Mutant soybean lipoxygenase LOX2 and its application in producing odorless soybeans

Abstract

The present invention provides a mutant of soybean lipoxygenase LOX2 and its application in creating odorless soybeans. The mutant is based on the parent amino acid sequence of SEQ ID NO.1. By introducing succinylation modifications at lysine residues 257, 263, and 472, the catalytic efficiency of LOX2 is reduced, reducing the oxidation of polyunsaturated fatty acids and effectively creating odorless soybeans. Compared with the wild type, the catalytic efficiency is reduced by 15.62%, providing a new basis for application in the food industry. In addition, by achieving molecular-level transformation through grafting breeding technology, the present invention not only improves soybean quality but also significantly reduces production costs, which is of great significance for soybean variety improvement and oil quality improvement.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a mutant of soybean lipoxygenase LOX2 and application thereof in creating odorless soybeans. Background Art

[0002] Soybeans are a globally important economic crop, providing a rich source of dietary protein and vegetable oil. They contain all essential amino acids for human nutrition and are used in a variety of foods, such as soy milk and tofu. However, soybeans are high in polyunsaturated fatty acids, which inevitably contribute to the unpleasant odor of soy products, known as beany odor. This odor is primarily due to the activation of lipoxygenases (LOXs) in soybeans by oxygen and water during crushing. Polyunsaturated fatty acids such as linoleic acid and linolenic acid are oxidized to form hydroperoxides, which are then degraded into a variety of volatile compounds with varying degrees of odor, including small alcohols, aldehydes, ketones, acids, and amines. Once formed, beany odor is difficult to remove, significantly impacting the taste and consumption of soy products. In the food industry, treatments such as heating, microwave treatment, and organic solvent extraction can be used to eliminate the beany odor in soy products, but these methods significantly increase production costs. Therefore, developing odor-free soybean varieties is a promising strategy to eliminate the odor and significantly reduce production costs.

[0003] Research has shown that mature soybean seeds primarily contain three lipoxygenase isoenzymes: LOX1, LOX2, and LOX3, encoded by Glyma13g347600, Glyma13g347500, and Glyma15g026300, respectively. Of these three isoenzymes, LOX2 is the primary isoenzyme responsible for beany odor. This suggests that modifying LOX2 activity in soybean seeds through breeding could yield higher-quality, odor-free soybean products and oils. However, current research on LOX2 molecular modification primarily focuses on elucidating the structure-function relationship, while reports on modifying LOX2 catalytic efficiency through novel breeding techniques are limited. Therefore, developing a mutant with reduced lipoxygenase catalytic activity, with the goal of low-cost industrial application, would be of great value for the production and application of odor-free soybeans.

[0004] It should be noted that the information disclosed in the above background technology section is only used to understand the background of this application, and therefore may include information that does not constitute prior art known to ordinary technicians in this field. Summary of the Invention

[0005] The main purpose of the present invention is to overcome the defects in the above-mentioned background technology and provide a mutant of soybean lipoxygenase LOX2 and its application in creating odorless soybeans.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A soybean lipoxygenase LOX2 mutant is based on the parent amino acid sequence shown in SEQ ID NO. 1, and succinylation modification is introduced into the lysine residues at positions 257, 263 and 472, thereby changing the amino acid residues of the wild type in the parent.

[0008] Furthermore, after the lysine residues at positions 257, 263 and 472 in the mutant are succinylated, the amino acid sequence of the protein encoded is shown in SEQ ID NO.2.

[0009] The mutant of soybean lipoxygenase LOX2 is used to reduce the activity of catalyzing polyunsaturated fatty acids and to create odorless soybeans.

[0010] Furthermore, the application includes the following steps:

[0011] Use grafting breeding technology to modify LOX2 activity in parent soybean varieties;

[0012] Grafting the parent soybean onto ginger, and harvesting grafted contemporary seeds;

[0013] After years of systematic breeding, a strain was selected in which the lysine residues 257, 263, and 472 in the parent amino acid sequence shown in SEQ ID NO.1 were succinylated;

[0014] Finally, a strain with excellent comprehensive traits was screened out, and the amino acid sequence of its LOX2 enzyme is shown in SEQ ID NO.2.

