Rice OsACC gene mutant for resisting ACCase inhibitor herbicides and application of rice OsACC gene mutant

Abstract

The invention relates to a rice OsACC gene mutant for resisting ACCase inhibitor herbicides and application of the rice OsACC gene mutant. The rice OsACC gene mutant is a deletion 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 20 bp to 27 bp, A in an initiation codon ATG of the OsACC gene is 0 site, the deleted nucleic acid occurs in 704 to 678 sites on the upstream of the initiation codon ATG, and at least bases on 700 to 681 sites on the upstream of the initiation codon ATG are deleted. The mutant can enable rice to obtain resistance to at least one of herbicides such as haloxyfop-R-methyl, fluazifop-p-butyl and clethodim.

Description

A rice OsACC gene mutant resistant to ACCase inhibitor herbicides and its application Technical Field

[0001] This invention relates to the field of nucleic acids, and in particular to a rice OsACC gene mutant that resists ACCase inhibitor herbicides and its application. 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 aspect of this invention provides a rice OsACC gene mutant, which is a deletion mutant. 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 in SEQ ID No. 1. The length of the deleted nucleic acid sequence is 20 bp to 27 bp, wherein the A in the start codon ATG of the OsACC gene is 0, and the deleted nucleic acid occurs at positions 704 to 678 upstream of the start codon ATG (its sequence is shown in SEQ ID No. 9), and at least positions 700 to 681 upstream of the start codon ATG are deleted (its sequence is shown in SEQ ID No. 10). That is, the longest deletion sequence can be SEQ ID No. 9 containing SEQ ID No. 10; it can also be any segment of SEQ ID No. 9 containing SEQ ID No. 10; or it can be a combination of any segment of SEQ ID No. 9 containing SEQ ID No. 10 and other sequences of any length in SEQ ID No. 9.

[0005] In one specific implementation, the length of the deleted nucleic acid sequence is 20 bp, with A in the start codon ATG of the OsACC gene being position 0, and the deleted nucleic acid occurring at positions 700 to 681 upstream of the start codon ATG.

[0006] In one specific implementation, the length of the deleted nucleic acid sequence is 27 bp, with A in the start codon ATG of the OsACC gene being position 0, and the deleted nucleic acid occurring at positions 704 to 678 upstream of the start codon ATG.

[0007] In one specific implementation, the length of the deleted nucleic acid sequence is 23 bp, with A in the start codon ATG of the OsACC gene being position 0, and the deleted nucleic acid occurring 700 to 678 positions upstream of the start codon ATG.

[0008] In one specific implementation, the length of the deleted nucleic acid sequence is 24 bp, with A in the start codon ATG of the OsACC gene being position 0, and the deleted nucleic acid occurring at positions 704 to 681 upstream of the start codon ATG.

[0009] The second invention provides the application of the rice OsACC gene mutant according to any one of the inventions in the use of rice ACCase inhibitor herbicides, wherein the rice variety is Nanjing 46 or Nipponbare.

[0010] In one specific embodiment, the ACCase inhibitor herbicide is at least one of haloxyfop-R-methyl, quizalofop-P-ethyl, and clethodim.

[0011] The third invention provides a method for enabling rice to acquire resistance to ACCase inhibitor herbicides. This method involves deleting a portion of the bases in the promoter of the OsACC gene in the rice genome through gene editing or homologous recombination to obtain a rice OsACC gene mutant as described in any one of the inventions, wherein the rice variety is Nanjing 46 or Nipponbare.

[0012] In one specific embodiment, the method includes the following steps: 1) obtaining 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 and pUbi-IEE-Cas12i3 into a single vector to obtain the pHZLib-Cas12i3 vector; 2) obtaining 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 as shown in SEQ ID No. 6) Positions 34 to 56 of the sequence shown; 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) Transform the pHZLib-Cas12i3-OsACCcrRNA or pUbi-IEE-Cas12i3-HZ33-OsACCSpacer into Agrobacterium and infect rice callus tissue to screen for rice lines resistant to ACCase inhibitor herbicides.

[0013] In one specific embodiment, the ACCase inhibitor herbicide is at least one of haloxyfop-R-methyl, quizalofop-P-ethyl, and clethodim.

[0014] The beneficial effects of this invention are as follows: This invention discovers that by deleting a portion of the sequence in the promoter of the rice OsACC gene and optionally inserting a portion of bases into the OsACC gene promoter, rice can acquire resistance to at least one of the herbicides haloxyfop-R-methyl, quizalofop-P-ethyl, and clethodim. This has significant application value in ensuring agricultural production safety and improving the efficiency of herbicide use. The mutant can be achieved using the CRISPR / Cas12i3 system, or through other CRISPR / Cas systems, gene editing, or homologous recombination. Attached Figure Description

[0015] Figure 1 is a schematic diagram of the target sites of the non-coding region regulatory elements of rice OsACC.

