WO2019024534A1 - Rice als mutant protein for conferring herbicide resistance to plants, and use thereof - Google Patents

Abstract

Disclosed are a rice ALS mutant protein for conferring herbicide resistance to plants, and a nucleic acid encoding the protein. The protein is derived from a rice mutant plant resistant to an ALS inhibitor herbicide, and compared to the ALS sequence in the genome of wild type rice, the protein sequence thereof is mutated at the Pro171 site and/or Asp350 site. The experiment results of spraying the ALS inhibitor herbicide, "Imazameth", in fields show that rice seedlings with 3-4 leaves and containing the herbicide-resistant ALS protein still grow normally and fructify after applying 3.3 mL of Imazameth/L of water, whereas wild type rice seedlings with 3-4 leaves show whole plant death after applying 1 mL of Imazameth/3 L of water for 30 days.

Description

Herbicide-resistant rice ALS mutant protein and application thereof Technical field

The invention belongs to the field of plant protein and plant herbicide resistance, relates to rice ALS mutant protein, gene and application thereof for herbicide resistance; in particular, the invention relates to rice acetolactate synthase (ALS) mutein The protein confers properties to plants, especially rice anti-acetolactate synthase inhibitor herbicides. The present invention discloses the sequences of the proteins and their use in the field of plant herbicide resistance.

Background technique

Weeds are unfavorable factors that restrict the stable production and high yield of agricultural production. The use of chemical herbicides is an efficient, simple and economical method of weed control compared to traditional methods of relying on cultivation, manual weeding and mechanical weeding.

The biosynthesis of plant and microbial branched-chain amino acids (valine, leucine and isoleucine) requires four enzymes to co-catalyze, acetolactate synthase (ALS), ketol reductase (ketol) -acid reductoisomerase), dihydroxyavid dehydratase, branched-chain amino acid transaminase. Acetolactate synthase is a key enzyme in the first stage of biosynthesis. In the synthesis of proline and leucine, two molecules of pyruvic acid are catalyzed to form acetolactate and carbon dioxide, and one molecule of acetone is catalyzed in the synthesis of isoleucine. The acid and one molecule of alpha butanone acid form 2-acetaldehyde-2-hydroxybutyric acid and carbon dioxide. ALS inhibitor herbicides inhibit the synthesis of branched-chain amino acids by inhibiting the activity of ALS enzymes in plants, leading to the destruction of protein synthesis, hindering DNA synthesis during cell division, and thus stopping the mitosis of plant cells in the G1 stage. The phase (DNA synthesis phase) and the M phase of the G2 phase interfere with the synthesis of DNA, and thus the cells cannot complete mitosis, which in turn causes the plants to stop growing, eventually leading to the death of the individual plants.

Inflammatory lactic acid 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 (SU), Imidazolinones (IMI), Triazolopyrimidines (TP), Pyrimidinylthio(or oxy)-benzoates, PTB; pyrimidinyl-carboxyherbicides; PCs and sulfonamide carbonyls 13 compounds such as Sulfonylamino-carbonyltriazolinones (SCT). Acetolactate synthase, which is present in the growth process of plants, can catalyze the pyruvate as acetolactate with high specificity and high catalytic efficiency, resulting in the biosynthesis of branched chain amino acids.

ALS inhibitor herbicides have the characteristics of strong selectivity, wide herbicidal spectrum, low toxicity and high efficiency, and have been widely used in large areas. However, these herbicides also cause phytotoxicity to crops which generally do not have the resistance to herbicides, which greatly limits their use time and space for use. For example, it is necessary to use herbicides for a period of time before crops are planted to avoid crops suffering from medicines. harm. Breeding resistant (resistance) herbicide crop varieties can reduce crop phytotoxicity and broaden the use of herbicides.

Currently, known anti-ALS inhibitor mutation sites in rice include Gly 95, Ala 96, Trp 548, and Ser 627. The level of ALS mutant herbicide resistance is related to the location of the ALS amino acid mutation and also to the number of amino acids after mutation and the number of mutant amino acids.

At present, the mechanism of action of ALS inhibitor herbicides has not been determined. It is difficult to predict in advance whether mutations in other amino acid sites of ALS protein will produce herbicide resistance. It can only rely on long-term and painstaking practice of researchers, and rely on some Luck can discover new herbicide resistance sites for ALS proteins.

Summary of the invention

OBJECT OF THE INVENTION The first technical problem to be solved by the present invention is to provide a protein which confers herbicide resistance to plants.

The technical problem to be solved by the present invention is to provide a nucleic acid encoding the protein, as well as an expression cassette, a vector, a cell and the like.

A technical problem to be solved by the present invention is to provide a method and application for obtaining a herbicide resistant plant.

A technical problem to be solved by the present invention is to provide an identification method for judging whether or not a plant is obtained by the method of the present invention.

Through long-term and arduous research, the present inventors have long-term and continuous screening of EMS mutant plants of the wild type rice, the conventional rice seed Heterogeneous sterile line, the common type rice seed R89, and the wild type Jiahua No. 1 rice. A series of ALS muteins have been discovered, including some of the known ALS muteins and novel ALS muteins described above, which are insensitive to ALS inhibitor herbicides, thereby rendering plants resistant to ALS inhibitor herbicides. The use of the present invention in plant breeding can be used to grow plants having herbicide resistance, especially crops, and the development of these proteins and their coding genes in transgenic or non-transgenic plants such as rice.

