> 2
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala AlaMet Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 151 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu ProThr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30 20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val SerAla Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45 35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg ProPro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60 50 55 60
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu AlaTrp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 8065 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly AlaLeu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95 85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr AsnSer Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110 100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly TyrHis Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125 115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly ProAla Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140 130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp SerGly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile GlyVal Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175 165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser IleThr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190 180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg ValThr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205 195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro ValIle Gln Glu Ala Phe Phe Leu Ala Ser Ser Ser Gly Arg Pro Gly Pro Val
210 215 220 210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro ValLeu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro LysTrp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255 245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly GluPro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270 260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser GlySer Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285 275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr ThrAsp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300 290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser LeuThr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val AspArg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335 325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg ValLys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350 340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His IleThr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365 355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val SerAsp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380 370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu LeuIle Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His AsnAsp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415 405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr PheGlu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430 420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu LeuGly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445 435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln MetThr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460 450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu SerTrp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala GlySer Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495 485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly AspAla Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510 500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile GluGly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525 515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly MetAsn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540 530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His ThrVal Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe ValTyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575 565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys LysThr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590 580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly ProSer Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605 595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro MetTyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620 610 615 620
Ile Pro Asn Glu Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp GlyIle Pro Asn Glu Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640625 630 635 640
Arg Thr Met TyrArg Thr Met Tyr
<210> 3<210> 3
<211> 1935<211> 1935
<212> DNA<212>DNA
<213> 水稻种子扬州恢复系抗除草剂突变体2 ALS基因<213> Herbicide-resistant mutant 2 ALS gene of rice seed Yangzhou restorer line
<220><220>
<221> CDS<221> CDS
<222> (1)..(1935)<222> (1)..(1935)
<400> 3<400> 3
atg gct acg acc gcc gcg gcc gcg gcc gcc acc ttg tcc gcc gcc gcg 48atg 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 AlaMet Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 151 5 10 15
acg gcc aag acc ggc cgt aag aac cac cag cga cac cac gtc ctt ccc 96acg 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 ProThr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30 20 25 30
gct cga ggc cgg gtg ggg gcg gcg gcg gtc agg tgc tcg gcg gtg tcc 144gct 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 SerAla Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45 35 40 45
ccg gtc acc ccg ccg tcc ccg gcg ccg ccg gcc acg ccg ctc cgg ccg 192ccg 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 ProPro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60 50 55 60
tgg ggg acg gcc gag ccc cgc aag ggc gcg gac atc ctc gtg gag gcg 240tgg 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 AlaTrp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 8065 70 75 80
ctg gag cgg tgc ggc gtc agc gac gtg ttc gcc tac ccg ggc ggc gcg 288ctg 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 AlaLeu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95 85 90 95
tcc atg gag atc cac cag gcg ctg acg cgc tcc ccg gtc atc acc aac 336tcc 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 AsnSer Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110 100 105 110
cac ctc ttc cgc cac gag cag ggc gag gcg ttc gcg gcg tcc ggg tac 384cac 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 TyrHis Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125 115 120 125
gcg cgc gcg tcc ggc cgc gtc ggg gtc tgc gtc gcc acc tcc ggc ccc 432gcg 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 ProAla Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140 130 135 140
