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CN106754810A - Rape protein, its encoding gene and its application with antiweed activity - Google Patents

Rape protein, its encoding gene and its application with antiweed activity Download PDF

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CN106754810A
CN106754810A CN201710069534.3A CN201710069534A CN106754810A CN 106754810 A CN106754810 A CN 106754810A CN 201710069534 A CN201710069534 A CN 201710069534A CN 106754810 A CN106754810 A CN 106754810A
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方军
朴钟泽
万常照
白建江
杨瑞芳
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Abstract

Rape protein, its encoding gene and application with antiweed activity, it is the mutein of rape Acetohydroxyacid synthase, its amino acid sequence is as shown in SEQ ID No.1 or SEQ ID No.3, the expression of protein source Acetohydroxyacid synthase gene in rapeseed gene group, through artificial reconstructed, rape Acetohydroxyacid synthase is obtained by external mutation, is the characteristics of its amino acid sequence:Compared with wild type rape Acetohydroxyacid synthase, missing Trp559 site amino acids, the polymorphic type herbicides such as mutant protein confrontation imidazolone type, sulfonylurea and the miazines are resistant, and the plant of its encoding gene conversion has the activity of the polymorphic type herbicide such as anti-imidazolone type, sulfonylurea and miazines.

Description

具有抗除草剂活性的油菜蛋白质、其编码基因及其应用Rapeseed protein with herbicide resistance activity, its coding gene and its application

技术领域technical field

本发明属于抗除草剂蛋白质领域,具体涉及一种具有抗除草剂活性的油菜蛋白质、其编码基因及其应用。The invention belongs to the field of herbicide-resistant proteins, and in particular relates to a rapeseed protein with herbicide-resistant activity, its coding gene and its application.

背景技术Background technique

油菜是我国四大主要油料作物之一(其他为大豆、花生、向日葵),是我国播种面积最大,分布最广的油料作物。Rapeseed is one of the four major oil crops in my country (the others are soybean, peanut, and sunflower), and it is the oil crop with the largest sowing area and the widest distribution in my country.

由于机械化直播油菜的推广,杂草对油菜产量的影响越来越大。杂草的发生对油菜产量一般减产20%~30%,严重时达50%以上,最严重的田块颗粒无收。油菜田中杂草种类复杂,一类是看麦娘、棒头草等禾本科杂草,另一类是繁缕、猪殃殃等阔叶杂草。同时防除这两类杂草涉及到除草剂混用问题。但是,混用除草剂需要的技术性非常强,由于除草剂具有高度选择性和强烈杀伤力,稍有不慎容易造成药害甚至毁坏作物。而种植抗除草剂油菜则可以避免这个问题。Due to the promotion of mechanized direct-seeding rapeseed, the impact of weeds on rapeseed yield is increasing. The occurrence of weeds generally reduces the yield of rapeseed by 20% to 30%, and when it is serious, it reaches more than 50%. The most serious field has no harvest. There are complex types of weeds in rapeseed fields, one type is grass weeds such as A. chinensis and club head grass, and the other type is broad-leaved weeds such as chickweed and pig's weed. Simultaneous control of these two types of weeds involves the use of mixed herbicides. However, mixing herbicides requires a very high level of technicality. Due to the high selectivity and strong lethality of herbicides, a little carelessness can easily cause phytotoxicity or even destroy crops. Planting herbicide-resistant canola avoids this problem.

油菜内源的抗除草剂乙酰羟基酸合成酶(acetohydroxy acid synthase,简称AHAS)蛋白质由ahas基因突变产生,表达抗除草剂AHAS蛋白质可以使植物获得抗除草剂特性,解决田块中的杂草问题。AHAS蛋白质主要分布在微生物和植物中,在动物中几乎不存在,AHAS抑制除草剂对人畜无害。因此,发掘和研究具有抗除草剂活性的油菜AHAS蛋白质具有良好的创新性和经济价值。The endogenous herbicide-resistant acetohydroxy acid synthase (AHAS) protein in rapeseed is produced by the mutation of the ahas gene, and the expression of the herbicide-resistant AHAS protein can make the plant acquire herbicide resistance and solve the weed problem in the field . AHAS proteins are mainly distributed in microorganisms and plants, and hardly exist in animals. AHAS-inhibiting herbicides are harmless to humans and animals. Therefore, discovering and researching rapeseed AHAS proteins with herbicide resistance activity has good innovation and economic value.

目前,培育抗AHAS抑制除草剂油菜的方法为以油菜种子或者植株为材料进行化学或者物理诱变,进行植物体内基因组突变。这种方法获得抗除草剂油菜的试验时间长(大于3年),获得新突变体难(突变结果为核苷酸单个碱基变化引起单个氨基酸的替换,容易与已经存在的突变重复),而且成功几率小(通常突变成功率小于百万分之一),很多实验室进行超过5年的研发无法获得新的突变体。At present, the method for cultivating rapeseed resistant to AHAS-inhibiting herbicides is to use rapeseed seeds or plants as materials to carry out chemical or physical mutagenesis to carry out genome mutation in plants. This method takes a long time to obtain herbicide-resistant rapeseed (greater than 3 years), and it is difficult to obtain new mutants (the mutation result is a single amino acid substitution caused by a single nucleotide base change, which is easy to repeat with the existing mutation), and The probability of success is small (usually the mutation success rate is less than one in a million), and many laboratories cannot obtain new mutants after more than 5 years of research and development.

发明内容Contents of the invention

本发明的目的在于提供一种具有抗除草剂活性的油菜蛋白质、其编码基因及应用,其为抗多类型除草剂活性的油菜乙酰羟基酸合成酶(即AHAS)突变体蛋白质,该蛋白质具有抗咪唑啉酮类、磺酰脲类或者嘧啶类等多类型除草剂的活性,其编码基因转化的植物具有抗咪唑啉酮类、磺酰脲类或者嘧啶类等多类型除草剂的特性。The object of the present invention is to provide a rapeseed protein with herbicide-resistant activity, its coding gene and application, which is a rapeseed acetohydroxyacid synthase (i.e. AHAS) mutant protein with anti-herbicide activity. The activity of multiple types of herbicides such as imidazolinones, sulfonylureas or pyrimidines, and the plants transformed with the coding genes have the characteristics of resistance to multiple types of herbicides such as imidazolinones, sulfonylureas or pyrimidines.

与目前通过体内突变获得的氨基酸替换突变体不同,本发明采用氨基酸删除策略,通过基因体外突变,改造油菜AHAS蛋白质,获得油菜AHAS突变体蛋白质,使其具有抗除草剂特性。Different from the amino acid replacement mutants obtained through in vivo mutation, the present invention adopts an amino acid deletion strategy to transform rapeseed AHAS protein through in vitro mutation to obtain rapeseed AHAS mutant protein, which has herbicide resistance.

为了达到上述目的,本发明提供如下技术方案:In order to achieve the above object, the present invention provides the following technical solutions:

具有抗除草剂活性的油菜蛋白质,其为油菜AHAS突变体蛋白质,其氨基酸序列如SEQ ID No.1或SEQ ID No.3所示。The rapeseed protein with herbicide resistance activity is a rapeseed AHAS mutant protein, and its amino acid sequence is shown in SEQ ID No.1 or SEQ ID No.3.

本发明其氨基酸序列如SEQ ID No.1所示的抗除草剂活性的蛋白质为前体全长蛋白质,该前体全长蛋白质删除信号肽后成为成熟蛋白质,氨基酸序列如SEQ ID No.3所示。The protein of the present invention whose amino acid sequence is as shown in SEQ ID No.1 and has anti-herbicide activity is a precursor full-length protein, and the precursor full-length protein becomes a mature protein after deleting the signal peptide, and the amino acid sequence is as shown in SEQ ID No.3. Show.

具有抗除草剂活性的基因,其为编码所述具有抗除草剂活性的油菜蛋白质的核苷酸序列。The gene with herbicide resistance activity is the nucleotide sequence encoding the rapeseed protein with herbicide resistance activity.

进一步,所述核苷酸序列如SEQ ID No.2或SEQ ID No.4所示。Further, the nucleotide sequence is shown as SEQ ID No.2 or SEQ ID No.4.

本发明所述具有抗除草剂活性的油菜蛋白质用于培育抗咪唑啉酮类、磺酰脲类和嘧啶类除草剂植物。The rapeseed protein with herbicide-resistant activity described in the invention is used for cultivating plants resistant to imidazolinones, sulfonylureas and pyrimidine herbicides.

一种获得抗咪唑啉酮类、磺酰脲类和嘧啶类等多类型除草剂植物的方法,包括,将所述油菜AHAS突变体蛋白质的编码基因转化到植物中,使植物产生具有抗除草剂活性的油菜AHAS突变体蛋白质。A method for obtaining multi-type herbicide-resistant plants such as imidazolinones, sulfonylureas and pyrimidines, comprising: transforming the coding gene of the rapeseed AHAS mutant protein into plants, so that the plants produce herbicide-resistant Active canola AHAS mutant protein.

进一步,所述编码基因的核苷酸序列如SEQ ID No.2或SEQ ID No.4所示。Further, the nucleotide sequence of the coding gene is shown as SEQ ID No.2 or SEQ ID No.4.

