Brassica campestris ALS natural variant gene, mutant protein and application thereof
By discovering and utilizing the ALS mutant gene BrALS3R in Chinese cabbage-type rapeseed, the problem of insufficient resistance of rapeseed to ALS herbicides was solved. Through hybridization breeding or transgenic methods, the gene was introduced into rapeseed, which improved the rapeseed's tolerance to ALS herbicides and enhanced rapeseed production efficiency.
Patent Information
- Application Number
- CN202511952395.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-02-13
AI Technical Summary
In the current technology, the resistance mutation sites of rapeseed to ALS herbicides are limited, chemical mutagenesis methods are limited and transgenic regulations are restricted, and the screening of natural mutation sites is random and uncertain, making it difficult to effectively improve the tolerance of rapeseed to ALS herbicides.
The ALS mutant gene BrALS3R of Chinese rapeseed was discovered and utilized. The gene has a mutation of C to A at the 1074th base, which causes the amino acid at the 358th position of the encoded protein to change from aspartic acid to glutamic acid. It was introduced into plants through hybridization breeding or transgenic methods to improve the tolerance of ALS herbicides.
Significant resistance to ALS-type herbicides was achieved in rapeseed, improving the plant's herbicide tolerance and enhancing rapeseed production efficiency.
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Figure CN121518508A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering and crop breeding technology, specifically relating to a type of rapeseed (Chinese cabbage). ALS Naturally mutated genes, mutant proteins, and their applications. Background Technology
[0002] Rapeseed is an important oilseed crop, providing high-quality edible vegetable oil and feed protein. Weeds in rapeseed fields mainly consist of grasses and broadleaf weeds, and the impact of wild mustard-type rapeseed (also known as mustard greens) on rapeseed cultivation is expanding. Grasses can be controlled with specific monocotyledonous herbicides, and since rapeseed is also a broadleaf weed, broadleaf weed control is key in rapeseed field weed management. ALS herbicides are effective against broadleaf weeds, with sulfonylurea (SU) herbicides being the most important. Therefore, cultivating SU-resistant rapeseed varieties will facilitate the use of SU herbicides, thereby effectively improving rapeseed production efficiency.
[0003] Acetolactate synthase (ALS), also known as acetylhydroxy acid synthase (AHAS), is a key rate-limiting enzyme in plants, bacteria, and fungi that catalyzes the biosynthesis of branched-chain amino acids (valine, leucine, and isoleucine). It catalyzes the condensation of pyruvate to acetolactate (a precursor to valine / leucine), or the condensation of pyruvate with 2-ketobutyrate to acetylhydroxybutyrate (a precursor to isoleucine). ALS is a core target of five major classes of herbicides, including sulfonylureas (SUs) and imidazolinones (IMIs). Inhibitors bind to the channel above the enzyme's active site, blocking the entry of the substrate (pyruvate), leading to the interruption of branched-chain amino acid synthesis, thereby inhibiting cell division and growth, ultimately severely suppressing plant growth and even causing death.
[0004] Current research results on numerous materials, including weeds and crops, indicate that... ALSVariations at eight amino acid sites in the gene can confer resistance to different ALS herbicides in plants, namely Ala122, Pro197, Ala205, Asp376, Arg377, Trp574, Ser653, and Gly654 (based on the amino acid sequence of the ALS protein in Arabidopsis thaliana). For example, replacing proline at position 197 of the ALS protein with amino acids such as threonine, leucine, serine, glutamine, and histidine can give plants different resistances to ALS-type herbicides; replacing aspartic acid at position 376 of the ALS protein with glutamic acid can give plants broad-spectrum resistance to ALS-type herbicides; replacing tryptophan at position 574 of the ALS protein with leucine, glycine, methionine, or arginine can give plants different resistances to ALS-type herbicides or broad-spectrum resistance to ALS-type herbicides; replacing serine at position 653 of the ALS protein with threonine, asparagine, or isoleucine can give plants strong resistance to IMIs-type herbicides and, in some weeds, moderate resistance to SU-type herbicides or strong resistance to pyrimidinyl benzoate herbicides.
