Paddy rice OsACC gene mutant resistant to fluazifop-p-butyl or clethodim and application of paddy rice OsACC gene mutant

Abstract

The invention relates to a rice OsACC gene mutant resistant to fluazifop-p-butyl or clethodim and application of the rice OsACC gene mutant. The rice OsACC gene mutant is a deletion mutant or a deletion and combination insertion mutant; wherein the deletion mutation occurs in the promoter of the rice OsACC gene, and the sequence of the nucleic acid before the deletion mutation is shown as SEQ ID No.1; the length of the sequence of the deleted nucleic acid is 16 bp, A in the initiation codon ATG of the OsACC gene is 0 site, and the deleted nucleic acid occurs at 455-440 sites of the upstream of the initiation codon ATG; a in the initiation codon ATG of the OsACC gene is the 0 site, and inserted nucleic acid occurs in the 455-440 sites on the upstream of the initiation codon ATG. According to the rice OsACC gene mutant disclosed by the invention, rice can obtain two kinds of resistance to herbicides, namely fluazifop-p-butyl and clethodim.

Description

Technical Field

[0001] This invention relates to the field of nucleic acids, and particularly to a rice resistant to quizalofop-P-ethyl or clethodim. OsACC Gene mutants and their applications. Background Technology

[0002] Field weeds are one of the major biological hazards in agricultural production, seriously affecting the high-quality development of the rice industry. In weed control strategies, the combined use of chemical control and herbicide-resistant varieties has shown excellent performance. Acetyl-CoA carboxylase (ACCase) inhibitor herbicides are mainly used to control gramineous weeds, and are characterized by high efficiency, low toxicity, and safety for subsequent crops.

[0003] Currently, the development of herbicide-resistant crops using ACCase inhibitors primarily relies on gene editing and other technologies to mutate the coding region of the target protein ACC, resulting in amino acid mutations. These mutations reduce the target protein's binding capacity to herbicides, thus preventing disruption to normal plant life processes and fostering herbicide resistance. However, such protein mutations often alter the enzyme activity of ACC itself, causing growth and developmental defects, which limits its application in production. Furthermore, frequent use of a single herbicide accelerates the evolution of weed resistance and can lead to phytotoxicity. These problems significantly restrict weed control in rice production. Summary of the Invention

[0004] One of the present inventions provides a rice OsACC Gene mutant, the rice OsACC The gene mutant is a deletion mutant; among them, the deletion mutation occurs in rice. OsACC In the gene promoter, the sequence of the nucleic acid before deletion mutation is shown in SEQ ID No. 1; the length of the deleted nucleic acid sequence is 16 bp, of which, OsACC The A in the gene start codon ATG is at position 0, and the deleted nucleic acid occurs at positions 455 to 440 upstream of the start codon ATG.

[0005] The second invention provides a rice OsACC Gene mutant, the rice OsACC Gene mutants are deletion-merged insertion mutants, where deletion-merged insertion mutations occur in rice. OsACC In the gene promoter, the sequence of the deleted and merged nucleic acid before the insertion mutation is shown in SEQ ID No. 1; the length of the deleted nucleic acid sequence is 16 bp, and the length of the inserted nucleic acid sequence is at least 1 to 6 bp; OsACCThe A in the gene start codon ATG is at position 0, and the deleted nucleic acid occurs at positions 455 to 440 upstream of the start codon ATG; OsACC The A in the gene start codon ATG is at position 0, and the inserted nucleic acid occurs at positions 455 to 440 upstream of the start codon ATG.

[0006] In one specific implementation, the inserted nucleic acid sequence is any 1 to 6 bp fragment of TAATTA.

[0007] The third invention provides rice according to any one of the first and second inventions. OsACC The application of gene mutants in rice ACCase inhibitor herbicides, wherein the rice variety is Nanjing 46 or Nipponbare.

[0008] In one specific embodiment, the ACCase inhibitor herbicide is quizalofop-p-ethyl and / or clethodim.