[0015] The present invention has the following beneficial effects:

[0016] This invention utilizes grafting breeding technology to molecularly engineer lipoxygenase, post-translationally modifying lysine residues at positions 257, 263, and 472 by succinylation, ultimately yielding a mutant lipoxygenase with reduced catalytic efficiency. Compared to the wild-type lipoxygenase, the catalytic efficiency of the mutant lipoxygenase provided by this invention is 15.62% lower, creating soybeans with reduced lipoxygenase activity and low or no odor, laying the foundation for their industrial application.

[0017] Other beneficial effects of the embodiments of the present invention will be further described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the identification of LOXs activity in the soybean line specifically for soymilk obtained by grafting breeding technology in the examples of the present invention.

[0019] Figure 2 is the content (μg / g) of volatile compounds in soybean seeds for soy milk according to the embodiment of the present invention.

[0020] Figure 3 The lysines at positions 257 and 263 of the LOX2 enzyme of the present invention are post-translationally modified.

[0021] Figure 4 The lysine at position 472 of the LOX2 enzyme of the present invention is post-translationally modified.

[0022] Figure 5 This is the transgenic rooting detection of an embodiment of the present invention.

[0023] Figure 6 This is the LOX2 enzyme activity assay of the transgenic hairy roots according to the embodiment of the present invention. DETAILED DESCRIPTION

[0024] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope of the present invention and its application.

[0025] Embodiments of the present invention provide a soybean lipoxygenase LOX2 mutant. This mutant is based on the parent amino acid sequence set forth in SEQ ID NO. 1, and has succinylated lysine residues at positions 257, 263, and 472, thereby altering the amino acid residues of the wild-type parent. In some embodiments, the amino acid sequence of the protein encoded by the succinylated lysine residues at positions 257, 263, and 472 in this mutant is set forth in SEQ ID NO. 2.

[0026] The first objective of the present invention is to provide a soybean lipoxygenase mutant with reduced catalytic efficiency. The mutant is based on the amino acid sequence of SEQ ID NO. 1, with epimutations at positions 257, 263, and 472. In one embodiment, the lysine residues at positions 257, 263, and 472 are epimutated by succinylation. In one embodiment, the amino acid sequence of the soybean lipoxygenase mutant is shown in SEQ ID NO. 2.

[0027] The present invention provides the enzymatic activity results of the soybean lipoxygenase mutant, such as Figure 6 As shown, the catalytic efficiency decreased by 15.62%.

[0028] The present invention also provides the use of the mutant soybean lipoxygenase LOX2 in reducing the activity of catalyzing polyunsaturated fatty acids and in creating odorless soybeans. A second object of the present invention is to create odorless soybeans containing the mutant soybean lipoxygenase.

[0029] In some embodiments, the application includes the following steps: using grafting breeding technology to modify the LOX2 activity in the parent soybean variety; grafting the parent soybean onto ginger and harvesting the grafted contemporary seeds; after years of systematic breeding, screening out strains in which the lysine residues at positions 257, 263, and 472 in the parent amino acid sequence shown in SEQ ID NO.1 are succinylated; and finally screening out strains with excellent comprehensive traits, whose LOX2 enzyme amino acid sequence is shown in SEQ ID NO.2.

[0030] The present invention uses genetic engineering techniques to create a mutant of soybean lipoxygenase LOX2. Based on the parent amino acid sequence shown in SEQ ID NO. 1, this mutant introduces succinylation modifications at lysine residues 257, 263, and 472, effectively reducing the catalytic efficiency of LOX2, thereby reducing the oxidation of polyunsaturated fatty acids in soybeans and significantly reducing the production of soy odor. Compared with wild-type lipoxygenase, the catalytic efficiency of this mutant enzyme is reduced by 15.62%, successfully creating a soybean variety with no or low odor. This method not only improves the quality of soybean products but also significantly reduces the production costs required to eliminate beany odor. For the food industry, this is a low-cost and efficient industrial application strategy. Furthermore, the present invention achieves precise modification of LOX2 activity through grafting breeding technology, providing an application of a new breeding technology and offering an innovative solution for future soybean variety improvement and oil quality enhancement.