[0005] Currently reported in rapeseed ALS The gene mutation sites are few, with only three amino acid substitutions, including Pro197, Trp574, and Ser653, all obtained through chemical mutagenesis or transgenic methods. For example, in some studies known to the inventors, Brassica napus has been reported. ALS1 The mutation (Pro-197-Ser) resulted in resistance to bensulfuron-methyl herbicides; in other studies known to the inventors, resistance was obtained by screening Brassica napus seeds after EMS treatment. ALS1 The mutant (Ser-574-Asp) exhibits resistance to IMI and SU class herbicides; ALS1 The mutant (Ser-653-Asp) possesses resistance to IMI class herbicides; ALS3 The mutant (Ser-574-Asp) exhibits resistance to IMI and SU class herbicides; in other studies known to the inventors, it was obtained through in vitro introduction of base variations. ALS1 The sequence of nucleotides with bases 544-546 mutated from CCT to AAC or GAA causes the amino acid at position 182 to mutate from proline (Pro) to asparagine (Asn) or glutamic acid (Glu), thus obtaining... ALS3 The nucleotide sequence at positions 535-537 was mutated from CCT to AAC or GAA, causing the amino acid at position 179 to be mutated from proline (Pro) to asparagine (Asn) or glutamic acid (Glu). This transgenic method was used to induce tolerance and resistance to sulfonylurea herbicides in rapeseed. In other studies known to the inventors, the same method was used to reconstruct the sequence in vitro.ALS1 The gene sequence was reconstructed by mutating TGG to GTC or TCC at positions 1675-1677 of the nucleotide sequence, corresponding to a mutation of tryptophan (Trp) to valine (Val) or serine at position 559 of the encoded protein. ALS3 The gene sequence was modified so that the bases at positions 1666-1668 were mutated from TGG to GTC or TCC, and the amino acid at position 556 of the corresponding encoded protein was mutated from tryptophan (Trp) to valine (Val) or serine. In this way, rapeseed was made to have tolerance and resistance to sulfonylurea herbicides through transgenic methods.
[0006] In summary, the number of mutation sites obtained through chemical mutagenesis is limited, while those obtained through artificial reconstruction is much greater. ALS The application of effective variants obtained through gene sequencing and transgenic methods is restricted under current regulations, and the screening for effective natural mutation sites is subject to chance and uncertainty. Summary of the Invention
[0007] The purpose of this invention is to provide a type of rapeseed similar to Chinese cabbage. ALS Naturally mutated genes, mutant proteins and their applications, in the case of the Chinese cabbage-type rapeseed ALS The introduction of naturally mutated genes into plants can improve their tolerance to ALS-type herbicides, which can then be used to cultivate herbicide-resistant plants.
[0008] This invention provides a type of rapeseed similar to Chinese cabbage. ALS Mutant genes BrALS3R The Chinese cabbage-type rapeseed ALS Mutant genes BrALS3R Compared to wild type ALS3 In the gene, the 1074th base is mutated from C to A, which is the Chinese cabbage-type rapeseed. ALS Mutant genes BrALS3R The nucleotide sequence is shown in SEQ ID NO:1.
[0009] The present invention also provides the Chinese cabbage-type rapeseed described in the above technical solution. ALS Mutant genes BrALS3R The encoded ALS mutant protein, compared to the wild type. ALS3 The protein encoded by the gene has an amino acid sequence of glutamic acid instead of aspartic acid at position 358. The amino acid sequence of the ALS mutant protein is shown in SEQ ID NO:2.