进一步,所述咪唑啉酮类除草剂为咪唑乙烟酸、甲氧咪草烟或者甲基咪草烟,但不限于这几种咪唑啉酮类除草剂。Further, the imidazolinone herbicide is imazethapyr, imazamox or imazapic, but not limited to these imidazolinone herbicides.

又,所述磺酰脲类除草剂为氯磺隆、吡嘧磺隆、氯嘧磺隆、甲嘧磺隆,但不限于这几种磺酰脲类除草剂。In addition, the sulfonylurea herbicides are chlorsulfuron-methyl, pyrazosulfuron-methyl, chlorimuron-methyl, and sulfonylurea-methyl, but not limited to these sulfonylurea herbicides.

优选地,所述嘧啶类除草剂为双草醚,但不限于这种嘧啶类除草剂。Preferably, the pyrimidine herbicide is bispyribac, but not limited to this pyrimidine herbicide.

进一步,所述植物为油菜、玉米或棉花,但不限于这几种植物。Further, the plant is rapeseed, corn or cotton, but not limited to these plants.

本发明抗除草剂活性的油菜蛋白质,其为油菜中经过人工改造的AHAS突变体蛋白质,源于油菜中ahas1基因的表达,将油菜ahas1基因通过体外突变,获得AHAS突变体蛋白质,其氨基酸序列的特点为:与野生型油菜AHAS蛋白质相比,缺失Trp559位点氨基酸,该突变体蛋白质对咪唑啉酮类、磺酰脲类和嘧啶类等多类型除草剂具有抗性。The herbicide-resistant rapeseed protein of the present invention is an artificially modified AHAS mutant protein in rapeseed, derived from the expression of the ahas1 gene in rapeseed, and the rapeseed ahas1 gene is mutated in vitro to obtain the AHAS mutant protein, and its amino acid sequence The characteristic is: compared with the wild-type rapeseed AHAS protein, the amino acid at the Trp559 site is missing, and the mutant protein is resistant to multiple types of herbicides such as imidazolinones, sulfonylureas and pyrimidines.

将本发明获得的AHAS突变体蛋白质的编码基因转化到植物中,在植物中进行表达,使植物中含有油菜AHAS突变体蛋白质,可使植物具有抗咪唑啉酮类、磺酰脲类和嘧啶羧酸类等多类型除草剂的特性。The coding gene of the AHAS mutant protein obtained in the present invention is transformed into a plant, and expressed in the plant, so that the plant contains the rapeseed AHAS mutant protein, which can make the plant resistant to imidazolinones, sulfonylureas and pyrimidine carboxyl Characteristics of many types of herbicides such as acids.

本发明的油菜AHAS突变体蛋白质的编码基因的核苷酸序列不限于油菜内源序列,可以是表达所述蛋白质的人工合成核苷酸序列。The nucleotide sequence of the coding gene of the rapeseed AHAS mutant protein of the present invention is not limited to the endogenous sequence of rapeseed, and may be an artificially synthesized nucleotide sequence expressing the protein.

在油菜、玉米或者棉花中表达油菜AHAS突变体,得到转基因油菜R-RA1M5、转基因玉米M-RA1M5、转基因棉花C-RA1M5,在转基因植物和野生型植物苗期喷洒抗咪唑啉酮类、磺酰脲类或者嘧啶羧酸类等多类型除草剂,野生型植物植株全部死亡,转基因植物全部存活。说明表达本发明AHAS突变体蛋白质的植物具有抗咪唑啉酮类、磺酰脲类或者嘧啶羧酸类等多类型除草剂的特性。Express rapeseed AHAS mutants in rapeseed, corn or cotton to obtain transgenic rapeseed R-RA1M5, transgenic corn M-RA1M5, and transgenic cotton C-RA1M5, and spray resistant imidazolinones, sulfonyl With multiple types of herbicides such as ureas or pyrimidine carboxylic acids, all wild-type plants died, and all transgenic plants survived. It shows that the plants expressing the AHAS mutant protein of the present invention have the characteristics of resistance to multiple types of herbicides such as imidazolinones, sulfonylureas or pyrimidine carboxylic acids.

与现有技术相比,本发明具有如下有益效果:Compared with the prior art, the present invention has the following beneficial effects:

1)本发明采用氨基酸删除策略,对氨基酸进行定点突变,可对删除氨基酸后的突变体蛋白质进行体外抗除草剂活性测定,为开发更多新型抗除草剂AHAS突变体蛋白质提供基础,具有操作简单,筛选过程简单、创新性强等优点。1) The present invention uses an amino acid deletion strategy to perform site-directed mutation on amino acids, and can perform in vitro herbicide-resistant activity assays on mutant proteins after amino acid deletion, providing a basis for the development of more new herbicide-resistant AHAS mutant proteins, with simple operation , the screening process is simple, and the innovation is strong.

2)本发明的AHAS突变体蛋白质是通过体外突变获得的,体外突变具有可设计性强、试验时间短、创新性强等体内突变无法比拟的优点,获得突变体时间小于2个月,同时,通过特定的突变设计,避免与已报道AHAS突变体蛋白质重复。2) The AHAS mutant protein of the present invention is obtained by in vitro mutation. The in vitro mutation has the incomparable advantages of strong designability, short test time, and strong innovation. The time to obtain the mutant is less than 2 months. At the same time, Through specific mutation design, avoid duplication with the reported AHAS mutant protein.

3)将本发明AHSA突变体蛋白质的编码基因表达后,获得的AHAS全长突变体蛋白质和AHAS无信号肽成熟突变体蛋白质均具有抗咪唑啉酮类、磺酰脲类和嘧啶羧酸类等多类型除草剂的特性。3) After expressing the gene encoding the AHSA mutant protein of the present invention, the obtained AHAS full-length mutant protein and the AHAS signal peptide-free mature mutant protein both have resistance to imidazolinones, sulfonylureas and pyrimidine carboxylic acids, etc. Properties of multiple types of herbicides.

附图说明Description of drawings

图1为本发明实施例1中纯化后的AHAS蛋白质SDS-PAGE电泳图;Fig. 1 is the AHAS protein SDS-PAGE electrophoresis figure after purification in the embodiment 1 of the present invention;

其中,泳道1:纯化后的RA1WS蛋白质;泳道2:纯化后的RA1M5S蛋白质;Mk:蛋白质Marker,分子量已在图上标出。Among them, lane 1: purified RA1WS protein; lane 2: purified RA1M5S protein; Mk: protein marker, the molecular weight has been marked on the figure.

图2-图3为本发明实施例2中RA1M5S及其对照RA1WS对咪唑啉酮类除草剂咪唑(乙烟酸和甲氧咪草烟)的抗性曲线图。Fig. 2-Fig. 3 are the resistance curves of RA1M5S and its control RA1WS to imidazolinone herbicides imidazoles (eniacin and imazamox) in Example 2 of the present invention.

图4为本发明实施例2中RA1M5S及其对照RA1WS对嘧啶羧酸类除草剂(双草醚)的抗性曲线图。Fig. 4 is a graph showing the resistance curves of RA1M5S and its control RA1WS to pyrimidine carboxylic acid herbicide (bispyribac) in Example 2 of the present invention.

图5-图8为本发明实施例2中RA1M5S及其对照RA1WS对磺酰脲类除草剂(氯磺隆、吡嘧磺隆、氯嘧磺隆、甲嘧磺隆)的抗性曲线图。Figures 5 to 8 are graphs showing the resistance curves of RA1M5S and its control RA1WS to sulfonylurea herbicides (chlorsulfuron-methyl, pyrazosulfuron-methyl, chlorimuron-methyl, and sulfursulfuron-methyl) in Example 2 of the present invention.

具体实施方式detailed description

以下结合具体实施例对本发明作进一步说明。The present invention will be further described below in conjunction with specific examples.

以下实施例中,如无特殊说明为《分子克隆实验指南》(科学出版社,2002年)所记载方法。In the following examples, unless otherwise specified, the methods described in the "Molecular Cloning Experiment Guide" (Science Press, 2002) were used.

引物合成和测序委托生工上海生物工程股份有限公司进行,试剂、菌株、载体等实验用品从杭州西格玛生物技术公司购买。Primer synthesis and sequencing were entrusted to Sangon Shanghai Bioengineering Co., Ltd., reagents, strains, vectors and other experimental supplies were purchased from Hangzhou Sigma Biotechnology Company.

