Acetyl coenzyme A carboxylase mutant and application thereof

Abstract

The invention belongs to the field of biotechnology and crop genetic breeding, and particularly relates to an acetyl coenzyme A carboxylase mutant and application thereof. The invention discloses a mutant acetyl-coenzyme A carboxylase (ACC) protein, a coding nucleic acid thereof and an application of the mutant acetyl-coenzyme A carboxylase (ACC) protein in plant breeding, and particularly provides the mutant acetyl-coenzyme A carboxylase (ACC) protein, and compared with a parent acetyl-coenzyme A carboxylase (ACC) protein, the mutant acetyl-coenzyme A carboxylase (ACC) protein has the advantages that the mutant acetyl-coenzyme A carboxylase (ACC) protein is more stable, and the yield of the mutant acetyl-coenzyme A carboxylase (ACC) protein is higher than that of the parent acetyl-coenzyme A carboxylase (ACC) protein; the rice after the mutation of the corresponding 1927 amino acid and the mutation of acetyl-CoA carboxylase (ACC) has very high herbicide resistance to quizalofop-p-ethyl and haloxyfop-R-methyl, and has a very wide application prospect in the cultivation of herbicide-resistant crops.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and crop genetic breeding, and specifically relates to an acetyl-CoA carboxylase mutant and an application thereof. Background Art

[0002] Rice (Oryza sativa) is one of the three most important food crops in the world and is the basic food for more than half of the world's population. Farmland weeds are one of the important biological disasters that threaten agricultural production. Farmland weeds compete with crops for light, water, fertilizer, and spread pests and diseases, which will lead to reduced crop yield and quality. In my country, the area of ​​rice field weeds that harm rice fields reaches about 300 million mu every year. Chemical weed control is the most important means of weed control and is also an indispensable measure in modern agriculture. Since 2,4-D began to be used in the 1940s, the herbicide industry has a development history of more than 60 years, and a large number of selective herbicides have been successfully developed so far. Research on ACC enzyme inhibitors began in the 1970s. ACC herbicides are divided into four types, namely, aryloxyphenoxy propionate (APP), cyclohexanedione oximes (CHD), aryloxyphenylcy-clohexanedione (APCHD) and cyclohexanedione (CTR). The first two types are the most common or commercialized. ACC herbicides can inhibit fatty acid synthesis in grass plants, have high selectivity, are transmitted in plants, and can control annual or perennial grass weeds after seedlings. It has the advantages of high efficiency, low toxicity, long application period, and safety for subsequent crops, so it occupies an important position in the herbicide market.

[0003] As an emerging genome editing technology, CRISPR / Cas9 can insert and delete DNA at specific sites in the genome and replace one or more bases. Single-base editing is a gene editing method that uses the CRISPR / Cas system to target deaminases to specific sites in the genome to modify specific bases. This method has achieved the creation of herbicide-resistant germplasm resources in a variety of crops.

[0004] Acetyl-CoA carboxylase (ACCase, ACC) is the rate-limiting enzyme in the de novo fatty acid synthesis pathway in plants, and is involved in the process of generating malonyl-CoA from acetyl-CoA as a precursor in the plastid. Since ACCase plays a key role in the normal growth and development of plants, it is a very important herbicide target.

[0005] CN 113755457 A discloses that plants containing mutant acetyl-CoA carboxylase (ACC) proteins have good resistance to fenpyroxil, sethoxydim and clethodim, but cannot develop tolerance to high-efficiency haloxyfop-P-ethyl or fenoxaprop-P-ethyl. Summary of the invention

[0006] In order to cultivate a new plant variety resistant to highly effective haloxyfop-butyl or fenoxaprop-butyl herbicides, the present invention provides the following technical solutions.

[0007] The invention discloses a mutant acetyl-CoA carboxylase DNA. The nucleotide sequence of the DNA encoding the acetyl-CoA carboxylase is shown in SEQ ID NO:1.