ggg gca acc aac ctc gtg tcc gcg ctc gcc gac gcg ctg ctc gac tcc 480ggg 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 SerGly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160145 150 155 160
gtc ccg atg gtc gcc atc acg ggc cag gtc ccc cgc cgc atg atc ggc 528gtc 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 GlyVal Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175 165 170 175
acc gac gcc ttc cag gag acg ccc ata gtc gag gtc acc cgc tcc atc 576acc 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 IleThr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190 180 185 190
acc aag cac aat tac ctt gtc ctt gat gtg gag gac atc ccc cgc gtc 624acc 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 ValThr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205 195 200 205
ata cag gaa gcc ttc ttc ctc gcg tcc tcg ggc cgt cct ggc ccg gtg 672ata 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 ValIle Gln Glu Ala Phe Phe Leu Ala Ser Ser Ser Gly Arg Pro Gly Pro Val
210 215 220 210 215 220
ctg gtc gac atc ccc aag gac atc cag cag cag atg gct gtg cca gtc 720ctg 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 ValLeu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240225 230 235 240
tgg gac acc tcg atg aat cta ccg ggg tac att gca cgc ctg ccc aag 768tgg 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 LysTrp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255 245 250 255
cca ccc gcg aca gaa ttg ctt gag cag gtc ttg cgt ctg gtt ggc gag 816cca 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 GluPro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270 260 265 270
tca cgg cgc ccg att ctc tat gtc ggt ggt ggc tgc tct gca tct ggt 864tca 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 GlySer Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285 275 280 285
gat gaa ttg cgc cgg ttt gtt gag ctg acc ggc atc cca gtt aca acc 912gat 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 ThrAsp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300 290 295 300
act ctg atg ggc ctc ggc aat ttc ccc agt gat gat ccg ttg tcc ctg 960act 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 LeuThr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320305 310 315 320
cgc atg ctt ggg atg cat ggc acg gtg tac gca aat tat gcg gtg gat 1008cgc 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 AspArg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335 325 330 335
aag gct gac ctg ttg ctt gca ttt ggc gtg cgg ttt gat gat cgt gtg 1056aag 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 ValLys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350 340 345 350
aca ggg aaa att gag gct ttt gca agc agg gcc aag att gtg cac att 1104aca 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 IleThr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365 355 360 365
gac att gat cca gcg gag att gga aag aac aag caa cca cat gtg tca 1152gac 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 SerAsp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380 370 375 380
att tgc gca gat gtt aag ctt gct tta cag ggc ttg aat gct ctg cta 1200att 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 LeuIle Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400385 390 395 400
gac cag agc aca aca aag aca agt tct gat ttt agt gca tgg cac aat 1248gac 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 AsnAsp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415 405 410 415
gag ttg gac cag cag aag agg gag ttt cct ctg ggg tac aag act ttt 1296gag 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 PheGlu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430 420 425 430
ggt gaa gag atc cca ccg caa tat gct att cag gtg ctg gat gag ctg 1344ggt 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 LeuGly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445 435 440 445
acg aaa ggg gag gca atc atc gct act ggt gtt gga cag cac cag atg 1392acg 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 MetThr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460 450 455 460
tgg gcg gca caa tat tac acc tac aag cgg cca cgg cag tgg ctg tct 1440tgg 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 SerTrp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480465 470 475 480
tcg gct ggt ctg ggc gca atg gga ttt ggg ctg cct gct gca gct ggt 1488tcg 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 GlySer Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495 485 490 495
gct tct gtg gct aac cca ggt gtc aca gtt gtt gat att gat ggg gat 1536gct 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 AspAla Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510 500 505 510
ggt agc ttc ctc atg aac att cag gag ttg gca ttg atc cgc att gag 1584ggt 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 GluGly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525 515 520 525
aac ctc ccg gtg aag gtg atg gtg ttg aac aac caa cat ttg ggt atg 1632aac 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 MetAsn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540 530 535 540
gtt gtg caa tgg gag gat agg ttt tac aag gca aat agg gcg cat aca 1680gtt 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 ThrVal Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560545 550 555 560
tac ttg ggc aac cca gaa tgt gag agc gag ata tat cca gat ttt gtg 1728tac 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 ValTyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575 565 570 575
act att gct aaa ggg ttc aat att cct gca gtc cgt gta aca aag aag 1776act 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 LysThr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590 580 585 590
agt gaa gtc cgt gcc gcc atc aag aag atg ctc gag acc cca ggg cca 1824agt 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 ProSer Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605 595 600 605
tac ttg ttg gat atc atc gtc cca cac cag gag cat gtg ctg cct atg 1872tac 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 MetTyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620 610 615 620
atc cca aat ggg ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920atc cca aat ggg ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920
Ile Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp GlyIle Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640625 630 635 640
agg act atg tat taa 1935agg act atg tat taa 1935
Arg Thr Met TyrArg Thr Met Tyr
<210> 4<210> 4
<211> 644<211> 644
<212> PRT<212> PRT
<213> 水稻种子扬州恢复系抗除草剂突变体2 ALS基因<213> Herbicide-resistant mutant 2 ALS gene of rice seed Yangzhou restorer line
<400> 4<400> 4
