LcCYP93A3 gene, protein, application of LcCYP93A3 gene, protein, overexpression vector and overexpression method

Abstract

The invention relates to the technical field of agricultural science and plant biological genetic engineering, in particular to an LcCYP93A3 gene, a protein, application of the LcCYP93A3 gene, an overexpression vector and an overexpression method. The nucleotide sequence of the LcCYP93A3 gene is as shown in SEQ ID No. 1 (sequence identifier number 1). According to the invention, the expression of LcCYP93A3 is enhanced by using a transgenic technology, an overexpression transgenic plant is constructed, and the drug resistance of the plant to cyhalofop-butyl can be obviously enhanced. The gene regulation strategy provided by the invention provides a gene resource for cultivating herbicide-resistant crops, and has important agricultural application value.

Description

Technical Field

[0001] The invention relates to the field of agricultural science and plant biological genetic engineering technology, and in particular to a LcCYP93A3 gene, protein and application thereof, an overexpression vector and a method. Background Art

[0002] With the development of global agriculture, chemical herbicides have become an important tool for weed control in farmland. However, long-term reliance on the use of a single type of herbicide has led to the gradual increase of weed resistance, bringing new challenges to crop production. The cultivation of herbicide-resistant crops is an effective weed management strategy. By introducing herbicide-resistant genes, crops can grow normally under specific herbicide treatments, which helps to achieve more efficient weed management.

[0003] Herbicide resistance genes are the core of herbicide-resistant crop breeding. Weeds are exposed to herbicides for a long time and gradually evolve resistance to herbicides under the pressure of natural selection. Therefore, they are ideal research objects for mining herbicide resistance genes. Leptochloa chinensis is one of the main malignant weeds in rice fields, posing a serious threat to rice yield. Cyhalofop-butyl is an acetyl-CoA carboxylase (ACCase) inhibitor widely used in chemical control of grass weeds. However, with the long-term use of cyhalofop-butyl, the problem of herbicide resistance in Leptochloa chinensis has gradually emerged, resulting in a decrease in herbicide efficacy, an increase in herbicide usage, environmental pollution, and crop damage. Plant resistance can be divided into two types: target resistance (TSR) and non-target resistance (NTSR). Target resistance is usually caused by mutations in herbicide target genes, while non-target resistance involves multiple complex gene networks, which are usually related to the metabolic degradation, efflux mechanism, and antioxidant response of herbicides.

[0004] With the development of omics technology, the discovery of herbicide-resistant genes has been significantly promoted. Transcriptomics and genomics technologies enable scientists to analyze the resistance mechanism of plants to herbicides in multiple dimensions such as gene expression levels, genome structure, and metabolic pathways. By performing transcriptome sequencing analysis on the whole genome expression of plants under herbicide treatment, resistance-related gene modules and regulatory networks can be identified. The identified potential resistance genes can be further verified by transgenic means. These genes are transferred into model plants or target crops, and the resistance function of the genes is verified by observing the growth performance of the plants under herbicide treatment. Therefore, the discovery of new resistance genes is an important prerequisite for the future breeding of herbicide-resistant crops. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a LcCYP93A3 gene, protein and application thereof, an overexpression vector and method, which clarify the function of the LcCYP93A3 gene in herbicide resistance and provide in-depth application in the subsequent breeding of herbicide-resistant crops.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a LcCYP93A3 gene, the nucleotide sequence of the LcCYP93A3 gene is shown as SEQ ID No.1.

[0008] The present invention also provides a protein encoded by the LcCYP93A3 gene in the above technical solution, and the amino acid sequence of the protein is shown in SEQ ID No.2.

[0009] The present invention also provides the use of the LcCYP93A3 gene described in the above technical solution or the protein described in claim 2 in enhancing the resistance of plants to cyhalofop-butyl.

[0010] Preferably, the plant includes Leptochloa chinensis.

[0011] The present invention also provides the application of the LcCYP93A3 gene described in the above technical solution in the breeding of herbicide-resistant crops.

[0012] Preferably, the herbicide comprises cyhalofop-butyl.

