Lccyp93a3 gene, protein and application thereof, overexpression vector and method

Abstract

The present application relates to the field of agricultural science and plant bioengineering technology, and particularly relates to a LcCYP93A3 gene, a protein and application, an overexpression vector and method.The nucleotide sequence of the LcCYP93A3 gene is shown as SEQ ID No.1.The present application enhances the expression of LcCYP93A3 by using transgenic technology, constructs an overexpression transgenic plant, and can significantly enhance the drug resistance of plants to cyhalofop-butyl.The gene regulation strategy provided by the present application provides a gene resource for cultivating herbicide-resistant crops, and has important agricultural application value.

Description

Technical Field

[0001] This invention relates to the fields of agricultural science and plant bioengineering technology, and in particular to an LcCYP93A3 gene, protein, its applications, overexpression vectors, and methods. Background Technology

[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 herbicide type has led to the gradual increase of herbicide resistance in weeds, posing new challenges to crop production. Breeding herbicide-resistant crops is an effective weed management strategy. By introducing herbicide-resistant genes, crops can grow normally under specific herbicide treatments, contributing to more efficient weed management.

[0003] Herbicide resistance genes are central to herbicide-resistant crop breeding. Weeds exposed to herbicides for extended periods evolve resistance under natural selection pressure, making them ideal research subjects for identifying herbicide resistance genes. *Leptochloa chinensis* is one of the major noxious weeds in rice paddies, posing a serious threat to rice yield. Cyhalofop-butyl, an acetyl-CoA carboxylase (ACCase) inhibitor, is widely used for the chemical control of grassy weeds. However, with the long-term use of cyhalofop-butyl, herbicide resistance in *Leptochloa chinensis* has gradually emerged, leading to decreased weed control efficacy, increased herbicide usage, environmental pollution, and crop damage. Plant herbicide 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, typically related to herbicide metabolic degradation, efflux mechanisms, and antioxidant responses.

[0004] With the development of omics technologies, the discovery of herbicide-resistant genes has been significantly advanced. Transcriptomics and genomics technologies enable scientists to analyze plant herbicide resistance mechanisms from multiple dimensions, including 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. Potential resistance genes identified can be further validated through transgenic methods. These genes are transferred into model plants or target crops, and the resistance function of the genes is verified by observing the plant's growth performance under herbicide treatment. Therefore, discovering new resistance genes is a crucial prerequisite for future breeding of herbicide-resistant crops. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides an LcCYP93A3 gene, protein, its applications, overexpression vector, and method, clarifying the function of the LcCYP93A3 gene in herbicide resistance, and enabling its in-depth application in the subsequent breeding of herbicide-resistant crops.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides an LcCYP93A3 gene, the nucleotide sequence of which is shown in SEQ ID No. 1.

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

[0009] This invention also provides the application of the LcCYP93A3 gene described in the above technical solution or the protein described in claim 2 in enhancing plant resistance to cyhalofop-butyl.

[0010] Preferably, the plant includes Echinopsis thunbergii.

[0011] This 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 includes 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 the plant expression vector pBWA.

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

[0015] 1) The overexpression vector described in the above technical solution is transferred into Agrobacterium to obtain the transformant bacteria;

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

[0017] Preferably, the herbicide includes cyhalofop-butyl, and the plant includes Echinochloa crus-galli.

[0018] Preferably, the infection conditions include: placing the plant callus tissue in the bacterial solution of the transforming bacteria and culturing it at 200 rpm and 28°C for 15–20 min;

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

[0020] This invention, through transcriptome sequencing and weighted co-expression network analysis (WGCNA), reveals the crucial role of the LcCYP93A3 gene in cyhalofop-butyl resistance. The study found that LcCYP93A3 gene expression is regulated by the diurnal rhythm gene LcRVE4; increased expression levels during specific time periods enhance plant resistance to cyhalofop-butyl. Enhancing LcCYP93A3 expression through transgenic technology and constructing overexpressing transgenic plants significantly improves plant resistance to cyhalofop-butyl. The gene regulation strategy proposed in this invention provides genetic resources for breeding herbicide-resistant crops and has significant agricultural application value.

[0021] The beneficial effects of this invention are:

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

[0023] This invention analyzed, identified, and cloned the LcCYP93A3 gene from *Echinochloa crus-galli* using bioinformatics methods, and verified its function through genetic transformation. By constructing transgenic lines overexpressing this gene, the mechanism of action of the LcCYP93A3 gene in resistance to cyhalofop-butyl was elucidated, providing valuable theoretical basis and genetic resources for the breeding of herbicide-resistant crops.