[0010] The present invention also provides a recombinant vector, comprising a base vector and a Chinese rapeseed (Brassica napus) inserted into the base vector. ALS Mutant genes BrALS3R The Chinese cabbage-type rapeseed ALS Mutant genesBrALS3R The nucleotide sequence is shown in SEQ ID NO:1.
[0011] The present invention also provides a transformant, the transformant comprising engineered bacteria, transgenic plant cell lines, or transgenic plant tissues; the transformant comprising Chinese rapeseed (Brassica napus) type rapeseed. ALS Mutant genes BrALS3R Or contains the aforementioned Chinese cabbage-type rapeseed ALS Mutant genes BrALS3R The recombinant vector, the Chinese cabbage-type rapeseed ALS Mutant genes BrALS3R The nucleotide sequence is shown in SEQ ID NO:1.
[0012] The present invention also provides the Chinese cabbage-type rapeseed described in the above technical solution. ALS Mutant genes BrALS3R The application of the ALS mutant protein, recombinant vector, or transformant described in the above technical solutions in improving herbicide resistance in plants and / or cultivating herbicide-resistant plants.
[0013] Preferably, the method for improving herbicide resistance in plants and / or cultivating herbicide-resistant plants includes: [using the Chinese cabbage-type rapeseed...] ALS Mutant genes BrALS3R Introduced into recipient plants.
[0014] Preferably, when carrying out the hybridization breeding, the rapeseed variety KD4 or a variety derived from rapeseed KD4 and containing the aforementioned rapeseed variety is used. ALS Mutant genes BrALS3R The plant material used as the parent is the Chinese cabbage-type rapeseed seed KD4, which has the preservation number CCTCC NO: P202534.
[0015] Preferably, the herbicide includes ALS-type herbicides.
[0016] Preferably, the plant includes plants of the Brassicaceae family.
[0017] This invention also provides a method for cultivating herbicide-resistant rapeseed, which involves hybridizing Chinese cabbage-type rapeseed KD4 and Brassica napus-type rapeseed Zheyou 50 as parents to obtain the F1 generation; Using Zheyou 50 as the backcross parent, four generations of backcrossing were performed with the F1 generation to obtain the BC4 generation. The BC4 generation was self-crossed to obtain the BC4F2 population; Genotypic positive plants were identified in the BC4F2 population using primer pairs shown in SEQ ID NO:7~SEQ ID NO:8 and SEQ ID NO:9~SEQ ID NO:10, thus obtaining the herbicide-resistant rapeseed.
[0018] Beneficial effects: This invention provides a type of rapeseed similar to Chinese cabbage. ALS Naturally mutated genes, mutant proteins and their applications, and the Chinese rapeseed variety in this invention. ALS Mutant genes BrALS3R The haplotype, first discovered in rapeseed, consists of 1959 base pairs and is a natural mutation encoding rapeseed acetolactate synthase III (ALS3), which is different from the wild type. ALS3 Compared to the gene, its 1074th base is mutated from C to A, resulting in the 358th amino acid of the mutant protein it encodes being mutated from aspartic acid to glutamic acid (D358E). BrALS3R Plants with the gene possess resistance to ALS herbicides. By introducing this gene into plants that are not resistant to ALS herbicides through hybridization breeding or transgenic methods, the resistance of the recipient plants to ALS herbicides can be improved.
[0019] Biological Preservation Information Chinese cabbage-type rapeseed seed KD4, biologically classified as Brassica rapa L. was deposited on November 26, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: P202534. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0021] Figure 1 This is the resistance performance of Chinese rapeseed KD4 to the SU herbicide thifensulfuron in Example 2; Figure 2 In Example 3 BrALS3R Resistance expression in genetically modified Arabidopsis thaliana; Figure 3 Imported in Example 4 BrALS3R Genotyping results of individual plants in the BC4F2 population of genetically modified Brassica napus; Figure 4 In Example 4, molecular marker-assisted selection was combined with conventional breeding methods to... BrALS3R Positive plants with resistance were obtained by gene transfer into herbicide-resistant Brassica napus. Detailed Implementation
[0022] This invention provides a type of rapeseed similar to Chinese cabbage. ALS Mutant genes BrALS3R The Chinese cabbage-type rapeseed ALS Mutant genes BrALS3R Compared to wild type ALS3 In the gene, the 1074th base is mutated from C to A, which is the Chinese cabbage-type rapeseed. ALS Mutant genes BrALS3R The nucleotide sequence is shown in SEQ ID NO:1.