咪唑烟乙酸除草剂为山东先达公司的“豆说好”,稀释浓度为稀释200倍。甲基咪草烟除草剂为德国巴斯夫公司的“百垄通”,稀释浓度为稀释1000倍。甲氧咪草烟除草剂为美国氰胺公司的“金豆”,稀释浓度为稀释100倍。氯磺隆除草剂为江苏省激素研究所股份有限公司的“氯磺隆”,稀释浓度为稀释20000倍。吡嘧磺隆除草剂为江苏省激素研究所股份有限公司的原药,稀释浓度为稀释2000倍。氯嘧磺隆除草剂为江苏省激素研究所股份有限公司的原药,稀释浓度为稀释5000倍。甲嘧磺隆除草剂为江苏瑞邦农药厂的“森草净”,稀释浓度为稀释5000倍。双草醚除草剂为江苏省激素研究所股份有限公司的“双草醚”,稀释浓度为稀释1000倍。The imazethapyr acetic acid herbicide is "Dou Shuo Hao" produced by Shandong Xianda Company, and the dilution concentration is 200 times. The imazethapyr herbicide is "Bailongtong" of BASF, Germany, and the dilution concentration is 1000 times. The imazethapyr herbicide is "Golden Bean" of American Cyanamid Company, and the dilution concentration is 100 times. The chlorsulfuron herbicide is "Chlorsulfuron" of Jiangsu Provincial Hormone Research Institute Co., Ltd., and the dilution concentration is 20000 times. Pyrazosulfuron-methyl herbicide is the original drug of Jiangsu Provincial Hormone Research Institute Co., Ltd., and the dilution concentration is 2000 times. Chlorsulfuron-methyl herbicide is the original drug of Jiangsu Provincial Hormone Research Institute Co., Ltd., and the dilution concentration is 5000 times. The sulfuron-methyl herbicide is "Sencaojing" from Jiangsu Ruibang Pesticide Factory, and the dilution concentration is 5000 times. The bispyribac herbicide is "Bisybroben" from Jiangsu Provincial Hormone Research Institute Co., Ltd., and the dilution concentration is 1000 times.

实施例1获得油菜AHAS突变体蛋白质,包括以下步骤:Embodiment 1 obtains rapeseed AHAS mutant protein, comprises the following steps:

1.构建删除Trp559氨基酸的ahas1基因1. Construction of the ahas1 gene that deletes the Trp559 amino acid

(1)通过多聚合链式反应(PCR)的方法,从油菜因组中扩增出ahas1基因(NCBI序列号:Z11524)。(1) The ahas1 gene (NCBI sequence number: Z11524) was amplified from the Brassica napus genome by multiple polymer chain reaction (PCR).

其中,进行PCR反应使用的引物为:Wherein, the primers used for PCR reaction are:

BN A1F:5’ctaaccatggcggcggcaacatcgtc;BN A1F: 5'ctaaccatggcggcggcaacatcgtc;

BN A1EcoR:5’gaattctcagtacttagtgcgaccatcccctt。BN A1 EcoR: 5' gaattctcagtacttagtgcgaccatccccctt.

PCR反应体系为:2×高保真聚合酶预混液50μl、引物BN A1F(10μM)4μl、引物BNA1EcoR(10μM)4μl、野生型油菜基因组DNA 1μl和去离子水41μl,总体积为100μl。The PCR reaction system was: 50 μl of 2×high-fidelity polymerase master mix, 4 μl of primer BNA1F (10 μM), 4 μl of primer BNA1EcoR (10 μM), 1 μl of wild-type rape genomic DNA and 41 μl of deionized water, with a total volume of 100 μl.

PCR程序:a)95℃,10分钟;b)95℃,40秒;c)52℃,40秒;d)68℃,2分钟;e)循环步骤2-4,25次;f)68℃,10分钟;g)16℃,保存。PCR program: a) 95°C, 10 minutes; b) 95°C, 40 seconds; c) 52°C, 40 seconds; d) 68°C, 2 minutes; e) cycle steps 2-4, 25 times; f) 68°C , 10 minutes; g) 16 ° C, save.

(2)通过琼脂糖电泳分离、纯化出步骤1)的PCR产物,即ahas1基因,将PCR产物进行T克隆(pGEMT,美国Promega公司),获得克隆T-RA1W。(2) The PCR product of step 1), that is, the ahas1 gene, was separated and purified by agarose electrophoresis, and the PCR product was subjected to T-cloning (pGEMT, Promega, USA) to obtain clone T-RA1W.

(3)以T-RA1W为模板,使用引物BN A1559WF和BN A1559WR进行PCR反应,将PCR产物进行T克隆,获得删除Trp559氨基酸的突变体克隆T-RA1M5。(3) Using T-RA1W as a template, the primers BN A1559WF and BN A1559WR were used for PCR reaction, and the PCR product was subjected to T cloning to obtain a mutant clone T-RA1M5 deleting the amino acid of Trp559.

其中,PCR反应体系和程序中,除模板和引物外其它条件与步骤1)相同。Wherein, in the PCR reaction system and procedure, other conditions are the same as step 1) except the template and primers.

引物的具体序列为:The specific sequence of the primer is:

BN A1559WF:5’catcttgggatggtcatgcaagaagatcggttctacaaagct;BN A1559WF: 5' catcttgggatggtcatgcaagaagatcggttctacaaagct;

BN A1559WR:5’agctttgtagaaccgatcttcttgcatgaccatcccaagatg。BN A1559WR: 5'agctttgtagaaccgatcttcttgcatgaccatcccaagatg.

其中,RA1M5的DNA序列如SEQ ID NO.2所示,氨基酸序列如SEQ ID NO.1所示。Wherein, the DNA sequence of RA1M5 is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1.

2.表达、纯化删除信号肽的AHAS成熟蛋白质2. Expression and purification of AHAS mature protein with signal peptide deleted

(1)以T-RA1W为模板,使用引物BN A1BamF和BN A1EcoR进行PCR反应,将PCR产物进行T克隆,获得删除信号肽的野生型T-RA1WS。(1) Using T-RA1W as a template, PCR reaction was performed using primers BN A1BamF and BN A1EcoR, and the PCR product was subjected to T-cloning to obtain wild-type T-RA1WS with the signal peptide deleted.

其中,PCR反应体系和程序中,除模板和引物外其它条件与上述步骤1(1)相同,引物的具体序列为:Wherein, in the PCR reaction system and program, except the template and primers, other conditions are the same as the above step 1 (1), and the specific sequences of the primers are:

BN A1BamF:5’ggatccacaatgactttcgtctcccgctacgctc;BN A1BamF: 5'ggatccacaatgactttcgtctcccgctacgctc;

BN A1EcoR:5’gaattctcagtacttagtgcgaccatcccctt。BN A1 EcoR: 5' gaattctcagtacttagtgcgaccatccccctt.

(2)构建蛋白质表达载体,将RA1WS克隆入pGEX4T2(美国GE生命科学公司),获得表达无信号肽的AHAS野生型蛋白质的载体4T2-RA1WS。(2) Constructing a protein expression vector, cloning RAIWS into pGEX4T2 (GE Life Sciences, USA) to obtain the vector 4T2-RA1WS expressing AHAS wild-type protein without signal peptide.

(3)将表达无信号肽的AHAS野生型蛋白质的载体转入表达菌株Rosetta中,培养、收获细胞,超声波破碎细胞,将离心获得上清液通过GST柱,清洗GST柱,最终洗脱获得成熟的野生型蛋白质RA1WS。(3) Transfer the vector expressing the AHAS wild-type protein without signal peptide into the expression strain Rosetta, culture and harvest the cells, disrupt the cells by ultrasonic, pass the supernatant obtained by centrifugation through the GST column, wash the GST column, and finally elute to obtain mature The wild-type protein RA1WS.

同理,采用与上述步骤2(1)-2(3)同样的方法,以删除Trp559氨基酸的T-RA1M5基因为模板,以引物BN A1Bam和BN A1EcoR为引物,进行PCR反应、T克隆、构建表达载体4T2-RA1M5S,将表达载体4T2-RA1M5S转入表达菌株Rosetta中进行表达,获得无信号肽而且删除Trp559氨基酸的成熟AHAS突变体蛋白质RA1M5S,其氨基酸序列如SEQ ID NO.3所示,DNA序列如SEQ ID NO.4所示。Similarly, use the same method as the above steps 2(1)-2(3), use the T-RA1M5 gene that deletes the Trp559 amino acid as a template, and use primers BN A1Bam and BN A1EcoR as primers to perform PCR reaction, T cloning, and construction The expression vector 4T2-RA1M5S, the expression vector 4T2-RA1M5S was transferred into the expression strain Rosetta for expression, and the mature AHAS mutant protein RA1M5S without signal peptide and Trp559 amino acid was deleted, and its amino acid sequence was shown in SEQ ID NO.3, DNA The sequence is shown in SEQ ID NO.4.

将获得的野生型蛋白质RA1WS和成熟的AHAS突变体蛋白质RA1M5S进行SDS-PAGE电泳检测,蛋白质胶参见图1。The obtained wild-type protein RA1WS and the mature AHAS mutant protein RA1M5S were detected by SDS-PAGE electrophoresis, see Figure 1 for the protein gel.

由图1可见,泳道1中的RA1WS和泳道2中的RA1M5S蛋白质在90kDa位置具有明显条带,此位置与蛋白质的理论大小相符。此外,该蛋白质条带在整个泳道的蛋白质条带中占主要比例,说明获得了高纯度的成熟油菜AHAS野生型蛋白质和成熟的油菜AHAS突变体蛋白质。It can be seen from Figure 1 that the RA1WS in lane 1 and the RA1M5S protein in lane 2 have obvious bands at the position of 90 kDa, which is consistent with the theoretical size of the protein. In addition, the protein band accounted for the main proportion of the protein bands in the entire swimming lane, indicating that high-purity mature rapeseed AHAS wild-type protein and mature rapeseed AHAS mutant protein were obtained.

