Mutant type of soybean lipoxygenase LOX2 and application of mutant type in creation of fishy-smell-free soybean

Abstract

The invention provides a mutant of soybean lipoxygenase LOX2 and application of the mutant in creation of fishy-smell-free soybeans, the mutant is based on a parent amino acid sequence of SEQ ID NO.1, succinylation modification is introduced into lysine residues at the 257th site, the 263rd site and the 472th site, the catalytic efficiency of LOX2 is reduced, oxidation of polyunsaturated fatty acid is reduced, and the yield of the soybean lipoxygenase LOX2 is increased. And the fishy-smell-free soybeans are effectively prepared. Compared with a wild type, the catalytic efficiency is reduced by 15.62%, and a new basis is provided for food industrial application. In addition, molecular transformation is realized through a grafting breeding technology, the soybean quality is improved, the production cost is remarkably reduced, and the method has important significance on soybean variety improvement and grease quality improvement.

Description

Technical Field

[0001] The 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] Soybean is an important economic crop in the world, providing humans with abundant dietary protein and vegetable oil. Soybean contains all the essential amino acids required for human nutrition and is made into a variety of foods, such as soy milk and tofu. However, the high content of polyunsaturated fatty acids in soybeans inevitably leads to the unpleasant smell of soybean products, namely soybean smell. This is mainly due to the activation of lipoxygenases (LOXs) in soybeans by oxygen and water when soybeans are crushed, and polyunsaturated fatty acids such as linoleic acid and linolenic acid are oxidized to generate hydroperoxides, which are then degraded into a variety of volatile compounds such as small molecular alcohols, aldehydes, ketones, acids and amines with different degrees of odor, thus forming soybean smell. Once the beany smell is formed, it is difficult to remove, which seriously affects the taste and consumption of bean products. In the food industry, treatment methods such as heating, microwave treatment and organic solvent extraction can be used to eliminate the beany smell of soybean products, but greatly increase the production cost of soybean products. Therefore, cultivating odorless soybean varieties is a promising strategy to eliminate the odor in soybean products and greatly reduce cost losses.

[0003] Studies have shown that mature soybean seeds mainly contain three lipoxygenase isozymes, LOX1, LOX2 and LOX3, which are encoded by Glyma13g347600, Glyma.13g347500, and Glyma15g026300, respectively. Among these three isozymes, LOX2 is the main isozyme responsible for the beany smell. It can be seen that the modification of LOX2 activity in soybean seeds through breeding methods can create better quality odorless soybean products and oils. However, the current research on LOX2 molecular modification is mainly focused on the analysis of the relationship between structure and function, while there are few reports on the modification of LOX2 catalytic efficiency through new breeding technologies. Therefore, with the goal of low-cost industrial application, providing a mutant that reduces the catalytic activity of lipoxygenase is of great value for the production and application of odorless soybeans.

[0004] It should be noted that the information disclosed in the above background technology section is only used for understanding the background of the present application, and therefore may include information that does not constitute prior art known to ordinary technicians in the 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 mutant of soybean lipoxygenase LOX2 is provided. 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 to change the wild-type amino acid residues in the parent.

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

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

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

[0011] Use grafting breeding techniques to modify LOX2 activity in parental soybean varieties;

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

[0013] After years of systematic breeding, strains were selected in which the lysine residues at positions 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] The present invention uses grafting breeding technology to molecularly modify lipoxygenase, and performs post-translational modification of lysine at positions 257, 263 and 472, namely succinylation, to finally obtain a lipoxygenase mutant with reduced catalytic efficiency. Compared with the catalytic efficiency of wild-type lipoxygenase, the catalytic efficiency of the lipoxygenase mutant provided by the present invention is reduced by 15.62% compared with the wild-type, creating soybeans with reduced lipoxygenase activity and low fishy smell, laying a foundation for its 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 of soybean strains for soy milk obtained by grafting breeding technology in the embodiments of the present invention.

[0019] Figure 2 is the content (μg / g) of volatile compounds in the 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 according to 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 and application of the present invention.

[0025] The embodiment of the present invention provides a mutant of soybean lipoxygenase LOX2, which is based on the parent amino acid sequence shown in SEQ ID NO.1, and the wild-type amino acid residues in the parent are changed by introducing succinylation modification at the lysine residues at positions 257, 263 and 472. In some embodiments, after the lysine residues at positions 257, 263 and 472 in the mutant are succinylated, the amino acid sequence of the protein encoded is as shown in SEQ ID NO.2.

[0026] The first object of the present invention is to provide a soybean lipoxygenase mutant with reduced catalytic efficiency, wherein the mutant has an amino acid sequence such as SEQ ID NO.1 as a parent, and epimutations are made at positions 257, 263 and 472 of the parent. In one embodiment, lysine at positions 257, 263 and 472 is subjected to an epimutation-succinylation modification. 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 embodiment of the present invention also provides the use of the mutant of soybean lipoxygenase LOX2 in reducing the activity of catalyzing polyunsaturated fatty acids and in creating odorless soybeans. The second purpose of the present invention is to create odorless soybeans containing the mutant of lipoxygenase.

