Peanut omega-3 fatty acid dehydrogenase gene combination and application thereof, plant overexpression vector and method

Abstract

The invention relates to the technical field of gene engineering, in particular to a peanut omega-3 fatty acid dehydrogenase gene combination, application thereof, a plant overexpression vector and a method. The omega-3 fatty acid dehydrogenase gene combination comprises omega-3 fatty acid dehydrogenase genes AhFAD3a, AhFAD3c, AhFAD7a and AhFAD7d, and the omega-3 fatty acid dehydrogenase gene combination comprises omega-3 fatty acid dehydrogenase genes AhFAD3a, AhFAD3c, according to the invention, four omega-3 fatty acid dehydrogenase genes, namely AhFAD3a, AhFAD3c, AhFAD7a and AhFAD7d, are cloned from peanuts, and the four omega-3 fatty acid dehydrogenase genes are co-transformed into arabidopsis thaliana. Results show that fatty acid components of transgenic arabidopsis thaliana overexpression strain seeds are greatly different from those of wild type seeds, the total fatty acid content is remarkably increased by 4.50%-9.00%, and the linolenic acid content is increased by 2.16%-14.29%. A research result provides a theoretical basis for a molecular mechanism of peanut fatty acid metabolism and provides a new clue for genetic improvement of peanut quality.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to a peanut ω-3 fatty acid dehydrogenase gene combination and application thereof, a plant overexpression vector and a method. Background Art

[0002] Peanut (Arachis hypogaea L.), also known as groundnut, longevity fruit, etc., belongs to the genus Arachis in the family Fabaceae, Fabaceae, and is an annual herbaceous plant with tetraploid (2n=4x=40). As one of the four major oil crops in the world, peanut occupies an important position in the world oil and fat market. At the same time, peanut is an important oil and cash crop in my country, and is an important source of edible oil and protein. [1] In recent years, my country's annual peanut planting area has exceeded 70 million mu, with a total output of about 18 million tons, ranking first in the world and accounting for 50% of my country's total oil crop output. More than 60% of peanuts will be used for oil extraction. In the fatty acid composition of peanuts, oleic acid and linoleic acid account for more than 80% of the total fatty acids. [2] However, the proportion of linolenic acid in the total fatty acids is extremely low and is almost undetectable in many varieties of peanut kernels. Linolenic acid belongs to ω-3 fatty acids, which are polyunsaturated fatty acids and have health functions for human growth and development, immune response, etc. [3] . Omega-3 fatty acids mainly include α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In higher plants, FAD3, FAD7 and FAD8 are key enzymes for the synthesis of ALA. They use linoleic acid as a precursor to synthesize ALA. At the same time, ALA is a precursor for the synthesis of the signaling molecule jasmonic acid. Jasmonic acid participates in regulating different biological metabolic pathways such as defense response and pollen abortion. [4] .

[0003] In recent years, ω-3FAD has been cloned and studied in many plants. In soybean and rapeseed, ω-3FAD related genes have been cloned. [5,6] , the FAD8 gene was also reported to be cloned in peanut [7] Tovuu et al. [8] It was found that knocking out the rice OsFAD8 gene reduced the trienoic acid level and membrane fluidity of the plant leaves, and the plants were more sensitive to low temperature stress in the short term compared with the wild type. Kodama et al. [9] The Arabidopsis AtFAD7 gene was introduced into tobacco, which significantly improved the cold tolerance of transgenic tobacco seedlings. Teresa Domínguez et al.

[10] Overexpression of tomato BnFAD3 and StFAD7 genes not only increases the C18:3 / C18:2 ratio in tomato leaves and fruits, but also causes significant changes in the content of C6 complexes. In addition, tomato plants overexpressing FAD are more tolerant to low temperatures. [4]Several ω-3 peanut FAD (ω-3AhFAD) genes and their promoters were cloned from the peanut cultivar Fenghua No. 1 (FH-1) and their functions were verified. [11,12] , including AhFAB2-1, AhSLD-1, four microsomal AhFAD2 and two chloroplast AhFAD6 genes [13,14] Among them, FAD2 is the most studied fatty acid desaturase gene. Their expression patterns in different tissues, different developmental stages and abiotic stress conditions have been preliminarily analyzed, and the functions of some FAD genes have been verified in algae and Saccharomyces cerevisiae. The de novo synthesis pathway of fatty acids in plastids is the basis for the accumulation of α-linolenic acid (ALA). Currently, a gene AhFAD3-A01 has been cloned in peanuts, and it has been confirmed that its expression can affect the accumulation of α-linolenic acid. Li et al. [1] Using the constitutive promoter P 35S and seed-specific promoter P AhWRI1 Overexpression of AhFAD3-A01 in Arabidopsis and peanut resulted in the inhibition of P 35S ::The ALA content of AhFAD3-A01 T1 seeds increased from 15.18% to 30.65%, P AhWRI1 ::The ALA content of AhFAD3-A01 seeds increased from 11.23% to 25.49%. Chi et al. [2] The researchers found that during seed development, AhFAD7-1 expression levels initially increased and then decreased. In the initial stages of seed development, AhFAD7-2 transcript levels were relatively high, then decreased and then increased. While the expression of some individual FAD genes has been demonstrated in peanut, the simultaneous overexpression of multiple FAD genes has not been reported. Furthermore, the co-overexpression of multiple ω-3 FAD genes has not been reported.