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala AlaMet Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 151 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu ProThr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30 20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val SerAla Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45 35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg ProPro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60 50 55 60
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu AlaTrp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 8065 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly AlaLeu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95 85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr AsnSer Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110 100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly TyrHis Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125 115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly ProAla Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140 130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp SerGly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile GlyVal Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175 165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser IleThr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190 180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg ValThr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205 195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro ValIle Gln Glu Ala Phe Phe Leu Ala Ser Ser Ser Gly Arg Pro Gly Pro Val
210 215 220 210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro ValLeu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro LysTrp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255 245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly GluPro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270 260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser GlySer Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285 275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr ThrAsp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300 290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser LeuThr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val AspArg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335 325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg ValLys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350 340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His IleThr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365 355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val SerAsp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380 370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu LeuIle Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His AsnAsp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415 405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr PheGlu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430 420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu LeuGly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445 435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln MetThr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460 450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu SerTrp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala GlySer Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495 485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly AspAla Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510 500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile GluGly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525 515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly MetAsn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540 530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His ThrVal Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe ValTyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575 565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys LysThr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590 580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly ProSer Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605 595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro MetTyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620 610 615 620
Ile Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp GlyIle Pro Asn Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640625 630 635 640
Arg Thr Met TyrArg Thr Met Tyr
<210> 5<210> 5
<211> 1935<211> 1935
<212> DNA<212>DNA
<213> 水稻种子扬州恢复系抗除草剂突变体3 ALS基因<213> Herbicide-resistant mutant 3 ALS gene of rice seed Yangzhou restorer line
<220><220>
<221> CDS<221> CDS
<222> (1)..(1935)<222> (1)..(1935)
<400> 5<400> 5
atg gct acg acc gcc gcg gcc gcg gcc gcc acc ttg tcc gcc gcc gcg 48atg 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 AlaMet Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 151 5 10 15
acg gcc aag acc ggc cgt aag aac cac cag cga cac cac gtc ctt ccc 96acg 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 ProThr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30 20 25 30
gct cga ggc cgg gtg ggg gcg gcg gcg gtc agg tgc tcg gcg gtg tcc 144gct 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 SerAla Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45 35 40 45
ccg gtc acc ccg ccg tcc ccg gcg ccg ccg gcc acg ccg ctc cgg ccg 192ccg 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 ProPro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60 50 55 60
tgg ggg acg gcc gag ccc cgc aag ggc gcg gac atc ctc gtg gag gcg 240tgg 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 AlaTrp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 8065 70 75 80
ctg gag cgg tgc ggc gtc agc gac gtg ttc gcc tac ccg ggc ggc gcg 288ctg 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 AlaLeu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95 85 90 95
tcc atg gag atc cac cag gcg ctg acg cgc tcc ccg gtc atc acc aac 336tcc 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 AsnSer Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110 100 105 110
cac ctc ttc cgc cac gag cag ggc gag gcg ttc gcg gcg tcc ggg tac 384cac 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 TyrHis Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125 115 120 125
gcg cgc gcg tcc ggc cgc gtc ggg gtc tgc gtc gcc acc tcc ggc ccc 432gcg 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 ProAla Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140 130 135 140
ggg gca acc aac ctc gtg tcc gcg ctc gcc gac gcg ctg ctc gac tcc 480ggg 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 SerGly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160145 150 155 160
gtc ccg atg gtc gcc atc acg ggc cag gtc ccc cgc cgc atg atc ggc 528gtc 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 GlyVal Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175 165 170 175