Technical Solution: In order to solve the above technical problems, the technical scheme adopted by the present invention is as follows: a plant having a herbicide-resistant rice ALS mutant protein, and the amino acid sequence of the ALS mutant protein has any one or more of the following mutations: It corresponds to mutation at amino acid 171 and/or amino acid 350 of the rice ALS mutant protein.

Among them, rice ALS mutant proteins which confer herbicide resistance to plants include:

(a) having an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 8 or SEQ ID NO: 12;

(b) A protein derived from (a) wherein the amino acid sequence in (a) is substituted and/or deleted and/or one or more amino acids are added and has acetolactate synthase activity.

Among them, the herbicide is an imidazolinone herbicide.

The present invention also encompasses nucleic acids or genes encoding the proteins.

The above nucleic acids or genes, including:

(a) it encodes the protein; or

(b) a nucleotide sequence which hybridizes under stringent conditions to (a) a defined nucleotide sequence and which encodes a protein having acetolactate synthase activity;

(c) Its nucleotide sequence is shown as SEQ ID NO: 1 or SEQ ID NO: 7 or SEQ ID NO: 11.

The stringent conditions are: highly stringent hybridization conditions in which a membrane is washed at 65 ° C for 15 minutes in a solution of 0.1 x SSC and 0.1% SDS.

The present invention also provides an ALS protein which confers herbicide resistance to a plant, wherein the amino acid at position 171 of the amino acid sequence is changed from proline to histidine, and the amino acid at position 350 is mutated from aspartic acid to glutamic acid. There have been no reports of mutants that are simultaneously mutated at the Pro171 and Asp350 sites of the ALS protein from the rice cultivar Hefei sterile line.

Preferably, the protein of the first aspect of the invention carries a mutant strain in which the Pro171 and Asp350 sites are simultaneously mutated, in addition to the mutant plant screening or amplification method described in the embodiment of the present invention, in the case where the protein sequence is known Those skilled in the art have the ability to prepare proteins substituted, added or deleted with amino acid residues by changing the coding sequence of a known protein and introducing it into a vector, and these methods are 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. The protein has been shown to be insensitive to the imidazolinone herbicide acetolactate synthase.

The invention also includes providing a nucleic acid or gene encoding the protein of the first aspect of the invention. In the present invention, the nucleic acid may be DNA or RNA, of which DNA is preferred. Those skilled in the art will be able to obtain and optimize the coding of the present invention by using a conventional codon correspondence and host expression frequency, using a PCR method, a DNA recombination method, or a synthetic method, knowing the encoded protein sequence or nucleic acid sequence. 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 by 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 invention is as shown in SEQ ID NO. Specifically, the DNA sequence of the present invention is the 512th nucleotide of the ALS gene sequence of the wild type rice cultivar conventional rice seed Heterogeneic line from C to nucleotide A, nucleotide 1050. From T to nucleotide A.

In addition, the wild-type rice before the mutation of the present invention is a conventional rice seed-fertilized sterile line of the indica type, and the nucleotide sequence of the wild type-fertility rice ALS of the ALS inhibitor herbicide-sensitive herbicide is as shown in SEQ ID NO. The amino acid is shown in SEQ ID NO.

The invention also includes an expression cassette, a recombinant vector or a cell comprising the nucleic acid or gene described above.

The present invention also encompasses the use of the herbicide-resistant ALS protein, nucleic acid or gene, expression cassette, recombinant vector or cell described above for plant herbicide resistance.

Among them, the above plants are rice, tobacco, Arabidopsis, cotton, etc., but are not limited to the above plants.

The invention also includes a method of obtaining a herbicide resistant plant comprising the steps of:

1) causing the plant to comprise a nucleic acid or gene according to the invention; or

2) causing the plant to express the protein.

Among them, the above methods include transgenic, hybrid, backcross or asexual reproduction steps.

The invention also includes a method of identifying a plant, wherein the plant is a plant comprising the nucleic acid, a plant expressing the protein, or a plant obtained by the method, specifically comprising the steps of:

1) determining whether the plant comprises the nucleic acid or gene; or

2) Determine whether the plant expresses the protein.

The present invention also encompasses ALS proteins which confer herbicide resistance to plants which are mutated from aspartic acid to glutamic acid only at amino acid position 350 of the amino acid sequence. The amino acid sequence of the ALS protein is shown in SEQ ID NO. Specifically, the DNA sequence of the present invention is that the nucleotide of the 1050th ALS gene sequence of the wild type rice is a 籼 type conventional rice seed R89 is changed from T to nucleotide A, and the nucleotide sequence is SEQ ID NO. .

The nucleotide sequence of the ALS of the wild-type indica type conventional rice seed R89 of the present invention is shown in SEQ ID NO. 9, and the amino acid sequence is shown in SEQ ID NO.

The present invention also encompasses ALS proteins which confer herbicide resistance to plants, which only change from valine to leucine at amino acid position 171 of the amino acid sequence. The amino acid sequence of the ALS protein is shown in SEQ ID NO. Specifically, the DNA sequence of the present invention is the nucleotide number 512 of the ALS gene sequence of the wild type rice cultivar conventional rice seed Jiahua No. 1 changed from C to T, and the nucleotide sequence is SEQ ID NO. .

The nucleotide sequence of the wild-type Jiahua No. 1 rice ALS of the present invention is shown in SEQ ID NO. 13, and the amino acid sequence is shown in SEQ ID NO.