[0004] [1]Li XN,Xue LL,Liu Han,Qu PY,Zhao HH,Luo DD,Wang XB,Huang BY,Zhang MN,Li CY,Zhang ZX,Dong WZ,Shi L,Zhang

[0005] [2]Chi Summary of the Invention

[0006] To address the above-mentioned issues, the present invention provides a peanut ω-3 fatty acid desaturase gene combination, its application, plant overexpression vector, and method. Eight copies of AhFAD3 and AhFAD7 were cloned. Four FAD genes, AhFAD3a, AhFAD3c, AhFAD7a, and AhFAD7d, were identified through expression pattern analysis and subcellular localization analysis. A plant overexpression vector driven by the CaMV35S promoter was constructed and genetically transformed into Arabidopsis thaliana, generating positive transgenic plants. The fatty acid composition and agronomic traits of these plants were then determined. This approach lays the foundation for further elucidating the biological functions of these genes and provides technical support for molecular breeding approaches to improve the quality traits of crops such as peanuts using the AhFAD3 and AhFAD7 genes.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a peanut ω-3 fatty acid dehydrogenase gene combination, comprising ω-3 fatty acid dehydrogenase gene AhFAD3a, ω-3 fatty acid dehydrogenase gene AhFAD3c, ω-3 fatty acid dehydrogenase gene AhFAD7a and ω-3 fatty acid dehydrogenase gene AhFAD7d.

[0009] Preferably, the nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD3a is shown as SEQ ID No. 1, and the amino acid sequence is shown as SEQ ID No. 2;

[0010] The nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD3c is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 4;

[0011] The nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD7a is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No. 6;

[0012] The nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD7d is shown in SEQ ID No. 7, and the amino acid sequence is shown in SEQ ID No. 8.

[0013] The present invention also provides the use of the peanut ω-3 fatty acid desaturase gene combination described in the above technical solution in increasing the fatty acid content of peanuts.

[0014] Preferably, the peanut fatty acid includes one or more of palmitic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidic acid, arachidic acid, arachidienoic acid and behenic acid.

[0015] The present invention also provides the use of the peanut ω-3 fatty acid desaturase gene combination described in the above technical solution in regulating peanut fatty acid metabolism-related genes.

[0016] Preferably, the expression of aldehyde dehydrogenase gene, plant stearoyl-acyl-carrier protein desaturase gene, GNS1 / SUR4 membrane protein family gene, acetyl-CoA acyltransferase 1 gene, peroxidase gene, acyl-CoA dehydrogenase gene, alcohol-forming fatty acid acyl-CoA reductase gene, fatty acid ω-hydroxylase gene, alcohol-forming fatty acid acyl-CoA reductase gene, aldehyde decarbonylase gene and acyl-CoA oxidase gene in peanut is upregulated.

[0017] Preferably, the expression of aldehyde decarbonylase gene, 3-ketoacyl-CoA synthetase gene and palmitoyl-protein thioesterase gene in peanut is down-regulated.

[0018] The present invention also provides a plant overexpression vector, which is obtained by connecting the peanut ω-3 fatty acid desaturase gene combination described in the above technical solution to the plant expression vector pCAMBIA2300.

[0019] Preferably, the ω-3 fatty acid dehydrogenase gene AhFAD3a, the ω-3 fatty acid dehydrogenase gene AhFAD3c, the ω-3 fatty acid dehydrogenase gene AhFAD7a and the ω-3 fatty acid dehydrogenase gene AhFAD7d in the ω-3 fatty acid dehydrogenase gene combination are sequentially connected in series to obtain a gene synthesis fragment, and the gene synthesis fragment is connected to the plant expression vector pCAMBIA2300;

[0020] The plant expression vector pCAMBIA2300 is ligated after the EGFP gene is cut out.

[0021] The present invention also provides a method for obtaining Arabidopsis thaliana plants with high fatty acid content, comprising the following steps:

[0022] 1) Transforming the plant overexpression vector described in the above technical solution into Agrobacterium strain GV3101 to obtain transformed bacteria;

[0023] 2) Infecting Arabidopsis inflorescences with the transformed bacteria obtained in step 1) to obtain Arabidopsis plants with high fatty acid content.

[0024] Beneficial effects of the present invention:

[0025] ω-3 fatty acid desaturase (ω-3FAD) catalyzes the conversion of linoleic acid to α-linolenic acid. Linolenic acid is a polyunsaturated fatty acid that has health benefits for human growth and development, immune response, and other health benefits. To explore the role of ω-3FAD in peanut growth and development, the present invention cloned four ω-3 fatty acid desaturase genes, AhFAD3a, AhFAD3c, AhFAD7a, and AhFAD7d, from peanut and co-transformed them into Arabidopsis thaliana. The results showed that the fatty acid composition of seeds of transgenic Arabidopsis overexpressing strains differed significantly from that of the wild type, with a significant increase in total fatty acid content of 4.50%-9.00%, including an increase in linolenic acid content of 2.16%-14.29%. Subcellular localization analysis showed that AhFAD3a and AhFAD3c are localized to the endoplasmic reticulum, while AhFAD7a and AhFAD7d are localized to the chloroplasts. GO annotation and KEGG enrichment results showed that differentially expressed genes were enriched in various biological metabolic processes, including lipid metabolism, amino acid metabolism, and carbohydrate metabolism. Eleven upregulated genes were enriched in the α-linoleic acid metabolic pathway, and a total of 15 differentially expressed genes were screened for fatty acid metabolism. These findings provide a theoretical basis for the molecular mechanisms of peanut fatty acid metabolism and offer new insights into genetic improvement of peanut quality. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0027] Figure 1 This is the backbone map of the plant expression vector pCAMBIA2300;