[0008] A CDS sequence of a DNA encoding acetyl-CoA carboxylase, wherein the CDS sequence is shown in SEQ ID NO: 2.

[0009] An acetyl-CoA carboxylase protein, the amino acid sequence of the protein is as shown in SEQ ID NO: 3.

[0010] Application of a mutant acetyl-CoA carboxylase DNA or its CDS sequence or the protein encoded by the mutant acetyl-CoA carboxylase in preparing a reagent or a kit for making plants resistant to herbicides.

[0011] Application of mutant acetyl-CoA carboxylase DNA or its CDS sequence or the protein encoded by it in breeding new herbicide-resistant plant varieties.

[0012] Preferably, the herbicide is at least one of haloxyfop-ethyl or quizalofop-ethyl.

[0013] Preferably, the plant is rice. Preferably, the rice is japonica rice variety Xiushui 134, indica rice variety Yuzhenxiang and Xizi 3.

[0014] Beneficial effects of the present invention:

[0015] The invention discloses a mutant acetyl-CoA carboxylase (ACC) protein and a nucleic acid encoding the same, and application thereof in plant breeding. Specifically, the invention provides a mutant acetyl-CoA carboxylase (ACC) protein, and compared with a parent acetyl-CoA carboxylase (ACC) protein, the mutant acetyl-CoA carboxylase (ACC) protein has a mutation in the corresponding amino acid at position 1927, and rice after the acetyl-CoA carboxylase (ACC) mutation has high herbicide resistance to quizalofop-p-ethyl and haloxyfop-p-ethyl, and has broad application prospects in cultivating herbicide-resistant crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Schematic diagram of evoAPOBEC1-SpRYCas9n and evoCDA1--SpRYCas9n base editor vectors;

[0017] Figure 2 To obtain resistant mutant materials;

[0018] Figure 3 Spray the stems and leaves with herbicides. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example 1 Library construction and screening of herbicide resistance mutation sites

[0021] 1. Construct evoCDA1-SpRYCas9n(CBE) and evo APOBE C1-SpRYCas9n(CBE) base editors targeting the endogenous ACCase gene in rice.

[0022] Base editors can achieve base conversion from C / G->T / A (CBE) within a certain sequence window, and evoCDA1 and evoAPOBEC1 are optimized based on the base editors studied by previous researchers, and the efficiency of targeted base editing has been further improved. SpRYCas9 is improved from SpCas9. SpCas9 mainly recognizes the PAM sequence of NGG, while SpRYCas9 greatly reduces the requirements for the PAM motif and can use almost any three bases as the PAM sequence, but the recognition efficiency of NRN is higher than that of NYN (R=G / A, Y=C / T). In order to further improve the efficiency of rice base editing and expand the scope of base editing, we fused the new deaminase and Cas9 protein together. Based on the previously developed Anc689BE4max-nCas9 base editor, we replaced the deaminase and Cas9 proteins to form the new evoAPOBEC1-SpRYCas9n (CBE) and evoCDA1-SpRYCas9n (CBE) base editors ( Figure 1 ).

[0023] The present invention uses evoAPOBEC1-SpRYCas9n (CBE) and evoCDA1-SpRYCas9n (CBE) as vectors, designs sgRNA (sgRNA shown in Table 1) in the endogenous OsACC gene of rice (LOC_Os05g22940), and clones them into the vectors respectively to form base editors targeting endogenous OsACC of rice.