acc gac gcc ttc cag gag acg ccc ata gtc gag gtc acc cgc tcc atc 576acc 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 IleThr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190 180 185 190
acc aag cac aat tac ctt gtc ctt gat gtg gag gac atc ccc cgc gtc 624acc 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 ValThr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205 195 200 205
ata cag gaa gcc ttc ttc ctc gcg tcc tcg ggc cgt cct ggc ccg gtg 672ata 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 ValIle Gln Glu Ala Phe Phe Leu Ala Ser Ser Ser Gly Arg Pro Gly Pro Val
210 215 220 210 215 220
ctg gtc gac atc ccc aag gac atc cag cag cag atg gct gtg cca gtc 720ctg 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 ValLeu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240225 230 235 240
tgg gac acc tcg atg aat cta ccg ggg tac att gca cgc ctg ccc aag 768tgg 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 LysTrp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255 245 250 255
cca ccc gcg aca gaa ttg ctt gag cag gtc ttg cgt ctg gtt ggc gag 816cca 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 GluPro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270 260 265 270
tca cgg cgc ccg att ctc tat gtc ggt ggt ggc tgc tct gca tct ggt 864tca 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 GlySer Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285 275 280 285
gat gaa ttg cgc cgg ttt gtt gag ctg acc ggc atc cca gtt aca acc 912gat 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 ThrAsp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300 290 295 300
act ctg atg ggc ctc ggc aat ttc ccc agt gat gat ccg ttg tcc ctg 960act 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 LeuThr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320305 310 315 320
cgc atg ctt ggg atg cat ggc acg gtg tac gca aat tat gcg gtg gat 1008cgc 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 AspArg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335 325 330 335
aag gct gac ctg ttg ctt gca ttt ggc gtg cgg ttt gat gat cgt gtg 1056aag 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 ValLys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350 340 345 350
aca ggg aaa att gag gct ttt gca agc agg gcc aag att gtg cac att 1104aca 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 IleThr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365 355 360 365
gac att gat cca gcg gag att gga aag aac aag caa cca cat gtg tca 1152gac 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 SerAsp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380 370 375 380
att tgc gca gat gtt aag ctt gct tta cag ggc ttg aat gct ctg cta 1200att 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 LeuIle Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400385 390 395 400
gac cag agc aca aca aag aca agt tct gat ttt agt gca tgg cac aat 1248gac 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 AsnAsp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415 405 410 415
gag ttg gac cag cag aag agg gag ttt cct ctg ggg tac aag act ttt 1296gag 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 PheGlu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430 420 425 430
ggt gaa gag atc cca ccg caa tat gct att cag gtg ctg gat gag ctg 1344ggt 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 LeuGly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445 435 440 445
acg aaa ggg gag gca atc atc gct act ggt gtt gga cag cac cag atg 1392acg 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 MetThr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460 450 455 460
tgg gcg gca caa tat tac acc tac aag cgg cca cgg cag tgg ctg tct 1440tgg 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 SerTrp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480465 470 475 480
tcg gct ggt ctg ggc gca atg gga ttt ggg ctg cct gct gca gct ggt 1488tcg 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 GlySer Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495 485 490 495
gct tct gtg gct aac cca ggt gtc aca gtt gtt gat att gat ggg gat 1536gct 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 AspAla Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510 500 505 510
ggt agc ttc ctc atg aac att cag gag ttg gca ttg atc cgc att gag 1584ggt 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 GluGly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525 515 520 525
aac ctc ccg gtg aag gtg atg gtg ttg aac aac caa cat ttg ggt atg 1632aac 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 MetAsn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540 530 535 540
gtt gtg caa tgg gag gat agg ttt tac aag gca aat agg gcg cat aca 1680gtt 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 ThrVal Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560545 550 555 560
tac ttg ggc aac cca gaa tgt gag agc gag ata tat cca gat ttt gtg 1728tac 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 ValTyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575 565 570 575
act att gct aaa ggg ttc aat att cct gca gtc cgt gta aca aag aag 1776act 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 LysThr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590 580 585 590
agt gaa gtc cgt gcc gcc atc aag aag atg ctc gag acc cca ggg cca 1824agt 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 ProSer Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605 595 600 605
tac ttg ttg gat atc atc gtc cca cac cag gag cat gtg ctg cct atg 1872tac 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 MetTyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620 610 615 620
atc cca agt gag ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920atc cca agt gag ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920
Ile Pro Ser Glu Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp GlyIle Pro Ser Glu Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640625 630 635 640
agg act atg tat taa 1935agg act atg tat taa 1935
Arg Thr Met TyrArg Thr Met Tyr
<210> 6<210> 6
<211> 644<211> 644
<212> PRT<212> PRT
<213> 水稻种子扬州恢复系抗除草剂突变体3 ALS基因<213> Herbicide-resistant mutant 3 ALS gene of rice seed Yangzhou restorer line
<400> 6<400> 6
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala AlaMet Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 151 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu ProThr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30 20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val SerAla Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45 35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg ProPro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60 50 55 60
