Application of overexpressed product of GmDREB2A2 gene in regulating content of fatty acid in plants

Abstract

This invention belongs to the field of genetic engineering technology, specifically involving GmDREB2A2 Application of genes in regulating fatty acid content in plants. Overexpression. GmDREB2A2 Gene regulation of fatty acid content in plants. This invention utilizes an exogenous gene sequence expression vector to express the coding sequence shown in SEQ ID NO.1. GmDREB2A2 Gene sequences are introduced into plants to enable GmDREB2A2 Gene overexpression. Under these conditions, the plant's linolenic acid, linoleic acid, and palmitic acid content were significantly higher than the control, while the content of eicosatrienoic acid and erucic acid was significantly reduced.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to overexpression. GmDREB2A2 Application of gene-based products in regulating the fatty acid content of plants. Background Technology

[0002] Fatty acids, as major structural components of biological membranes, important forms of energy storage, and precursors of various bioactive molecules, play a central role in plant growth and development, stress resistance, and seed quality formation. Oleic acid, linoleic acid, and linolenic acid are essential fatty acids for human health. The composition of these fatty acids is crucial to the overall quality, nutritional value, stability, and health of oils and fats. However, in natural oilseed crops, the high content of these beneficial fatty acids is often accompanied by the coexistence of some harmful fatty acids. For example, erucic acid, abundant in traditional oilseeds such as rapeseed, is strictly limited by international standards due to its potential food safety risks, which greatly restricts its direct development as edible oil. Therefore, synergistically regulating fatty acid composition through technological means to significantly reduce the content of harmful fatty acids while increasing the content of beneficial fatty acids has become a key approach to improving the overall quality and functional value of oils and fats. Harmful fatty acids are those associated with an increased risk of chronic diseases, such as cardiovascular disease.

[0003] The acquisition of plant fatty acids is highly dependent on large-scale agricultural production of oil crops, and is extracted and processed through physical pressing or chemical extraction techniques. However, the yield and composition of oils are severely limited by the genetic background of the species itself. Summary of the Invention

[0004] This invention is intended to provide GmDREB2A2 The application of genes in regulating the content of fatty acids in plants aims to address the technical problem that oil yield and composition are severely limited by the genetic background of the species in existing technologies.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: overexpression GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content, the aforementioned GmDREB2A2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; oil-related genes were screened using the soybean young pod transcriptome database. GmDREB2A2 Specific primers were designed, RNA from young pods was extracted and cloned for sequencing, and the sequenced sequences were compared to confirm the integrity and accuracy of the coding frame (ORF). The final accurate coding sequence is SEQ ID NO.1.

[0006] Furthermore, the biological product includes: a recombinant plasmid or recombinant Agrobacterium containing the gene shown in SEQ ID NO.1; and the establishment of a transgenic plant through genetic engineering methods.

[0007] Furthermore, the fatty acids include palmitic acid, palmitoleic acid, stearic acid, linoleic acid, linolenic acid, oleic acid, myristic acid, behenic acid, erucic acid, arachidonic acid, icosanoic acid, and arachidiol. These 12 fatty acids cover the main representatives of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids. They are core indicators for evaluating the quality of oils and fats, such as nutritional value, oxidative stability, and industrial properties. Moreover, different fatty acids have different economic values.

[0008] Furthermore, the recombinant plasmid is obtained by inserting the gene shown in SEQ ID NO.1 into a plant binary expression vector. The binary vector system is the most commonly used and mature vector system in Agrobacterium-mediated plant transgenic technology. It contains two replication origins, one for replication in Escherichia coli and the other for replication in Agrobacterium. At the same time, there are T-DNA boundary sequences, LB and RB, on both sides. Exogenous genes and selection marker genes, such as herbicide resistance genes, located between these two T-DNA boundary sequences can be efficiently cut and transferred into the plant genome when Agrobacterium infects the plant, while the vector backbone will not enter the plant cell.

[0009] Furthermore, the method of insertion into the plant binary expression vector is as follows: the insertion into the plant binary expression vector includes the insertion between the BamHI sites of the pCAMBIA3301-eGFP vector; the specific sticky ends generated by the BamHI restriction endonuclease can achieve directional and efficient ligation of the target fragment with the linearized vector. Since the BamHI site is located in the T-DNA region of the vector and is associated with the eGFP reporter gene, the constructed recombinant vector can stably integrate the target gene into the plant genome through Agrobacterium-mediated transformation.