实施例2检测AHAS突变体蛋白质对除草剂的抗性Example 2 Detection of AHAS mutant protein resistance to herbicides

分别以实施例1获得的野生型蛋白质RA1WS和突变体蛋白质RA1M5S为检测主体,水为空白对照处理,以浓度在0-100μM的不同品种除草剂(咪唑烟乙酸、甲氧咪草烟、双草醚、氯嘧磺隆、吡嘧磺隆、甲嘧磺隆)为影响因素,进行抗除草剂生物测定。The wild-type protein RA1WS obtained in Example 1 and the mutant protein RA1M5S were respectively used as detection subjects, and water was treated as a blank control, and different kinds of herbicides (imazethapyr, imazethapyr, and bismuth) were used at a concentration of 0-100 μM Ether, chlorimuron-methyl, pyrazosulfuron-methyl, and sulfuron-methyl) were the influencing factors, and the herbicide resistance bioassay was carried out.

在2mL的离心管中配置溶液反应体系:450μl AHAS活性测定缓冲液,10μlAHAS蛋白质(RA1WS或RA1M5S,0.5μg/μl),40μl影响因素溶液(以水为空白对照处理),总体积500μl。Prepare the solution reaction system in a 2mL centrifuge tube: 450 μl AHAS activity assay buffer, 10 μl AHAS protein (RA1WS or RA1M5S, 0.5 μg/μl), 40 μl influencing factor solution (with water as the blank control), the total volume is 500 μl.

AHAS活性测定缓冲液:100mM丙酮酸钠,10mM氯化镁,1mM TPP,50μM FAD,50mM磷酸盐,pH7.4。AHAS activity assay buffer: 100 mM sodium pyruvate, 10 mM magnesium chloride, 1 mM TPP, 50 μM FAD, 50 mM phosphate, pH 7.4.

反应程序:将含有不同影响因素反应体系的离心管置于37℃反应1小时;20μl30%硫酸终止反应。60℃反应0.5小时;加入250μl 1.7%α-萘酚,0.17%肌酸,60℃显色0.5小时。从离心管中取出200μl,检测含有不同影响因素的溶液在525nm吸光度值。Reaction procedure: put the centrifuge tube containing the reaction system of different influencing factors at 37° C. for 1 hour; 20 μl of 30% sulfuric acid to stop the reaction. React at 60°C for 0.5 hours; add 250 μl of 1.7% α-naphthol and 0.17% creatine, and develop color at 60°C for 0.5 hours. Take out 200 μl from the centrifuge tube, and detect the absorbance value at 525nm of the solutions containing different influencing factors.

检测结果:以除草剂浓度为0(影响因素溶液为水对照)的野生型蛋白质RA1WS处理的525nm吸光度值为100%活性,计算野生型和突变体蛋白质在不同影响因素条件下的活性比例,绘制曲线图,结果参见图2-图8。Test results: the 525nm absorbance value of the wild-type protein RA1WS treated with herbicide concentration as 0 (influencing factor solution is water control) is 100% active, calculate the activity ratio of wild-type and mutant protein under different influencing factor conditions, and draw For the graph, see Figure 2-Figure 8 for the results.

由图2-图8可见,随着咪唑啉酮类、磺酰脲类或者嘧啶羧酸类等多类型除草剂的增加,RA1WS活性降低至0;而RA1M5S活性在高浓度除草剂(氯磺隆和吡嘧磺隆除外)中依然保持接近100%活性,在高浓度氯磺隆中保持70%活性,在高浓度吡嘧磺隆中保持50%活性。因此,RA1M5S对咪唑啉酮类、磺酰脲类或者嘧啶羧酸类等多类型除草剂具有抗性。As can be seen from Figure 2-Figure 8, with the increase of imidazolinones, sulfonylureas or pyrimidine carboxylic acids and other multi-type herbicides, the activity of RA1WS decreased to 0; and pyrazosulfuron-methyl) still maintain close to 100% activity, maintain 70% activity in high concentration chlorsulfuron-methyl, and maintain 50% activity in high concentration pyrazosulfuron-methyl. Therefore, RA1M5S is resistant to multiple types of herbicides such as imidazolinones, sulfonylureas or pyrimidine carboxylic acids.

因此,本发明获得的RA1M5S对咪唑啉酮类、磺酰脲类或者嘧啶羧酸类等多类型除草剂具有抗性。Therefore, the RA1M5S obtained in the present invention is resistant to multiple types of herbicides such as imidazolinones, sulfonylureas or pyrimidine carboxylic acids.

实施例3油菜ahas1突变体基因在植物中的转化及表达Embodiment 3 Transformation and expression of rapeseed ahas1 mutant gene in plants

1.构建AHAS突变体蛋白质的表达元件1. Construction of expression elements for AHAS mutant proteins

为了在单子叶和双子叶植物中表达AHAS突变体蛋白质,以pCambia1300载体(简称1300)为载体,用辣椒斑驳病毒35S(简称35S)启动子和终止子构建AHAS表达元件,其中,35S启动子和终止子DNA序列见http://www.snapgene.com/resources/plasmid_files/plant_vectors/pCAMBIA1300/,具体步骤如下:In order to express AHAS mutant protein in monocotyledon and dicotyledonous plant, take pCambia1300 vector (abbreviation 1300) as carrier, construct AHAS expression element with capsicum mottle virus 35S (abbreviation 35S) promoter and terminator, wherein, 35S promoter and See http://www.snapgene.com/resources/plasmid_files/plant_vectors/pCAMBIA1300/ for the DNA sequence of the terminator, and the specific steps are as follows:

(1)以实施例1获得的T-RA1M5为模板,使用引物BN A1BamF和BN A1SacR,通过PCR和BamH1/Sac1双酶切,获得ahas突变体片段RA1M5T;(1) Using the T-RA1M5 obtained in Example 1 as a template, using primers BN A1BamF and BN A1SacR, by PCR and BamH1/Sac1 double digestion, the ahas mutant fragment RA1M5T was obtained;

其中,引物序列为:Among them, the primer sequence is:

BN A1BamF:5’ggatccacaatgactttcgtctcccgctacgctc;BN A1BamF: 5'ggatccacaatgactttcgtctcccgctacgctc;

BN A1SacR:5’gagctctcagtacttagtgcgaccatcccctt。BN A1 SacR: 5'gagctctcagtacttagtgcgaccatcccctt.

(2)以1300载体为模板,使用引物35S-Hind和35S-Bam,通过PCR和BamH1/Hind3双酶切,获得启动子片段35S;(2) Use the 1300 vector as a template, use primers 35S-Hind and 35S-Bam, and obtain the promoter fragment 35S by PCR and BamH1/Hind3 double digestion;

其中,引物序列为:Among them, the primer sequence is:

35S-Hind:5’gcgaagcttcatggagtcaaagattcaaa;35S-Hind: 5' gcgaagcttcatggagtcaaagattcaaa;

35S-Bam:5’gtgggatccagtcccccgtgttctctccaaatgaa。35S-Bam: 5'gtgggatccagtcccccgtgttctctccaaatgaa.

(3)以1300载体为模板,使用引物ter-Sac和ter-Kpn,通过PCR和BamH1/Hind3双酶切,获得终止子片段ter;(3) Using the 1300 vector as a template, using primers ter-Sac and ter-Kpn, by PCR and BamH1/Hind3 double digestion, the terminator fragment ter was obtained;

其中,引物序列为:Among them, the primer sequence is:

ter-Sac:5’gtggagctcagtagatgccgaccg gatctgt;ter-Sac: 5'gtggagctcagtagatgccgaccggatctgt;

ter-Kpn:5’cagggtacccgccgaattaattcggggga。ter-Kpn: 5' cagggtacccgccgaattaattcggggga.

(4)将1300进行HindIII/SacI双酶切,获得片段1300HS,与35S和RA1M5T进行连接、克隆,获得1300-35S-RA1M5。(4) 1300 was subjected to HindIII/SacI double enzyme digestion to obtain fragment 1300HS, which was ligated with 35S and RA1M5T and cloned to obtain 1300-35S-RA1M5.

(5)将1300-35S-RA1M5进行SacI/KpnI双酶切,与ter连接、克隆,获得表达油菜AHAS突变体的载体1300-RA1M5。(5) 1300-35S-RA1M5 was subjected to SacI/KpnI double enzyme digestion, ligated with ter, and cloned to obtain the vector 1300-RA1M5 expressing rapeseed AHAS mutant.

(6)将载体1300-RA1M5转入农杆菌菌株LBA4044中,获得表达AHAS突变体蛋白质的植物转化农杆菌。(6) Transforming the vector 1300-RA1M5 into the Agrobacterium strain LBA4044 to obtain a plant-transformed Agrobacterium expressing the AHAS mutant protein.

2.获得表达AHAS突变体蛋白质的植物2. Obtaining plants expressing AHAS mutant proteins

转基因植物的获得方法为现有成熟技术,转基因植物制备委托上海博祈生物科技有限公司进行。The method of obtaining transgenic plants is an existing mature technology, and the preparation of transgenic plants is entrusted to Shanghai Boqi Biotechnology Co., Ltd.