[0028] Figure 2 is a schematic diagram of the co-expression vector pFAD3 / 7;

[0029] Figure 3 The expression patterns of AhFAD3s and AhFAD7s genes in different peanut tissues;

[0030] Figure 4 This is the subcellular localization analysis of the gene AhFAD. Note: GFP: green fluorescence; ER-mCherry: endoplasmic reticulum-localized mCherry red fluorescence; Chloroplast: chloroplast fluorescence; Bright Field: bright field; Merged: fused photo;

[0031] Figure 5 The effect of co-overexpression of AhFADs on fatty acids in Arabidopsis thaliana, Note: * indicates 0.05 significance level; ** indicates 0.01 significance level;

[0032] Figure 6 Volcano plot of differentially expressed genes;

[0033] Figure 7 GO enrichment analysis of differentially expressed genes;

[0034] Figure 8 KEGG pathway enrichment analysis of differentially expressed genes. DETAILED DESCRIPTION

[0035] The present invention provides a peanut ω-3 fatty acid dehydrogenase gene combination, including ω-3 fatty acid dehydrogenase gene AhFAD3a, ω-3 fatty acid dehydrogenase gene AhFAD3c, ω-3 fatty acid dehydrogenase gene AhFAD7a and ω-3 fatty acid dehydrogenase gene AhFAD7d. In the present invention, the nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD3a is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2; the nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD3c is shown in SEQ ID No.3, and the amino acid sequence is shown in SEQ ID No.4; the nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD7a is shown in SEQ ID No.5, and the amino acid sequence is shown in SEQ ID No.6; the nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD7d is shown in SEQ ID No.7, and the amino acid sequence is shown in SEQ ID No.8.

[0036] The present invention also provides the use of the peanut ω-3 fatty acid desaturase gene combination described in the above technical solution to increase the fatty acid content of peanuts. In the present invention, the peanut fatty acids preferably include one or more of palmitic acid, oleic acid, linoleic acid, linolenic acid, eicosanoic acid, arachidic acid, arachidic acid, arachidienoic acid, and behenic acid.

[0037] The present invention also provides the use of the peanut ω-3 fatty acid dehydrogenase gene combination described in the above technical solution in regulating peanut fatty acid metabolism-related genes. The present invention preferably upregulates the expression of peanut aldehyde dehydrogenase genes, plant stearoyl-acyl-carrier protein desaturase genes, GNS1 / SUR4 membrane protein family genes, acetyl-CoA acyltransferase 1 genes, peroxidase genes, acyl-CoA dehydrogenase genes, alcohol-forming fatty acid acyl-CoA reductase genes, fatty acid ω-hydroxylase genes, alcohol-forming fatty acid acyl-CoA reductase genes, aldehyde decarbonylase genes, and acyl-CoA oxidase genes. The present invention preferably downregulates the expression of peanut aldehyde decarbonylase genes, 3-ketoacyl-CoA synthetase genes, and palmitoyl-protein thioesterase genes.

[0038] The present invention also provides a plant overexpression vector, comprising the peanut ω-3 fatty acid dehydrogenase gene combination described in the above technical solution being ligated to the plant expression vector pCAMBIA2300 to obtain a plant overexpression vector. In the present invention, the ω-3 fatty acid dehydrogenase genes AhFAD3a, AhFAD3c, AhFAD7a, and AhFAD7d in the ω-3 fatty acid dehydrogenase gene combination are sequentially connected in series to obtain a gene synthesis fragment, which is then connected to the plant expression vector pCAMBIA2300. The present invention does not specifically limit the method for connecting the genes in series; those skilled in the art can use conventional methods for connection. In the present invention, the plant expression vector pCAMBIA2300 is preferably cleaved of the EGFP gene before connection. The present invention does not specifically limit the method for cleaving the EGFP gene; those skilled in the art can use conventional methods. The present invention does not specifically limit the method for connecting the genes in series; those skilled in the art can use conventional methods.

[0039] The present invention also provides a method for obtaining a high-fat acid Arabidopsis plant, comprising the following steps:

[0040] 1) Transforming the plant overexpression vector described in the above technical solution into Agrobacterium strain GV3101 to obtain transformed bacteria;

[0041] 2) Infecting Arabidopsis inflorescences with the transformed bacteria obtained in step 1) to obtain Arabidopsis plants with high fatty acid content.

[0042] The present invention transforms the plant overexpression vector described in the above technical solution into Agrobacterium tumefaciens to obtain a transformed bacterium. In the present invention, the Agrobacterium tumefaciens is preferably Agrobacterium strain GV3101. The present invention does not particularly limit the method for transforming the plant overexpression vector into Agrobacterium tumefaciens; conventional methods used by those skilled in the art can be used.

[0043] The present invention uses the obtained transformed bacteria to infect Arabidopsis inflorescences to obtain Arabidopsis plants with high fatty acid content. The present invention does not specifically limit the method of infecting Arabidopsis inflorescences with the transformed bacteria, and those skilled in the art can simply follow conventional operations.