[0010] Furthermore, the recombinant Agrobacterium is obtained by introducing a recombinant plasmid into engineered bacteria; the introduction method is heat shock; the heat shock method changes the cell membrane structure through temperature changes, low temperature treatment makes the cells enter the competent state, 42°C heat shock makes the membrane form micropores, exogenous DNA enters the cell, after the temperature is restored, the cell membrane recovers, the plasmid replicates and expresses in the culture medium, thereby screening the successfully transformed Agrobacterium.

[0011] Furthermore, the engineered bacteria include Escherichia coli DH5α and Agrobacterium GV3101; Escherichia coli DH5α, with its genetic characteristics of recombination deficiency and endonuclease deficiency, is an ideal host for efficient cloning, stable amplification and sequence verification of recombinant plasmids, while Agrobacterium tumefaciens GV3101 carries a disarmed helper plasmid, which can trans-provide all Vir protein functions required for T-DNA transfer, and is an efficient and safe delivery vector for realizing plant genetic transformation.

[0012] Furthermore, overexpression GmDREB2A2 The method of gene regulation of fatty acid content is to infect dicotyledonous plants with recombinant Agrobacterium.

[0013] The steps for infecting dicotyledonous plants with recombinant Agrobacterium are as follows: Recombinant Agrobacterium was cultured to obtain an inoculum. During the flowering period of dicotyledonous plants, the inflorescences of these plants were immersed in the inoculum to obtain seeds. The seeds were then cultured to obtain overexpressed [genes / products]. GmDREB2A2 Dicotyledonous plants with genes that enable the control of genes in dicotyledonous plants. GmDREB2A2 Gene overexpression; the principle of the above inflorescence-based transfection method is to utilize the high plasticity of reproductive meristem cells during the flowering period of plants, allowing Agrobacterium to directly introduce T-DNA into reproductive precursor cells, and then obtain non-chimeric transgenic offspring through sexual reproduction. In specific operations, the preparation of high-activity inducing bacterial solution aims to maximize the transfer efficiency of T-DNA; selecting inflorescences for transfection during the flowering period is to precisely target the cells most easily transformed and capable of forming seeds; and resistance screening after seed harvesting is to efficiently and accurately isolate stable integrated genes from the offspring population. GmDREB2A2 Genetically modified plants. This series of steps works in concert to achieve... GmDREB2A The stable overexpression of gene 2 in the whole plant, especially in seeds, provides a direct and efficient technical approach for subsequent fatty acid trait analysis.

[0014] Furthermore, the dicotyledonous plants include soybean and Arabidopsis thaliana; the soybean variety includes Jinong 18; using Arabidopsis thaliana as a model plant, relying on its mature genetic system and short life cycle advantage, the study can quickly and efficiently verify... GmDREB2A2 The study aims to investigate the basic functions and molecular mechanisms of gene regulation of fatty acid metabolism, using soybean Jinong 18 as the target crop. The study will directly evaluate the actual improvement effect of this gene on key agronomic traits such as seed oil content and fatty acid composition, ensuring the direct correlation between research results and industrial applications.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides GmDREB2A2 Application of genes in regulating plant fatty acid content, overexpression GmDREB2A2Gene regulation of fatty acid content in plants. This invention utilizes an exogenous gene sequence expression vector to express the coding sequence shown in SEQ ID NO.1. GmDREB2A2 Gene sequences are introduced into plants to enable GmDREB2A2 Gene overexpression. Under these conditions, the plant's linolenic acid, linoleic acid, and palmitic acid content were significantly higher than the control, while the content of eicosatrienoic acid and erucic acid was significantly reduced.

[0016] (2) This invention regulates GmDREB2A2 The expression of gene sequences in plants can be used to breed new plant varieties with controllable fatty acid content, providing new ideas for breeding new crop varieties and having great application value in the field of molecular breeding.

[0017] (3) Based on the results of bioinformatics prediction and relative expression analysis in different tissues, this invention successfully cloned differentially expressed fatty acid-related genes. GmDREB2A2 They also constructed a plant overexpression vector.

[0018] (4) This invention uses Arabidopsis thaliana as a genetic transformation recipient material to explore GmDREB2A2 The biological function of genes in plant oil biosynthesis lays a theoretical foundation for in-depth research on plant lipid metabolism, and also provides resources for discovering and creating new high-oil and high-yield soybean germplasm. Attached Figure Description

[0019] Figure 1 for GmDREB2A2 Electrophoresis diagram of gene amplification products; where M represents DL2000bp DNA marker; (-) represents negative control; lanes 1, 2 and 3 are the amplified fragments of the target gene.