The invention also includes a method of controlling weeds comprising: applying an effective amount of a herbicide to a field in which the crop is grown, the crop comprising the nucleic acid or gene or the expression cassette, recombinant vector or cell, The herbicide is an imidazolinone herbicide.

The present invention also encompasses a method for protecting a plant from damage caused by a herbicide, comprising: applying an effective amount of a herbicide to a field in which the crop is grown, the crop comprising the nucleic acid or gene or the described The expression cassette and the recombinant vector are introduced into the plant, and the introduced plant produces a herbicide resistance protein, and the herbicide is an imidazolinone herbicide.

The present invention also encompasses a herbicide protectant comprising the ALS mutant protein.

Advantageous Effects: Compared with the prior art, the present invention has the following advantages:

1) The results of the experiment of spraying the ALS inhibitor herbicide "Bai Luntong" in the field of the present invention indicate that the rice 3-4 seedlings containing the herbicide-resistant ALS protein of the present invention are administered with 3.3 mL of ridges. /L water (10 times recommended concentration), the plant is still normal growth and development, while the wild type rice 3-4 seedlings are treated as whole after 30 days of application of 1 mL of ridges/3 L of water (1 times recommended concentration). The plant died.

2) The ALS enzyme activity of the transgenic rice containing the ALS gene of the Hefei sterile line herbicide resistant mutant of the present invention is 4.7 times higher than that of the wild type ALS, and the ALS containing the Hefei sterile line herbicide resistant mutant of the present invention The ALS activity of the transgenic tobacco progeny plants of the gene was 5.2 times higher than that of the wild type ALS.

3) The Hefei sterile line mutant of the present invention and the herbicide-resistant mutant plant of CGMCC No. 12265 in Patent No. 201610226003.6 were subjected to ALS activity assay, and the ALS enzyme activity ratio of the Hefei sterile line mutant in the present patent application was found. CGMCC No.12265 is 18% higher.

4) The ALS enzyme activity of the transgenic rice containing the ALS gene resistant to herbicide mutant 2 and mutant 3 of the present invention is 4.96-5.25 times higher than that of the wild type ALS, and the herbicide-containing mutant 2 and mutation of the present invention are contained. The ALS enzyme activity of the transgenic tobacco progeny plants of the ALS gene of the body 3 was 5.84-6.3 times higher than that of the wild type ALS enzyme activity.

5) The herbicide-resistant mutant 2 and mutant 3 of the present invention and the herbicide-resistant mutant plant of CGMCC No. 12265 of Patent No. 201610226003.6 were subjected to ALS activity assay, and 2 and mutant 3 of the present patent application were found. The ALS enzyme activity was 15.7%-18.6% higher than CGMCC No. 12265.

DRAWINGS

Figure 1 shows a resistant rice mutant obtained by screening a herbicide;

Figure 2 is a graph showing the results of PCR amplification of ALS genes. A, the first lane is Marker; the molecular weight of Marker is 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp from top to bottom, and the second lane is Hefei sterile wild-type rice DNA, and the third lane is anti- The DNA of the herbicide mutant, the target fragment was 2091 bp in length. B, the first lane is Marker; Marker molecular weight is 5kb, 3kb, 2kb, 1.5kb, 750bp, 500bp, 250bp from top to bottom, the second lane is R89 wild type rice DNA, and the third lane is R89 herbicide resistance mutation. DNA of the body, the fourth lane is the wild type rice DNA of Jiahua No.1, and the fifth lane is the DNA of the Jiahua No.1 herbicide resistant mutant, and the target fragment is 2091 bp in length;

Figure 3. Determination of the absorbance of ALS enzyme activity inhibition of wild-type and herbicide-resistant mutants by Bailangtong herbicide;

Fig. 4 BamHI/SacI double-cut restriction analysis of the expression vector of ALS gene resistant to herbicide mutant; A, the first lane is Marker; the molecular weight of Marker is 5 kb, 3 kb, 2 kb, 1 kb, 750 bp, 500 bp from top to bottom. 250bp, 100bp; the second lane is the undigested recombinant vector, and the third lane is the Hess sterile line mutant ALS gene fragment and plasmid fragment DNA produced by double digestion of the recombinant vector by BamHI/SacI. It is expected that the vector is successfully constructed; B, the first lane is Marker; the molecular weight of Marker is 15kb, 8kb, 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp from top to bottom; the second lane is uncut. The recombinant vector, Lane 3 is the R89 mutant ALS gene fragment and plasmid fragment DNA produced by double digestion of the recombinant vector by BamHI/SacI, and the fourth lane is the Jiahua No. 1 produced by double digestion of the recombinant vector by BamHI/SacI. Mutant ALS gene fragment and plasmid fragment DNA, the size of the gene fragment was in line with expectations, which proved that the vector was successfully constructed;

Fig.5 PCR detection map of mutant ALS gene rice; A, the first lane is Marker; Marker molecular weight is 5kb, 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 a plasmid DNA containing the mutant sequence of Hefei sterile line ALS as a template, and as a positive control, lanes 4 to 6 are transgenic plants DNA transformed with the herbicide-resistant mutant ALS gene; B, first The 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, and the third lane is plasmid DNA containing the R89 ALS mutation sequence. As a template, as a positive control, the fourth lane is a plasmid DNA containing the Jiahua No. 1 ALS mutation sequence as a template, and as a positive control, lanes 5 to 6 are transgenic plant DNAs which transform the R89 herbicide-resistant mutant ALS gene; Lanes 7-8 are transgenic plant DNAs that transform the Jiahua No. 1 herbicide-resistant mutant ALS gene;