[0044] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] 1 Materials and Methods

[0047] 1.1 Test materials

[0048] The peanut variety used in this example is Huayu 917, bred by our research group. It has a crude fat content of 55.8%, a crude protein content of 20.3%, an oleic acid content of 77.7%, a linoleic acid content of 6.62%, and medium seed dormancy, medium drought resistance, and medium waterlogging tolerance. Huayu 917 was planted at the Laixi Experimental Station, and a mixed sample of its roots, stems, leaves, nodules, and pods was collected for cloning AhFAD3a, AhFAD3c, AhFAD7a, and AhFAD7d. Transgenic Arabidopsis thaliana was of the Columbia ecotype (Col-0). The transgenic strains and wild-type plants were grown under the same artificial climate chamber conditions, with a 16h / 8h photoperiod, a daytime temperature of 23°C, a nighttime temperature of 21°C, and a relative humidity of 60%.

[0049] 1.2 Test methods

[0050] 1.2.1 Total RNA extraction and reverse transcription synthesis of first-strand cDNA

[0051] Total RNA was extracted from various peanut tissues using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Catalog No. DP441) from Tiangen Biochemical Technology (Beijing) Co., Ltd. To prevent RNA degradation, the entire extraction process was performed on ice. First-strand cDNA was synthesized using the extracted total RNA as a template using the PrimeScriper 1st strand cDNA Synthesis Kit (Catalog No. 6110A) from TaKaRa. A 25-μL reaction system contained 2 μg of RNA. After a 60-min reverse transcription reaction at 42°C, the reverse transcribed product was placed on ice for 5 minutes and then stored in a -20°C freezer until ready for use.

[0052] 1.2.2 Gene cloning

[0053] Peanut FAD3 and FAD7 were searched in the Peanut Genome Database (PeanutBase, https: / / www.peanutbase.org / ). Specific primers were designed using Primer Premier 5 software based on the retrieved sequences. The target gene was amplified by PCR using the cDNA from 1.2.1 as a template. LA Taq™ DNA polymerase (TaKaRa) was used in a 25-μL PCR system: 2.5 μL 10× PCR buffer (containing MgCl2); 2.5 μL 10 mM dNTPs; 1 μL cDNA template; 0.5 μL LA polymerase; and 17.5 μL ddH2O. PCR reaction conditions were: (a) 94°C for 5 min; (b) 94°C for 45 s; 55°C for 45 s; 72°C for 90 s, for a total of 35 cycles; and (c) 72°C for 10 min. The PCR products were separated by 1% agarose gel electrophoresis and purified using a gel extraction kit (Axygen). The purified products were ligated into pMD18-T Easy vector (Takara) and sequenced (Sangon, Shanghai).

[0054] 1.2.3 Construction of multigene plant expression vectors

[0055] The vector was constructed by Shaanxi Jiyinjia Biotechnology Co., Ltd. The four ω-3FAD genes were synthesized into a large fragment by tandem gene sequencing. The EGFP gene on the plant expression vector pCAMBIA2300 was cut out and the synthesized large fragment was ligated into it. This position provides the first promoter and the last terminator for the tandem structure, resulting in a recombinant plasmid. The order of the four ω-3FAD genes is AhFAD3a, AhFAD3c, AhFAD7a, and AhFAD7d, and it is labeled pFAD3 / 7 ( Figure 2 ).

[0056] The backbone map of the plant expression vector pCAMBIA2300 is shown in Figure 1 The design framework of the four ω-3FAD synthesis genes is as follows:

[0057] CaMV 35S promoter-AhFAD3a-NOS terminator;

[0058] -MAS promoter-AhFAD3c-CaMV poly(A)signal;

[0059] -NOS promoter-AhFAD7a-CaMV poly(A)signal;

[0060] -CaMV 35S promoter-AhFAD7d-E9 terminator.

[0061] 1.2.4 Arabidopsis genetic transformation

[0062] The multi-gene plant overexpression vector was introduced into Agrobacterium strain GV3101 using the freeze-thaw method. After transformation, single clones were picked for PCR verification, and positive recombinant Agrobacterium single colonies were selected and preserved. Arabidopsis thaliana was transformed using the Floral-dip method.

[15] Arabidopsis inflorescences are immersed in a positive recombinant Agrobacterium suspension for 20–30 seconds. The infected plants are then bagged and placed horizontally for incubation in a highly humid environment, protected from light, for 18–24 hours. Thereafter, they are incubated under normal light conditions. Seeds are collected after natural maturation. The collected T0 generation seeds are seeded on MS medium containing kanamycin for kanamycin resistance screening. Positive plants are transferred to nutrient soil for further incubation. After maturity, T1 generation seeds are collected.

[0063] 1.2.5 Analysis of FAD3 and FAD7 gene expression patterns

[0064] From the cultivated allotetraploid peanut genome database PeanutBase ( https: / / peanutbase.org / home ) to obtain the full-growing-stage transcriptional data of FAD3 and FAD7, and then a heat map was made in TB tools based on the obtained data.

[0065] 1.2.6 Subcellular localization analysis

[0066] The pAN580 plasmid vector was cut with restriction endonucleases, and the two amplified genes were ligated into pAN580 (P 35S ::MCS::GFP) plasmid vector. Constitute the fusion expression vector: P 35S ::AhFAD3a::GFP, P 35S ::AhFAD3c::GFP, P 35S ::AhFAD7a::GFP and P 35S ::AhFAD7d::GFP, with the empty vector pAN580 as the control group. The fusion expression vector was introduced into Arabidopsis protoplasts using PEG, and the green fluorescence signal was observed under a confocal microscope.