[0029] In some embodiments, the application comprises 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 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 creates a mutant of soybean lipoxygenase LOX2 through genetic engineering technology. The mutant is based on the parent amino acid sequence shown in SEQ ID NO.1, and by introducing succinylation modification at the lysine residues at positions 257, 263 and 472, the catalytic efficiency of LOX2 is effectively reduced, thereby reducing the oxidation of polyunsaturated fatty acids in soybeans and significantly reducing the production of soybean odor. Compared with the wild-type lipoxygenase, the catalytic efficiency of the mutant enzyme is reduced by 15.62%, and a soybean variety with no or low odor is successfully created. This method not only improves the quality of soybean products, but also greatly reduces the production cost required to eliminate the bean odor. For the food industry, this is a low-cost and efficient industrial application strategy. In addition, the present invention achieves precise modification of LOX2 activity through grafting breeding technology, provides an application of a new breeding technology, and provides an innovative solution for future soybean variety improvement and oil quality improvement.

[0031] The 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 it was 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] 2. The grafted mutant offspring were systematically selected and bred for many years according to the pedigree selection method. The grafting combination was configured in 2015, and the grafted first generation G1 was planted in 2016. After the individual plants were harvested, the G2 generation was planted in Sanya, Hainan Province. In 2017, the grafted third generation G3 was planted; in 2018, the grafted fourth generation G4 was planted, and excellent individual plants with good yield and strong stress resistance were continued to be selected; in 2019, the grafted fifth generation G5 was planted, and excellent strains with comprehensive traits were selected; in 2020, the strain identification test was carried out to select stable strains with good yield, excellent quality and green seed coat color.

[0035] 3. According to the method in the group standard (T / CNFIA 138-2022) 'Whole Bean Soy Milk' issued by the China Food Industry Association, we selected customers of different ages to conduct taste tests on the soybean grafted stable strains, and screened out some special soybeans suitable for making soy milk, which have the following characteristics: uniform and consistent milky white or light yellow appearance color; inherent taste of soy milk, rich fragrance, no astringency; inherent aroma of soy milk, no other odor; delicate, non-granular, and rich taste; thick, glossy, and delicate and uniform texture of soy milk. Among them, there are 4 strains that are most popular with customers, named '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 strain with better quality and taste than the grafted soybean 'Heinong 48' before grafting.

[0036] Fourth, the LOXs enzyme activity of the four new soymilk 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) When detecting soybean lipoxygenase isozyme LOX1: a. Take 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. Take 1 ml of the supernatant and add 0.5 ml of LS1 indicator solution, which is composed of: 125 mM sodium borate buffer (PH 9.0), 12.5 μM methylene blue, 1.375 mM sodium linoleate.

[0039] 2) When detecting soybean lipoxygenase isozyme LOX2: a. Take 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 clarified supernatant; b. Take 1 ml of the supernatant and add 0.5 ml of LS2 indicator solution, which is composed of: 125 mM sodium phosphate buffer (pH 6.0), 12.5 umol methylene blue, 1.375 mM sodium linoleate substrate, 25 mM dithiothreitol, and 12.5% ​​acetone.

[0040] 3) When detecting soybean lipoxygenase isozyme LOX3: a. Take 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. Take 1 ml of the supernatant and add 0.5 ml of LS3 indicator solution, which is composed of: 125 mM sodium phosphate buffer (pH 6.6), 1.375 mM sodium linoleate substrate, 12.5% ​​50% saturation β-carotene.

[0041] The reduction of LOXs enzyme activity is the main reason for soybeans to have no / low fishy smell. In order to better explore the LOXs enzyme activity in the grafted soybean lines for soy milk, we purchased two widely used local soybeans for soy milk without fishy smell (LBS1 and LBS2) from the market as positive controls. The color reaction and colorimetry were used to determine whether these grafted lines had LOXs activity. Figure 1 The figure shows the identification of LOXs activity of soybean lines for soy milk obtained by grafting breeding technology. The absorbance value and the corresponding coloring reaction (under the absorbance value) are used to detect the enzyme activities of LOX1, LOX2 and LOX3, respectively. CK is the blank solution. HN48 is the negative control. LBS1 and LBS2 are the positive controls. Compared with the negative control group (HN48), Figure 1 The 'DS222', 'DS224', 'DS225' and 'DS229' shown in the figure still clearly maintained the color of the LOX2 substrate solution (the solution was still blue), while there was no difference in the color of the LOX1 and LOX3 substrates, indicating that the LOX2 content was low. The coloration reaction and absorbance of these lines were similar to those of LBS1 and LBS2, respectively. These results indicate that grafting breeding has altered the lipoxygenase trait and produced a novel low-LOX2 mutation.