使用农杆菌介导法将载体1300-RA1M5分别转入油菜、玉米和棉花中,获得转基因油菜R-RA1M5,转基因玉米M-RA1M5、转基因棉花C-RA1M5。The vector 1300-RA1M5 was transformed into rapeseed, corn and cotton by Agrobacterium-mediated method to obtain transgenic rapeseed R-RA1M5, transgenic corn M-RA1M5 and transgenic cotton C-RA1M5.

在获得的转基因植物转基因油菜R-RA1M5、转基因玉米M-RA1M5、转基因棉花C-RA1M5和对应野生型植物的苗期喷除草剂,十天后,统计死亡率,结果参见表1。The obtained transgenic plants, transgenic rapeseed R-RA1M5, transgenic corn M-RA1M5, transgenic cotton C-RA1M5, and corresponding wild-type plants were sprayed with herbicides at the seedling stage, and the mortality was counted ten days later. The results are shown in Table 1.

表1Table 1

由表1可知,野生型植物植株全部死亡,而转基因植物全部存活,说明表达本发明的油菜AHAS突变体蛋白质的植物具有抗咪唑啉酮类、磺酰脲类或者嘧啶羧酸类等多类型除草剂活性。As can be seen from Table 1, all the wild-type plants died, while all the transgenic plants survived, indicating that the plants expressing the rapeseed AHAS mutant protein of the present invention have resistance to imidazolinones, sulfonylureas or pyrimidine carboxylic acids and other types of herbicides. agent activity.

<110> 上海市农业科学院<110> Shanghai Academy of Agricultural Sciences

<120> 具有抗除草剂活性的油菜蛋白质、其编码基因及其应用<120> Rapeseed protein with herbicide resistance activity, its coding gene and its application

<130> 1711032<130> 1711032

<160> 4<160> 4

<170> PatentIn version 3.5<170> PatentIn version 3.5

<210> SEQ ID NO.1<210> SEQ ID NO.1

<211> 654<211> 654

<212> PRT<212> PRT

<213> 油菜(Brassica napus)<213> Rapeseed (Brassica napus)

<400> 1<400> 1

Met Ala Ala Ala Thr Ser Ser Ser Pro Ile Ser Leu Thr Ala Lys ProMet Ala Ala Ala Thr Ser Ser Ser Ser Pro Ile Ser Leu Thr Ala Lys Pro

1 5 10 151 5 10 15

Ser Ser Lys Ser Pro Leu Pro Ile Ser Arg Phe Ser Leu Pro Phe SerSer Ser Lys Ser Pro Leu Pro Ile Ser Arg Phe Ser Leu Pro Phe Ser

20 25 30 20 25 30

Leu Thr Pro Gln Lys Asp Ser Ser Arg Leu His Arg Pro Leu Ala IleLeu Thr Pro Gln Lys Asp Ser Ser Arg Leu His Arg Pro Leu Ala Ile

35 40 45 35 40 45

Ser Ala Val Leu Asn Ser Pro Val Asn Val Ala Pro Pro Ser Pro GluSer Ala Val Leu Asn Ser Pro Val Asn Val Ala Pro Pro Ser Pro Glu

50 55 60 50 55 60

Lys Thr Asp Lys Asn Lys Thr Phe Val Ser Arg Tyr Ala Pro Asp GluLys Thr Asp Lys Asn Lys Thr Phe Val Ser Arg Tyr Ala Pro Asp Glu

65 70 75 8065 70 75 80

Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala Leu Glu Arg Gln GlyPro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala Leu Glu Arg Gln Gly

85 90 95 85 90 95

Val Glu Thr Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile HisVal Glu Thr Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His

100 105 110 100 105 110

Gln Ala Leu Thr Arg Ser Ser Thr Ile Arg Asn Val Leu Pro Arg HisGln Ala Leu Thr Arg Ser Ser Thr Ile Arg Asn Val Leu Pro Arg His

115 120 125 115 120 125

Glu Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr Ala Arg Ser Ser GlyGlu Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr Ala Arg Ser Ser Ser Gly

130 135 140 130 135 140

Lys Pro Gly Ile Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn LeuLys Pro Gly Ile Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu

145 150 155 160145 150 155 160

Val Ser Gly Leu Ala Asp Ala Met Leu Asp Ser Val Pro Leu Val AlaVal Ser Gly Leu Ala Asp Ala Met Leu Asp Ser Val Pro Leu Val Ala

165 170 175 165 170 175

Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe GlnIle Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln

180 185 190 180 185 190

Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn TyrGlu Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr

195 200 205 195 200 205

Leu Val Met Asp Val Asp Asp Ile Pro Arg Ile Val Gln Glu Ala PheLeu Val Met Asp Val Asp Asp Ile Pro Arg Ile Val Gln Glu Ala Phe

210 215 220 210 215 220

Phe Leu Ala Thr Ser Gly Arg Pro Gly Pro Val Leu Val Asp Val ProPhe Leu Ala Thr Ser Gly Arg Pro Gly Pro Val Leu Val Asp Val Pro

225 230 235 240225 230 235 240

Lys Asp Ile Gln Gln Gln Leu Ala Ile Pro Asn Trp Asp Gln Pro MetLys Asp Ile Gln Gln Gln Leu Ala Ile Pro Asn Trp Asp Gln Pro Met

245 250 255 245 250 255

Arg Leu Pro Gly Tyr Met Ser Arg Leu Pro Gln Pro Pro Glu Val SerArg Leu Pro Gly Tyr Met Ser Arg Leu Pro Gln Pro Pro Glu Val Ser

260 265 270 260 265 270

Gln Leu Gly Gln Ile Val Arg Leu Ile Ser Glu Ser Lys Arg Pro ValGln Leu Gly Gln Ile Val Arg Leu Ile Ser Glu Ser Lys Arg Pro Val

275 280 285 275 280 285

Leu Tyr Val Gly Gly Gly Ser Leu Asn Ser Ser Glu Glu Leu Gly ArgLeu Tyr Val Gly Gly Gly Ser Leu Asn Ser Ser Ser Glu Glu Leu Gly Arg

290 295 300 290 295 300

Phe Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly LeuPhe Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu

305 310 315 320305 310 315 320

Gly Ser Tyr Pro Cys Asn Asp Glu Leu Ser Leu Gln Met Leu Gly MetGly Ser Tyr Pro Cys Asn Asp Glu Leu Ser Leu Gln Met Leu Gly Met

325 330 335 325 330 335

His Gly Thr Val Tyr Ala Asn Tyr Ala Val Glu His Ser Asp Leu LeuHis Gly Thr Val Tyr Ala Asn Tyr Ala Val Glu His Ser Asp Leu Leu

340 345 350 340 345 350

Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu GluLeu Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu Glu

355 360 365 355 360 365

Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp Ile Asp Ser AlaAla Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp Ile Asp Ser Ala

370 375 380 370 375 380

Glu Ile Gly Lys Asn Lys Thr Pro His Val Ser Val Cys Gly Asp ValGlu Ile Gly Lys Asn Lys Thr Pro His Val Ser Val Cys Gly Asp Val

385 390 395 400385 390 395 400

Lys Leu Ala Leu Gln Gly Met Asn Lys Val Leu Glu Asn Arg Ala GluLys Leu Ala Leu Gln Gly Met Asn Lys Val Leu Glu Asn Arg Ala Glu

405 410 415 405 410 415

Glu Leu Lys Leu Asp Phe Gly Val Trp Arg Ser Glu Leu Ser Glu GlnGlu Leu Lys Leu Asp Phe Gly Val Trp Arg Ser Glu Leu Ser Glu Gln

420 425 430 420 425 430

Lys Gln Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile ProLys Gln Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro

435 440 445 435 440 445

Pro Gln Tyr Ala Ile Gln Ile Leu Asp Glu Leu Thr Glu Gly Lys AlaPro Gln Tyr Ala Ile Gln Ile Leu Asp Glu Leu Thr Glu Gly Lys Ala

450 455 460 450 455 460

Ile Ile Ser Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln PheIle Ile Ser Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Phe

465 470 475 480465 470 475 480

Tyr Lys Tyr Arg Lys Pro Arg Gln Trp Leu Ser Ser Ser Gly Leu GlyTyr Lys Tyr Arg Lys Pro Arg Gln Trp Leu Ser Ser Ser Ser Gly Leu Gly

485 490 495 485 490 495

Ala Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ser Val Ala AsnAla Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ser Val Ala Asn

500 505 510 500 505 510

Pro Asp Ala Ile Val Val Asp Ile Asp Gly Asp Gly Ser Phe Ile MetPro Asp Ala Ile Val Val Asp Ile Asp Gly Asp Gly Ser Phe Ile Met

515 520 525 515 520 525

Asn Val Gln Glu Leu Ala Thr Ile Arg Val Glu Asn Leu Pro Val LysAsn Val Gln Glu Leu Ala Thr Ile Arg Val Glu Asn Leu Pro Val Lys

530 535 540 530 535 540

Ile Leu Leu Leu Asn Asn Gln His Leu Gly Met Val Met Gln Glu AspIle Leu Leu Leu Asn Asn Gln His Leu Gly Met Val Met Gln Glu Asp