[0031] Specific embodiments of the present invention are further described below.

[0032] Example 1: Screening of odorless soybeans

[0033] First, the parent 'Heinong 48' (abbreviated as 'HN48') was used as the experimental material and grafted onto ginger according to the soybean distant grafting mutagenesis technique of Pan Xiangwen et al. (2012), and the grafted contemporary seeds were harvested;

[0034] Second, the grafted mutant offspring were systematically selected and bred using pedigree selection methods over many years. The grafting combination was established in 2015, and the first-generation G1 was planted in 2016. After harvesting individual plants, the second-generation G2 was planted in Sanya, Hainan Province. In 2017, the third-generation G3 was planted; in 2018, the fourth-generation G4 was planted, continuing to select for high-yield and strong stress resistance. In 2019, the fifth-generation G5 was planted, selecting strains with excellent comprehensive traits. In 2020, strain identification trials were conducted to select stable strains with high yield, excellent quality, and a green seed coat.

[0035] Third, based on the methods outlined in the China National Food Industry Association's group standard (T / CNFIA 138-2022) for "Whole-Bean Soymilk," we conducted taste tests on our selected stable grafted soybean lines with consumers of varying ages. We identified specialized soybeans suitable for soymilk production. These soybeans exhibit the following characteristics: a uniform, milky white or light yellow appearance; a rich, fragrant, and astringent soymilk flavor; a naturally fragrant soymilk aroma without any other odors; a smooth, non-grainy, and rich mouthfeel; and a rich, glossy, and smooth texture. Four of these lines are particularly popular with customers: 'Dongsheng 222,' 'Dongsheng 224,' 'Dongsheng 225,' and 'Dongsheng 229,' abbreviated as 'DS222,' 'DS224,' 'DS225,' and 'DS229,' respectively. This indicates that grafting breeding has created a new soybean milk-specific strain with better quality and taste than the ungrafted scion soybean 'Heinong 48'.

[0036] Fourth, the LOXs enzyme activity of the four new soymilk-specific strains selected in the above three was determined by colorimetry. The detection method is as follows:

[0037] First, take dried mature soybean seeds and grind them into powder separately.

[0038] 1) Detection of soybean lipoxygenase isozyme LOX1: a. To 15 mg of soybean powder, add 1.5 ml of 200 mM sodium borate buffer (pH 9.0), 1.5 ml of 200 mM sodium phosphate buffer (pH 6.0), and 1.5 ml of 200 mM sodium phosphate buffer (pH 6.6). Centrifuge (12,000 rpm, 5 min, 4°C) and collect the clarified supernatant. b. Aspirate 1 ml of the supernatant and add 0.5 ml of LS1 indicator solution, which consists of 125 mM sodium borate buffer (pH 9.0), 12.5 μM methylene blue, and 1.375 mM sodium linoleate.

[0039] 2) Detection of soybean lipoxygenase isozyme LOX2: a. To 30 mg of soybean powder, add 1.5 ml of 200 mM sodium borate buffer (pH 9.0), 1.5 ml of 200 mM sodium phosphate buffer (pH 6.0), and 1.5 ml of 200 mM sodium phosphate buffer (pH 6.6). Centrifuge (12,000 rpm, 5 min, 4°C) and collect the clear supernatant. b. Aspirate 1 ml of the supernatant and add 0.5 ml of LS2 indicator solution, which consists of 125 mM sodium phosphate buffer (pH 6.0), 12.5 μl of methylene blue, 1.375 mM sodium linoleate substrate, 25 mM dithiothreitol, and 12.5% ​​acetone.