Fig. 6 Determination of the absorbance of ALS activity of wild type rice and transgenic rice containing herbicide resistant mutant ALS gene by Baidoutong herbicide;

Figure 7 is a graph showing the determination of the absorbance of ALS enzyme activity in wild type tobacco and transgenic tobacco containing the herbicide resistant mutant ALS gene;

Figure 8 is a graph showing the determination of the absorbance of ALS enzyme activity against herbicide mutants and CGMCC No. 12265;

Fig. 9 Jiahua No. 1 resistant rice mutant obtained by screening Baidoutong herbicide;

Fig. 10 R89 resistant rice mutant obtained by screening of ridges and herbicides;

Figure 11 is a graph showing the determination of the absorbance of ALS enzyme activity against wild type and herbicide resistant mutant 2 and herbicide resistant mutant 3;

Figure 12: Determination of the absorbance of ALS activity of wild type rice, transgenic rice containing herbicide resistant mutant 2 ALS gene and transgenic rice containing herbicide resistant mutant 3 ALS gene;

Figure 13 is a graph showing the determination of the absorbance of ALS activity of wild type tobacco and transgenic tobacco containing wild type tobacco and ALS gene containing herbicide resistant mutant 2 and herbicide resistant mutant 3;

Figure 14. Determination of the absorbance of ALS enzyme activity against herbicide mutant 2, herbicide resistant mutant 3 and CGMCC No. 12265.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, however, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

Example 1: Obtaining the sequence of rice Hefei sterile line, Jiahua No.1 and R89 wild type

The primers were designed for amplification by Nipponbare ALS gene (NCBI: XM_015770973) in NCBI, and the ALS gene of rice Heterogene sterile line, Jiahua No.1 and R89 wild type was amplified by Takara PrimerSTAR Max DNA Polymerase polymerase (purchased from Takara). The reaction system is as follows:

2×PrimerSTAR Max Premix 10.0μl

10μM forward primer 1.0μl

10μM reverse primer 1.0μl

20-30ng/μL rice genomic DNA 1.0μl

Add sterile water to a total volume of 20μl

The PCR amplification reaction procedure uses a two-step process, annealing and extension together, using 68 degrees.

The procedure was as follows: pre-denaturation: 98 ° C for 3 min; 35 cycles: denaturation 98 ° C for 10 sec; extension 68 ° C for 2 min; incubation: 72 ° C for 10 min.

2 μl of the PCR product was detected by 1% agarose gel electrophoresis. After the fragment of the expected size was found, the remaining PCR product was cleaned and recovered by PCR cleaning kit (purchased from Axygen) and cloned into pMD19-T vector (purchased from pMD19-T vector) Takara), then transformed E. coli. Each transformation randomly picked 12 E. coli monoclonals for PCR detection, and took 6 monoclonal clones positive for PCR. They were sent to Nanjing Biotech Co., Ltd. for sequencing, and the mutant ALS gene sequence was obtained. The length of the gene fragment was 2091 bp.

The sequencing results of the wild type ALS sequence gene fragment of Hefei sterile line are as follows:

The sequencing results of the R89 wild type ALS sequence gene fragment are as follows:

The sequencing results of the Jiahua No. 1 wild type ALS sequence gene fragment are as follows:

Example 2: Rice imidazole ketone herbicide mutant acquisition process (Hundred Ridge)

1. Indica type conventional rice seed Hefei sterile line (Hua K01S, provided by Anhui Huakai Agricultural Science and Technology Co., Ltd.) (this is M0, soaked in water for 2 hours) 150kg divided into 6 times with 0.5-1.0% (w/w) Ethyl methanesulfonate (EMS) was immersed for 6-9 hours at room temperature, during which the seeds were shaken every 1 hour; the EMS solution was discarded, the seeds were immersed in tap water for 5 times for 5 minutes, and then the seeds were rinsed overnight with tap water. Carry out field planting and carry out conventional fertilizer management (this is M1). After the plants are mature, the seeds are mixed, dried, and preserved in winter. Sowing the fields the following year. When the seedlings of rice (this is M2) grow to the 3-4 leaf stage, the spray is 3.3 mL of ridges/L water ("Bailutong" is a water-based imidazolinone herbicide produced by BASF, Germany. The recommended minimum concentration is 1 mL of ridges/1.5 to 3 L of water), and after 30 days, normal green plants are also rice mutants resistant to imidazolinone herbicides (Fig. 1). A total of 128 M2 plants with herbicide resistance were obtained. These resistant plants were managed by conventional fertilizer and water. 101 M2 plants were normal and firm. After the seeds were mature, the plants were harvested, dried, and preserved in winter.