[0023] The Chinese cabbage-type rapeseed of this invention ALS Mutant genes BrALS3R Composed of 1959 bases, the nucleotide sequence is as follows: A CGTGTCACGGGAAAGCTCGAGGCGTTTGCGAGCAGGGCTAAGATTGTGCACATAGACATTGATTCTGCTGAGATTGGGAAGAATAAGACACCTCACGTGTCTGTGTGTGGTGATGTAAAGCTGGCTTTGCAAGGGATGAACAAGGTTCTTGAGAACCGGGCGGAGGAGCTCAAGCTTGATTTCGGTGTTTGGAGGAGTGAGTTGAGCGAGCAGAAACAGAAGTTCCCGTTGAGCTTCAAAACGTTTGGAGAAGCCATTCCTCCGCAGTACGCGATTCAGGTCCTAGACGAGCTAACCCAAGGGAAGGCAATTATCAGTACTGGTGTTGGACAGCATCAGATGTGGGCGGCGCAGTTTTACAAGTACAGGAAGCCGAGGCAGTGGCTGTCGTCCTCAGGACTCGGAGCTATGGGTTTCGGACTTCCTGCTGCGATTGGAGCGTCTGTGGCGAACCCTGATGCGATTGTTGTGGACATTGACGGTGATGGAAGCTTCATAATGAACGTTCAAGAGCTGGCCACAATCCGTGTAGAGAATCTTCCTGTGAAGATACTCTTGTTAAACAACCAGCATCTTGGGATGGTCATGCAATGGGAAGATCGGTTCTACAAAGCTAACAGAGCTCACACTTATCTCGGGGACCCGGCAAGGGAGAACGAGATCTTCCCTAACATGCTGCAGTTTGCAGGAGCTTGCGGGATTCCAGCTGCGAGAGTGACGAAGAAAGAAGAACTCCGAGAAGCTATTCAGACAATGCTGGATACACCTGGACCGTACCTGTTGGATGTCATCTGTCCGCACCAAGAACATGTGTTACCGATGATCCCAAGTGGTGGCACTTTCAAAGATGTAATAACCGAAGGGGATGGTCGCACTAAGTACTGA; The bases shown in italics, bold, and underlined in SEQ ID NO:1 are the mutated bases.
[0024] The present invention also provides the Brassica rapa described in the above technical solution ALS mutant geneBrALS3R The encoded ALS mutant protein, compared to the wild type. ALS3 The protein encoded by the gene has an amino acid sequence of glutamic acid instead of aspartic acid at position 358. The amino acid sequence of the ALS mutant protein is shown in SEQ ID NO:2.
[0025] The ALS mutant protein described in this invention consists of 652 amino acids, with the following specific amino acid sequence: MAAATSSSPISLTAKPSSKSPLPISRFSLPFSLTPQKPSSRLHRPLAISAVLNSPVNVAPEKTDKIKTFISRYAPDEPRKGADILVEALERQGVETVFAYPGGASMEIHQALTRSSTIRNVLPRHEQGGVFAAEGYARSSGKPGICIATSGPGATNLVSGLADAMLDSVPLVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVMDVDDIPRIVQEAFFLATSGRPGPVLVDVPKDIQQQLAIPNWDQPMRLPGYMSRLPQPPEVSQLGQIVRLISESKRPVLYVGGGSLNSSEELGRFVELTGIPVASTLMGLGSYPCNDELSLQMLGMHGTVYANYAVEHSDLLLAFGVRFD E The amino acids shown in italics and underlined in SEQ ID NO:2 are the mutated amino acids.