[0009] The fourth invention provides a method for enabling rice to acquire resistance to ACCase inhibitor herbicides, which involves deleting certain components from the rice genome through gene editing or homologous recombination. OsACC A subset of bases in the gene promoter, and optionally directed towards the... OsACC A portion of bases is inserted into the gene promoter to obtain rice as described in any one of the first and second inventions. OsACC The rice variety is Nanjing 46 or Nipponbare, which is achieved through gene mutants.

[0010] In one specific embodiment, the method includes the following steps: 1) Obtain the pHZLib-Cas12i3 vector, or the pHZ33 and pUbi-IEE-Cas12i3 vector; wherein, the pHZLib-Cas12i3 vector is constructed by the following operation: replacing the DR-crRNA-BsaI-BsaI-DR element in pHZ33 with the suicide gene ccdB to obtain the pHZ33-ccdB vector; integrating pHZ33-ccdB with pUbi-IEE-Cas12i3 into a vector to obtain the pHZLib-Cas12i3 vector; 2) Obtain a crRNA sequence or target sequence for gene editing, wherein the crRNA sequence is shown in SEQ ID No. 6; and the target sequence is located at positions 34 to 56 of the sequence shown in SEQ ID No. 6. 3) Replace the ccdB gene on the pHZLib-Cas12i3 vector with the crRNA, thereby cloning the crRNA sequence into the pHZLib-Cas12i3 vector to obtain pHZLib-Cas12i3-OsACCcrRNA; or clone the target sequence into the pHZ33 vector to obtain the pHZ33-OsACCSpacer vector, and then integrate the pHZ33-OsACCSpacer with the pUbi-IEE-Cas12i3 vector into a single vector to obtain pUbi-IEE-Cas12i3-HZ33-OsACCSpacer; 4) pHZLib-Cas12i3-OsACCcrRNA or pUbi-IEE-Cas12i3-HZ33-OsACCSpacer were transformed into Agrobacterium and infected rice callus tissue to screen for rice lines resistant to ACCase inhibitor herbicides.

[0011] In one specific embodiment, the ACCase inhibitor herbicide is quizalofop-p-ethyl and / or clethodim.

[0012] Beneficial effects of the present invention: The present invention discovers that by deleting rice OsACC A portion of the sequence in the promoter of a gene, and optionally directed to... OsACC Inserting a partial base into the gene promoter can confer resistance to two herbicides, quizalofop-P-ethyl and clethodim, in rice. This is of great value in ensuring agricultural production safety and improving the efficiency of herbicide use. Mutants can be created using the CRISPR / Cas12i3 system, or through other CRISPR / Cas systems, gene editing, or homologous recombination. Attached Figure Description

[0013] Figure 1 For rice OsACC A schematic diagram of the target sites for the non-coding region regulatory elements.

[0014] Figure 2 A schematic diagram of the main components of the knockout vector pHZLib-Cas12i3 and the editing vector library pHZLib-Cas12i3-OsACCcrRNA after inserting crRNA into the CRISPR / Cas12i3 system.

[0015] Figure 3 Sanger sequencing was used to identify allelic variation types and resistance phenotypes of T1 generation plants to quizalofop-p-ethyl by screening resistant plants A3-#3.

[0016] Figure 4The resistance phenotype of A3-#3 T1 generation plants after spraying with quizalofop-P-ethyl or clethodim. Detailed Implementation

[0017] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0018] Unless otherwise specified, the plasmids and reagents used in the embodiments of this invention can be purchased commercially.

[0019] For details on the pHZLib-Cas12i3 plasmid, please refer to CN202511501754.X.

[0020] All synthesized nucleic acids were outsourced to Beijing Qingke Xinyue Biotechnology Co., Ltd.

[0021] Example 1: Rice OsACC Construction of gene plasmid libraries.