[0013] The present invention also provides an overexpression vector, which is obtained by inserting the LcCYP93A3 gene described in the above technical solution into a plant expression vector pBWA.

[0014] The present invention also provides a method for obtaining herbicide-resistant plants, comprising the following steps:

[0015] 1) Transforming the overexpression vector described in the above technical solution into Agrobacterium to obtain transformed bacteria;

[0016] 2) Infecting plant callus with the transformed bacteria obtained in step 1) to obtain herbicide-resistant plants.

[0017] Preferably, the herbicide includes cyhalofop-butyl and the plant includes Leptochloa chinensis.

[0018] Preferably, the infection conditions include: placing the plant callus in a bacterial solution of the transformation bacteria, and culturing at 200 rpm and 28° C. for 15 to 20 minutes;

[0019] The OD of the bacterial solution 600 The value is 0.5~0.6.

[0020] The present invention reveals the important role of LcCYP93A3 gene in cyhalofop-butyl resistance through transcriptome sequencing analysis and weighted gene co-expression network analysis (WGCNA). Studies have found that the expression of LcCYP93A3 gene is regulated by the circadian rhythm gene LcRVE4, and the expression level increases in a specific time period, which can improve the resistance of plants to cyhalofop-butyl. Using transgenic technology to enhance the expression of LcCYP93A3 and construct overexpression transgenic plants can significantly enhance the resistance of plants to cyhalofop-butyl. The gene regulation strategy proposed in the present invention provides gene resources for cultivating herbicide-resistant crops and has important agricultural application value.

[0021] Beneficial effects of the present invention:

[0022] 1. Revealing the resistance function of LcCYP93A3 gene at the molecular level

[0023] The present invention analyzes, identifies and clones the LcCYP93A3 gene in Leptochloa chinensis by bioinformatics methods, and verifies its function by genetic transformation. By constructing an overexpression transgenic strain of the gene, the mechanism of action of the LcCYP93A3 gene in cyhalofop-butyl resistance is clarified, providing a valuable theoretical basis and gene resources for the cultivation of herbicide-resistant crops.

[0024] 2. Improve the herbicide resistance of crops and create new resistant varieties

[0025] The expression of LcCYP93A3 gene significantly improved the plant's resistance to cyfluthrin herbicide, and cultivated new germplasm with resistance, which can maintain a good growth state under herbicide treatment conditions. This result not only reflects the important role of LcCYP93A3 gene in plant resistance, but also provides new ideas and resources for molecular breeding of crop resistance to herbicides. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0027] Figure 1 This is a graph showing the resistance results of transferring the LcCYP93A3 gene into cyhalofop-butyl in Leptochloa chinensis. DETAILED DESCRIPTION

[0028] The present invention provides a LcCYP93A3 gene, the nucleotide sequence of the LcCYP93A3 gene is shown in SEQ ID No. 1,

[0029] SEQ ID No.1:

[0030]

[0031] The present invention also provides a protein encoded by the LcCYP93A3 gene in the above technical solution, wherein the amino acid sequence of the protein is shown in SEQ ID No. 2,

[0032] SEQ ID No.2:

[0033] MAEEAAVLVAVAAIVLVLVLWRRSRRTTGKLPPSPLGLPLIGHLHLIRPPPHRAFDRIISRYGPLVYLRLGPSTHCVVAGTADAARDLLRFEASIPERPLTAVTRHLAYDSAGFAFAPYGPHWRFMK RLCMSELLGPRTVDQLRPVREAELAAVLGAARDAAARGEALDVSRQLIRLANNAIMRMVATDLPGDMTEEARDCAKQVAEVVGAFNLEDYVALCKGWDLQGLTRRTREVRDRFDALMEIMIKAKEER RREHATKEEDLLDILMDAAEDRKADVKLTRENIKAFILDIFTAGSDTTATSVEWMLAHLINNPACLRKLRQELDDVVGKSRLVAEHDVARLPYLNAVFKETLRLQPPAVFAQRETIEPVHVRGYTIP AKTSVFFNIFSIGRDPACWDQPLLFRPERFMPGGEGAAIDPKGQHMQLMPFGSGRRACPGMGLAMQAVPAFLAAMVQCFDWAVPVPQGQSKAPPLDMEEAEGLVSARKQPLLLFPTQRIHPLPMS*.