2. The conventional rice seed R89 (provided by Anhui Luyi Seed Co., Ltd.) and the conventional rice seed Jiahua No. 1 (a gift from Jiangsu Agricultural Germplasm Resources Protection and Utilization Platform) (this is M0, soaked in water) 2 hours) 150kg divided into 6 times with 0.5-1.0% (w/w) ethyl methanesulfonate (EMS) for 6-9 hours at room temperature, during which the seeds are shaken every 1 hour; the EMS solution is discarded, and the tap water is soaked to soak the seeds. 5 times, 5 minutes each time, then rinse the seeds with tap water overnight, seeding the field the next day, and carry out conventional fertilizer management (this is M1). After the plants are mature, the seeds are mixed, dried, and preserved in winter. Sowing the fields the following year. When the seedlings of rice (this is M2) grow to the 3-4 leaf stage, the spray is 3.3 mL of ridges/L water ("Bailutong" is a water-based imidazolinone herbicide produced by BASF, Germany. The recommended minimum concentration is 1 mL of ridges/1.5 to 3 L of water), and after 30 days, normal green plants are also rice mutant 2 and mutant 3 resistant to imidazolinone herbicides (Fig. 9, Fig. 10). After the herbicide-resistant mutant 2 and mutant 3 seeds are mature, the individual plants are harvested, dried, and preserved in winter.

Example 3: Analysis of mutation sites of rice mutants resistant to imidazolinone herbicides

The herbicide-tolerant rice mutant plants obtained in the above Example 2 were selected from the leaves of the mutant plants, and the genomic DNA was extracted and sent to Nanjing Biotechnology Co., Ltd. for genome sequencing. Compared with the wild-type ALS gene of Hefei sterile line, the sequencing results showed that the herbicide-resistant rice mutant had two mutations in the ALS gene, and the bases of the 512th and 1050th mutations were changed from C to A. , T becomes A, resulting in the 171, 350 position of the corresponding encoded amino acid sequence from valine to histidine, asparagine to glutamic acid, that is, the nucleus of the ALS gene resistant to herbicide mutants The nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded ALS protein is shown in SEQ ID NO.

The herbicide-resistant mutant 2 obtained in the above Example 2 was selected from the leaves of the mutant plants, and the genomic DNA was extracted and sent to Nanjing Biotechnology Co., Ltd. for genome sequencing. Compared with the conventional rice seed R89 wild-type ALS gene, the sequencing results showed that the herbicide-resistant rice mutant 2 only had one site mutation in the ALS gene, and the 1050th base mutation occurred only by T. Into A, the 350th position of the corresponding encoded amino acid sequence is changed from asparagine to glutamic acid, that is, the nucleotide sequence of the ALS gene resistant to herbicide mutant 2 is as shown in SEQ ID NO. The amino acid sequence of the encoded ALS protein is set forth in SEQ ID NO.

The herbicide-resistant mutant 3 obtained in the above Example 2 was selected from the leaves of the mutant plants, and the genomic DNA was extracted and sent to Nanjing Biotechnology Co., Ltd. for genome sequencing. The sequencing results were compared with the wild type ALS gene of the conventional rice seed Jiahua No.1, and it was found that the herbicide-resistant rice mutant 3 had a mutation at the ALS gene, and the 512th base was mutated. C becomes T, resulting in the 171th position of the corresponding encoded amino acid sequence being changed from valine to leucine, that is, the nucleotide sequence of the ALS gene resistant to herbicide mutant 3 is as shown in SEQ ID NO. The amino acid sequence of the encoded ALS protein is shown in SEQ ID NO.

The herbicide-tolerant rice mutant of the present invention is classified as rice seed HF-407 (Oryza sativa Indica Group HF-407), which was deposited with the China Center for Type Culture Collection (CCTCC) on June 2, 2017. Address: Wuhan University Depository Center, Wuchang District, Wuhan City, Hubei Province (opposite to the First Affiliated Primary School of Wuhan University), Zip Code: 430072, with the accession number CCTCC No: P201714.

The R89 herbicide resistant rice mutant 2 of the present invention is classified as rice seed R28 (Oryza sativa Indica Group R28), and the material has been deposited with the China Center for Type Culture Collection (CCTCC) on July 30, 2017, Address: Hubei Wuhan University Wuchang District Wuhan University Depository Center (opposite to the First Affiliated Primary School of Wuhan University), Zip Code: 430072, with the accession number CCTCC No: P201718.

The Jiahua No. 1 herbicide resistant rice mutant 3 of the present invention is classified as rice seed JH10 (Oryza sativa Japonica Group JH10), and the material has been deposited with the China Center for Type Culture Collection (CCTCC) on July 30, 2017. Address: Wuhan University Depository Center, Wuchang District, Wuhan City, Hubei Province (opposite to the First Affiliated Primary School of Wuhan University), Zip Code: 430072, with the accession number CCTCC No: P201720.

Example 4: Anti-imidazolinone herbicide rice mutant ALS gene cloning

Genomic DNA was extracted from the herbicide-resistant rice mutant, the herbicide-resistant rice mutant 2, and the herbicide-resistant rice mutant 3, respectively. The specific primers for the full length of the ALS gene were designed based on the wild type ALS gene sequence of Hefei sterile line, the conventional rice seed R89 wild type ALS gene, and the conventional rice seed Jiahua No. 1 wild type ALS gene: forward primer F5'-TCGCCCAAACCCAGAAACCC-3', reverse primer R 5'-CTCTTTATGGGTCATTCAGGTC-3'.

The ALS gene was amplified using Takara PrimerSTAR Max DNA Polymerase polymerase (purchased from Takara), and the reaction system was as follows:

The PCR amplification reaction procedure uses a two-step process, annealing and extension together, using 68 degrees.