[0020] Figure 2 This is an electrophoresis image of the pCAMBIA3301-eGFP single enzyme digestion product; where M represents the DL10000bp DNA marker; lanes 1 and 2 represent GmDREB2A2 Gene.

[0021] Figure 3 for GmDREB2A2 Effects on the expression of regulatory factors related to key genes involved in lipid regulation; ***p<0.0001 statistical difference, ****p<0.0001 statistical difference; wild-type Arabidopsis thaliana is represented; overexpression of 2A2 represents T2 generation overexpression. GmDREB2A2 Plant.

[0022] Figure 4 The relative percentage contents of palmitic acid, palmitoleic acid, stearic acid, linoleic acid, linolenic acid, and oleic acid in T3 generation transgenic Arabidopsis seeds were determined; wild-type Arabidopsis seeds were also included; and overexpression of 2A2 was used to represent T3 generation overexpression.GmDREB2A2 Seeds of plants with gene sequences.

[0023] Figure 5 The relative percentage contents of myristic acid, behenic acid, erucic acid, arachidonic acid, cocoic acid, and arachidonic acid in T3 generation transgenic Arabidopsis seeds were determined; wild-type Arabidopsis seeds were also included; overexpression of 2A2 represented T3 generation overexpression. GmDREB2A2 Seeds of plants with gene sequences.

[0024] Figure 6 This is a schematic diagram of the structure of the pCAMBIA3301-eGFP vector. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials, reagents, etc. used are all commercially available.

[0026] PCR stands for Polymerase Chain Reaction. FATB The gene, whose full name is acyl-ACP thioesterase B; FAD2 The gene, short for fatty acid dehydrogenase; LPAT5 The gene, whose full name is lysophosphatidyl acyltransferase 5; LACS8 The gene, whose full name is long-chain acyl-CoA synthase 8; GmDREB2A2 Genes in Figure 3 , Figure 4 and Figure 5 The abbreviation is 2A2. The Basta solution comes from Yuanye Biotechnology Co., Ltd. The original concentration was 20%, which was diluted with water to 0.15% to obtain a 0.15% Basta solution.

[0027] Example 1 GmDREB2A2 The application of genes in regulating the fatty acid content of plants is as follows: S1. Constructing overexpression GmDREB2A2 carrier: Using young pods of soybean variety Jinong 18 "JN18" 30 days after flowering as material, RNA was extracted, and the cDNA transcribed from the RNA was used as a template. Primer F with homologous arms was then used. 5'-acacgctgagtgtcaggatATGGGTGCTTATGATCAAGTT-3', SEQ ID NO.3; And primer R with homologous arms: 5'-tccaaacgcatgcagggatCTAGTCATCCTTCCTTGCTTG-3', SEQ ID NO.4; The lowercase nucleotide sequences in SEQ ID NO.3 and SEQ ID NO.4 represent seamless cloning homologous arm sequences.

[0028] PCR amplification was performed, yielding a 1170bp image with homologous arms. GmDREB2A2 The CDS sequence of the gene and the PCR reaction are shown in Table 1. GmDREB2A2 The original gene sequence is shown in SEQ ID NO.1. The amplified sequence contains homologous arms. GmDREB2A2 The gene sequence is shown in SEQ ID NO.2.

[0029] Table 1 PCR reaction system

[0030]

[0031] Cloned containing homologous arms GmDREB2A2 The gene sequence is 1170 bp in length.

[0032] PCR products were identified by size using agarose gel electrophoresis, and then the gel was recovered. The identification results are as follows: Figure 1 As shown, the target segment is obtained.

[0033] The pCAMBIA3301-eGFP vector was digested with BamHI, and the digestion results are as follows: Figure 2 As shown, the pCAMBIA3301-eGFP vector is as follows: Figure 6 As shown, gel recovery was then performed. The procedure utilized a specific recombinase, 2xBasic Assembly Mix, and the principle of homologous recombination; detailed reaction procedures are shown in Table 2. The reaction system was placed in a PCR instrument and incubated at 50°C for 15 minutes. After the reaction, the ligation product was cooled on ice. It was then transformed into *E. coli* using a heat shock method at 42°C. Positive clones were screened on LB agar containing kanamycin. Colonies were picked, and positive clones were further screened by PCR and agarose gel electrophoresis. The selected positive clones were sent to the company for sequencing, and the desired product was successfully obtained. GmDREB2A2 The overexpression vector plasmid pCAMBIA3301-eGFP-DREB2A2.