[0034] The present invention also provides the use of the LcCYP93A3 gene described in the above technical solution or the protein described in the above technical solution in enhancing the resistance of plants to cyhalofop-butyl. In the present invention, the plant preferably includes Leptochloa chinensis. In the present invention, the plant is also preferably applicable to genes having the gene or homologous thereto, and is particularly suitable for gramineous crops such as rice, corn, wheat, etc. In the present invention, the plant is also preferably including but not limited to: Arabidopsis thaliana, and all genes having the gene or homologous thereto are applicable.

[0035] The present invention also provides the use of the LcCYP93A3 gene described in the above technical solution in herbicide-resistant crop breeding. In the present invention, the herbicide preferably includes cyhalofop-butyl.

[0036] The present invention also provides an overexpression vector obtained by inserting the LcCYP93A3 gene described in the above technical solution into the plant expression vector pBWA. The present invention has no special limitation on the method of inserting the LcCYP93A3 gene into the plant expression vector pBWA, and those skilled in the art can perform conventional operations, which will not be described in detail here.

[0037] The present invention also provides a method for obtaining herbicide-resistant plants, comprising the following steps:

[0038] 1) Transforming the overexpression vector described in the above technical solution into Agrobacterium to obtain transformed bacteria;

[0039] 2) Infecting plant callus with the transformed bacteria obtained in step 1) to obtain herbicide-resistant plants.

[0040] The present invention transfers the overexpression vector described in the above technical solution into Agrobacterium to obtain a transformed bacterium. The present invention does not specifically limit the type of the Agrobacterium, and a strain used for conventional transformation can be used, such as Agrobacterium EHA105 strain. The present invention does not specifically limit the method for transferring the overexpression vector into Agrobacterium, and those skilled in the art can operate according to conventional methods, which will not be repeated here.

[0041] The present invention infects plant callus with the obtained transformed bacteria to obtain herbicide-resistant plants. In the present invention, the herbicide preferably includes cyhalofop-butyl, and the plant preferably includes Leptochloa chinensis. In the present invention, the infection conditions preferably include: placing the plant callus in a bacterial solution of the transformed bacteria, and culturing at 200 rpm and 28° C. for 15 to 20 minutes. In the present invention, the OD of the bacterial solution is 600 The value is preferably 0.5 to 0.6.

[0042] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] Cloning of LcCYP93A3 gene and acquisition of gene overexpression plant materials:

[0045] Based on the genome sequence information of Leptochloa chinensis, specific primers for amplifying the LcCYP93A3 gene were designed.

[0046] The primer sequences are as follows: forward primer, LcCYP93A3-F (SEQ ID No. 3):

[0047] 5′-ATGGCGGAGGAAGCCGCC-3′;

[0048] Reverse primer, LcCYP93A3-R (SEQ ID No. 4):

[0049] 5'-GGACATGGGAAGCGATGGATG-3'.

[0050] The complete coding sequence (CDS) of LcCYP93A3 gene (SEQ ID No. 1) was amplified from the cDNA of Leptochloa chinensis. The PCR amplification conditions were: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 30 seconds, annealing temperature of 58°C for 30 seconds, extension at 72°C for 1 minute, a total of 35 cycles, and a final extension at 72°C for 5 minutes. The amplified product was purified after confirmation by agarose gel electrophoresis.

[0051] The obtained LcCYP93A3 gene CDS was inserted into the plant expression vector pBWA, and the expression of the gene was achieved under the drive of the 35S promoter to generate the LcCYP93A3 overexpression (LcCYP93A3-OE) vector. The vector construction was achieved by restriction endonuclease digestion and T4 DNA ligase ligation, and the constructed recombinant vector was verified by sequencing. These recombinant vectors were used for Agrobacterium-mediated genetic transformation of Leptochloa chinensis. The transgenic vector was transferred into the Agrobacterium EHA105 strain, and then the callus was placed in the Agrobacterium suspension (OD600 = 0.5-0.6) and cultured on a shaker at 200 rpm and 28°C for 15-20 minutes. After two generations, hygromycin-resistant callus was selected on the selection medium. After 2-3 weeks, the callus was separated into fresh callus induction medium and 1-2 generations of callus were cultured.