The procedure was as follows: pre-denaturation: 98 ° C for 3 min; 35 cycles: denaturation 98 ° C for 10 sec; extension 68 ° C for 2 min; incubation: 72 ° C for 10 min.

2 μl of the PCR product was detected by 1% agarose gel electrophoresis and found to have the expected size of the fragment (Fig. 2A and Fig. 2B). The remaining PCR product was cleaned and recovered by PCR cleaning kit (purchased from Axygen) and cloned to The pMD19-T vector (purchased from Takara) was then transformed into E. coli. Each transformation randomly picked 12 E. coli monoclonals for PCR detection, and took 6 monoclonal clones positive for PCR. They were sent to Nanjing Biotech Co., Ltd. for sequencing, and the mutant ALS gene sequence was obtained. The length of the gene fragment was 2091 bp. The sequencing results of the sterile line herbicide-resistant rice mutant 1 ALS gene fragment are as follows:

The sequencing results of the ALS gene fragment of R89 herbicide resistant rice mutant 2 are as follows:

The sequencing results of the ALS gene fragment of Jiahua No. 1 herbicide resistant rice mutant 3 are as follows:

Example 5: Rice M3 mutant anti-imidazolinone herbicide (Hundred Ridge)

Seeds of Hefei sterile line herbicide resistant mutants were sown and emerged (this is M3). When M3 rice seedlings grow to 3-4 leaf stage, spray 3.3 mL of ridges/L water (the recommended minimum concentration is 1 mL of ridges / 3 L of water, equivalent to 10 times the concentration). After 15 days of spraying the herbicide, the resistant seedling M3 was normal green and could continue to grow to 20-30 cm. The non-resistant seedling leaves lost green or even partially yellow, and the plant did not grow tall, only 5-9 cm. After 30 days, the M3 resistant strains were all normal green plants, while the wild type rice sprayed with the same concentration of herbicides had all died, indicating that the rice mutants were resistant to at least 10 times the concentration of the thyroid herbicide.

The seeds of the herbicide-resistant rice mutant 2 and the herbicide-resistant rice mutant 3 were separately sown (this is M3), and when the M3 rice seedlings were grown to the 3-4 leaf stage, 3.3 mL of ridges/L was sprayed. Water (recommended minimum concentration is 1mL ridges / 3L water, equivalent to 10 times concentration). After 15 days of spraying the herbicide, the resistant seedling M3 was normal green and could continue to grow to 20-30 cm. The non-resistant seedling leaves lost green or even partially yellow, and the plant did not grow tall, only 5-9 cm. After 30 days, the M3 resistant strains were all normal green plants, and the wild type rice sprayed with the same concentration of herbicides all died, indicating that the herbicide resistant rice mutant 2 and the herbicide resistant rice mutant 3 were all resistant to at least 10 A multi-concentration of the ridge-passing herbicide.

Example 6: Determination of ALS activity in rice M3 herbicide resistant mutants

In order to verify whether the herbicide resistance of the rice mutant is caused by the ALS mutation, the inventors performed the ALS enzyme activity assay. For the measurement method, refer to the method of Singh et al. (Singh B. K., Stidham M. A., Shaner D. L. Assay of acetohydroxyacid synthase. Analytical Biochemistry, 1988, 171: 173-179.). Specifically, 0.2 g of wild type, Hefei male sterile line herbicide resistant mutant, herbicide resistant mutant 2 and herbicide resistant mutant 3 M3 plants were ground and ground in a mortar with liquid nitrogen, and 2 mL was added. Extract (100 mM K2HPO4, pH 7.5, 10 mM sodium pyruvate, 5 mM EDTA, 1 mM valine, 1 mM leucine, 10 mM cysteine, 0.1 mM flavin adenine dinucleotide, 5 mM magnesium chloride, 10% ( V/V) glycerin, 1% (w/v) polyvinylpyrrolidone), after the extract was thawed, it was further milled for about 1 min. Centrifuge at 12000 rpm, 4 ° C for 30 min, aspirate the supernatant, add ammonium sulfate to 50% saturation, place on ice for half an hour, centrifuge at 12000 rpm, 4 ° C for 30 min, discard the supernatant, and dissolve the precipitate in 0.2 mL of reaction buffer. (100 mM K2HPO4, pH 7.0, 1 mM EDTA, 10 mM magnesium chloride, 100 mM sodium pyruvate, 1 mM thiamine pyrophosphate, 0.1 mM flavin adenine dinucleotide), and ALS extracts of each plant were obtained.

Add 10 μL of herbicide “Bai Lian Tong” (water agent, active ingredient 240 g/L) to the ALS extract, mix, incubate at 37 ° C for 1 h, add 0.1 ml of 3 M sulfuric acid to terminate the reaction, and react the reaction mixture at 60 ° C. 30 minutes for decarboxylation. Then 0.4 mL of the color developing solution (0.09 g/L 1-naphthol and 0.009 g/L creatine was dissolved and dissolved in 2.5 M NaOH). The mixture was incubated at 37 ° C for 30 minutes for color development (ALS catalyzed the formation of acetolactate by two pyruvic acids, decarboxylation of acetolactate to form 3-hydroxybutanone, and then formed a pink complex with creatine and 1-naphthol, the complex The maximum absorption value at 530 nm), followed by measuring the absorbance at 530 nm, ALS activity is expressed by the absorbance of A530, and the level of absorbance of A530 reflects the level of ALS activity. In the experiment, water was used as a control, and wild type, Hefei sterile line herbicide resistant mutant, herbicide resistant mutant 2 and herbicide resistant mutant 3 mutant were tested for 5 individuals.