[0034] Table 2 Reaction System

[0035] S2, Obtain GmDREB2A2 Transgenic Arabidopsis plants: Take 1 μg of pCAMBIA3301-eGFP-DREB2A2 plasmid and add it to 100 μL of Agrobacterium tumefaciens competent cells GV3101. Gently tap the bottom of the tube with your finger to mix the contents. Then, perform the following steps in sequence: incubate on ice for 10 min, place in liquid nitrogen for 5 min, place in a 37°C water bath for 5 min, and then incubate on ice for 5 min. After that, add 900 μL of antibiotic-free YEP liquid medium and incubate at 28°C and 200 rpm for 2 h. Finally, spread the culture evenly on LB medium containing 50 μg / mL kanamycin and incubate upside down at 28°C for 2 days.

[0036] A single colony was picked and inoculated into 2 mL of LB medium containing 50 μg / mL rifampin and 50 μg / mL kanamycin, and incubated overnight at 28°C and 200 rpm. Subsequently, primer F was used... 5'-ATGGGTGCTTATGATCAAGTTTCT-3', SEQ ID NO.5; PCR verification was performed using primer R: 5'-GCTTTCCCCTCATTGTCTTT-3', SEQ ID NO.6. The specific PCR reaction system is detailed in Table 3.

[0037] Table 3 PCR reaction system

[0038] Preparation of dyeing solution: The bacterial suspension was prepared by adding 4.4g MS powder, 100g sucrose, 2mL of 1000× B5 vitamin, 20μL of 1mg / mL 6-BA, 400μL of Silweet L-77, 180μL of 1M NaOH, and 1950mL of ddH2O to maintain the OD value of Agrobacterium tumefaciens in the range of 0.8 to 1.0. The measured OD value was 0.9.

[0039] Prepare a beaker containing Agrobacterium-mediated infection solution. Invert the flowering Arabidopsis thaliana to completely submerge the inflorescence in the solution for 5 minutes. Afterward, carefully remove the Arabidopsis and place it sideways in a clean plastic tray. Cover with plastic film to protect from light and maintain humidity for 24 hours. Then, transfer it to normal light conditions for cultivation. Seeds can be harvested approximately 4 weeks later, when the siliques are completely yellowed and about to split open.

[0040] Seeds were cultured to obtain seedlings at the four-leaf stage. These seedlings were then sprayed with a 0.15% Basta solution every two days. After some Arabidopsis seedlings showed signs of yellowing and wilting, the healthy seedlings were transplanted into new pots, thus obtaining the transgenic plant pCAMBIA3301-eGFP-DREB2A2, a T2 generation overexpression. GmDREB2A2 Plant.

[0041] Experiment 1 Overexpression GmDREB2A2 Gene sequences affect the expression levels of key fatty acid-related enzyme genes.

[0042] The relative expression of acyl-ACP thioesterase B, fatty acid dehydrogenase, lysophosphatidyl acyltransferase 5, and long-chain acyl-CoA synthase 8 in transgenic and wild-type Arabidopsis thaliana was investigated. Changes in the expression levels of these genes in wild-type and transgenic Arabidopsis thaliana were used as indicators. qPCR analysis was performed using cDNA as a template, and specific primers were designed using NCBI.

[0043] LACS8 Primer F: 5'-ATGGAAGTTGGTTTGGTGCC-3', SEQ ID NO.7; and LACS8Primer R: 5'-CGTTGCGAATCAGGAGACAG-3', SEQ ID NO.8.

[0044] FAD2 Primer F: 5'-ATGGGTGCAGGTGGAAGAATGCCGG-3', SEQ ID NO.9; and FAD2 Primer R: 5'-CCGGCATTCTTCCACCTGCACCCAT-3', SEQ ID NO.10.

[0045] FATB Primer F: 5'-TTTTCTTAGCGGCTGAGAAACA-3', SEQ ID NO.11; and FATB Primer R: 5'-CAAGGCCATCCTGAACAATTC-3', SEQ ID NO.12.

[0046] LPAT5 Primer F: 5'-GTTTGTGGCTAAGGATGCTCTG-3', SEQ ID NO.13; and LPAT5 Primer R: 5'-CAAGGGACTTTATGGGACGG-3', SEQ ID NO.14.