[0024] 2. Enhance crop herbicide resistance and create new resistant varieties.

[0025] Expression of the LcCYP93A3 gene significantly enhanced plant resistance to cyhalofop-butyl herbicide, leading to the development of new resistant germplasm that maintained good growth under herbicide treatment conditions. This result not only demonstrates the important role of the LcCYP93A3 gene in plant resistance but also provides new ideas and resources for molecular breeding of herbicide-resistant crops. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 The figure shows the resistance results of cyhalofop-butyl after LcCYP93A3 gene was transferred into Echinochloa crus-galli. Detailed Implementation

[0028] This invention provides an LcCYP93A3 gene, the nucleotide sequence of which 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 described in the above-mentioned technical solution, the amino acid sequence of which is shown in SEQ ID No. 2.

[0032] SEQ ID No. 2:

[0033] MAEEAAVLVAVAAIVLVLVLWRRSRRTTGKLPPSPLGLPLIGHLHLIRPPPHRAFDRIISRYGPLVYLRLGPSTHCVVAGTADAARDLLRFEASIPERPLTAVTRHLAYDSAGFAFAPYGPHWRFMK RLCMSELLGPRTVDQLRPVREAELAAVLGAARDAAARGEALDVSRQLIRLANNAIMRMVATDLPGDMTEEARDCAKQVAEVVGAFNLEDYVALCKGWDLQGLTRRTREVRDRFDALMEIMIKAKEER RREHATKEEDLLDILMDAAEDRKADVKLTRENIKAFILDIFTAGSDTTATSVEWMLAHLINNPACLRKLRQELDDVVGKSRLVAEHDVARLPYLNAVFKETLRLQPPAVFAQRETIEPVHVRGYTIP AKTSVFFNIFSIGRDPACWDQPLLFRPERFMPGGEGAAIDPKGQHMQLMPFGSGRRACPGMGLAMQAVPAFLAAMVQCFDWAVPVPQGQSKAPPLDMEEAEGLVSARKQPLLLFPTQRIHPLPMS*.

[0034] This invention also provides the application of the LcCYP93A3 gene or the protein described in the above-mentioned technical solutions in enhancing plant resistance to cyhalofop-butyl. In this invention, the plant preferably includes *Echinochloa crus-galli*. In this invention, the plant is also preferably suitable for those possessing this gene or a homologous gene, especially suitable for gramineous crops such as rice, maize, and wheat. In this invention, the plant is also preferably including, but not limited to, *Arabidopsis thaliana*, and any plant possessing this gene or a homologous gene is suitable.

[0035] This invention also provides the application of the LcCYP93A3 gene described in the above-mentioned technical solution in the breeding of herbicide-resistant crops. In this invention, the herbicide preferably includes cyhalofop-butyl.

[0036] This invention also provides an overexpression vector, obtained by inserting the LcCYP93A3 gene described in the above-described technical solution into the plant expression vector pBWA. This invention does not impose any particular limitation on the method for inserting the LcCYP93A3 gene into the plant expression vector pBWA; those skilled in the art can follow conventional procedures, and further details are omitted here.

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

[0038] 1) The overexpression vector described in the above technical solution is transferred into Agrobacterium to obtain the transformant bacteria;

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

[0040] This invention involves transferring the overexpression vector described in the above-mentioned technical solution into Agrobacterium to obtain a transformed bacterium. This invention does not specifically limit the type of Agrobacterium; any strain commonly used for transformation, such as Agrobacterium EHA105, can be used. This invention also does not specifically limit the method of transferring the overexpression vector into Agrobacterium; those skilled in the art can operate according to conventional methods, and will not be elaborated further here.

[0041] This invention involves infecting plant callus tissue with the obtained transforming bacteria to obtain herbicide-resistant plants. In this invention, the herbicide preferably includes cyhalofop-butyl, and the plant preferably includes *Erigeron breviscapus*. In this invention, the infection conditions preferably include: placing the plant callus tissue in a bacterial solution of the transforming bacteria and culturing it at 200 rpm and 28°C for 15–20 min. In this invention, the OD of the bacterial solution... 600 The preferred value is 0.5 to 0.6.