[0067] 1.2.7 Determination of fatty acid content in Arabidopsis seeds

[0068] The absolute content of fatty acids in Arabidopsis seeds was determined by gas chromatography. The specific method was referred to Chen Silong et al.

[16] .

[0069] 1.2.8 Transcriptome Sequencing Analysis

[0070] Wild-type Arabidopsis Columbia seeds and transgenic plant seeds were collected 13 days after flowering and quick-frozen in liquid nitrogen, with three biological replicates set for each treatment. They were sent to Qingdao Biomark Biotechnology Co., Ltd. for total RNA extraction and cDNA library construction. After the library passed the inspection, it was sequenced on the Illumina HiSeq platform. After the transcriptome sequencing was completed, the raw sequencing data was filtered to ensure the accuracy of subsequent analysis. First, reads containing adapters and low-quality reads were removed (including removing reads with an N ratio greater than 10%; removing reads with a quality value Q≤10 that accounted for more than 50% of the entire read). Finally, clean reads were obtained. Clean reads were aligned with the reference genome sequence (Arabidopsis_thaliana.Araport11_JGI.genome.fa). Based on the genomic files and reads mapped to genomic locations, the number of reads per gene (i.e., expression level) was counted. Differentially expressed genes were then screened using DESeq2 (1.20.0) differential analysis software. Differentially expressed genes between samples were selected based on a fold change (|log2(fold change)|>1) and a significance level (P-value < 0.05). The overall distribution of differentially expressed genes was visualized using a volcano plot. Finally, functional annotation and GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment tests were performed on the differentially expressed genes using the Uniprot and KEGG databases, respectively, to obtain GO and KEGG enrichment sets for the differentially expressed genes.

[0071] 2 Results and Analysis

[0072] 2.1 Analysis of ω-3FAD gene expression patterns

[0073] In order to analyze the expression pattern of ω-3FAD gene in different tissues of peanut, the

[17] The transcriptional expression profiles of AhFAD3s and AhFAD7s were obtained from the seed. The results showed that AhFAD3a (Arahy.0JDQ22) was highly expressed in developing embryos (seed Pat.5, 6, 7), roots, pistils, stamens and fruit (fruit Pat.1), AhFAD3b (Arahy.40PHQK) was highly expressed in developing embryos (seed Pat.5, 6), roots, capsules, pistils, stamens and fruit, while AhFAD3c (Arahy.ZDHF3I) and AhFAD3d (Arahy.BC0JZ1) were highly expressed in stamens, fruit stalk tips and developing embryos (seed Pat.5, 6). AhFAD7a (Arahy.722ASC) and AhFAD7b (Arahy.WLZ7Z3) were highly expressed in leaves, pistils and fruit stalk tips. 1), while AhFAD7c (Arahy.9ET73H) and AhFAD7d (Arahy.1R706V) were highly expressed in roots, nodules, capsules, pistils, stamens, stalk tips, fruits, and pericarps ( Figure 3 These genes in peanut may be related to seed formation, development, and the synthesis and metabolism of fatty acids.

[0074] 2.2 Subcellular localization of the ω-3FAD gene

[0075] P 35S ::AhFAD3a::GFP, P 35S ::AhFAD3c::GFP, P 35S ::AhFAD7a::GFP and P 35S ::AhFAD7d::GFP were respectively combined with the nuclear localization marker (P 35S ::NLS::mCherry) were co-transformed into wild-type Arabidopsis protoplasts. The distribution of fluorescent protein was then examined using a laser confocal scanning microscope. The results are shown in the figure ( Figure 4 ) : The green fluorescent protein in the empty vector control group is distributed throughout the cell; the red fluorescence of the endoplasmic reticulum-localized marker ER::mCherry overlaps with the green fluorescence of the fusion proteins AhFAD3a::GFP and AhFAD3c::GFP, indicating that AhFAD3a and AhFAD3c are localized to the endoplasmic reticulum. The green fluorescence of the proteins AhFAD7a::GFP and AhFAD7d::GFP overlaps with the red fluorescence of the chloroplasts, indicating that the proteins AhFAD7a and AhFAD7d are localized to the chloroplasts.

