Herbicide-tolerant nucleic acid molecules and uses thereof

Abstract

The invention provides a novel nucleic acid molecule and application thereof in cultivating herbicide-resistant plants. The nucleic acid molecule provided by the invention can improve the tolerance of corn to quizalofop-p-ethyl and 2, 4-D herbicides, can be used for cultivating novel herbicide-resistant crops, and provides a solution for controlling the growth of weeds in agricultural production.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology. Specifically, the present invention provides a novel nucleic acid molecule and its application in cultivating herbicide-tolerant plants. Background Art

[0002] Quizalofop-p-ethyl is a selective post-emergence herbicide for dry fields. By inhibiting the synthesis of fatty acids in weeds, it prevents weeds from growing normally and ultimately causes them to die. It is mainly used to control annual gramineous weeds such as barnyard grass, Alopecurus aequalis, green foxtail, Polypogon fugax, Digitaria sanguinalis, Eleusine indica, Leptochloa chinensis, Phragmites australis, and Avena fatua. 2,4-D herbicides are absorbed into plants through roots, stems, and leaves and have slow metabolism. After accumulating a certain concentration, they interfere with the balance of active hormones in plants, disrupt protein and nucleic acid metabolism, and inhibit or promote the growth of certain organs of plants. They are mainly used to control dicotyledonous broad-leaved weeds such as Cyperus rotundus, Commelina communis, Alternanthera philoxeroides, Eichhornia crassipes, Ammannia baccifera, Sagittaria trifolia, Scirpus planiculmis, Polygonum spp., Vicia sativa, Descurainia sophia, Lithospermum officinale, Galium aparine, and Ranunculus japonicus, as well as gramineous weeds in the germination stage. Quizalofop-p-ethyl and 2,4-D herbicides are safe for crops such as wheat and corn, and can be decomposed by microorganisms in the soil. They have the advantages of low dosage, low cost, and high economic benefits, so they are widely used in agricultural production and are often important targets in the research and development of herbicide-tolerant crops.

[0003] The aad1 (aryloxyalkanoate dioxygenase 1) gene can be isolated from the herbicide-degrading bacterium Sphingobium herbicidovorans. This bacterium has a special enzyme, aryloxyalkanoate dioxygenase (AAD), which can degrade or convert these herbicides into substances that are harmless or of low toxicity to plants, thus losing their herbicidal activity. Through genetic engineering techniques, scientists have transferred the aad1 gene into other plants such as soybeans, cotton, and corn, thereby cultivating transgenic crops that can tolerate quizalofop-p-ethyl and 2,4-D herbicides.

[0004] In the herbicide tolerance genes of the original microbial hosts, there are many genetic elements that may cause unstable expression in plant cells, such as non-plant-specific transcription termination signals, splicing sites, and sequences with high AT content, which may lead to the instability of the mRNA encoded by the gene in plants. Compared with plant genes, there are significant differences in the GC content and codon usage preferences of these microbial genes, which limits their efficient expression in plants. When these original genes are transferred into corn, the expression of herbicide tolerance is often weak, and the tolerance level of corn plants to quizalofop-p-ethyl and 2,4-D herbicides is low, making it difficult to meet the needs of agricultural production.

[0005] By adjusting the codon usage of genes to make it more in line with the expression preferences of the host, and at the same time optimizing the structure of mRNA to reduce instability factors, the expression efficiency of genes in maize can be improved. Codon optimization requires comprehensive consideration of various factors to achieve good results, such as the codon usage preferences of the host, as well as mRNA secondary structure, restriction enzyme cleavage sites, and GC content. Simply adjusting the synonymous codons of foreign genes to the codons with high expression abundance and high usage frequency in host cells does not necessarily improve the translation efficiency of proteins, and sometimes the effect may be counterproductive. Therefore, codon optimization is not a simple experiment. In addition, nucleic acid sequence optimization for plants, especially monocotyledonous plants, is more difficult and complex. First, plants originated earlier than animals, and the genome size is much larger than that of microorganisms. The evolutionary history of plant genomes is long and the evolutionary process is more complex, with high heterozygosity, highly repetitive sequences, and complex polyploid phenomena, etc. Second, the research on plant genomes by humans started relatively late and is more difficult, so the existing data information on plant genome structure and gene expression is not rich enough. Therefore, for a specific foreign protein, it is not very certain what kind of coding method can achieve higher protein functional efficiency.