[0022] Reference rice ( Oryza sativa L) Genome Database OsACC The gene sequence (accession number LOC_Os05g22940, rice variety Nipponbare) was amplified and sequenced using the extracted genome of Nanjing 46 as a template to obtain the Nanjing 46 gene sequence. OsACC The gene and its upstream and downstream nucleic acid sequences indicate that Nanjing 46 OsACC The gene and its upstream and downstream nucleic acid sequences are similar to those of Nipponbare. OsACC The gene and its upstream and downstream nucleic acid sequences are completely identical, among which, OsACC The intron and exon sequence length of the gene is 13489. (Nanjing 46) OsACC The gene start codon and its upstream nucleic acid sequence are shown in SEQ ID No. 1, where the bases from positions 1 to 1967 from the 5' to 3' ends are... OsACC In the promoter section, bits 1968 to 1970 are the start codon ATG.

[0023] for OsACC The gene editing library for the non-coding region (promoter) of the gene is divided into three libraries (Pool01 covers bases 1 to 610 of the sequence shown in SEQ ID No. 1, Pool02 covers bases 611 to 1214, and Pool03 covers bases 1215 to 1967). Each library contains 20 to 25 crRNA sequences (including conserved repetitive sequences in the CRISPR / Cas12i3 system guide RNA located at both ends of the target sequence, and further sequences flanking the conserved repetitive sequences to facilitate PCR amplification), for a total of 70 sequences. A schematic diagram showing the location of the target sequence in the sequence shown in SEQ ID No. 1 is provided below. Figure 1 .

[0024] Using an equal volume and concentration of a mixture of all crRNA oligonucleotide sequences from each library as templates, and using Array-F (as shown in SEQ ID No. 2) and Array-R (as shown in SEQ ID No. 3) as primers, PCR amplification was performed using I-5™ 2×High-Fidelity Master Mix. The PCR products were recovered to obtain the results for each library. OsACC crRNA of the gene. For the vector pHZLib-Cas12i3... Bsa I enzyme digestion yielded approximately 15.8 kb of the vector backbone (releasing the ccdB gene fragment). The fragments were then processed using 2×MultiF Seamless Assembly Mix to separate the contents of each library. OsACC The crRNA of the gene was cloned into a 15.8 kb vector backbone (that is, the ccdB gene on the pHZLib-Cas12i3 vector was replaced with the ccdB gene in each library). OsACC The crRNAs of the gene were named pHZLib-Cas12i3-OsACCcrRNA(Pool01), pHZLib-Cas12i3-OsACCcrRNA(Pool02), and pHZLib-Cas12i3-OsACCcrRNA(Pool03) respectively. A schematic diagram of the main elements in their plasmids is shown below. Figure 2 As shown in the figure, 40 colonies were randomly selected from each library for sequencing, with an accuracy rate of 98%.

[0025] Example 2: Rice transformation of plasmid library and screening for resistance to ACCase inhibitor herbicides.

[0026] 1) Rice callus induction: hulled mature Nanjing 46 rice seeds were treated with 50% commercial disinfectant for 30 minutes; washed 3 to 5 times with sterile water, then transferred to sterile petri dishes to remove excess water; seeds were placed on MSD plates (4.43 g / L MS powder (Murashige and Skoog medium); 30 g / L sucrose; 2 ml / L 2,4-dichlorophenoxyacetic acid (2,4-D); 8 g / L agar powder; pH 5.7) and cultured in a light-controlled environment for 10 days to induce callus formation; the embryo and bud of the seeds were removed, and the callus tissue was transferred to a new MSD petri dish and cultured for 4 days until it was ready for Agrobacterium transformation.

[0027] 2) Agrobacterium transformation: Three plasmid libraries, pHZLib-Cas12i3-OsACCcrRNA(Pool01) to pHZLib-Cas12i3-OsACCcrRNA(Pool03), were transformed into Agrobacterium strain EHA105 using electroporation, constructing three EHA105 strain libraries containing all the above crRNA plasmid libraries. The three Agrobacterium libraries were then eluted with sterile water and subjected to OD245-distillation. 600 Between steps 1 and 2, Agrobacterium was collected from each bacterial cell bank by centrifugation, resuspended in MSD solution, and its OD value was adjusted. 600 =0.2, to obtain the bacterial suspension for each bacterial cell.