[0076] 2.3 Analysis of fatty acid content in overexpressed Arabidopsis seeds

[0077] The vector fused with the ω-3 FAD gene was transformed into Arabidopsis thaliana, and a total of three overexpression strains were obtained: FAD-OX#1, FAD-OX#2, and FAD-OX#3. The absolute contents of different fatty acid components were significantly different between the overexpression strains and the wild type. The palmitic acid (C16:0) contents of the three overexpression strains were significantly higher than that of the wild type, with an increase of 4.97%-8.02%. There was no significant difference in stearic acid (C18:0) between the three overexpression strains and the wild type. The C18:0 content of FAD-OX#2 was higher than that of the wild type, the C18:0 content of FAD-OX#3 was lower than that of the wild type, and the content of FAD-OX#1 was similar to that of the wild type. The oleic acid (C18:1) contents of FAD-OX#1, FAD-OX#2, and FAD-OX#3 were significantly higher than that of the wild type, with increases of 7.20%, 7.71%, and 3.05%, respectively. The linoleic acid (C18:2) contents of the three overexpression strains were all higher than that of the wild type, with an increase of 6. .14%-6.87%; the linolenic acid (C18:3) contents of the three overexpression lines were also higher than that of the wild type, increasing by 4.51%, 14.29% and 2.16%, respectively, among which the C18:3 content differences of FAD-OX#1 and FAD-OX#2 reached an extremely significant level; the arachidic acid (C20:0) contents of the three overexpression lines were extremely significantly higher than that of the wild type, increasing by 4.64%, 11.30% and 9.06%, respectively; the arachidonic acid (C20:1) contents of FAD-OX#1, FAD-OX#2 and FAD-OX#3 were significantly higher than that of the wild type, increasing by 5.99%, 9.42% and 4.89%, respectively; the arachidienoic acid (C20:2) contents of the three overexpression lines were significantly higher than that of the wild type, with increases ranging from 4.95% to 7.11%. The contents of behenic acid (C22:0) in the three overexpression lines were all higher than that in the wild type; there was no significant difference in erucic acid (C22:1) between the three overexpression lines and the wild type. Except for FAD-OX#2, the C22:1 contents of the other two lines were lower than that of the wild type. The total fatty acid contents of the three overexpression lines were significantly higher than those in the wild type, with increases of 5.47%, 9.00% and 4.50% respectively, indicating that FAD3 and FAD7 have a promoting effect on the accumulation of fatty acids. In addition, the C18 content, C20 content, unsaturated fatty acid content and C18 / C20 content of FAD-OX#1, FAD-OX#2 and FAD-OX#3 were significantly or extremely significantly higher than those in the wild type ( Figure 5 ).

[0078] 2.4 Quality analysis of transcriptome library sequencing results

[0079] To explore the molecular mechanism of ω-3FAD gene regulation of fatty acid biosynthesis and plant growth and development, high-throughput transcriptome sequencing was performed on seeds of overexpressing Arabidopsis thaliana lines and wild-type plants. Raw data (Raw reads) were filtered to remove low-quality reads, reads with adapter contamination, and reads with unknown base N content greater than 5%, and 6 filtered reads of approximately 25×10 6 The GC content of clean reads was 46.26% to 46.79%, the percentage of Q20 bases was as high as 96.95% and above, and the percentage of Q30 bases was 92.18% and above (Table 1).

[0080] Table 1 Sequencing data statistics

[0081]

[0082]

[0083] 2.5 Analysis of differentially expressed genes in the transcriptomes of overexpression lines and wild-type seeds

[0084] Based on the transcriptome sequencing results, significantly differentially expressed genes were screened and 1291 genes were identified that were differentially expressed between the overexpression strains and wild-type seeds (fold increase ≥ 1.5, FDR < 0.05). Among them, 678 genes were up-regulated and 613 genes were down-regulated in the seeds of the overexpression strains ( Figure 6 In order to screen genes involved in fatty acid biosynthesis pathway and regulating plant growth and development, gene function annotation analysis (GO) was performed on the differentially expressed genes in the seeds of the overexpression lines. The results showed that the differentially expressed genes were mainly involved in three categories: biological process, cellular component and molecular function. Figure 7 In terms of biological processes, metabolic processes, single-organism processes, and cellular processes were significantly enriched in transgenic line seeds. In terms of molecular function, differentially expressed genes were primarily enriched in catalytic activity and binding. In terms of cellular components, upregulated genes were significantly enriched in membranes and membrane components, while downregulated genes were more enriched in cells and cellular components.

[0085] 2.6 KEGG enrichment analysis of differentially expressed genes

[0086] According to the KEGG database, this example performed enrichment analysis on differentially expressed genes and found that they were enriched in 25 pathways ( Figure 8 ). Among them, the differentially expressed genes were mostly enriched in metabolic classification pathways, accounting for 48.23%, and the least enriched in cellular processes, accounting for only 2.09%; the differentially expressed genes enriched in environmental information processing, gene information processing, and biological organism system pathways were 19.84%, 9.6%, and 10.65%, respectively. The up-regulated genes were most enriched in plant-pathogen interaction, including 27 genes, and the down-regulated genes were mainly enriched in the plant hormone signal transduction pathway, including 26 genes. The pathway related to fatty acid metabolism is alpha-linolenic acid metabolism, which is enriched with 11 up-regulated genes and no down-regulated genes ( Figure 8 , Table 2). Among the 11 differentially expressed genes, 5 genes encode lipases (putative triglyceride lipase; phospholipase A1; 3-ketoacyl-CoA thiolase 2; triacylglycerol lipase; acyl-CoA oxidase 2), and the remaining 6 genes encode lipoxygenase, NADH oxidase family, jasmonic acid carboxyl methyltransferase, alpha-dioxygenase 1, acetyl CoA:(Z)-3-hexen-1-ol acetyltransferase, and alleneoxide cyclase 3, respectively.