[0024] Table 1 sgRNA sequences targeting rice ACC (guide sequences)

[0025] sgRNA ID guide-PAM sequence (5'-3') SG1-F ACCAGCCTATTATTCTTAC SG1-R GTAAGAATAATAGGCTGGT

[0026] 2. Rice genetic transformation and identification of transgenic seedlings

[0027] The base editing library constructed above was transformed into callus tissue of rice japonica variety Xiushui 134 by Agrobacterium, and transgenic positive callus was selected by hygromycin. During the differentiation stage of positive callus, we added 0.2 mg / L high-efficiency haloxyfop-R-methyl (Sigma) targeting ACCase to the culture medium. On this culture medium, callus tissue transferred with CBE base editor, when OsACC gene achieved base editing (CC was replaced by TT at positions 11878-11879 of the parent, and the specific nucleotide sequence after mutation was shown in SEQ ID NO:1, and its CDS sequence was shown in SEQ ID NO:2) leading to amino acid mutation (amino acid mutation occurred at position 1927, from proline to phenylalanine, and the specific amino acid of the protein after mutation was shown in SEQ ID NO:3), and the mutation site was able to produce resistance to high-efficiency haloxyfop-R-methyl, the corresponding mutant plants could differentiate normally to form seedlings and the roots could normally penetrate into the culture medium with herbicide added ( Figure 2 ).

[0028] 3. Genotyping of resistant mutants

[0029] The CBE base transmitter obtained dozens of T0 generation positive seedlings through Agrobacterium transformation. We extracted leaf DNA from the screened resistant plants, identified the target region of the OsACC gene by PCR and Sanger sequencing, and identified the above herbicide-resistant plants by PCR and sequencing. It was found that the resistant plants had the expected base substitution within the target range, and the specific editing type was P1927F.

[0030] 4. Tolerance of resistant mutants to high-efficiency halpyr and quizalofop-p-ethyl

[0031] The homozygous mutant strains isolated from T-DNA of the T2 generation were subjected to herbicide tolerance experiments. For the P1927F mutant planted in the culture room, stem and leaf spray treatment was carried out at the 3-4 leaf stage. The high-efficiency halpyr-methyl was applied at 40.5 g ai / ha, and the quizalofop-p-ethyl was applied at 63.4 g ai / ha. After 14 days, the edited plants treated with the drugs showed resistance to both the high-efficiency halpyr-methyl and the quizalofop-p-ethyl compared with the wild-type control treated with the drugs ( Figure 3 ). For the P1927F mutant in the planting field, stem and leaf spray treatment was carried out at the 3-4 leaf stage. The high-efficiency halpyr-methyl was applied at 40.5 g ai / ha and the quizalofop-p-ethyl was applied at 63.4 g ai / ha. After 14 days, the edited plants treated with the drugs also developed resistance to both the high-efficiency halpyr-methyl and the quizalofop-p-ethyl compared with the wild-type control treated with the drugs.

Claims

1. A DNA encoding acetyl-CoA carboxylase, characterized in that: The nucleotide sequence of the mutant acetyl-CoA carboxylase DNA is shown in SEQ ID NO:

1.

2. A CDS sequence of a mutant acetyl-CoA carboxylase DNA according to claim 1, characterized in that: The CDS sequence is shown in SEQ ID NO:

2.

3. A protein encoded by the DNA encoding acetyl-CoA carboxylase according to claim 1 or the CDS sequence according to claim 2, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO:

3.

4. Use of the DNA encoding acetyl-CoA carboxylase according to claim 1, the CDS sequence according to claim 2, or the protein according to claim 3 in preparing a reagent or a kit for making plants resistant to herbicides.

5. The use according to claim 4, characterized in that The herbicide is at least one of haloxyfop-ethyl or quizalofop-ethyl.

6. Use according to claim 5, characterized in that The plant is rice.

7. The use according to claim 6, characterized in that The rice is the japonica rice variety Xiushui 134, and the indica rice varieties Yuzhenxiang and Xizi No.

3.

8. Use of the mutant acetyl-CoA carboxylase DNA according to claim 1, the CDS sequence according to claim 2, or the protein according to claim 3 in breeding new herbicide-resistant plant varieties.

9. The use according to claim 8, characterized in that The new plant variety is a new rice variety.

10. The use according to claim 9, characterized in that The herbicide is at least one of haloxyfop-ethyl or quizalofop-ethyl.

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