[0026] The present invention also provides a recombinant vector, comprising a base vector and a Chinese rapeseed (Brassica napus) inserted into the base vector. ALS Mutant genes BrALS3R The Chinese cabbage-type rapeseed ALS Mutant genes BrALS3RThe nucleotide sequence is shown in SEQ ID NO:1. As one embodiment, the base vector can be a plasmid vector; the plasmid vector can be, but is not limited to, pFGC5941. This invention does not impose any particular limitation on the construction method of the recombinant vector; conventional construction methods for recombinant vectors in the art can be used.
[0027] The present invention also provides a transformant, the transformant comprising engineered bacteria, transgenic plant cell lines, or transgenic plant tissues; the transformant comprising Chinese rapeseed (Brassica napus) type rapeseed. ALS Mutant genes BrALS3R Or contains the aforementioned Chinese cabbage-type rapeseed ALS Mutant genes BrALS3R The recombinant vector, the Chinese cabbage-type rapeseed ALS Mutant genes BrALS3R The nucleotide sequence is shown in SEQ ID NO:1. As one embodiment, the initial strain in the engineered bacteria is Agrobacterium; the Agrobacterium can be Agrobacterium GV3101. This invention does not specifically limit the method for constructing the transformant; conventional methods for constructing transformants in the art can be used.
[0028] The present invention also provides the Chinese cabbage-type rapeseed described in the above technical solution. ALS Mutant genes BrALS3R The application of the ALS mutant protein, recombinant vector, or transformant described in the above technical solutions in improving herbicide resistance in plants and / or cultivating herbicide-resistant plants.
[0029] As one implementation method, the method for improving herbicide resistance in plants and / or cultivating herbicide-resistant plants includes: using hybridization breeding or transgenic methods to improve the herbicide resistance of the Chinese rapeseed variety. ALS Mutant genes BrALS3R The substance is introduced into a recipient plant. As one embodiment, the Chinese rapeseed (Brassica napus) is used. ALS Mutant genes BrALS3R The mixture is introduced into the cells or tissues of the recipient plant. As one embodiment, when carrying out the hybridization breeding, the mixture is made of *Brassica napus* KD4 or a *Brassica napus* derived from *Brassica napus* KD4 that includes the aforementioned *Brassica napus*. ALS Mutant genes BrALS3R The plant material used as the parent is the Chinese rapeseed KD4, whose preservation number is CCTCC NO: P202534. The hybridization breeding method described in this invention is not particularly limited, and can include single cross, compound hybridization, or backcrossing. As one embodiment, it includes ALS-type herbicides; the ALS-type herbicides include sulfonylurea herbicides. This invention uses the Chinese rapeseed... ALS Mutant genes BrALS3RIntroducing these herbicides into plants resistant to ALS-type herbicides can improve the plants' tolerance to ALS-type herbicides. As one embodiment, the plants can be cruciferous plants, including but not limited to rapeseed and / or Arabidopsis thaliana.
[0030] This invention also provides a method for cultivating herbicide-resistant rapeseed, which involves hybridizing Chinese cabbage-type rapeseed KD4 and Brassica napus-type rapeseed Zheyou 50 as parents to obtain the F1 generation; Using Zheyou 50 as the backcross parent, four generations of backcrossing were performed with the F1 generation to obtain the BC4 generation. The BC4 generation was self-crossed to obtain the BC4F2 population; Genotypic positive plants were identified in the BC4F2 population using primer pairs shown in SEQ ID NO:7~SEQ ID NO:8 and SEQ ID NO:9~SEQ ID NO:10, thus obtaining the herbicide-resistant rapeseed.