545 550 555 560545 550 555 560

Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asp Pro AlaArg Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asp Pro Ala

565 570 575 565 570 575

Arg Glu Asn Glu Ile Phe Pro Asn Met Leu Gln Phe Ala Gly Ala CysArg Glu Asn Glu Ile Phe Pro Asn Met Leu Gln Phe Ala Gly Ala Cys

580 585 590 580 585 590

Gly Ile Pro Ala Ala Arg Val Thr Lys Lys Glu Glu Leu Arg Glu AlaGly Ile Pro Ala Ala Arg Val Thr Lys Lys Glu Glu Leu Arg Glu Ala

595 600 605 595 600 605

Ile Gln Thr Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val IleIle Gln Thr Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile

610 615 620 610 615 620

Cys Pro His Gln Glu His Val Leu Pro Met Ile Pro Ser Gly Gly ThrCys Pro His Gln Glu His Val Leu Pro Met Ile Pro Ser Gly Gly Thr

625 630 635 640625 630 635 640

Phe Lys Asp Val Ile Thr Glu Gly Asp Gly Arg Thr Lys TyrPhe Lys Asp Val Ile Thr Glu Gly Asp Gly Arg Thr Lys Tyr

645 650 654 645 650 654

<210> SEQ ID NO.2<210> SEQ ID NO.2

<211> 1965<211> 1965

<212> DNA<212>DNA

<213> 油菜(Brassica napus)<213> Rapeseed (Brassica napus)

<400> 2<400> 2

atggcggcgg caacatcgtc ttctccgatc tccttaaccg ctaaaccttc ttccaaatcc 60atggcggcgg caacatcgtc ttctccgatc tccttaaccg ctaaaccttc ttccaaatcc 60

cctctaccca tttccagatt ctcccttccc ttctccttaa ccccacagaa agactcctcc 120cctctaccca tttccagatt ctcccttccc ttctccttaa ccccacagaa agactcctcc 120

cgtctccacc gtcctctcgc catctccgcc gttctcaact cacccgtcaa tgtcgcacct 180cgtctccacc gtcctctcgc catctccgcc gttctcaact cacccgtcaa tgtcgcacct 180

ccttcccctg aaaaaaccga caagaacaag actttcgtct cccgctacgc tcccgacgag 240ccttcccctg aaaaaaccga caagaacaag actttcgtct cccgctacgc tcccgacgag 240

ccccgcaagg gtgctgatat cctcgtcgaa gccctcgagc gtcaaggcgt cgaaaccgtc 300ccccgcaagg gtgctgatat cctcgtcgaa gccctcgagc gtcaaggcgt cgaaaccgtc 300

tttgcttatc ccggaggtgc ttccatggag atccaccaag ccttgactcg ctcctccacc 360tttgcttatc ccggaggtgc ttccatggag atccaccaag ccttgactcg ctcctccacc 360

atccgtaacg tccttccccg tcacgaacaa ggaggagtct tcgccgccga gggttacgct 420atccgtaacg tccttccccg tcacgaacaa ggaggagtct tcgccgccga gggttacgct 420

cgttcctccg gcaaaccggg aatctgcata gccacttcgg gtcccggagc taccaacctc 480cgttcctccg gcaaaccggg aatctgcata gccacttcgg gtcccggagc taccaacctc 480

gtcagcgggt tagcagacgc gatgcttgac agtgttcctc ttgtcgccat tacaggacag 540gtcagcgggt tagcagacgc gatgcttgac agtgttcctc ttgtcgccat tacaggacag 540

gtccctcgcc ggatgatcgg tactgacgcc ttccaagaga caccaatcgt tgaggtaacg 600gtccctcgcc ggatgatcgg tactgacgcc ttccaagaga caccaatcgt tgaggtaacg 600

aggtctatta cgaaacataa ctatttggtg atggatgttg atgacatacc taggatcgtt 660aggtctatta cgaaacataa ctatttggtg atggatgttg atgacatacc taggatcgtt 660

caagaagctt tctttctagc tacttccggt agacccggac cggttttggt tgatgttcct 720caagaagctt tctttctagc tacttccggt agacccggac cggttttggt tgatgttcct 720

aaggatattc agcagcagct tgcgattcct aactgggatc aacctatgcg cttacctggc 780aaggatattc agcagcagct tgcgattcct aactgggatc aacctatgcg cttacctggc 780

tacatgtcta ggttgcctca gcctccggaa gtttctcagt taggtcagat cgttaggttg 840tacatgtcta ggttgcctca gcctccggaa gtttctcagt taggtcagat cgttaggttg 840

atctcggagt ctaagaggcc tgttttgtac gttggtggtg gaagcttgaa ctcgagtgaa 900atctcggagt ctaagaggcc tgttttgtac gttggtggtg gaagcttgaa ctcgagtgaa 900

gaactgggga gatttgtcga gcttactggg atccccgttg cgagtacttt gatggggctt 960gaactgggga gatttgtcga gcttactggg atccccgttg cgagtacttt gatggggctt 960

ggctcttatc cttgtaacga tgagttgtcc ctgcagatgc ttggcatgca cgggactgtg 1020ggctcttatc cttgtaacga tgagttgtcc ctgcagatgc ttggcatgca cgggactgtg 1020

tatgctaact acgctgtgga gcatagtgat ttgttgctgg cgtttggtgt taggtttgat 1080tatgctaact acgctgtgga gcatagtgat ttgttgctgg cgtttggtgt taggtttgat 1080

gaccgtgtca cgggaaagct cgaggctttc gctagcaggg ctaaaattgt gcacatagac 1140gaccgtgtca cgggaaagct cgaggctttc gctagcaggg ctaaaattgt gcacatagac 1140

attgattctg ctgagattgg gaagaataag acacctcacg tgtctgtgtg tggtgatgta 1200attgattctg ctgagattgg gaagaataag acacctcacg tgtctgtgtg tggtgatgta 1200

aagctggctt tgcaagggat gaacaaggtt cttgagaacc gggcggagga gctcaagctt 1260aagctggctt tgcaagggat gaacaaggtt cttgagaacc gggcggagga gctcaagctt 1260

gatttcggtg tttggaggag tgagttgagc gagcagaaac agaagttccc tttgagcttc 1320gatttcggtg tttggaggag tgagttgagc gagcagaaac agaagttccc tttgagcttc 1320

aaaacgtttg gagaagccat tcctccgcag tacgcgattc agatcctcga cgagctaacc 1380aaaacgtttg gagaagccat tcctccgcag tacgcgattc agatcctcga cgagctaacc 1380

gaagggaagg caattatcag tactggtgtt ggacagcatc agatgtgggc ggcgcagttt 1440gaagggaagg caattatcag tactggtgtt ggacagcatc agatgtgggc ggcgcagttt 1440

tacaagtaca ggaagccgag acagtggctg tcgtcatcag gcctcggagc tatgggtttt 1500tacaagtaca ggaagccgag acagtggctg tcgtcatcag gcctcggagc tatgggtttt 1500

ggacttcctg ctgcgattgg agcgtctgtg gcgaaccctg atgcgattgt tgtggatatt 1560ggacttcctg ctgcgattgg agcgtctgtg gcgaaccctg atgcgattgt tgtggatatt 1560

gacggtgatg gaagcttcat aatgaacgtt caagagctgg ccacaatccg tgtagagaat 1620gacggtgatg gaagcttcat aatgaacgtt caagagctgg ccacaatccg tgtagagaat 1620

cttcctgtga agatactctt gttaaacaac cagcatcttg ggatggtcat gcaagaagat 1680cttcctgtga agatactctt gttaaacaac cagcatcttg ggatggtcat gcaagaagat 1680

cggttctaca aagctaacag agctcacact tatctcgggg acccggcaag ggagaacgag 1740cggttctaca aagctaacag agctcacact tatctcgggg acccggcaag ggagaacgag 1740

atcttcccta acatgctgca gtttgcagga gcttgcggga ttccagctgc gagagtgacg 1800atcttcccta acatgctgca gtttgcagga gcttgcggga ttccagctgc gagagtgacg 1800

aagaaagaag aactccgaga agctattcag acaatgctgg atacaccagg accatacctg 1860aagaaagaag aactccgaga agctattcag acaatgctgg atacaccagg accatacctg 1860

ttggatgtga tatgtccgca ccaagaacat gtgttaccga tgatcccaag tggtggcact 1920ttggatgtga tatgtccgca ccaagaacat gtgttaccga tgatcccaag tggtggcact 1920

ttcaaagatg taataacaga aggggatggt cgcactaagt actga 1965ttcaaagatg taataacaga aggggatggt cgcactaagt actga 1965

<210> SEQ ID NO.3<210> SEQ ID NO.3

<211> 584<211> 584

<212> PRT<212> PRT

<213> 油菜(Brassica napus)<213> Rapeseed (Brassica napus)

<400> 3<400> 3

Thr Phe Val Ser Arg Tyr Ala Pro Asp Glu Pro Arg Lys Gly Ala AspThr Phe Val Ser Arg Tyr Ala Pro Asp Glu Pro Arg Lys Gly Ala Asp