A530 absorbance measurement results showed that when the ALS extract of wild-type and Hefei sterile line herbicide-resistant mutants had no ALS inhibitor, their A530 absorbance values were between 1.2 and 1.4, indicating wild type and There was no significant difference in ALS enzyme activity between the mutants (Fig. 3). After adding the ALS inhibitor Bailutong, the wild type A530 had an absorbance of only 0.25, and the Hefei sterile line herbicide resistant mutant A530 had an absorbance of 1.21. The wild-type ALS enzyme activity was only 18.9% of the control, while the Hefei sterile line herbicide-resistant mutant had more than 89% ALS activity (Fig. 3), indicating that the Hefei sterile line was resistant to herbicide mutants. ALS is not sensitive to ridges and thus confers resistance.

As a result of measuring the absorbance of A530, it was found that when the ALS extract of wild type, herbicide resistant mutant 2 and herbicide resistant mutant 3 had no ALS inhibitor, their A530 absorbance values were between 1.2 and 1.4. It was shown that there was no significant difference in ALS activity between wild type and mutant (Fig. 11). After adding ALS inhibitor, the wild type A530 had an absorbance of only 0.19 to 0.23, herbicide resistant mutant 2 and herbicide resistance. The A530 absorbance of the mutant 3 was 1.16 - 1.21. That is, the ALS activity of the wild type was only 18.9% when the ridge was not added, and the ALS activity of the herbicide resistant mutant 2 and the herbicide resistant mutant 3 was The enzyme activity was 5.25 times and 4.49 times higher than that of the wild type ALS (Fig. 11).

Example 7: Transgenic ALS rice resistance

Specific primers 5'-CGCGGATCCATCCGAGCCACACATCGCCTC-3' and 5'-TCCCCGCGGCCTACGGAAAACAACACAC-3' were designed, and 5' were added to the BamHI and SacI digestion sites, respectively. Referring to the method of Example 4, the mutant ALS gene was amplified from the genomic DNA of the above-mentioned rice mutant Heterotrophic line resistant herbicide mutant, herbicide resistant mutant 2 and herbicide resistant mutant 3 by PCR, and the sequencing was correct. Thereafter, the ALS gene fragment and the plant expression vector pCAMBIA1301 plasmid (purchased from pcambia) were digested with BamHI and SacI, respectively, and the digested product was ligated with T4-DNase (purchased from TaKaRa), and the ligated product was transformed into Escherichia coli. DNA was extracted from the recombinant plasmid and verified by double digestion with BamHI and SacI to generate large plasmid fragments and small gene fragments, which proved that the nucleotide sequences were respectively SEQ ID NO. 1 (Fig. 4A) and SEQ ID NO. (Fig. 4B), the ALS gene shown in SEQ ID NO. 11 (Fig. 4B) was cloned into the plant expression vector pCAMBIA1301 plasmid (purchased from pcambia). The constructed plasmid vector was transformed into Agrobacterium EHA105, and the cells were cultured. The conventional Agrobacterium-mediated transformation of indica rice Nipponbare (purchased from the Jiangsu Province agricultural germplasm resources protection and utilization platform), after the transgenic plants were harvested, the progeny plants were grown to the 3-4 leaf stage, and the transgenic plants were detected by PCR. (Fig. 5A and Fig. 5B).

The PCR detection primer was a forward primer 35SF 5'-ATGGTTAGAGAGGCTTACGC-3', a reverse primer 5R 5'-AGCAACAGGTCAGCCTTATCCAC-3', and the amplified fragment encompassed the CaMV35S promoter and the 5'-end sequence of the ALS gene, and the size was about 2 kb. The PCR amplification reaction system was referred to Example 4. The PCR amplification reaction procedure was carried out in a two-step process, annealing and extension together, using 68 degrees. The amplification procedure was as follows: pre-denaturation: 98 ° C for 3 min; 30 cycles: denaturation 98 ° C for 10 sec; extension 68 ° C for 2 min; incubation: 72 ° C for 10 min. After positive PCR identification, spray 3.3 mL of latitude/L water (10 times recommended concentration). After 7 days, the ALS activity was determined by the method described in Example 6. It was found that the ALS activity of the transgenic rice was significantly higher than that. Non-GMO rice; 30 days later, the transgenic rice was found to be in good growth condition, while the non-transgenic Nipponbare rice was all dead. Comparing the ALS enzyme activities of transgenic rice progeny plants transformed with Hefei sterile line herbicide resistant mutant, herbicide resistant mutant 2 and herbicide resistant mutant 3 ALS gene, and found to contain Hefei sterile line herbicide resistant mutants The ALS activity of the ALS gene transgenic plants was 4.7 times higher than that of the wild type ALS (Fig. 6), and the transgenic plants of the ALS gene resistant to herbicide mutant 2 and herbicide resistant mutant 3 were found to have ALS activity compared to wild type. The ALS enzyme activity was 4.96 and 5.04 times higher (Fig. 12).