[0040] 3) Detection of soybean lipoxygenase isozyme LOX3: a. To 15 mg of soybean powder, add 1.5 ml of 200 mM sodium borate buffer (pH 9.0), 1.5 ml of 200 mM sodium phosphate buffer (pH 6.0), and 1.5 ml of 200 mM sodium phosphate buffer (pH 6.6). Centrifuge (12,000 rpm, 5 min, 4°C) and collect the clear supernatant. b. Aspirate 1 ml of the supernatant and add 0.5 ml of LS3 indicator solution, which consists of 125 mM sodium phosphate buffer (pH 6.6), 1.375 mM sodium linoleate substrate, and 12.5% ​​50% saturation β-carotene.

[0041] Reduced LOX enzyme activity is the primary cause of low or no fishy odor in soybeans. To better investigate LOX enzyme activity in grafted soybean lines specifically developed for soymilk production, we purchased two commercially available, locally widely used, odorless soybeans for soymilk production (LBS1 and LBS2) as positive controls. These grafted lines were assayed for LOX activity using a colorimetric assay and a chromogenic reaction. Figure 1 This figure shows the identification of LOX activity in soybean lines specifically for soymilk production obtained using grafting breeding technology. The absorbance values ​​and corresponding color reactions (at the absorbance values) were used to detect the enzyme activities of LOX1, LOX2, and LOX3, respectively. CK is a blank solution. HN48 is a negative control. LBS1 and LBS2 are positive controls. Compared with the negative control (HN48), Figure 1 The strains 'DS222', 'DS224', 'DS225', and 'DS229' shown in Figure 1 clearly retained the color of the LOX2 substrate solution (the solution remained blue), while the colors of the LOX1 and LOX3 substrates remained unchanged, indicating low LOX2 content. The coloration reaction and absorbance of these strains were similar to those of LBS1 and LBS2, respectively. These results indicate that grafting altered lipoxygenase traits and generated novel low-LOX2 mutations.

[0042] 5. Gas chromatography-mass spectrometry (GC-MS) was used to identify the volatile compounds in the screened soybean seeds for soy milk production: 1.0 g of ground soybean sample was accurately weighed and placed in a 20 mL headspace bottle, and 10 μL of internal standard ethyl decanoate (0.174 mg / mL) was added. The bottle was sealed with a lid and placed in a 60°C water bath for equilibration for 15 minutes. Then, an aged extraction head was inserted above the sample in the headspace bottle and extracted for 60 minutes. After removing the extraction head, the sample was directly injected and thermally decomposed at 230°C at the injection port for 5 minutes.

[0043] Quantification and calculation of aroma activity values: Quantitative analysis: ethyl decanoate was used as the internal standard and the internal standard method was used for quantification.

[0044]

[0045] Where: C represents the concentration of your internal standard, μg / mL; V represents the volume of your internal standard, μL; m represents the sample amount; A1 represents the peak area; A2 represents the peak area of ​​the internal standard.

[0046] The gas chromatography detection method is:

[0047] Equipment model: Gas chromatograph-mass spectrometer AGILENT6890-5973N Agilent Technologies, USA

[0048] Chromatographic column: Agilent HP-5MS quartz flexible capillary column (30.0μm×250μm×0.25μm)

[0049] The following conditions were set: carrier gas was high-purity helium (99.999%) at a flow rate of 1.0 mL / min; GC conditions: HP-5MS quartz elastic capillary column (30.0 μm × 250 μm × 0.25 μm); heating program: injection port temperature 250°C; the initial temperature of the chromatographic column was 50°C (maintained for 1.0 min), then increased at 3°C / min to 120°C (maintained for 2 min), and finally increased at 4°C / min to 210°C (maintained for 10 min) for non-split injection.

[0050] MS conditions: EI ion source at 70 eV, 230°C, interface temperature at 250°C, mass scan range from 10 to 450 m / z; solvent delay time of 10 min.