[0028] 3) Agrobacterium infection of rice callus: Place the callus tissue in the suspension of each of the above three bacterial banks for 30 minutes; remove the suspension, transfer the callus tissue to sterile absorbent paper to remove excess suspension, and then transfer the callus tissue to a new MSD medium containing 100 μmol / L acetylsyl syringone and incubate at room temperature in the dark for 2 to 3 days.

[0029] 4) Screening for resistant rice callus: After dark culture, callus tissue was transferred to MSD medium (100 mg / L termethin; 50 mg / L hygromycin B) and cultured for 2 weeks to 2 months until resistant callus appeared on the surface of the callus tissue. The medium was changed every 2 weeks.

[0030] 5) Screening of rice ACCase inhibitor-resistant callus and acquisition of regenerated plants: Resistant callus tissue was transferred to regeneration medium (4.43 g / L MS powder; 30 g / L sucrose; 25 g / L sorbitol; 0.5 mg / L 1-naphthaleneacetic acid (NAA); 3 mg / L 6-benzylaminopurine (6-BA); 100 mg / L termethin; 50 mg / L hygromycin B; 12 g / L agar powder; pH=5.7). After 7 to 10 days, the callus was transferred to regeneration medium containing 5 μmol / L quizalofop-P-ethyl, 1 μmol / L haloxyfop-P-ethyl, and 0.8 μmol / L clethodim, respectively, to screen for rice callus tissue resistant to the corresponding herbicides. The callus was then transferred every 7 to 10 days until seedlings were formed. Seedlings were then transferred to 1 / 2 MS medium (2.21 g / L MS powder). Rooting was achieved using MS powder; 15 g / L sucrose; 8 g / L agar powder; pH 5.7. After screening, five T0 generation rice regenerated plants resistant to quizalofop-P-ethyl (A2-#1, A3-#3, A3-#13, A3-#16, and A3-#20) were obtained; two T0 generation rice regenerated plants resistant to quizalofop-P-ethyl (A3-#1 and A3-#17) were obtained; and one T0 generation rice regenerated plant resistant to clethodim (A3-#7) was obtained. These eight resistant T0 generation rice plants were grown in a greenhouse until self-pollination and T0 generation seeds were harvested.

[0031] 6) Screening of T1 generation ACCase inhibitor-resistant rice: Seeds from A3-#3 T0 generation plants and wild-type control were treated with 50% commercial disinfectant for 30 minutes; washed 3 to 5 times with sterile water; then the seeds were sown on 1 / 2 MS medium containing 0.8 μmol / L quizalofop-P-ethyl for screening of quizalofop-P-ethyl resistant T1 generation mutant lines. The plants were cultured in a light-controlled incubator for 10 days, and seed germination and plant development were observed. Results are shown in […]. Figure 3 The results showed that wild-type materials could germinate but not grow on 1 / 2 MS medium containing 0.8 μmol / L quizalofop-P-ethyl, while seeds of A3-#3 could not only germinate but also grow into plants on 1 / 2 MS medium containing 0.8 μmol / L quizalofop-P-ethyl. This indicates that A3-#3 obtained through screening possesses quizalofop-P-ethyl resistance.

[0032] Example 3: Rice with ACCase inhibitor herbicides OsACC Molecular identification of mutants.

[0033] 1) Genomic DNA Extraction: During the growth period of T0 and T1 generation rice plants, approximately 0.1 g of leaves from resistant T0 and T1 generation plants were harvested, flash-frozen in liquid nitrogen, and then ground using a grinder. 600 μL of 2× cetyltrimethylammonium bromide (CTAB) DNA extraction buffer (containing 1 / 1000 β-mercaptoethanol) was added, and the mixture was vortexed and lysed at 65°C for 45 min. 500 μL of chloroform was added, and the mixture was vigorously shaken to form an emulsion. The emulsion was centrifuged at 14000 rpm for 10 min. After centrifugation, the supernatant was transferred to a 1.5 ml centrifuge tube, and an equal volume of isopropanol was added. The mixture was inverted and centrifuged at 14000 rpm for 10 min. The supernatant was discarded, and the white precipitate was washed with 700 μL of 70% ethanol. The tube was centrifuged at 14000 rpm for 5 min, and the supernatant was discarded. The tube was then air-dried in a fume hood for 10 min. 30 μL of ultrapure water (ddH2O) was added to dissolve the DNA. The DNA solution should be stored at -20 degrees Celsius for later use.