[0042] 5. Use gas chromatography-mass spectrometry (GC-MS) to identify the volatile compounds in the screened soybean seeds for soy milk: accurately weigh 1.0 g of ground soybean sample and put it into a 20 mL headspace bottle, and add 10 μL of internal standard ethyl decanoate (0.174 mg / mL), cover and seal, put it in a 60°C water bath for equilibration for 15 minutes, insert the aged extraction head above the sample in the headspace bottle, extract for 60 minutes, take out the extraction head and directly inject the sample, and thermally decompose it 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] In the formula: 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] Set conditions: carrier gas is high-purity helium (99.999%) with 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 is 50°C (maintained for 1.0 min), increased to 120°C at 3°C / min (maintained for 2 min), and finally increased to 210°C at 4°C / min (maintained for 10 min) for non-split injection.

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

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

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

[0053] According to the results in Example 1, the LOX2 protein in soybeans for no / low fishy smell soy milk 'DS222' and 'DS224' was extracted and mass spectrometry was performed, and it was found that the lysine at positions 257, 263 and 472 of the LOX2 enzyme had epigenetic mutations, a post-translational modification type - succinylation. The mass spectrometry results are shown in 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 ', amino acid sequence is shown in SEQ ID NO. 2. The possible conclusion is that post-translational modification of LOX2 protein causes a decrease in LOX2 enzyme activity, resulting in a new soybean variety with no or low fishy smell.

[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, namely, simulating complete succinylation by mutating lysine residues to glutamine residues Q. Then, we constructed two plant expression vectors fused with GFP (LOX2:GFP and LOX2 Suc :GFP), 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 HSDNA Polymerase operating instructions, using forward primer F1 and reverse primer R1 as amplification primers, forward primer F1: 5'-ATGTTTTCAGTTCCAGGGGTG-3', reverse primer R1: 5'-TTAGATAGAGATGCTATTAGGAATCCC-3', amplify the coding region of the wild-type LOX2 gene, the sequence of which is shown in SEQ ID NO.3; based on the succinylation site of LOX2 after mutation, synthesize the LOX2 nucleic acid sequence, the sequence of which is shown in SEQ ID NO.4, and perform site-directed mutagenesis on LOX2.

[0057] Second, the above-mentioned amplified fragment and synthetic 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 target vectors are transformed into Agrobacterium K599 respectively, and the soybean hairy root transformation method mediated by Agrobacterium is to infect soybean seedlings with the fusion vectors constructed in the above step 2 respectively to obtain soybean hairy roots:

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

[0060] (1) The pG2RNAi-LOX2-GFP and pG2RNAi-LOX2 Suc 5 μL of each of the Agrobacterium rhizogenes K599 culture medium containing -GFP and the blank control culture medium (Agrobacterium rhizogenes K599 containing the original plasmid vector pG2RNAi) were placed in 25 mL of YEP medium (with kanamycin and streptomycin contents of 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 (the content of kanamycin and streptomycin is 50 μg / mL each). Cultivate at 28°C and 180 r / min until OD600 = 0.6-0.8, and then resuspend in double distilled water.

[0062] (3) Sowing 'Dongnong 50' soybeans in vermiculite. After 4 days, take the cotyledons and hypocotyls of healthy seedlings, cut them off 1.5 cm from the cotyledon node, and pierce three holes under 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 it 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 from 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 could be observed in the hairy roots infected with each vector, and the LOX2 enzyme activity was identified in the target hairy roots.

[0068] Figure 5 Transgenic hairy root assays are shown, 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 identification

[0070] By using the LOX2 enzyme activity identification method in Example 1, it was found that the LOX2 enzyme activity after LOX2 mutation was significantly reduced by 291.7 pg / ml compared with the infected wild-type root, and the catalytic efficiency was reduced by 15.62%. This fully demonstrates that the present invention identifies a new mutation type by identifying the lipoxygenase in the grafted odorless soybean; after site-directed mutagenesis, the lipoxygenase mutant with reduced catalytic efficiency is screened and obtained. The odorless soybean obtained by significantly reducing the catalytic efficiency of lipoxygenase lays a 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 listing of the above SEQ ID NO.1 to 4 is 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 contents are further detailed descriptions of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can also make several substitutions or modifications to these described embodiments without departing from the concept of the present invention, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention.

Claims

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

2.

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

4. The use according to claim 3, characterized in that The steps include: Use grafting breeding techniques to modify LOX2 activity in parental soybean varieties; Grafting the parent soybean onto ginger and harvesting grafted contemporary seeds; After years of systematic breeding, strains were selected in which the lysine residues at positions 257, 263, and 472 in the parent amino acid sequence shown in SEQ ID NO.1 were succinylated; 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.

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