[0051] Figure 2Results showed that the total volatile matter content of the soymilk-specific lines 'DS222' and 'DS224' decreased by 2410.81 to 3231.78 μg / g, or 66.47% to 89.11%, compared to the control (3626.69 μg / g). Furthermore, the contents of various substances, such as alcohols, alkene, acids, and esters, decreased by 380.57 μg / g, 118.37 to 142.13 μg / g, 1731.03 to 2685.67 μg / g, and 157.08 to 198.03 μg / g, respectively, compared to the control HN48, with corresponding reductions of 100%, 83.28 to 100%, 62.27 to 96.61%, and 48.48 to 61.12%, respectively. These two lines are also the most popular new soybean lines among the "three" soybean varieties. The results showed that new soybean varieties with no or low odor were developed by reducing LOX2 activity in soybeans specially cultivated for soy milk through grafting breeding.

[0052] Example 2: Identification of LOX mutations in odorless soybeans

[0053] Based on the results in Example 1, LOX2 protein in soybeans 'DS222' and 'DS224' for soymilk with no or low odor was extracted and analyzed by mass spectrometry. It was found that lysine at positions 257, 263 and 472 of the LOX2 enzyme had epimutations, and a post-translational modification type, succinylation, occurred. The mass spectrometry results are shown in FIG. Figure 3-4 . Figure 3 Lysines at positions 257 and 263 of the LOX2 enzyme are shown to be post-translationally modified. Figure 4 The amino acid sequence of LOX2 in the control variety HN48 is shown in SEQ ID NO.1. Suc The amino acid sequence is shown in SEQ ID NO. 2. Therefore, it is possible to conclude that post-translational modification of LOX2 protein leads to a decrease in LOX2 enzyme activity, resulting in a new soybean variety with no or low fishy odor.

[0054] Example 3: Identification of enzyme activity of LOX mutants

[0055] To verify the conclusion in Example 2, we performed site-directed mutagenesis on the three modified lysine residues in LOX2, simulating complete succinylation by mutating lysine residues to glutamine residues Q. We then constructed two GFP-fused plant expression vectors (LOX2:GFP and LOX2: Suc :GFP) and used to transform hard roots, and the enzyme activities were compared.

[0056] 1. Vector construction: Using the cDNA of soybean variety 'Heinong 48' as template, refer to TaKaRa's According to the HSDNA Polymerase operating instructions, forward primer F1 (5'-ATGTTTTCAGTTCCAGGGGTG-3') and reverse primer R1 (5'-TTAGATAGAGATGCTATTAGGAATCCC-3') were used as amplification primers to amplify the coding region of the wild-type LOX2 gene (sequence shown in SEQ ID NO. 3). Based on the succinylation site of the mutated LOX2, a LOX2 nucleic acid sequence (sequence shown in SEQ ID NO. 4) was synthesized, and site-directed mutagenesis of LOX2 was performed.

[0057] Second, the amplified fragment and the synthesized fragment were cloned into the plant expression vector pG2RNAi (provided by the Soybean Molecular Breeding Laboratory, Northeast Center of Geography and Agriculture, Chinese Academy of Sciences) to form a CaMV 35S promoter-driven LOX2-GFP fusion plasmid, denoted as pG2RNAi-LOX2-GFP and pG2RNAi-LOX2 Suc -GFP.

[0058] 3. Two destination vectors are transformed into Agrobacterium K599 respectively, and the soybean hairy roots are obtained by Agrobacterium-mediated transformation method. The fusion vectors constructed in the above step 2 are respectively infected into soybean seedlings to obtain soybean hairy roots:

[0059] The following steps were performed to infect soybean seedlings with Agrobacterium K599 bacterial solution to obtain soybean hairy roots.

[0060] (1) The cells containing pG2RNAi-LOX2-GFP and pG2RNAi-LOX2 Suc 5 μL each of -GFP Agrobacterium rhizogenes K599 bacterial solution and blank control bacterial solution (Agrobacterium rhizogenes K599 containing the original plasmid vector pG2RNAi) were placed in 25 mL YEP medium (kanamycin and streptomycin content were both 50 μg / mL) and cultured at 28°C and 180 r / min for 12 h.