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu AlaTrp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 8065 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly AlaLeu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95 85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr AsnSer Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110 100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly TyrHis Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125 115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly ProAla Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140 130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp SerGly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile GlyVal Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175 165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser IleThr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190 180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg ValThr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205 195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro ValIle Gln Glu Ala Phe Phe Leu Ala Ser Ser Ser Gly Arg Pro Gly Pro Val
210 215 220 210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro ValLeu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro LysTrp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255 245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly GluPro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270 260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser GlySer Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285 275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr ThrAsp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300 290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser LeuThr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val AspArg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335 325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg ValLys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350 340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His IleThr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365 355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val SerAsp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380 370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu LeuIle Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His AsnAsp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415 405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr PheGlu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430 420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu LeuGly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445 435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln MetThr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460 450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu SerTrp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala GlySer Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495 485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly AspAla Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510 500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile GluGly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525 515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly MetAsn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540 530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His ThrVal Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe ValTyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575 565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys LysThr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590 580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly ProSer Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605 595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro MetTyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620 610 615 620
Ile Pro Ser Glu Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp GlyIle Pro Ser Glu Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640625 630 635 640
Arg Thr Met TyrArg Thr Met Tyr
<210> 7<210> 7
<211> 1935<211> 1935
<212> DNA<212>DNA
<213> 水稻种子扬州恢复系野生型ALS基因<213> Rice seed Yangzhou restorer line wild-type ALS gene
<220><220>
<221> CDS<221> CDS
<222> (1)..(1935)<222> (1)..(1935)
<400> 7<400> 7
atg gct acg acc gcc gcg gcc gcg gcc gcc acc ttg tcc gcc gcc gcg 48atg 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 AlaMet Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 151 5 10 15
acg gcc aag acc ggc cgt aag aac cac cag cga cac cac gtc ctt ccc 96acg 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 ProThr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30 20 25 30
gct cga ggc cgg gtg ggg gcg gcg gcg gtc agg tgc tcg gcg gtg tcc 144gct 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 SerAla Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45 35 40 45
ccg gtc acc ccg ccg tcc ccg gcg ccg ccg gcc acg ccg ctc cgg ccg 192ccg 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 ProPro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60 50 55 60
tgg ggg acg gcc gag ccc cgc aag ggc gcg gac atc ctc gtg gag gcg 240tgg 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 AlaTrp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 8065 70 75 80
ctg gag cgg tgc ggc gtc agc gac gtg ttc gcc tac ccg ggc ggc gcg 288ctg 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 AlaLeu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95 85 90 95
tcc atg gag atc cac cag gcg ctg acg cgc tcc ccg gtc atc acc aac 336tcc 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 AsnSer Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110 100 105 110
cac ctc ttc cgc cac gag cag ggc gag gcg ttc gcg gcg tcc ggg tac 384cac 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 TyrHis Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125 115 120 125
gcg cgc gcg tcc ggc cgc gtc ggg gtc tgc gtc gcc acc tcc ggc ccc 432gcg 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 ProAla Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140 130 135 140
ggg gca acc aac ctc gtg tcc gcg ctc gcc gac gcg ctg ctc gac tcc 480ggg 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 SerGly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160145 150 155 160
gtc ccg atg gtc gcc atc acg ggc cag gtc ccc cgc cgc atg atc ggc 528gtc 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 GlyVal Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175 165 170 175
acc gac gcc ttc cag gag acg ccc ata gtc gag gtc acc cgc tcc atc 576acc 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 IleThr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190 180 185 190