[0047] T2 generation overexpression was detected by qRT-PCR. GmDREB2A2 Expression levels of key enzyme genes in broodstock and wild-type plants. Results are as follows: Figure 3 As shown, compared with wild-type Arabidopsis thaliana, GmDREB2A2 In the plant LACS8 , FAD2 , FATB and LPAT5 Gene expression levels were significantly higher.

[0048] This indicates GmDREB2A2 It directly or indirectly affects the expression levels of key fatty acid-related enzyme genes, thus confirming that... GmDREB2A2 It participates in fatty acid synthesis and metabolism in Arabidopsis thaliana.

[0049] Experiment 2 Overexpression GmDREB2A2 Detection of fatty acid content in genetically modified plants.

[0050] This embodiment uses Method 3 of GB 5009.168-2016 "National Food Safety Standard - Determination of Fatty Acids in Food" to detect T3 generation overexpression. GmDREB2A2 Fatty acid content in the seeds of both the gene sequence-seeded and wild-type Arabidopsis thaliana plants. Results are as follows:Figure 4 and Figure 5 As shown, overexpression GmDREB2A2 The gene sequence showed significantly higher levels of linoleic acid, linolenic acid, and palmitic acid in the plants compared to the control, while the levels of eicosatrienoic acid (EPA) and erucic acid (EEA) were significantly lower. Excessive EEA may interfere with the body's utilization and metabolism of Omega-3 fatty acids, which have a proven cardiovascular protective effect, potentially indirectly weakening the beneficial physiological functions of Omega-3 fatty acids in terms of anti-inflammatory and lipid-lowering effects. Erucic acid accumulates in the myocardium, leading to myocardial lipid deposition and myocardial fibrosis, thereby impairing cardiac function. Overexpression GmDREB2A2 After gene modification, the content of linoleic acid in transgenic Arabidopsis seeds increased by 2.6%, the content of linolenic acid increased by 2.4%, and the content of palmitoleic acid increased by 42%.

[0051] The results show that GmDREB2A2 Genes are involved in the regulation of fatty acids in Arabidopsis thaliana.

[0052] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. Overexpression GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content is characterized by, The GmDREB2A2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The overexpression according to claim 1 GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content is characterized by, The fatty acids include palmitic acid, palmitoleic acid, stearic acid, linoleic acid, linolenic acid, oleic acid, myristic acid, behenic acid, erucic acid, arachidonic acid, icosanoic acid, and arachidiol.

3. The overexpression according to claim 1 GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content is characterized by, The biological product includes: a recombinant plasmid or recombinant Agrobacterium containing the gene shown in SEQ ID NO.

1.

4. The overexpression according to claim 3 GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content is characterized by, The recombinant plasmid was obtained by inserting the gene shown in SEQ ID NO.1 into a plant binary expression vector.

5. The overexpression according to claim 4 GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content is characterized by, The method for insertion into a plant binary expression vector is as follows: the insertion is performed between the BamHI sites of the pCAMBIA3301-eGFP vector.

6. The overexpression according to claim 3 GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content is characterized by, The recombinant Agrobacterium was obtained by introducing a recombinant plasmid into engineered bacteria; the introduction method was heat shock.

7. The overexpression according to claim 6 GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content is characterized by, The engineered bacteria include Agrobacterium GV3101 and Escherichia coli DH5α.

8. The overexpression according to claim 6 GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content is characterized by, overexpression GmDREB2A2 The method of gene regulation of fatty acid content is to infect dicotyledonous plants with recombinant Agrobacterium.

9. The overexpression according to claim 8 GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content is characterized by, The steps for infecting dicotyledonous plants with recombinant Agrobacterium are as follows: Recombinant Agrobacterium was cultured to obtain an inoculum. During the flowering period of dicotyledonous plants, the inflorescences of these plants were immersed in the inoculum to obtain seeds. The seeds were then cultured to obtain overexpressed [genes / products]. GmDREB2A2 Dicotyledonous plants with genes that enable the control of genes in dicotyledonous plants. GmDREB2A2 Gene overexpression.

10. The overexpression according to claim 8 GmDREB2A2 The application of gene-based bioproducts in regulating plant fatty acid content is characterized by, The dicotyledonous plants include soybean and Arabidopsis thaliana; the soybean variety includes Ginnon 18.

Images