[0087] Table 2 Differentially expressed genes in fatty acid metabolism pathway

[0088]

[0089]

[0090] 2.7 Screening of genes related to fatty acid metabolism

[0091] Based on the annotation results of differentially expressed genes, a total of 15 key genes involved in fatty acid synthesis and degradation were identified (Table 3). Stearoyl ACP desaturase (FAB2), a key enzyme catalyzing the conversion of stearic acid to oleic acid, was upregulated in transgenic seeds (AT1G43800), with expression levels 1.65-fold higher than in the wild type. 3-Ketoacyl-CoA synthase (KCS), a key gene regulating the biosynthesis of long-chain fatty acids, was identified. Two KCS genes (AT1G07720 and AT2G46720) were downregulated, with expression levels of 0.64- and 0.51-fold, respectively. The GNS1 / SUR4 membrane protein family, involved in the elongation of very long chain fatty acids, was upregulated in transgenic seeds (AT1G75000), with expression levels 2.29-fold higher than in the wild type. Two fatty acid reductases (AT3G44540 and AT5G22500) catalyze the production of fatty alcohols, and their expression was upregulated. The expression levels in the transgenic strain seeds were 2.81 times and 1.74 times higher than those in the wild type, respectively.

[0092] Table 3 Fatty acid metabolism related genes

[0093]

[0094]

[0095]

[0096] 3 Discussions

[0097] ALA is a ω-3 polyunsaturated fatty acid, which is a beneficial fatty acid. Because it can only be synthesized in plants, humans can only consume it from plant-derived foods.

[18] It has many functions, including promoting human development, anti-diabetes, regulating cardiovascular and cerebrovascular, anti-inflammatory, controlling blood lipids and promoting fat metabolism regulation.

[19] . However, the ALA content of plants in nature is generally low, and our common bulk vegetable oils also have this problem, with very low ALA content, accounting for less than 1%. Therefore, increasing the content of nutrients such as ALA in seeds has gradually become one of the research hotspots for improving the quality of oilseed vegetable oils. In this study, four ω-3 fatty acid desaturase genes AhFAD3a, AhFAD3c, AhFAD7a and AhFAD7d were cloned from peanut leaves. Expression pattern analysis showed that AhFAD3s was highly expressed in the early stage of seed production, indicating that AhFAD3s made an important contribution to the accumulation of ALA in seeds, while AhFAD7s was highly expressed in the peel, indicating that AhFAD7s was mainly involved in the formation of linolenic acid in the peel, which is consistent with Hernández et al.

[20] The subcellular localization showed that AhFAD3s was in the endoplasmic reticulum and AhFAD7s was in the chloroplast, which is consistent with the existing conclusions.[21,22] .

[0098] In higher plants, the most common unsaturated fatty acids are three 18-carbon (C18) species: 18:1 (oleic acid), 18:2 (linoleic acid) and 18:3 (α-linolenic acid). By overexpressing AhFAD3s and AhFAD7s in Arabidopsis, it was found that the ALA (C18:3) content of transgenic plants was significantly increased, indicating that AhFAD3 and AhFAD7 play an important role in the accumulation of ALA. Kadama et al. [9] The chloroplast ω-3 fatty acid desaturase gene was introduced into tobacco, and the content of hexadecatrienoic acid and octadecatrienoic acid in transgenic tobacco was significantly increased; Wakita et al.

[23] When NtFAD3 was introduced into sweet potato, the C18:3 content of the transgenic plants increased; the above results are consistent with our current research results. At the same time, the expression of ω-3 fatty acid desaturase genes is regulated by plant hormones and the environment. For example, after rapeseed embryos were treated with ABA, the endoplasmic reticulum ω-3 fatty acid desaturase transcripts accumulated.

[24] Auxin treatment of mung bean increased the transcript level of endoplasmic reticulum ω-3 fatty acid desaturase

[25] The expression of maize ZmFAD7 is affected by temperature and salt concentration.

[26] The expression of the Arabidopsis chloroplast ω-3 fatty acid desaturase gene FAD7 is induced by light and damage

[27] ; KIRSCH switched wheat from darkness to light and the expression of plastid ω-3 fatty acid desaturase transcripts increased rapidly

[28] Hajiahmadi et al.

[29] reported that cold stress significantly increased the expression levels of TaFAD3.4, TaFAD3.5, and TaFAD3.6, while the expression level of ShFAD3.1 was significantly increased under drought and heat stress.

[30] Zoong et al.

[31] reported that heat stress impairs the heat tolerance of peanuts by reducing the content of 18:3 fatty acids and lowering lipid unsaturation levels.

[32] Changes in the C18:3 content of maize leaf fatty acid composition are closely related to drought resistance. In addition, increasing molecular evidence also shows that heterologous expression of ω-3AhFAD3 isolated from peanut in Arabidopsis can improve survival under salt stress.

[33] .

[0099] The de novo fatty acid synthesis pathway in plastids is the basis for the accumulation of α-linolenic acid. It has been confirmed in many plants that the expression of FAD3 can affect the accumulation of α-linolenic acid. For the first time, FAD3 was found to be responsible for the synthesis of α-linolenic acid in the Arabidopsis fad3 mutant.

[21] . Then in rice

[34] ,Sesame

[35] ,flax

[36] The role of FAD3 has been demonstrated in plants such as . Similarly, the FAD7 gene was first identified in Arabidopsis thaliana. Arabidopsis transgenic experiments have shown that overexpression of the FAD7 gene can increase the linolenic acid content in Arabidopsis thaliana.