[0031] In one implementation, when breeding rapeseed using the Chinese cabbage-type rapeseed KD4, the Chinese cabbage-type rapeseed KD4 can be used as either the male or female parent; in another implementation, the rapeseed includes Brassica napus or Chinese cabbage-type rapeseed. In yet another implementation, the breeding method is a method for converting Brassica napus to Chinese cabbage-type rapeseed, preferably using Chinese cabbage-type rapeseed seed KD4 as the male parent.
[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0033] Unless otherwise specified, the methods used in the following embodiments are conventional methods.
[0034] Example 1 Chinese cabbage-type rapeseed KD4 and ALS3 Discovery of mutant alleles During the trial demonstration of the herbicide-resistant variety Huayouza 50HR, the inventors observed that when thifensulfuron-methyl was sprayed in the field, most of the rapeseed plants died, while a few individual wild-type rapeseed plants showed no obvious phytotoxicity. These wild-type rapeseed materials were named KD4. Further analysis was conducted on its A1 chromosome... BrALS3 Sequencing the gene revealed that its 1074th base was mutated from C to A, causing the amino acid at position 358 of the encoded ALS3 protein sequence to change from aspartic acid to glutamic acid.
[0035] Further testing revealed that KD4 continued to grow normally under treatment with thifensulfuron-methyl herbicide at 4 times the field concentration (4 g / mu in 30 L of water, i.e., 133 mg / L). To the inventors' knowledge, the mutant gene in KD4 of Chinese cabbage-type rapeseed originates from natural variation and is a previously unreported mutation. ALS3 Mutant allele type.
[0036] Example 2 Chinese cabbage-type rapeseed herbicide-resistant gene BrALS3R Screening and identification KD4 rapeseed (Chinese cabbage type) could grow normally under field application of thifensulfuron-methyl herbicides. Further testing of herbicide resistance under greenhouse conditions showed that: ordinary Chinese cabbage-type rapeseed gradually wilted and turned yellow after spraying with 133 mg / L thifensulfuron-methyl, eventually dying; KD4, however, could still grow normally after spraying with 533 mg / L thifensulfuron-methyl. Figure 1 As shown, a represents KD4 plants (two plants on the left) and wild-type plants (two plants on the right) that were not sprayed with herbicide; b represents KD4 plants (two plants on the left) and wild-type plants (two plants on the right) that were treated with thifensulfuron.
[0037] According to previous reports, there are two [specific genes] on the A genome of Brassica napus. ALS The genes are located on chromosomes A1 and A6, respectively, with the gene on chromosome A6 being the most prominent. ALS The gene is expressed only in flowers and siliques and is located on chromosome A1. ALS3 The gene is expressed in roots, stems, leaves, flowers, and siliques; therefore, the KD4 material... ALS3 Gene sequence analysis was performed. The data was downloaded from the database. ALS3 Upstream and downstream sequences of the gene (https: / / yanglab.hzau.edu.cn / BnIR, BnaA01G0223300ZS PCR amplification primers were designed upstream and downstream of the gene, with the forward primer being 5'-CAAAACCCTCACAAGCCTCG-3' (SEQ ID NO:3) and the reverse primer being 5'-CCTTGCATTGAGTCCCAAAC-3' (SEQ ID NO:4).
[0038] DNA was extracted from the leaves of KD4 material using the conventional CTAB method, and KOD One was used. TM PCR Master Mix (Toyobo, Code No. KMM-101S, KMM-101) ALS3After gene amplification, and confirmation by agarose gel electrophoresis that the amplified bands were single, clear, and of the expected size, the PCR products were sent to Wuhan Tianyi Huayu Company for sequencing. Sequencing results showed that, compared to the double 11 reference sequence in *Chiifurum chinense* (Gene ID 103874743 in the NCBI database) or *Brassica napus* (Gene ID 106353715 in the NCBI database), KD4... ALS3 The gene changes from C to A at position 1074 (as shown in SEQ ID NO:1), resulting in the corresponding amino acid at position 358 changing from aspartic acid to glutamic acid (as shown in SEQ ID NO:2).