acc aag cac aat tac ctt gtc ctt gat gtg gag gac atc ccc cgc gtc 624acc 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 ValThr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205 195 200 205
ata cag gaa gcc ttc ttc ctc gcg tcc tcg ggc cgt cct ggc ccg gtg 672ata 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 ValIle Gln Glu Ala Phe Phe Leu Ala Ser Ser Ser Gly Arg Pro Gly Pro Val
210 215 220 210 215 220
ctg gtc gac atc ccc aag gac atc cag cag cag atg gct gtg cca gtc 720ctg 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 ValLeu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240225 230 235 240
tgg gac acc tcg atg aat cta ccg ggg tac att gca cgc ctg ccc aag 768tgg 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 LysTrp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255 245 250 255
cca ccc gcg aca gaa ttg ctt gag cag gtc ttg cgt ctg gtt ggc gag 816cca 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 GluPro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270 260 265 270
tca cgg cgc ccg att ctc tat gtc ggt ggt ggc tgc tct gca tct ggt 864tca 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 GlySer Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285 275 280 285
gat gaa ttg cgc cgg ttt gtt gag ctg acc ggc atc cca gtt aca acc 912gat 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 ThrAsp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300 290 295 300
act ctg atg ggc ctc ggc aat ttc ccc agt gat gat ccg ttg tcc ctg 960act 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 LeuThr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320305 310 315 320
cgc atg ctt ggg atg cat ggc acg gtg tac gca aat tat gcg gtg gat 1008cgc 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 AspArg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335 325 330 335
aag gct gac ctg ttg ctt gca ttt ggc gtg cgg ttt gat gat cgt gtg 1056aag 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 ValLys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350 340 345 350
aca ggg aaa att gag gct ttt gca agc agg gcc aag att gtg cac att 1104aca 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 IleThr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365 355 360 365
gac att gat cca gcg gag att gga aag aac aag caa cca cat gtg tca 1152gac 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 SerAsp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380 370 375 380
att tgc gca gat gtt aag ctt gct tta cag ggc ttg aat gct ctg cta 1200att 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 LeuIle Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400385 390 395 400
gac cag agc aca aca aag aca agt tct gat ttt agt gca tgg cac aat 1248gac 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 AsnAsp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415 405 410 415
gag ttg gac cag cag aag agg gag ttt cct ctg ggg tac aag act ttt 1296gag 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 PheGlu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430 420 425 430
ggt gaa gag atc cca ccg caa tat gct att cag gtg ctg gat gag ctg 1344ggt 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 LeuGly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445 435 440 445
acg aaa ggg gag gca atc atc gct act ggt gtt gga cag cac cag atg 1392acg 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 MetThr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460 450 455 460
tgg gcg gca caa tat tac acc tac aag cgg cca cgg cag tgg ctg tct 1440tgg 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 SerTrp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480465 470 475 480
tcg gct ggt ctg ggc gca atg gga ttt ggg ctg cct gct gca gct ggt 1488tcg 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 GlySer Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495 485 490 495
gct tct gtg gct aac cca ggt gtc aca gtt gtt gat att gat ggg gat 1536gct 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 AspAla Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510 500 505 510
ggt agc ttc ctc atg aac att cag gag ttg gca ttg atc cgc att gag 1584ggt 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 GluGly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525 515 520 525
aac ctc ccg gtg aag gtg atg gtg ttg aac aac caa cat ttg ggt atg 1632aac 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 MetAsn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540 530 535 540
gtt gtg caa tgg gag gat agg ttt tac aag gca aat agg gcg cat aca 1680gtt 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 ThrVal Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560545 550 555 560
tac ttg ggc aac cca gaa tgt gag agc gag ata tat cca gat ttt gtg 1728tac 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 ValTyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575 565 570 575
act att gct aaa ggg ttc aat att cct gca gtc cgt gta aca aag aag 1776act 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 LysThr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590 580 585 590
agt gaa gtc cgt gcc gcc atc aag aag atg ctc gag acc cca ggg cca 1824agt 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 ProSer Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605 595 600 605
tac ttg ttg gat atc atc gtc cca cac cag gag cat gtg ctg cct atg 1872tac 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 MetTyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620 610 615 620
atc cca agt ggg ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920atc 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 GlyIle Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640625 630 635 640
agg act atg tat taa 1935agg act atg tat taa 1935
Arg Thr Met TyrArg Thr Met Tyr
<210> 8<210> 8
<211> 644<211> 644
<212> PRT<212> PRT
<213> 水稻种子扬州恢复系野生型ALS基因<213> Rice seed Yangzhou restorer line wild-type ALS gene
<400> 8<400> 8
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala AlaMet Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 151 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu ProThr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30 20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val SerAla Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45 35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg ProPro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60 50 55 60