[37] We co-transformed AhFAD3a, AhFAD3c, AhFAD7a, and AhFAD7d into Arabidopsis thaliana. The contents of different fatty acid components in the seeds of the transgenic positive lines were generally increased compared with those in the wild-type seeds. This may be due to the up-regulation or down-regulation of genes involved in fatty acid metabolism in the transgenic line seeds. KEGG enrichment analysis found that 11 up-regulated genes were enriched in the α-linoleic acid metabolic pathway ( Figure 8 ), catalyzing linoleic acid to linolenic acid, causing the accumulation of linolenic acid in the seeds of transgenic positive strains, thereby promoting the synthesis of long-chain fatty acids. Studies have shown that FAB2 plays a role in improving the fatty acid composition of oilseed crops. Compared with the wild type, the content of unsaturated fatty acids in tobacco plants with the FAB2 gene is increased.

[38] The GNS1 / SUR4 membrane protein family is a condensing enzyme that synthesizes long-chain fatty acids (VLCFAs), which are the main fatty acids in plant sphingolipids.

[39] . This study screened a total of 15 key genes for fatty acid synthesis and degradation, of which 6 differentially expressed genes were enriched in the cutin, suberin and wax synthesis pathways, 4 differentially expressed genes were involved in the extension and elongation of fatty acids, and 4 differentially expressed genes were related to fatty acid degradation. The above results show that fatty acid metabolism is a complex process regulated by many genes. The research results can provide a theoretical basis for the study of the metabolic mechanism of plant fatty acids and provide new genetic resources for peanut quality breeding, but the complex regulatory network and function of related genes need further verification.

[0100] 4 Conclusion

[0101] This study cloned four ω-3 fatty acid desaturases from peanut and co-transformed them into Arabidopsis thaliana. Subcellular localization analysis revealed that AhFAD3a and AhFAD3c were localized to the endoplasmic reticulum, while AhFAD7a and AhFAD7d were localized to the chloroplasts. GO annotation and KEGG enrichment revealed that differentially expressed genes were enriched in biological metabolic processes such as lipid metabolism, amino acid metabolism, and carbohydrate metabolism. Eleven upregulated genes were enriched in the α-linoleic acid metabolic pathway. A total of 15 differentially expressed genes were identified that were involved in fatty acid metabolism. This study provides a theoretical basis for the molecular mechanisms of peanut fatty acid metabolism and offers a new gene resource for peanut quality breeding.

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[0151] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A peanut ω-3 fatty acid desaturase gene combination, characterized in that: Including ω-3 fatty acid dehydrogenase gene AhFAD3a, ω-3 fatty acid dehydrogenase gene AhFAD3c, ω-3 fatty acid dehydrogenase gene AhFAD7a and ω-3 fatty acid dehydrogenase gene AhFAD7d.

2. The peanut ω-3 fatty acid desaturase gene combination according to claim 1, characterized in that The nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD3a is shown in SEQ ID No. 1; The nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD3c is shown in SEQ ID No. 3; The nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD7a is shown in SEQ ID No. 5; The nucleotide sequence of the ω-3 fatty acid dehydrogenase gene AhFAD7d is shown in SEQ ID No.

7.

3. Use of the peanut ω-3 fatty acid desaturase gene combination according to claim 1 or 2 in increasing the fatty acid content of peanuts.

4. The use according to claim 3, characterized in that The peanut fatty acid includes one or more of palmitic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidic acid, arachidic acid, arachidienoic acid and behenic acid.

5. Use of the peanut ω-3 fatty acid desaturase gene combination according to claim 1 or 2 in regulating genes related to peanut fatty acid metabolism.

6. The use according to claim 5, characterized in that The expression of aldehyde dehydrogenase gene, plant stearoyl-acyl-carrier protein desaturase gene, GNS1 / SUR4 membrane protein family gene, acetyl-CoA acyltransferase 1 gene, peroxidase gene, acyl-CoA dehydrogenase gene, alcohol-forming fatty acid acyl-CoA reductase gene, fatty acid ω-hydroxylase gene, alcohol-forming fatty acid acyl-CoA reductase gene, aldehyde decarbonylase gene and acyl-CoA oxidase gene in peanut was upregulated.

7. The use according to claim 5, characterized in that Down-regulate the expression of aldehyde decarbonylase genes, 3-ketoacyl-CoA synthetase genes and palmitoyl-protein thioesterase genes in peanut.

8. A plant overexpression vector, characterized in that: The peanut ω-3 fatty acid desaturase gene combination according to claim 1 or 2 is connected to the plant expression vector pCAMBIA2300 to obtain a plant overexpression vector.

9. The plant overexpression vector according to claim 8, characterized in that The ω-3 fatty acid dehydrogenase gene AhFAD3a, ω-3 fatty acid dehydrogenase gene AhFAD3c, ω-3 fatty acid dehydrogenase gene AhFAD7a and ω-3 fatty acid dehydrogenase gene AhFAD7d in the peanut ω-3 fatty acid dehydrogenase gene combination are sequentially connected in series to obtain a gene synthesis fragment, and the gene synthesis fragment is connected to the plant expression vector pCAMBIA2300; The plant expression vector pCAMBIA2300 is ligated after the EGFP gene is cut out.

10. A method for obtaining Arabidopsis plants with high fatty acid content, characterized in that: The following steps are involved: 1) introducing the plant overexpression vector according to claim 8 or 9 into Agrobacterium strain GV3101 to obtain a transformed bacterium; 2) Infecting Arabidopsis inflorescences with the transformed bacteria obtained in step 1) to obtain Arabidopsis plants with high fatty acid content.

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