[0061] (2) Take 5 mL of activated bacterial solution and place it in 50 mL of YEP medium (kanamycin and streptomycin content are both 50 μg / mL). Cultivate at 28°C and 180 rpm until OD600 = 0.6-0.8, and resuspend in double-distilled water.

[0062] (3) Sow 'Dongnong 50' soybeans in vermiculite. After 4 days, take the cotyledons and hypocotyls of healthy seedlings, cut them 1.5 cm from the cotyledonary nodes, and pierce three holes in each node with a syringe needle.

[0063] (4) The treated seedlings were immersed in K599 bacterial solution containing empty vector, pG2RNAi-LOX2-GFP and pG2RNAi-LOX2 Suc -GFP K599 bacterial suspension for 30 min.

[0064] (5) Add moist vermiculite to the pot and cover with a layer of filter paper. Place the infected seedlings on the filter paper, seal with plastic wrap to keep it moist, and culture in an intelligent artificial climate chamber (16 h light / 8 h dark, 28°C / 26°C).

[0065] (6) When new roots 2 cm long grow at the bottom of the hypocotyl, transfer the seedlings to vermiculite for further cultivation.

[0066] 4. Identification of Transgenic Hairy Roots

[0067] The vectors pG2RNAi, pG2RNAi-LOX2-GFP and pG2RNAi-LOX2 were transformed respectively. SUC -GFP soybean hairy roots were placed in a dynamic plant imaging system (Maikelun, Beijing, China) for transgenic identification. Figure 5 As shown, GFP was observed in the hairy roots infected with each vector, and LOX2 enzyme activity was detected in the target hairy roots.

[0068] Figure 5 Transgenic hairy root assays are shown, with empty vector (GFP), LOX2 wild-type tobacco (LOX2:GFP), and fully succinylated (LOX2 Suc :GFP) GFP signal in infected roots.

[0069] 5. LOX2 enzyme activity assay

[0070] Using the LOX2 enzyme activity assay described in Example 1, we found that the LOX2 enzyme activity in the mutant LOX2 roots was significantly lower by 291.7 pg / ml compared to that in infected wild-type roots, and the catalytic efficiency was reduced by 15.62%. This demonstrates that the present invention, through the identification of lipoxygenase in grafted, odor-free soybeans, has discovered a new mutation type. Through site-directed mutagenesis, we screened for lipoxygenase mutants with reduced catalytic efficiency. This significantly reduced catalytic efficiency of lipoxygenase, resulting in odor-free soybeans, lays the foundation for its application in the food industry.

[0071] Figure 6 Shown is the LOX2 enzyme activity assay of transgenic hairy roots.

[0072] The sequence listings of the above SEQ ID NOs. 1 to 4 are as follows:

[0073] >SEQ ID NO.1

[0074] MFSVPGVSGILNRGGGHKIKGTVVLMRKNVLDFNSVADLTKGNVGGLIGTGLNVVGSTLDNLTAFLGRSVALQLISATKPLANGKGKVGKDTFLEGIIVSLPTLGAGESAFNIQFEWDESMGIPGAFYIKNYMQVEFYLKSLTLEDVPNQGTIRFVCNSWVYNTKLYKSVRIFFANHTYVPSETPAALVGYREEELKNLRGDGKGERKEHDRIYDYDVYNDLGNPDHGENFARPILGGSSTHPYPRRGRTGRYPTRKDQNSEKPGEVYVPRDENFGHLKSSDFLAYGIKSLSQYVLPAFESVFDLNFTPNEFDSFQDVRDLHEGGIKLPTEVISTIMPLPVVKELFRTDGEQVLKFPPPHVIQVSKSAWMTDEEFAREMVAGVNPCVIRGLQEFPPKSNLDPTIYGEQTSKITADALDLDGYTVDEALASRRLFMLD YHDVFMPYIRRINQTYAKAYATRTILFLRENGTLKPVAIELSLPHPAGDLSGAVSQVILPAKEGVESTIWLLAKAYVVVNDSCYHQLMSHWLNTHAVIEPFIIATNRHLSALHPIYKLLTPHYRDTMNINALARQSLINADGIIEKSFLPSKHSVEMSSAVYKNWVFTDQALPADLIKRGVAIKDPSAPHGLRLLIEDYPYAVDGLEIWAAIKTWVQEYVSLYYARDDDVKPDSELQQWWKEAVEKGHGDLKDKPWWPKLQTIEELVEICTIIIWTASALHAAVNFGQYPYGGFILNRPTSSRRLLPEKGTPEYEEMVKSHQKAYLRTITSKFQTLVDLSVIEILSRHASDEVYLGQRDNPHWTSDSKALQAFQKFGNKLKEIEEKLARKNNDQSLSNRLGPVQLPYTLLHPNSEEGLTCRGIPNSISI*