[0006] In order to obtain the aad1 gene with high expression and stable inheritance in maize, the present invention finally screened out a nucleic acid molecule with the best herbicide tolerance effect in maize through a large number of bioinformatics analyses, gene syntheses, and experimental verifications, providing strong support for cultivating maize varieties resistant to quizalofop-p-ethyl and 2,4-D herbicides. Summary of the Invention

[0007] The present invention provides a nucleic acid molecule, characterized in that the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID No.6 or its reverse complementary sequence.

[0008] The present invention also provides an expression vector, characterized in that the expression vector contains the above-mentioned nucleic acid molecule.

[0009] The present invention also provides a host cell, characterized in that the host cell contains the above-mentioned nucleic acid molecule, or the above-mentioned expression vector; the host cell is a microbial cell or a non-renewable animal or plant cell.

[0010] In some embodiments, the above-mentioned host cell is an Agrobacterium cell or an Escherichia coli cell or a non-renewable maize plant cell.

[0011] The present invention also provides a method for producing herbicide-tolerant transgenic maize, characterized in that the above-mentioned nucleic acid molecule, or expression vector, or host cell is used to transform maize to obtain transformed plant cells, and then the transformed plant cells are cultured into transgenic plants.

[0012] The present invention also provides the use of the above nucleic acid molecule, expression vector, host cell, and method in the production of herbicide-tolerant maize.

[0013] The present invention also provides a method for protecting maize plants from damage caused by herbicides, which is characterized by comprising applying an effective dose of quizalofop-P-ethyl or 2,4-D herbicides to a field planted with at least one transgenic maize plant, wherein the transgenic plant cells contain the above nucleic acid molecule or the above expression vector in their genomes; and the transgenic maize plants have tolerance to quizalofop-P-ethyl and 2,4-D herbicides.

[0014] The beneficial effects of the present invention are as follows: For the aryloxyalkanoate dioxygenase gene aad1, through a large amount of bioinformatics analysis and specific experimental tests, a nucleic acid molecule with the best herbicide tolerance effect in maize is obtained. This nucleic acid molecule can be used to cultivate new herbicide-tolerant maize.

[0015] The technical solutions of the present invention will be further described in detail below with reference to the drawings and examples. Description of the Drawings

[0016] Figure 1 Physical map of the expression vector pBWA(V)HS-bar-aad1, taking the P5 vector as an example. Detailed Description of the Invention

[0017] The following definitions and methods are provided to better define the present application and guide those of ordinary skill in the art in the practice of the present application. Unless otherwise specified, the terms are understood according to the conventional usage of those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards, and other publicly published materials cited herein are incorporated herein by reference in their entirety.

[0018] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or substitution of the methods, steps, or conditions of the present invention belongs to the scope of the present application. Unless otherwise specified, the examples are carried out under conventional experimental conditions, such as those described in Molecular Cloning: A Laboratory Manual by Sambrook et al. (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or according to the conditions recommended by the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those of ordinary skill in the art.

[0019] Example 1 Optimization of the aad1 Gene Sequence

[0020] There is codon preference in the translation process of organisms. The most basic principle of codon optimization is to replace the codons in the exogenous mRNA sequence with synonymous codons that are frequently used in host cells to ensure that the codons in the exogenous mRNA sequence and the codon usage bias of the host cell are more consistent, thereby improving the expression level of the protein. The degree of consistency is often expressed by the Codon Adaptation Index (CAI), which reflects the degree to which the synonymous codons in the coding region are consistent with the optimal use of codons in a specific species. The value range is between 0-1 and is species-specific. In theory, the closer this value is to 1, the higher the protein expression of the exogenous mRNA in the host cell. In addition, the secondary structure of mRNA has a certain influence on the translation efficiency, and the restriction enzyme cutting site will also affect the operation of constructing the expression vector. The GC content is related to the stability of DNA. Too low or too high a GC content may indirectly affect the expression regulation of the gene.