[0042] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

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

[0045] Based on the genome sequence information of *Euphorbia lathyris*, 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 the LcCYP93A3 gene (SEQ ID No. 1) was amplified from the cDNA of *Euonymus alatus*. The PCR amplification conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1 min, for a total of 35 cycles, followed by a final extension at 72℃ for 5 min. 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 gene was expressed under the drive of the 35S promoter, generating the LcCYP93A3 overexpression (LcCYP93A3-OE) vector. Vector construction was achieved through restriction endonuclease digestion and T4 DNA ligase ligation. The constructed recombinant vector was verified by sequencing. These recombinant vectors were used for Agrobacterium-mediated genetic transformation of *Echinochloa crus-galli*. The transgenic vector was transformed into *Agrobacterium* strain EHA105, and then callus tissue was placed in *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 tissue was selected on selective medium. Two to three weeks later, the callus tissue was isolated into fresh callus induction medium and cultured for 1-2 generations.

[0052] Example 2

[0053] The LcCYP93A3 gene influences cyhalofop-butyl resistance in *Echinochloa crus-galli*.

[0054] The transgenic Echinochloa crus-galli plants (LcCYP93A3-OE1 and LcCYP93A3-OE2) with the LcCYP93A3 gene introduced in Example 1 were compared with wild-type Echinochloa crus-galli (WT) in terms of functional characterization.

[0055] When the plants reach the 4-5 leaf stage, apply the field-recommended dose of cyhalofop-butyl (105g A.I.·ha). -1 Foliar spraying was performed on both transgenic and WT plants. Two weeks after spraying, the transgenic and WT groups were photographed and measured under the same conditions. Two weeks after spraying with cyhalofop-butyl, the transgenic *Echinochloa crus-galli* plants (LcCYP93A3-OE1 and LcCYP93A3-OE2) showed stronger resistance than the WT plants.

[0056] The growth of WT group plants was significantly inhibited, and the leaves turned yellow and withered, while the leaves of transgenic plants remained healthy and green. Figure 1 The plant height of transgenic and WT plants in the cyhalofop-butyl treatment group and the control group was measured. The results showed that after cyhalofop-butyl treatment, the plant height of the WT group was significantly inhibited, with an inhibition rate of approximately 38%; while the inhibition rate of plant height for LcCYP93A3-OE1 was only 11%, and for LcCYP93A3-OE2 it was only 3%, indicating that the growth of transgenic plants was not significantly inhibited. Figure 1 Simultaneously, the fresh weight of the plants was 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%, respectively. Figure 1 ).

[0057] The above results indicate that overexpression of the LcCYP93A3 gene significantly enhances the resistance of *Echinochloa crus-galli* to cyhalofop-butyl. Overexpression of the LcCYP93A3 gene significantly reduced the inhibition rate of plant height and fresh weight after cyhalofop-butyl application, further confirming the role of this gene in conferring cyhalofop-butyl resistance in plants. This also provides a new gene resource for the study of herbicide resistance genes, contributing to the breeding of herbicide-resistant crops.

[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A kind LcCYP93A3 Genes, characterized by, The LcCYP93A3 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The one described in claim 1 LcCYP93A3 Gene-encoded proteins are characterized by, The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. The claim 1 LcCYP93A3 The application of the gene or the protein of claim 2 in enhancing plant resistance to cyhalofop-butyl; The plant in question is *Echinochloa crus-galli*.

4. The claim 1 LcCYP93A3 Application of genes in herbicide-resistant crop breeding, wherein the crop is *Echinochloa crus-galli* and the herbicide is cyhalofop-butyl.

5. An overexpression vector, characterized in that, By the method described in claim 1 LcCYP93A3 The gene was obtained by inserting it into the plant expression vector pBWA.

6. A method for obtaining herbicide-resistant plants, characterized in that, Includes the following steps: 1) The overexpression vector described in claim 5 is transferred into Agrobacterium to obtain the transformant bacteria; 2) Infect plant callus tissue with the transforming bacteria obtained in step 1) to obtain herbicide-resistant plants; The herbicide is cyhalofop-butyl, and the plant is Echinochloa crus-galli.

7. The method for obtaining according to claim 6, characterized in that, The infection conditions include: placing the plant callus tissue in the bacterial solution of the transforming bacteria and culturing it at 200 rpm and 28°C for 15-20 min; The OD of the bacterial solution 600 The value is 0.5~0.6.

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