[0039] Example 3 resistance gene BrALS3R Functional verification 1. Construction of recombinant vectors resistance BrALS3R The gene was ligated into the expression vector pFGC5941.
[0040] First, primers were designed, and homologous arms (underlined) were added to the 5' end of each primer. The BrALS3R forward primer was 5'-CATTTACAATTACCATGGATGGCGGCGGCAACATCG-3' (SEQ ID NO:5); the BrALS3R reverse primer was 5'-TAGACTCACCTAGGATCCTCAGTACTTAGTGCGACCATCC-3' (SEQ ID NO:6).
[0041] Using the PCR product from Example 2 as a template, PCR amplification was performed using the primers described above, and the PCR product was recovered to obtain... BrALS3R Target gene fragment. Utilization Bam HⅠ and Nco I. The prepared pFGC5941 vector was digested and recovered to obtain a linearized vector. Using the Novizan ClonExpress Ultra One Step Cloning Kit V3 solution, the linearized vector was... BrALS3R The gene was ligated into the expression vector pFGC5941, and the recombinant expression vector PFGC5941-35S:: was obtained by sequencing verification. BrALS3R Then, the recombinant expression vector PFGC5941-35S:: was converted using an electroporation method. BrALS3R The cells were transformed into Agrobacterium GV3101 competent cells, and recombinant Agrobacterium strains carrying the target plasmid were screened by PCR.
[0042] 2. BrALS3R Functional validation of transgenic Arabidopsis thaliana Wild-type Arabidopsis thaliana (Col.) was infected using an Agrobacterium-mediated flower-dip method. T0 generation seeds were then sown on MS medium resistant to ampicillin for germination. Positive plants were selected and transplanted to harvest T1 generation seeds. T1 generation positive plants were grown in a greenhouse, and a 2 μg / mL thifensulfuron-methyl spraying experiment was conducted. The results are as follows: Figure 2 As shown, a represents a transgenic positive plant that has been sprayed with herbicide; b represents a transgenic negative plant that has been sprayed with herbicide.
[0043] Figure 2 The results showed that the transgenic positive plants were resistant to thifensulfuron.
[0044] Example 4 Transgenic breeding of resistance sites in Brassica napus To further verify BrALS3R The resistance gene in Brassica napus was introduced into Brassica napus through distant hybridization.
[0045] First, KD4 was crossed with the conventional wild-type rapeseed material Zheyou 50 to obtain F1. Then, Zheyou 50 was used as the recurrent parent for backcrossing, and resistance site selection was carried out in the segregating generation to finally construct the BC4F2 population.
[0046] For the BC4F2 population, 5'-CCGGACCGGTTTTGGTTGAC-3' (SEQ ID NO.7) and 5'-CGAGCTTTCCCGTGACACGT-3' (SEQ ID NO.8) were used for amplification. BrALS3R The first primer pair of the gene and 5'-CGCTTATCCCGGAGGTGCT-3' (SEQ ID NO. 9) and 5'-GAGCTTTCCCGTGACACGG-3' (SEQ ID NO. 10) are shown for amplifying conventional non-mutated genes. BnALS3 Genotyping analysis was performed using the second primer pair of the gene, and the results are as follows: Figure 3 As shown in the figure, 1-8 represent different individual plants of the BC4F2 population. Among them, 1-4 are the amplification results of the first primer pair, with an amplified fragment size of 408 bp, and 5-8 are the amplification results of the second primer pair, with a fragment size of 799 bp. It can be seen that homozygous positive and homozygous negative individual plants can be obtained by the above-mentioned far hybridization method.