1 5 10 151 5 10 15

Ile Leu Val Glu Ala Leu Glu Arg Gln Gly Val Glu Thr Val Phe AlaIle Leu Val Glu Ala Leu Glu Arg Gln Gly Val Glu Thr Val Phe Ala

20 25 30 20 25 30

Tyr Pro Gly Gly Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg SerTyr Pro Gly Gly Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser

35 40 45 35 40 45

Ser Thr Ile Arg Asn Val Leu Pro Arg His Glu Gln Gly Gly Val PheSer Thr Ile Arg Asn Val Leu Pro Arg His Glu Gln Gly Gly Val Phe

50 55 60 50 55 60

Ala Ala Glu Gly Tyr Ala Arg Ser Ser Gly Lys Pro Gly Ile Cys IleAla Ala Glu Gly Tyr Ala Arg Ser Ser Ser Gly Lys Pro Gly Ile Cys Ile

65 70 75 8065 70 75 80

Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu Val Ser Gly Leu Ala AspAla Thr Ser Gly Pro Gly Ala Thr Asn Leu Val Ser Gly Leu Ala Asp

85 90 95 85 90 95

Ala Met Leu Asp Ser Val Pro Leu Val Ala Ile Thr Gly Gln Val ProAla Met Leu Asp Ser Val Pro Leu Val Ala Ile Thr Gly Gln Val Pro

100 105 110 100 105 110

Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val GluArg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu

115 120 125 115 120 125

Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu Val Met Asp Val AspVal Thr Arg Ser Ile Thr Lys His Asn Tyr Leu Val Met Asp Val Asp

130 135 140 130 135 140

Asp Ile Pro Arg Ile Val Gln Glu Ala Phe Phe Leu Ala Thr Ser GlyAsp Ile Pro Arg Ile Val Gln Glu Ala Phe Phe Leu Ala Thr Ser Gly

145 150 155 160145 150 155 160

Arg Pro Gly Pro Val Leu Val Asp Val Pro Lys Asp Ile Gln Gln GlnArg Pro Gly Pro Val Leu Val Asp Val Pro Lys Asp Ile Gln Gln Gln

165 170 175 165 170 175

Leu Ala Ile Pro Asn Trp Asp Gln Pro Met Arg Leu Pro Gly Tyr MetLeu Ala Ile Pro Asn Trp Asp Gln Pro Met Arg Leu Pro Gly Tyr Met

180 185 190 180 185 190

Ser Arg Leu Pro Gln Pro Pro Glu Val Ser Gln Leu Gly Gln Ile ValSer Arg Leu Pro Gln Pro Pro Glu Val Ser Gln Leu Gly Gln Ile Val

195 200 205 195 200 205

Arg Leu Ile Ser Glu Ser Lys Arg Pro Val Leu Tyr Val Gly Gly GlyArg Leu Ile Ser Glu Ser Lys Arg Pro Val Leu Tyr Val Gly Gly Gly

210 215 220 210 215 220

Ser Leu Asn Ser Ser Glu Glu Leu Gly Arg Phe Val Glu Leu Thr GlySer Leu Asn Ser Ser Ser Glu Glu Leu Gly Arg Phe Val Glu Leu Thr Gly

225 230 235 240225 230 235 240

Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ser Tyr Pro Cys AsnIle Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ser Tyr Pro Cys Asn

245 250 255 245 250 255

Asp Glu Leu Ser Leu Gln Met Leu Gly Met His Gly Thr Val Tyr AlaAsp Glu Leu Ser Leu Gln Met Leu Gly Met His Gly Thr Val Tyr Ala

260 265 270 260 265 270

Asn Tyr Ala Val Glu His Ser Asp Leu Leu Leu Ala Phe Gly Val ArgAsn Tyr Ala Val Glu His Ser Asp Leu Leu Leu Ala Phe Gly Val Arg

275 280 285 275 280 285

Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg AlaPhe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala

290 295 300 290 295 300

Lys Ile Val His Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys Asn LysLys Ile Val His Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys Asn Lys

305 310 315 320305 310 315 320

Thr Pro His Val Ser Val Cys Gly Asp Val Lys Leu Ala Leu Gln GlyThr Pro His Val Ser Val Cys Gly Asp Val Lys Leu Ala Leu Gln Gly

325 330 335 325 330 335

Met Asn Lys Val Leu Glu Asn Arg Ala Glu Glu Leu Lys Leu Asp PheMet Asn Lys Val Leu Glu Asn Arg Ala Glu Glu Leu Lys Leu Asp Phe

340 345 350 340 345 350

Gly Val Trp Arg Ser Glu Leu Ser Glu Gln Lys Gln Lys Phe Pro LeuGly Val Trp Arg Ser Glu Leu Ser Glu Gln Lys Gln Lys Phe Pro Leu

355 360 365 355 360 365

Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile GlnSer Phe Lys Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln

370 375 380 370 375 380

Ile Leu Asp Glu Leu Thr Glu Gly Lys Ala Ile Ile Ser Thr Gly ValIle Leu Asp Glu Leu Thr Glu Gly Lys Ala Ile Ile Ser Thr Gly Val

385 390 395 400385 390 395 400

Gly Gln His Gln Met Trp Ala Ala Gln Phe Tyr Lys Tyr Arg Lys ProGly Gln His Gln Met Trp Ala Ala Gln Phe Tyr Lys Tyr Arg Lys Pro

405 410 415 405 410 415

Arg Gln Trp Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly LeuArg Gln Trp Leu Ser Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu

420 425 430 420 425 430

Pro Ala Ala Ile Gly Ala Ser Val Ala Asn Pro Asp Ala Ile Val ValPro Ala Ala Ile Gly Ala Ser Val Ala Asn Pro Asp Ala Ile Val Val

435 440 445 435 440 445

Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln Glu Leu AlaAsp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln Glu Leu Ala

450 455 460 450 455 460

Thr Ile Arg Val Glu Asn Leu Pro Val Lys Ile Leu Leu Leu Asn AsnThr Ile Arg Val Glu Asn Leu Pro Val Lys Ile Leu Leu Leu Asn Asn

465 470 475 480465 470 475 480

Gln His Leu Gly Met Val Met Gln Glu Asp Arg Phe Tyr Lys Ala AsnGln His Leu Gly Met Val Met Gln Glu Asp Arg Phe Tyr Lys Ala Asn

485 490 495 485 490 495

Arg Ala His Thr Tyr Leu Gly Asp Pro Ala Arg Glu Asn Glu Ile PheArg Ala His Thr Tyr Leu Gly Asp Pro Ala Arg Glu Asn Glu Ile Phe

500 505 510 500 505 510

Pro Asn Met Leu Gln Phe Ala Gly Ala Cys Gly Ile Pro Ala Ala ArgPro Asn Met Leu Gln Phe Ala Gly Ala Cys Gly Ile Pro Ala Ala Arg

515 520 525 515 520 525

Val Thr Lys Lys Glu Glu Leu Arg Glu Ala Ile Gln Thr Met Leu AspVal Thr Lys Lys Glu Glu Leu Arg Glu Ala Ile Gln Thr Met Leu Asp

530 535 540 530 535 540

Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile Cys Pro His Gln Glu HisThr Pro Gly Pro Tyr Leu Leu Asp Val Ile Cys Pro His Gln Glu His

545 550 555 560545 550 555 560

Val Leu Pro Met Ile Pro Ser Gly Gly Thr Phe Lys Asp Val Ile ThrVal Leu Pro Met Ile Pro Ser Gly Gly Thr Phe Lys Asp Val Ile Thr

565 570 575 565 570 575

Glu Gly Asp Gly Arg Thr Lys TyrGlu Gly Asp Gly Arg Thr Lys Tyr

580 584 580 584

<210> SEQ ID NO.4<210> SEQ ID NO.4

<211> 1755<211> 1755

<212> DNA<212>DNA

<213> 油菜(Brassica napus)<213> Rapeseed (Brassica napus)