[0034] 2) PCR amplification and sequencing of crRNA sequences in T0 generation rice: Primer pairs U6p-F (as shown in SEQ ID No. 4) and pENTR4-R (as shown in SEQ ID No. 5) were designed for A3-#3 to identify the crRNA sequences. Using genomic DNA solutions from T0 generation plants as templates, and U6p-F and pENTR4-R as primer pairs, PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The PCR products were then directly sequenced using Sanger sequencing.

[0035] Sanger sequencing results showed that the crRNA sequence used in A3-#3 was OsACC-Pool03-crRNA59 from Pool03 (as shown in SEQ ID No. 6), see Figure 3 .

[0036] 3) PCR amplification and sequencing detection of the mutation site: Specific amplification primers OsACC-F (as shown in SEQ ID No. 7) and OsACC-R (as shown in SEQ ID No. 8) were designed for A3-#3. Genomic DNA solutions from T0 and T1 generation plants were used as templates, and corresponding plant primer pairs were used as primers. PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (purchased from Nanjing Novizan Biotechnology Co., Ltd.), and the PCR products were directly sequenced by Sanger sequencing.

[0037] Sanger sequencing results from generation T0 of A3-#3, compared with wild-type results, showed that A3-#3 contained insertion / deletion mutations. Specifically, it exhibited a homozygous insertion / deletion mutation of +6-16 bp, i.e., a mutation of 6-16 bp. OsACC In the start codon ATG, position A is 0. A homozygous mutation occurs between positions 440 and 455 upstream of A, involving a 6 bp insertion and a 16 bp deletion. The inserted 6 bp sequence is TAATTA. (See results below.) Figure 3 In T1 generation plants resistant to quizalofop-P-ethyl, the A3-#3+6-16 mutation, a homozygous insertion and deletion mutation of +6-16 bp / +6-16 bp occurring 440 to 455 upstream of the start codon, was detected.

[0038] Example 4: Rice OsACC Mutant anti-ACCase inhibitor herbicides spectrum.

[0039] The agricultural herbicide quizalofop-p-ethyl EC, with an active ingredient content of 15%, was purchased from Ningbo Shiyuan Jinniu Agricultural Technology Co., Ltd. The recommended field dose of quizalofop-p-ethyl EC is 34 g ai / ha, which is recorded as 1 × 1 times the recommended field dose.

[0040] The agricultural commercial herbicide clethodim suspension, with an active ingredient content of 24%, was purchased from Anhui Huaxing Chemical Co., Ltd. The recommended field dose of clethodim suspension is 72 g ai / ha, which is recorded as 1 × 1 times the recommended field dose.

[0041] The quizalofop-P-ethyl emulsifiable concentrate and clethodim suspension were diluted with tap water to obtain 2x (2×), 4x (4×) and 8x (8×) quizalofop-P-ethyl dilutions, and 0.5x (0.5×), 1x (1×), 1.5x (1.5×) and 2x (2×) clethodim dilutions, respectively.

[0042] T0 generation seeds (A3-#3) were sown in nutrient soil in seedling pots and cultured in a greenhouse to obtain corresponding T1 generation plants. Wild-type rice was used as a negative control. Each treatment had 30 seedlings. When the rice plants reached 2-3 leaves in the greenhouse, they were sprayed with diluted quizalofop-P-ethyl or clethodim at various concentrations using a mobile sprayer. After herbicide application, the plants continued to be cultured in the greenhouse. The growth of the T1 generation plants was observed after 14 days. Figure 4 . Figure 4 The results showed that wild-type rice seedlings withered and died under the influence of quizalofop-P-ethyl diluted at 4 times the field recommended dose and clethodim diluted at 1 times the field recommended dose, while the T1 generation plants of A3-#3 all showed normal growth and were green. This indicates that this mutation led to herbicide resistance in rice.