[0021] For aryloxyalkanoate dioxygenase AAD1 (amino acid sequence as shown in SEQ ID NO.1), in order to obtain a coding nucleic acid molecule with better herbicide resistance effect, the present invention counted the codon usage between different genes of monocotyledonous plants such as corn, and focused on the CAI (codon adaptability index) value of codons of highly expressed genes in different species, while paying attention to parameters such as relative usage of synonymous codons (RSCU) and number of effective codons (Nc), and combined with consideration of various factors such as mRNA secondary structure, species repeat sequence structure, rare codons, hidden editing sites, GC content and enzyme cutting sites, and preliminarily screened dozens of nucleotide sequences. Then, 6 sequences with high CAI value (CAI value ≥ 0.75), moderate GC content (between 50%-70%) and small Nc value (Nc ≤ 25) were selected, and named aad1-1 to aad1-6 in sequence, and the sequence-related parameter information is shown in Table 1.

[0022] Table 1aad1 nucleic acid molecule sequence list

[0023]

[0024]

[0025] Example 2 Obtaining transgenic plants and identifying their herbicide tolerance

[0026] Six candidate nucleic acid molecules aad1-1 to aad1-6 in Synthetic Example 1, and nucleic acid molecules aad1-7 to aad1-10 of four published aad1 genes (corresponding to SEQ ID NO: 37, 39, 41 of Patent No. MY161583A and SEQ ID NO: 3 of WO2005107437A3 in sequence), were respectively ligated with the NOS promoter and the NOS terminator to construct 10 plant expression vectors pBWA(V)HS-bar-aad1 (the bar gene expression cassette was used as a selection marker), numbered P1 to P10 in sequence. The element information of the expression cassette in the T-DNA region is shown in Table 2, and the physical map of the vector is shown in Figure 1 (taking the P5 vector as an example).

[0027] Table 2 Element Information Table of the T-DNA Region of 10 Expression Vectors

[0028]

[0029] After transforming the above expression vectors into Escherichia coli DH5α, the plasmids were extracted and then transferred into Agrobacterium tumefaciens EHA105 or LBA4404. By the Agrobacterium-mediated method, the above 10 vectors were respectively transformed into the maize inbred line B104. Glufosinate was used as a selection agent, and finally a batch of maize positive transgenic plants were obtained (the vector construction and plant genetic transformation were carried out by the general methods in the art), and the herbicide tolerance of the T1 generation transgenic positive plants was identified in the field.

[0030] 1. Identification of the tolerance to quizalofop-p-ethyl herbicide

[0031] The quizalofop-p-ethyl herbicide used in this experiment has the trade name Dunjian. The content of the active ingredient quizalofop-p-ethyl is 20%, the dosage form is emulsifiable concentrate, and the manufacturer is Hunan Nongda Hite Agrochemical Co., Ltd. The recommended dosage of the product is 17 - 25 mL / acre, the medium dosage is 21 mL / acre, and the water volume per acre is 30 kg. It is evenly sprayed on the stems and leaves at the 3 - 5 leaf stage of maize plants.

[0032] One week after spraying clear water (0×) and quizalofop-p-ethyl herbicide at 4 times the medium dosage of the recommended field dosage (4×, 84 mL / acre) on the above transgenic maize and the control B104 planted in the field, the phytotoxicity rate of all maize plants was investigated and counted. The classification of phytotoxicity symptoms is shown in Table 3.

[0033] Table 3 Standard for the Phytotoxicity Grade of Quizalofop-p-ethyl Herbicide to Maize

[0034]

[0035] The herbicide injury rate is calculated according to formula (1).

[0036]

[0037] Where: X represents the damage rate, in percentage (%); N represents the number of damaged plants at the same level; S represents the number of levels; T represents the total number of plants; M represents the highest level.

[0038] Table 4 Tolerance of Maize Plants to Quizalofop-P-Ethyl Herbicides

[0039]

[0040]

[0041] The test results are shown in Table 4. When no herbicide was sprayed, the receptor control B104 and all transgenic maize plants grew normally without phytotoxicity. When spraying the quizalofop-p-ethyl herbicide at the medium dose of 4 times the field recommended dosage, the phytotoxicity rate of the receptor control was the highest, reaching 96.7%; transgenic maize with different vectors showed varying degrees of damage. Among them, the transgenic maize with the P5 vector had the least damage, with a phytotoxicity rate of 16.0%, which was much lower than that of the control and transgenic maize with other vectors.