[0016] Figure 2 shows the main components of the knockout vector pHZLib-Cas12i3 and the edited vector library pHZLib-Cas12i3-OsACCcrRNA after insertion of OsACC gene crRNA in the CRISPR / Cas12i3 system.

[0017] Figure 3 shows the allele variation type identified by Sanger sequencing of the screened resistant plant A3-#1 and the resistance phenotype of the T1 generation plants to high-efficiency flupyradifurone.

[0018] Figure 4 shows the resistance phenotypes of A3-#1 T1 generation plants after spraying with high-efficiency flupyridine, quizalofop-p-ethyl, and clethodim. Detailed Implementation

[0019] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0020] Unless otherwise specified, the plasmids and reagents used in the embodiments of this invention can be purchased commercially.

[0021] For details on the pHZLib-Cas12i3 plasmid, please refer to CN202511501754.X.

[0022] All synthesized nucleic acids were outsourced to Beijing Qingke Xinyue Biotechnology Co., Ltd.

[0023] Example 1: Construction of rice OsACC gene plasmid library.

[0024] Using the OsACC gene sequence (accession number LOC_Os05g22940, Nipponbare rice variety) from the Oryza sativa L. genome database as a template, the extracted genome of Nanjing 46 was amplified and sequenced to obtain the Nanjing 46 OsACC gene and its upstream and downstream nucleic acid sequences. The results showed that the Nanjing 46 OsACC gene and its upstream and downstream nucleic acid sequences were completely identical to the Nipponbare OsACC gene and its upstream and downstream nucleic acid sequences. The intron and exon sequence length of the OsACC gene was 13489. The start codon of the Nanjing 46 OsACC gene and its upstream nucleic acid sequence are shown in SEQ ID No. 1. The bases from positions 1 to 1967 from the 5' to 3' end constitute the OsACC promoter portion, and positions 1968 to 1970 are the start codon ATG.

[0025] The gene editing library for the non-coding region (promoter) of the OsACC 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 of the position of the target sequence in the sequence shown in SEQ ID No. 1 is shown in Figure 1.

[0026] Using an equal volume and concentration of each crRNA oligonucleotide sequence from each library as a template, and with 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 crRNA of the OsACC gene in each library. The vector pHZLib-Cas12i3 was digested with BsaI, and approximately 15.8 kb of the vector backbone was recovered (releasing the ccdB gene fragment). The crRNA of the OsACC gene from each library was cloned into a 15.8 kb vector backbone using 2×MultiF Seamless Assembly Mix (i.e., the ccdB gene in the pHZLib-Cas12i3 vector was replaced with the crRNA of the OsACC gene in each library), and named pHZLib-Cas12i3-OsACCcrRNA(Pool01), pHZLib-Cas12i3-OsACCcrRNA(Pool02), and pHZLib-Cas12i3-OsACCcrRNA(Pool03), respectively. A schematic diagram of the main elements in the plasmids is shown in Figure 2. Forty colonies were randomly selected from each library for sequencing, achieving an accuracy of 98%.

[0027] Example 2: Rice transformation of plasmid library and screening for resistance to ACCase inhibitor herbicides.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 6) Screening of T1 generation ACCase inhibitor-resistant rice: Seeds from A3-#1 T0 generation plants and wild-type controls were treated with 50% commercial disinfectant for 30 minutes; washed 3 to 5 times with sterile water; and then sown separately on 1 / 2 MS medium containing 0.3 μmol / L haloxyfop-R-methyl to screen for haloxyfop-R-methyl resistant T1 generation plants. The plants were cultured in a light-controlled chamber for 10 days, and germination and plant development were observed. The results are shown in Figure 3. The results showed that wild-type materials germinated but could not grow on 1 / 2 MS medium containing 0.3 μmol / L haloxyfop-R-methyl, while A3-#1 seeds not only germinated but also grew into plants on 1 / 2 MS medium containing 0.3 μmol / L haloxyfop-R-methyl. This indicates that A3-#1 obtained through screening possesses haloxyfop-R-methyl resistance.

[0034] Example 3: Molecular identification of rice OsACC mutants resistant to ACCase inhibitor herbicides.

[0035] 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.

[0036] 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 identifying the crRNA sequences in A3-#1. 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.

[0037] Sanger sequencing results showed that the crRNA sequence used in A3-#1 was OsACC-Pool03-crRNA49 from Pool03 (as shown in SEQ ID No. 6), as shown in Figure 3.

[0038] 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-#1. 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.

[0039] The Sanger sequencing results of A3-#1 T0 generation, compared with the wild type, showed that A3-#1 contained a deletion mutation. Specifically, it had a -24bp / -23bp bicelestem deletion mutation, that is, with the A in the start codon ATG of the OsACC gene set to position 0, 24bp was deleted at positions 681 to 704 upstream of A, and 23bp was deleted at positions 678 to 700 upstream of A. The results are shown in Figure 4. In T1 generation plants with high resistance to flupyridine, A3-#1-24, a homozygous deletion mutation of -24bp / -24bp occurring at positions 681 to 704 upstream of the start codon, A3-#1-23, a homozygous deletion mutation of -23bp / -23bp occurring at positions 678 to 700 upstream of the start codon, and A3-#1-24 / -23, a biallelic deletion mutation of -24bp / -23bp occurring at positions 681 to 704 and 678 to 700 upstream of the start codon, were detected.