<400> 4<400> 4

actttcgtct cccgctacgc tcccgacgag ccccgcaagg gtgctgatat cctcgtcgaa 60actttcgtct cccgctacgc tcccgacgag ccccgcaagg gtgctgatat cctcgtcgaa 60

gccctcgagc gtcaaggcgt cgaaaccgtc tttgcttatc ccggaggtgc ttccatggag 120gccctcgagc gtcaaggcgt cgaaaccgtc tttgcttatc ccggaggtgc ttccatggag 120

atccaccaag ccttgactcg ctcctccacc atccgtaacg tccttccccg tcacgaacaa 180atccaccaag ccttgactcg ctcctccacc atccgtaacg tccttccccg tcacgaacaa 180

ggaggagtct tcgccgccga gggttacgct cgttcctccg gcaaaccggg aatctgcata 240ggaggagtct tcgccgccga gggttacgct cgttcctccg gcaaaccggg aatctgcata 240

gccacttcgg gtcccggagc taccaacctc gtcagcgggt tagcagacgc gatgcttgac 300gccacttcgg gtcccggagc taccaacctc gtcagcgggt tagcagacgc gatgcttgac 300

agtgttcctc ttgtcgccat tacaggacag gtccctcgcc ggatgatcgg tactgacgcc 360agtgttcctc ttgtcgccat tacaggacag gtccctcgcc ggatgatcgg tactgacgcc 360

ttccaagaga caccaatcgt tgaggtaacg aggtctatta cgaaacataa ctatttggtg 420ttccaagaga caccaatcgt tgaggtaacg aggtctatta cgaaacataa ctatttggtg 420

atggatgttg atgacatacc taggatcgtt caagaagctt tctttctagc tacttccggt 480atggatgttg atgacatacc taggatcgtt caagaagctt tctttctagc tacttccggt 480

agacccggac cggttttggt tgatgttcct aaggatattc agcagcagct tgcgattcct 540agacccggac cggttttggt tgatgttcct aaggatattc agcagcagct tgcgattcct 540

aactgggatc aacctatgcg cttacctggc tacatgtcta ggttgcctca gcctccggaa 600aactgggatc aacctatgcg cttacctggc tacatgtcta ggttgcctca gcctccggaa 600

gtttctcagt taggtcagat cgttaggttg atctcggagt ctaagaggcc tgttttgtac 660gtttctcagt taggtcagat cgttaggttg atctcggagt ctaagaggcc tgttttgtac 660

gttggtggtg gaagcttgaa ctcgagtgaa gaactgggga gatttgtcga gcttactggg 720gttggtggtg gaagcttgaa ctcgagtgaa gaactgggga gatttgtcga gcttactggg 720

atccccgttg cgagtacttt gatggggctt ggctcttatc cttgtaacga tgagttgtcc 780atccccgttg cgagtacttt gatggggctt ggctcttatc cttgtaacga tgagttgtcc 780

ctgcagatgc ttggcatgca cgggactgtg tatgctaact acgctgtgga gcatagtgat 840ctgcagatgc ttggcatgca cgggactgtg tatgctaact acgctgtgga gcatagtgat 840

ttgttgctgg cgtttggtgt taggtttgat gaccgtgtca cgggaaagct cgaggctttc 900ttgttgctgg cgtttggtgt taggtttgat gaccgtgtca cgggaaagct cgaggctttc 900

gctagcaggg ctaaaattgt gcacatagac attgattctg ctgagattgg gaagaataag 960gctagcaggg ctaaaattgt gcacatagac attgattctg ctgagattgg gaagaataag 960

acacctcacg tgtctgtgtg tggtgatgta aagctggctt tgcaagggat gaacaaggtt 1020aacacctcacg tgtctgtgtg tggtgatgta aagctggctt tgcaagggat gaacaaggtt 1020

cttgagaacc gggcggagga gctcaagctt gatttcggtg tttggaggag tgagttgagc 1080cttgagaacc gggcggagga gctcaagctt gatttcggtg tttggaggag tgagttgagc 1080

gagcagaaac agaagttccc tttgagcttc aaaacgtttg gagaagccat tcctccgcag 1140gagcagaaac agaagttccc tttgagcttc aaaacgtttg gagaagccat tcctccgcag 1140

tacgcgattc agatcctcga cgagctaacc gaagggaagg caattatcag tactggtgtt 1200tacgcgattc agatcctcga cgagctaacc gaagggaagg caattatcag tactggtgtt 1200

ggacagcatc agatgtgggc ggcgcagttt tacaagtaca ggaagccgag acagtggctg 1260ggacagcatc agatgtgggc ggcgcagttt tacaagtaca ggaagccgag acagtggctg 1260

tcgtcatcag gcctcggagc tatgggtttt ggacttcctg ctgcgattgg agcgtctgtg 1320tcgtcatcag gcctcggagc tatgggtttt ggacttcctg ctgcgattgg agcgtctgtg 1320

gcgaaccctg atgcgattgt tgtggatatt gacggtgatg gaagcttcat aatgaacgtt 1380gcgaaccctg atgcgattgt tgtggatatt gacggtgatg gaagcttcat aatgaacgtt 1380

caagagctgg ccacaatccg tgtagagaat cttcctgtga agatactctt gttaaacaac 1440caagagctgg ccacaatccg tgtagagaat cttcctgtga agatactctt gttaaacaac 1440

cagcatcttg ggatggtcat gcaagaagat cggttctaca aagctaacag agctcacact 1500cagcatcttg ggatggtcat gcaagaagat cggttctaca aagctaacag agctcacact 1500

tatctcgggg acccggcaag ggagaacgag atcttcccta acatgctgca gtttgcagga 1560tatctcgggg acccggcaag ggagaacgag atcttcccta acatgctgca gtttgcagga 1560

gcttgcggga ttccagctgc gagagtgacg aagaaagaag aactccgaga agctattcag 1620gcttgcggga ttccagctgc gagagtgacg aagaaagaag aactccgaga agctattcag 1620

acaatgctgg atacaccagg accatacctg ttggatgtga tatgtccgca ccaagaacat 1680acaatgctgg atacaccagg accatacctg ttggatgtga tatgtccgca ccaagaacat 1680

gtgttaccga tgatcccaag tggtggcact ttcaaagatg taataacaga aggggatggt 1740gtgttaccga tgatcccaag tggtggcact ttcaaagatg taataacaga aggggatggt 1740

cgcactaagt actga 1755cgcactaagt actga 1755

Claims (10)

1.具有抗除草剂活性的油菜蛋白质,其为油菜乙酰羟基酸合成酶的突变体蛋白质,其氨基酸序列如SEQ ID No.1或SEQ ID No.3所示。1. Rapeseed protein with herbicide-resistant activity, which is a mutant protein of rapeseed acetohydroxyacid synthase, and its amino acid sequence is shown in SEQ ID No.1 or SEQ ID No.3. 2.具有抗除草剂活性的基因,其为编码权利要求1所述蛋白质的核苷酸序列。2. A gene with herbicide resistance activity, which is a nucleotide sequence encoding the protein of claim 1. 3.如权利要求2所述的具有抗除草剂活性的基因,其特征在于,所述核苷酸序列如SEQID No.2或SEQ ID No.4所示。3. The gene with herbicide resistance activity according to claim 2, characterized in that said nucleotide sequence is as shown in SEQ ID No.2 or SEQ ID No.4. 4.如权利要求1具有抗除草剂活性的油菜蛋白质用于培育抗咪唑啉酮类、磺酰脲类和嘧啶类除草剂植物。4. The rapeseed protein having herbicide-resistant activity as claimed in claim 1 is used for cultivating plants resistant to imidazolinones, sulfonylureas and pyrimidine herbicides. 5.一种获得抗咪唑啉酮类、磺酰脲类和嘧啶类除草剂植物的方法,包括,将权利要求1所述油菜蛋白质的编码基因转化到植物中,使所述植物产生具有抗除草剂活性的油菜乙酰羟基酸合成酶的突变体蛋白质。5. A method for obtaining plants resistant to imidazolinones, sulfonylureas and pyrimidine herbicides, comprising, transforming the coding gene of rapeseed protein according to claim 1 into plants, so that said plants produce herbicide-resistant Agent active mutant protein of rapeseed acetohydroxyacid synthase. 6.根据权利要求5所述获得抗咪唑啉酮类、磺酰脲类和嘧啶类除草剂植物的方法,其特征在于,所述编码基因的核苷酸序列如SEQ ID No.2或SEQ ID No.4所示。6. according to claim 5, obtain the method for resistant imidazolinones, sulfonylureas and pyrimidine herbicide plants, it is characterized in that, the nucleotide sequence of described coding gene is as SEQ ID No.2 or SEQ ID Shown in No.4. 7.根据权利要求5所述获得抗咪唑啉酮类、磺酰脲类和嘧啶类除草剂植物的方法,其特征在于,所述咪唑啉酮类除草剂为咪唑乙烟酸、甲氧咪草烟和甲基咪草烟中的至少一种。7. according to the method for obtaining imidazolinones, sulfonylureas and pyrimidine herbicide plants according to claim 5, it is characterized in that, described imidazolinones herbicides are imazethapyr, imazethapyr and formazan At least one of imazamox. 8.根据权利要求5所述获得抗咪唑啉酮类、磺酰脲类和嘧啶类除草剂植物的方法,其特征在于,所述磺酰脲类除草剂为氯磺隆、吡嘧磺隆、氯嘧磺隆和甲嘧磺隆中的至少一种。8. obtain the method for resistant imidazolinones, sulfonylureas and pyrimidine herbicide plants according to claim 5, it is characterized in that, described sulfonylurea herbicides are chlorsulfuron-methyl, pyrazosulfuron-methyl, At least one of chlorimuron-methyl and metsulfuron-methyl. 9.根据权利要求5所述获得抗咪唑啉酮类、磺酰脲类和嘧啶类除草剂植物的方法,其特征在于,所述嘧啶类除草剂为双草醚。9. The method for obtaining plants resistant to imidazolinones, sulfonylureas and pyrimidine herbicides according to claim 5, wherein the pyrimidine herbicide is bispyribac. 10.根据权利要求5-9任一项所述获得抗咪唑啉酮类、磺酰脲类和嘧啶类除草剂植物的方法,其特征在于,所述植物为油菜、玉米或棉花。10. The method for obtaining plants resistant to imidazolinones, sulfonylureas and pyrimidine herbicides according to any one of claims 5-9, wherein the plants are rapeseed, corn or cotton.
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Application publication date: 20170531