[0042] 2. Identification of Tolerance to 2,4-D Herbicides

[0043] The 2,4-D herbicide used in this test has the trade name Dingyuanfang. The content of the active ingredient 2,4-D isooctyl ester is 87.5%, the dosage form is emulsifiable concentrate, and the manufacturer is Weihai Hanfu Biochemical Pharmaceutical Co., Ltd. The recommended dosage of the product is 40 - 50 mL / acre, the medium dose is 45 mL / acre, and the amount of water added per acre is 35 kg. It is evenly sprayed on the stems and leaves at the 3 - 5 leaf stage of maize plants.

[0044] One week after spraying clear water (0×) and the 2,4-D isooctyl ester herbicide at 4 times the medium dose of the field recommended dosage (4×, 180 mL / acre) on the above-mentioned transgenic maize and control B104 planted in the field, the phytotoxicity rate of all maize plants was investigated and statistically analyzed. The grading standard of phytotoxicity symptoms is shown in Table 5.

[0045] Table 5 Grade Standard of Phytotoxicity of 2,4-D Isooctyl Ester Herbicides to Maize

[0046]

[0047]

[0048] The herbicide damage rate is calculated according to formula (1).

[0049]

[0050] Where: X represents the damage rate, in percentage (%); N represents the number of damaged plants at the same level; S represents the number of levels; T represents the total number of plants; M represents the highest level.

[0051] Table 6 Tolerance of Maize Plants to the Herbicide Isooctyl 2,4-D

[0052]

[0053]

[0054] The test results of the herbicide are shown in Table 6. When no herbicide was sprayed, all the receptor control B104 and all transgenic maize plants grew normally without phytotoxicity. When spraying the medium dose of isooctyl 2,4-D herbicide at 4× the field recommended dosage, the phytotoxicity rate of the receptor control was the highest, reaching 68.9%; transgenic maize with different vectors showed different degrees of damage. Among them, the transgenic maize with the P5 vector had the least damage, and the damage rate was 7.3%, which was much lower than that of the control and transgenic maize with other vectors.

[0055] The experimental results show that the transgenic maize with the P5 vector has significantly higher tolerance to quizalofop-p-ethyl and 2,4-D herbicides than the control, and is also superior to the other transgenic materials. It can be seen that the nucleic acid molecule aad1-5 improves the herbicide tolerance of maize plants better than the nucleic acid molecules of the other 9 aad1 genes.

[0056] In summary, through the optimization of the aad1 coding sequence, the present invention obtained a nucleic acid molecule aad1-5 (the sequence is SEQ ID NO: 6) with the strongest resistance to quizalofop-p-ethyl and 2,4-D herbicides in maize. After identification, the transgenic maize produced using this nucleic acid molecule has good tolerance to quizalofop-p-ethyl and 2,4-D herbicides.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nucleic acid molecule, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No. 6 or its reverse complementary sequence.

2. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 1.

3. A host cell, characterized in that The host cell contains the nucleic acid molecule of claim 1 or the expression vector of claim 2; The host cell is a microbial cell or a non-renewable animal or plant cell.

4. The host cell according to claim 3, characterized in that The host cell is an Agrobacterium cell or an Escherichia coli cell or a non-renewable corn plant cell.

5. A method for producing transgenic corn, characterized in that: The nucleic acid molecule of claim 1, or the expression vector of claim 2, or the host cell of any one of claims 3-4 is transformed into corn to obtain transformed corn cells, and then the transformed corn cells are cultured into transgenic corn.

6. Use of the nucleic acid molecule according to claim 1, or the expression vector according to claim 2, or the host cell according to any one of claims 3 to 4, or the method according to claim 5 in producing herbicide-resistant corn.

7. A method for protecting corn plants from damage caused by herbicides, characterized in that The invention comprises applying an effective dose of quizalofop-p-ethyl and / or a 2,4-D herbicide to a field planted with at least one transgenic corn plant, wherein the transgenic corn plant cell comprises the nucleic acid molecule of claim 1 or the expression vector of claim 2 in its genome; and the transgenic corn plant has tolerance to quizalofop-p-ethyl and 2,4-D herbicides.

Images