[0040] Example 4: Spectrum of herbicides against ACCase inhibitors in rice OsACC mutants.

[0041] 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.

[0042] The agricultural commercial pesticide, high-efficiency flupyradifurone EC, with an active ingredient of 10.08%, was purchased from Weifang Xinlv Chemical Co., Ltd. The recommended field dose of high-efficiency flupyradifurone EC is 48.6 g ai / ha, which is recorded as 1 × 1 times the recommended field dose.

[0043] 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.

[0044] The quizalofop-P-ethyl EC, quizalofop-P-ethyl EC, and clethodim suspension were diluted with tap water to obtain 2×, 4×, and 8× quizalofop-P-ethyl dilutions, 2× quizalofop-P-ethyl dilution, and 0.5×, 1×, 1.5×, and 2× clethodim dilutions, respectively.

[0045] T0 generation seeds of A3-#1 were sown in nutrient soil in nutrient pots and cultured in a greenhouse to obtain corresponding T1 generation plants. Tap water (0×) was used as a negative control, and wild-type rice was included as a control. Each treatment had 30 seedlings. When the rice plants grew to 2-3 leaves in the greenhouse, they were sprayed with diluted quizalofop-P-ethyl, haloxyfop-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 (see Figure 4). Figure 4 shows that wild-type rice seedlings withered and died under the influence of quizalofop-P-ethyl diluted at 4 times the field recommended dose, haloxyfop-P-ethyl diluted at 2 times the field recommended dose, and clethodim diluted at 1 times the field recommended dose, while the T1 generation plants of A3-#1 all showed normal growth and were green.

[0046] The results above indicate that the A3-#1 mutant possesses highly efficient resistance to three herbicides: flupyradifurone, quizalofop-P-ethyl, and clethodim.

Claims

1. A rice OsACC gene mutant, wherein the rice OsACC gene mutant is a deletion mutant; wherein, The deletion mutation occurred in the promoter of the rice OsACC gene. The sequence of the nucleic acid before the deletion mutation is shown in SEQ ID No.

1. The length of the deleted nucleic acid sequence is 20 bp to 27 bp. The deleted nucleic acid is located at position 0 with A in the start codon ATG of the OsACC gene. The deleted nucleic acid is located at positions 704 to 678 upstream of the start codon ATG, and at least 700 to 681 bases upstream of the start codon ATG are deleted.

2. The rice OsACC gene mutant according to claim 1, characterized in that, The deleted nucleic acid sequence is 20 bp in length, with position 0 of the A in the OsACC gene start codon ATG, and the deleted nucleic acid is located 700 to 681 upstream of the start codon ATG; and / or the deleted nucleic acid sequence is 27 bp in length, with position 0 of the A in the OsACC gene start codon ATG, and the deleted nucleic acid is located 704 to 678 upstream of the start codon ATG; and / or the deleted nucleic acid sequence is 23 bp in length, with position 0 of the A in the OsACC gene start codon ATG, and the deleted nucleic acid is located 700 to 678 upstream of the start codon ATG; and / or the deleted nucleic acid sequence is 24 bp in length, with position 0 of the A in the OsACC gene start codon ATG, and the deleted nucleic acid is located 704 to 681 upstream of the start codon ATG.

3. The application of the rice OsACC gene mutant according to claim 1 or 2 in rice ACCase inhibitor herbicides, wherein the rice variety is Nanjing 46 or Nipponbare.

4. The application according to claim 3, characterized in that, The ACCase inhibitor herbicide is at least one of haloxyfop-R-methyl, quizalofop-P-ethyl, and clethodim.

5. A method for enabling rice to acquire resistance to ACCase inhibitor herbicides, which is achieved by deleting a portion of the bases in the promoter of the OsACC gene in the rice genome through gene editing or homologous recombination to obtain the rice OsACC gene mutant as described in claim 1 or 2, wherein the rice variety is Nanjing 46 or Nipponbare.

6. The method according to claim 5, characterized in that, The method includes the following steps: 1) Obtaining 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 and pUbi-IEE-Cas12i3 into a single vector to obtain the pHZLib-Cas12i3 vector; 2) Obtaining 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 as shown in SEQ ID No. 6) Positions 34 to 56 of the sequence shown; 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) Transform the pHZLib-Cas12i3-OsACCcrRNA or pUbi-IEE-Cas12i3-HZ33-OsACCSpacer into Agrobacterium and infect rice callus tissue to screen for rice lines resistant to ACCase inhibitor herbicides.

7. The method according to claim 5 or 6, characterized in that, The ACCase inhibitor herbicide is at least one of haloxyfop-R-methyl, quizalofop-P-ethyl, and clethodim.

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