[0075] >SEQ ID NO.2

[0076] MFSVPGVSGILNRGGGHKIKGTVVLMRKNVLDFNSVADLTKGNVGGLIGTGLNVVGSTLDNLTAFLGRSVALQLISATKPLANGKGKVGKDTFLEGIIVSLPTLGAGESAFNIQFEWDESMGIPGAFYIKNYMQVEFYLKSLTLEDVPNQGTIRFVCNSWVYNTKLYKSVRIFFANHTYVPSETPAALVGYREEELKNLRGDGKGERKEHDRIYDYDVYNDLGNPDHGENFARPILGGSSTHPYPRRGRTGRYPTRQDQNSEQPGEVYVPRDENFGHLKSSDFLAYGIKSLSQYVLPAFESVFDLNFTPNEFDSFQDVRDLHEGGIKLPTEVISTIMPLPVVKELFRTDGEQVLKFPPPHVIQVSKSAWMTDEEFAREMVAGVNPCVIRGLQEFPPKSNLDPTIYGEQTSKITADALDLDGYTVDEALASRRLFMLDYHDVFMPYIRRINQTYAKAYATRTILFLRENGTLQPVAIELSLPHPAGDLSGAVSQVILPAKEGVESTIWLLAKAYVVVNDSCYHQLMSHWLNTHAVIEPFIIATNRHLSALHPIYKLLTPHYRDTMNINALARQSLINADGIIEKSFLPSKHSVEMSSAVYKNWVFTDQALPADLIKRGVAIKDPSAPHGLRLLIEDYPYAVDGLEIWAAIKTWVQEYVSLYYARDDDVKPDSELQQWWKEAVEKGHGDLKDKPWWPKLQTIEELVEICTIIIWTASALHAAVNFGQYPYGGFILNRPTSSRRLLPEKGTPEYEEMVKSHQKAYLRTITSKFQTLVDLSVIEILSRHASDEVYLGQRDNPHWTSDSKALQAFQKFGNKLKEIEEKLARKNNDQSLSNRLGPVQLPYTLLHPNSEEGLTCRGIPNSISI*

[0077] >SEQ ID NO.3

[0078]

[0079] >SEQ ID NO.4

[0080]

[0081] The above is a further detailed description of the present invention in conjunction with specific / preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. Those skilled in the art to which the present invention relates may make various substitutions or modifications to the described embodiments without departing from the scope of the present invention, and such substitutions or modifications should be considered to fall within the scope of protection of the present invention.

Claims

1. A soybean lipoxygenase LOX2 mutant, characterized in that: The mutant is based on the parent amino acid sequence shown in SEQ ID NO.1, and succinylation modification is introduced into the lysine residues at positions 257, 263 and 472, thereby changing the wild-type amino acid residues in the parent.

2. The soybean lipoxygenase LOX2 mutant according to claim 1, characterized in that After the lysine residues at positions 257, 263 and 472 in the mutant are succinylated, the amino acid sequence of the protein encoded is shown in SEQ ID NO.

2.

3. Use of the soybean lipoxygenase LOX2 mutant according to claim 1 or 2 in reducing the activity of soybean lipoxygenase LOX2 in catalyzing the oxidation of polyunsaturated fatty acids.

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