[0043] The above results indicate that the A3-#3 mutant possesses resistance to both quizalofop-p-ethyl and clethodim.

Claims

1. A type of rice OsACC Gene mutant, the rice OsACC Gene mutants are deletion mutants; among them, Deletion mutations occur in rice OsACC In the gene promoter, the sequence of the nucleic acid before deletion mutation is shown in SEQ ID No. 1; the length of the deleted nucleic acid sequence is 16 bp, of which, OsACC The A in the gene start codon ATG is at position 0, and the deleted nucleic acid occurs at positions 455 to 440 upstream of the start codon ATG.

2. A type of rice OsACC Gene mutant, the rice OsACC Gene mutants are deletion-merged insertion mutants, in which... Deletion-merge-insertion mutations occur in rice OsACC The sequence of the gene promoter containing the deleted and merged nucleic acid before the insertion mutation is shown in SEQ ID No. 1; The length of the deleted nucleic acid sequence is 16 bp, and the length of the inserted nucleic acid sequence is at least 1 to 6 bp; OsACC The A in the gene start codon ATG is at position 0, and the deleted nucleic acid occurs at positions 455 to 440 upstream of the start codon ATG; OsACC The A in the gene start codon ATG is at position 0, and the inserted nucleic acid occurs at positions 455 to 440 upstream of the start codon ATG.

3. The rice according to claim 2 OsACC Gene mutants, characterized by, The inserted nucleic acid sequence is any 1 to 6 bp fragment of TAATTA.

4. The rice according to any one of claims 1 to 3 OsACC The application of gene mutants in rice ACCase inhibitor herbicides, wherein the rice variety is Nanjing 46 or Nipponbare.

5. The application according to claim 4, characterized in that, The ACCase inhibitor herbicides are quizalofop-p-ethyl and / or clethodim.

6. A method for conferring resistance to ACCase inhibitor herbicides on rice, which involves deleting certain components from the rice genome through gene editing or homologous recombination. OsACC A subset of bases in the gene promoter, and optionally directed towards the... OsACC The gene promoter inserts a partial base to obtain the rice as described in any one of claims 1 to 3. OsACC The rice variety is Nanjing 46 or Nipponbare, which is achieved through gene mutants.

7. The method according to claim 6, characterized in that, The method includes the following steps: 1) Obtain the pHZLib-Cas12i3 vector, or the pHZ33 and pUbi-IEE-Cas12i3 vector; wherein, the pHZLib-Cas12i3 vector is constructed by the following operation: replacing the DR-crRNA-BsaI-BsaI-DR element in pHZ33 with the suicide gene ccdB to obtain the pHZ33-ccdB vector; integrating pHZ33-ccdB with pUbi-IEE-Cas12i3 into a vector to obtain the pHZLib-Cas12i3 vector; 2) Obtain a crRNA sequence or target sequence for gene editing, wherein the crRNA sequence is shown in SEQ ID No. 6; and the target sequence is located at positions 34 to 56 of the sequence shown in SEQ ID No.

6. 3) Replace the ccdB gene on the pHZLib-Cas12i3 vector with the crRNA, thereby cloning the crRNA sequence into the pHZLib-Cas12i3 vector to obtain pHZLib-Cas12i3-OsACCcrRNA; or clone the target sequence into the pHZ33 vector to obtain the pHZ33-OsACCSpacer vector, and then integrate the pHZ33-OsACCSpacer with the pUbi-IEE-Cas12i3 vector into a single vector to obtain pUbi-IEE-Cas12i3-HZ33-OsACCSpacer; 4) pHZLib-Cas12i3-OsACCcrRNA or pUbi-IEE-Cas12i3-HZ33-OsACCSpacer were transformed into Agrobacterium and infected rice callus tissue to screen for rice lines resistant to ACCase inhibitor herbicides.

8. The method according to claim 6 or 7, characterized in that, The ACCase inhibitor herbicides are quizalofop-p-ethyl and / or clethodim.

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