Example 8: Transgenic ALS Tobacco

Specific primers 5'-CGCGGATCCATCCGAGCCACACATCGCCTC-3' and 5'-TCCCCGCGGCCTACGGAAAACAACACAC-3' were designed, and 5' were added to the BamHI and SacI digestion sites, respectively. The mutant ALS gene was amplified from the genomic DNA of the Hepatic Sterile Line herbicide resistant mutant, the herbicide resistant mutant 2 and the herbicide resistant mutant 3 by PCR, and after sequencing, the nucleoside was determined by the method of Example 7. The ALS gene having the acid sequence as shown in SEQ ID NO. 1, SEQ ID NO. 7, and SEQ ID NO. 11 was cloned into the plant expression vector pCAMBIA2301 plasmid (purchased from pcambia), respectively. The positive clones were selected to transform Agrobacterium tumefaciens EHA105, and the Agrobacterium tumefaciens-mediated transformation method was used to transform the tobacco leaves. After the transgenic plants were harvested, the progeny plants grew to 3-4 leaf stage, and after PCR identification was positive, spray 3.3. mL ridges/L water (10 times recommended concentration), after 7 days, the ALS activity was determined by the method described in Example 6, and it was found that the ALS activity of transgenic tobacco was significantly higher than that of non-transgenic tobacco; the growth of transgenic tobacco was found after 30 days. In good condition, non-GM tobaccos are all dead. Comparison of ALS activity of transgenic tobacco progeny plants transformed with Hefei sterile line herbicide resistant mutant, herbicide resistant mutant 2 and herbicide resistant mutant 3 ALS gene, and found to contain Hefei sterile line herbicide resistant mutants The ALS activity of the ALS gene transgenic plants was 5.2 times higher than that of the wild type ALS (Fig. 7), and the transgenic plants containing the ALS gene resistant to herbicide mutant 2 and herbicide resistant mutant 3 were found to have ALS activity compared to wild type. The ALS enzyme activity was 5.84 and 6.0 times higher (Fig. 13).

Example 9: Comparison and identification of resistant materials

In order to detect whether the resistant plants having the two mutation sites mentioned in the present application are more resistant to herbicide tolerance than the resistant plants mentioned in the prior patent application (201610226003.6), we will not use Hefei in Example 2 The cultivar mutant and the herbicide-resistant mutant plant of CGMCC No. 12265 in Patent No. 201610226003.6 were subjected to ALS activity assay, and it was found that the ALS enzyme activity of the Hefei sterile line mutant in the present invention was 18% higher than that of CGMCC No. 12265 (Fig. 8).

In order to test whether the resistant plants having the herbicide-resistant mutant 2 and the herbicide-resistant mutant 3 mentioned in the present application are more resistant to herbicide tolerance than the resistant plants mentioned in the prior patent application (201610226003.6), we The mutant of Example 2 and the herbicide-resistant mutant plant of CGMCC No. 12265 of Patent No. 201610226003.6 were subjected to ALS activity assay, and it was found that the herbicide-resistant mutant ALS enzyme activity in the present invention was higher than CGMCC No. 12265. % and 10.7% (Figure 14).

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in accordance with the teachings of the invention, which are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (11)

A rice ALS mutant protein that confers herbicide resistance to plants, including: (a) having an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 8 or SEQ ID NO: 12; (b) A protein derived from (a) wherein the amino acid sequence in (a) is substituted and/or deleted and/or one or more amino acids are added and has acetolactate synthase activity. The rice ALS mutant protein according to claim 1, wherein the herbicide is an imidazolinone herbicide. A nucleic acid or gene encoding the protein of any one of claims 1 to 2. A nucleic acid or gene according to claim 3 comprising: (a) encoding the protein of any one of claims 1 to 2; or (b) a nucleotide sequence which hybridizes under stringent conditions to (a) a defined nucleotide sequence and which encodes a protein having acetolactate synthase activity; (c) Its nucleotide sequence is shown as SEQ ID NO: 1 or SEQ ID NO: 7 or SEQ ID NO: 11. An expression cassette, recombinant vector or cell comprising the nucleic acid or gene of claim 3 or 4. A protein according to any one of claims 1 to 2, a nucleic acid or gene according to claim 3 or 4, an expression cassette according to claim 5, a recombinant vector or a cell for use in a plant herbicide resistance. A method for obtaining a herbicide-resistant plant, comprising the steps of: 1) causing the plant to comprise the nucleic acid or gene of claim 3 or 4; or 2) A plant expressing the protein according to any one of claims 1 to 2. The method of claim 7, comprising the step of editing, transgenic, hybridizing, backcrossing or vegetative propagation of the craspr gene. A method for identifying a plant, wherein the plant is a plant comprising the nucleic acid according to claim 3 or 4, a plant expressing the protein according to any one of claims 1 to 2, or the method according to any one of claims 7 to 8 The plant is characterized in that it comprises the following steps: 1) determining whether the plant comprises the nucleic acid or gene of claim 3 or 4; or 2) It is determined whether the plant expresses the protein of any one of claims 1 to 2. A method for controlling weeds, comprising: applying an effective amount of a herbicide to a field for growing crops, the crop comprising the nucleic acid or gene of claim 3 or 4 or the expression cassette of claim 5. And a recombinant vector or cell, wherein the herbicide is an imidazolinone herbicide. A method for protecting a plant from damage caused by a herbicide, comprising: applying an effective amount of a herbicide to the field of the crop plant, the crop comprising the nucleic acid or gene of claim 3 or Or the expression cassette according to claim 5 or a recombinant vector introduced into the plant, wherein the introduced plant produces a herbicide resistance protein, and the herbicide is an imidazolinone herbicide.

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