[0052] Example 2

[0053] LcCYP93A3 gene affects cyhalofop-butyl resistance in Leptochloa chinensis

[0054] Functional characterization was performed using the transgenic Leptochloa chinensis plants (LcCYP93A3-OE1 and LcCYP93A3-OE2) into which the LcCYP93A3 gene was introduced in Example 1 and the wild-type Leptochloa chinensis (WT) for comparison.

[0055] When the plants grow to 4-5 leaves, the recommended field dose of cyhalofop-butyl (105 g A.I. ha -1 ) were foliar sprayed on transgenic plants and WT plants. Two weeks after spraying, the transgenic and WT groups were photographed and measured under the same conditions. Two weeks after spraying cyhalofop-butyl, transgenic LcCYP93A3-OE1 and LcCYP93A3-OE2 showed stronger resistance than WT.

[0056] The growth of the WT group plants was significantly inhibited, and the leaves turned yellow, while the leaves of the transgenic plants remained healthy and green ( Figure 1 ). The plant heights of transgenic and WT plants in the cyhalofop-butyl treated and control groups were measured. The results showed that after cyhalofop-butyl treatment, the plant height of the WT group was significantly inhibited, with an inhibition rate of about 38%; while the plant height inhibition rates of LcCYP93A3-OE1 and LcCYP93A3-OE2 were only 11% and 3%, respectively, indicating that the growth of transgenic plants was not significantly inhibited ( Figure 1 ). The fresh weight of the plants was also measured to assess growth inhibition. The fresh weight inhibition rate of WT plants was approximately 59%; in contrast, the fresh weight inhibition rates of LcCYP93A3-OE1 and LcCYP93A3-OE2 plants were only 16% and 17% ( Figure 1 ).

[0057] The above results show that overexpression of the LcCYP93A3 gene significantly enhances the resistance of Leptochloa chinensis to cyhalofop-butyl. Overexpression of the LcCYP93A3 gene significantly reduced the inhibition rate of plant height and fresh weight after spraying cyhalofop-butyl, further confirming the role of this gene in conferring cyhalofop-butyl resistance to plants. It also provides new gene resources for studying herbicide-resistant genes, which is helpful for breeding herbicide-resistant crops.

[0058] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A LcCYP93A3 gene, characterized in that: The nucleotide sequence of the LcCYP93A3 gene is shown in SEQ ID No.

1.

2. A protein encoded by the LcCYP93A3 gene according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. Use of the LcCYP93A3 gene according to claim 1 or the protein according to claim 2 in enhancing the resistance of plants to cyhalofop-butyl.

4. The use according to claim 3, characterized in that: The plants include Leptochloa chinensis.

5. Use of the LcCYP93A3 gene according to claim 1 in breeding of herbicide-resistant crops.

6. The use according to claim 5, characterized in that: The herbicide includes cyhalofop-butyl.

7. An overexpression vector, characterized in that: The LcCYP93A3 gene according to claim 1 is inserted into the plant expression vector pBWA to obtain the LcCYP93A3 gene.

8. A method for obtaining herbicide-resistant plants, characterized in that: The following steps are involved: 1) Transforming the overexpression vector according to claim 7 into Agrobacterium to obtain transformed bacteria; 2) Infecting plant callus with the transformed bacteria obtained in step 1) to obtain herbicide-resistant plants.

9. The acquisition method according to claim 8, characterized in that: The herbicide includes cyhalofop-butyl, and the plant includes Leptochloa chinensis.

10. The acquisition method according to claim 8, characterized in that: The infection conditions include: placing the plant callus in a bacterial solution of the transformation bacteria, and culturing at 200 rpm and 28° C. for 15 to 20 minutes; The OD of the bacterial solution 600 The value is 0.5~0.6.

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