[0047] Thiophanate-sulfuron spraying experiments were conducted on homozygous positive and homozygous negative plants (the total amount of pesticide used was approximately four times that used in the field), and the results were as follows: Figure 4 As shown, a represents a single oilseed plant before herbicide application, and b represents a single oilseed plant after herbicide application. In a and b, the upper row represents negative-affected plants, and the lower row represents positive-affected plants.
[0048] Depend on Figure 4 The results showed that positive plants grew normally after herbicide application, while negative plants gradually developed yellowing and wilting leaves, proving that the resistance sites in KD4 were... BrALS3R Introducing Brassica napus into rapeseed can induce resistance to ALS-type herbicides.
[0049] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A type of rapeseed similar to Chinese cabbage ALS Mutant genes BrALS3R Its characteristics are, The Chinese cabbage-type rapeseed ALS Mutant genes BrALS3R Compared to wild type ALS3 In the gene, the 1074th base is mutated from C to A, which is the Chinese cabbage-type rapeseed. ALS Mutant genes BrALS3R The nucleotide sequence is shown in SEQ ID NO:
1.
2. The Chinese cabbage-type rapeseed as described in claim 1 ALS Mutant genes BrALS3R The encoded ALS mutant protein is characterized by, The ALS mutant protein compared to the wild type ALS3 The protein encoded by the gene has an amino acid sequence of glutamic acid instead of aspartic acid at position 358. The amino acid sequence of the ALS mutant protein is shown in SEQ ID NO:
2.
3. A recombinant vector, characterized in that, Includes a base carrier and Chinese cabbage-type rapeseed inserted into the base carrier. ALS Mutant genes BrALS3R The Chinese cabbage-type rapeseed ALS Mutant genes BrALS3R The nucleotide sequence is shown in SEQ ID NO:
1.
4. A transformant, characterized in that, The transformant includes engineered bacteria, transgenic plant cell lines, or transgenic plant tissues; the transformant includes Chinese rapeseed. ALS Mutant genes BrALS3R Or contains the aforementioned Chinese cabbage-type rapeseed ALS Mutant genes BrALS3R The recombinant vector, the Chinese cabbage-type rapeseed ALS Mutant genes BrALS3R The nucleotide sequence is shown in SEQ ID NO:
1.
5. The Chinese cabbage-type rapeseed according to claim 1 ALS Mutant genes BrALS3R The application of the ALS mutant protein of claim 2, the recombinant vector of claim 3, or the transformant of claim 4 in improving herbicide resistance in plants and / or cultivating herbicide-resistant plants.
6. The application according to claim 5, characterized in that, The method for improving herbicide resistance in plants and / or cultivating herbicide-resistant plants includes: [using the Chinese cabbage-type rapeseed...] ALS Mutant genes BrALS3R Introduced into recipient plants.
7. The application according to claim 6, characterized in that, When carrying out the aforementioned hybridization breeding, Chinese cabbage-type rapeseed ( Brassica rapa L.)KD4 or derived from and including the aforementioned Brassica rapa type KD4. ALS Mutant genes BrALS3R The plant material used as the parent is described in the Chinese cabbage-type rapeseed KD4, which has the preservation number CCTCC NO: P202534.
8. The application according to claim 5, characterized in that, The herbicides include ALS-type herbicides.
9. The application according to any one of claims 5 to 8, characterized in that, The plants mentioned include plants of the Brassicaceae family.
10. A method for cultivating herbicide-resistant rapeseed, characterized in that, The F1 generation was obtained by crossing Chinese cabbage-type rapeseed KD4 and Brassica napus-type rapeseed Zheyou 50 as parents. Using Zheyou 50 as the backcross parent, four generations of backcrossing were performed with the F1 generation to obtain the BC4 generation. The BC4 generation was self-crossed to obtain the BC4F2 population; Genotypic positive plants were identified in the BC4F2 population using primer pairs shown in SEQ ID NO:7~SEQ ID NO:8 and SEQ ID NO:9~SEQ ID NO:10, thus obtaining the herbicide-resistant rapeseed.