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu AlaTrp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 8065 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly AlaLeu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95 85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr AsnSer Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110 100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly TyrHis Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125 115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly ProAla Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140 130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp SerGly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile GlyVal Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175 165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser IleThr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190 180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg ValThr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205 195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro ValIle Gln Glu Ala Phe Phe Leu Ala Ser Ser Ser Gly Arg Pro Gly Pro Val
210 215 220 210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro ValLeu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro LysTrp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255 245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly GluPro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270 260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser GlySer Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285 275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr ThrAsp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300 290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser LeuThr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val AspArg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335 325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg ValLys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350 340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His IleThr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365 355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val SerAsp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380 370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu LeuIle Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His AsnAsp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415 405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr PheGlu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430 420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu LeuGly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445 435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln MetThr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460 450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu SerTrp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala GlySer Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495 485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly AspAla Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510 500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile GluGly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525 515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly MetAsn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540 530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His ThrVal Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe ValTyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575 565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys LysThr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590 580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly ProSer Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605 595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro MetTyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620 610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp GlyIle Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640625 630 635 640
Arg Thr Met TyrArg Thr Met Tyr
<210> 9<210> 9
<211> 30<211> 30
<212> DNA<212>DNA
<213> 上游引物<213> upstream primer
<400> 9<400> 9
cgcggatcca tccgagccac acatcgcctc 30cgcggatcca tccgagccac acatcgcctc 30
<210> 10<210> 10
<211> 28<211> 28
<212> DNA<212>DNA
<213> 下游引物<213> downstream primer
<400> 10<400> 10
tccccgcggc ctacggaaaa caacacac 28tccccgcggc ctacggaaaa caacacac 28
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| CN201710486852.XA CN107090447A (en) | 2017-06-23 | 2017-06-23 | Make plant that there is paddy rice ALS muteins, gene and its application of Herbicid resistant |
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| CN107964543A (en) * | 2017-12-25 | 2018-04-27 | 江苏省农业科学院 | Rice herbicide resistance ALS muteins, nucleic acid and its application |
| CN109097346A (en) * | 2018-09-06 | 2018-12-28 | 江苏省农业科学院 | Application based on the ALS mutein and its gene of gene editing technology in plant breeding |
| CN111411098A (en) * | 2020-05-07 | 2020-07-14 | 海南波莲水稻基因科技有限公司 | Rice A L S mutant gene, plant transgenic screening vector pCA L Sm2 containing gene and application thereof |
| WO2021155753A1 (en) * | 2020-02-07 | 2021-08-12 | 山东舜丰生物科技有限公司 | Herbicide-resistant gene, polypeptide, and application thereof in plant breeding |
| CN114317481A (en) * | 2022-02-08 | 2022-04-12 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | ALS mutant gene of rice and application of protein thereof in herbicide resistance |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107964543A (en) * | 2017-12-25 | 2018-04-27 | 江苏省农业科学院 | Rice herbicide resistance ALS muteins, nucleic acid and its application |
| CN109097346A (en) * | 2018-09-06 | 2018-12-28 | 江苏省农业科学院 | Application based on the ALS mutein and its gene of gene editing technology in plant breeding |
| WO2020048135A1 (en) * | 2018-09-06 | 2020-03-12 | 江苏省农业科学院 | Use of als mutant-type protein and gene thereof based on gene editing technology in plant breeding |
| CN109097346B (en) * | 2018-09-06 | 2021-07-13 | 江苏省农业科学院 | Application of ALS mutant protein and its gene in plant breeding based on gene editing technology |
| WO2021155753A1 (en) * | 2020-02-07 | 2021-08-12 | 山东舜丰生物科技有限公司 | Herbicide-resistant gene, polypeptide, and application thereof in plant breeding |
| CN111411098A (en) * | 2020-05-07 | 2020-07-14 | 海南波莲水稻基因科技有限公司 | Rice A L S mutant gene, plant transgenic screening vector pCA L Sm2 containing gene and application thereof |
| CN111411098B (en) * | 2020-05-07 | 2022-03-04 | 海南波莲水稻基因科技有限公司 | Rice ALS mutant gene, plant transgenic screening vector pCALSm2 containing gene and application thereof |
| CN114317481A (en) * | 2022-02-08 | 2022-04-12 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | ALS mutant gene of rice and application of protein thereof in herbicide resistance |
| CN114317481B (en) * | 2022-02-08 | 2023-10-27 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Application of ALS mutant gene and protein thereof in herbicide resistance |
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