A fatty acid desaturase gene from olea europaea and its use in improving cold resistance of plants

Abstract

The present application relates to the field of plant molecular biology and genetic breeding technology, and belongs to the field of plant fatty acid desaturase gene function utilization and adversity resistance improvement, and more particularly to a fatty acid desaturase gene from Olea europaea and application thereof in improving plant low temperature resistance, wherein the nucleotide coding sequence of the gene is shown as SEQ ID NO:1, and the coding protein amino acid sequence is shown as SEQ ID NO:2, the plant expression vector, plasmid and agrobacterium competent cell containing the fatty acid desaturase gene are constructed, and are introduced into plants such as Arabidopsis thaliana and Brassica, so that the expression amount of the fatty acid desaturase gene in the leaves of the transgenic plants is significantly improved. The low temperature treatment test shows that after the overexpression plants of the fatty acid desaturase gene are stressed at 4 DEG C and then recovered, the survival rate is obviously improved, the leaf yellowing rate and membrane damage degree are reduced, the proportion of unsaturated fatty acids is increased, and the low temperature resistance of the plants is significantly improved.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biology and genetic breeding technology, specifically to the functional utilization of plant fatty acid desaturase genes and the improvement of stress resistance, and more specifically, to a fatty acid desaturase gene derived from olive and its application in improving plant low-temperature resistance. Background Technology

[0002] Plants are frequently subjected to low-temperature stress in their natural environment. Low temperatures cause cell membrane lipids to transition from a liquid crystal state to a gel state, leading to membrane lipid "phase separation." This disrupts the selective permeability and integrity of the membrane, forming a crucial physiological basis for low-temperature damage and frost injury in plants. Numerous studies have shown that the degree of unsaturation of fatty acids in membrane lipids (especially the proportion of polyunsaturated fatty acids such as linoleic acid and α-linolenic acid) is one of the key factors determining the membrane lipid phase transition temperature and thus influencing plant low-temperature resistance. Fatty acid desaturases (FADs) are rate-limiting enzymes that catalyze the introduction of double bonds at specific sites in fatty acids, regulating the degree of fatty acid unsaturation. Previous reviews have summarized plant FADs into various types, including the Δ9, Δ12, and Δ15 desaturase families located in chloroplasts or endoplasmic reticulum. It is generally believed that altering FAD gene expression can significantly change membrane lipid composition and improve plant cold tolerance.

[0003] Against this backdrop, several patents have attempted to utilize FAD genes to improve the low-temperature resistance and fatty acid composition of plants. Chinese patent CN1142853A discloses a gene encoding a fatty acid desaturase with Δ9 desaturation activity, derived from microorganisms such as cyanobacteria, and systematically describes methods for using vectors containing this gene, transforming plant cells, and regenerating plants. This patent explicitly proposes that by increasing Δ9 desaturase activity, the content of saturated fatty acids in plant membrane lipids can be reduced, while the proportion of unsaturated fatty acids can be increased, thereby endowing plants with higher cold resistance. It also provides examples of introducing this gene into plants such as tobacco, where the plants exhibited strong cold resistance after low-temperature treatment, verifying the feasibility of improving plant cold resistance through exogenous Δ9 fatty acid desaturase. However, the genes in this patent are mainly derived from microorganisms such as cyanobacteria, focusing on the general construction and application of Δ9 desaturase.

[0004] Chinese patent CN100510083C discloses an ω-3 fatty acid desaturase gene and its expression vector in halophytes, as well as plant cells and plants transformed with this gene. This patent utilizes the ω-3FAD gene cloned from the halophyte *Suaeda salsa*, constructs a eukaryotic expression vector, and introduces it into plant cells, causing the gene to be overexpressed in the plant, thereby significantly increasing the content of ω-3 fatty acids such as α-linolenic acid in the leaves. Experiments show that transgenic plants overexpressing this gene have significantly improved cold tolerance, salt tolerance, and fungal resistance compared to the control, while antisense inhibition of gene expression can reduce α-linolenic acid content and improve the plant's tolerance to high temperatures. This patent, by mining the ω-3FAD gene from halophyte materials, mainly focuses on the regulation of polyunsaturated fatty acid content and stress tolerance, providing an important example of the application of fatty acid desaturase genes in plant stress resistance improvement.

[0005] Chinese patent CN104195169A discloses a method for constructing cold-resistant transgenic tobacco based on a fatty acid desaturase gene. The patent first amplifies the stearoyl-ACP desaturase gene (SAD) from spinach using RT-PCR, clones it into the plant expression vector pBI121, and constructs a recombinant vector after sequencing verification. The recombinant vector is then transformed into Agrobacterium, and tobacco explants are transformed using the leaf disc method. Kanamycin-resistant plants are screened and molecularly identified, ultimately obtaining a stably inherited transgenic tobacco line. By measuring the electrical conductivity and chlorophyll content of the transgenic tobacco under low-temperature stress conditions, the patent demonstrates that overexpression of the spinach SAD gene can significantly improve the cold resistance of tobacco.

[0006] Overall, existing patented technologies have isolated and applied various fatty acid desaturase genes from microorganisms, halophytes, and certain herbaceous crops to improve the low-temperature or frost resistance of plants by changing the unsaturation of membrane lipids. For example, Chinese patent CN1142853A introduces the Δ9 desaturase gene of blue-green algae to endow plants with cold and frost resistance, CN100510083C improves the α-linolenic acid content and cold and salt tolerance of plants through the ω-3FAD gene of halophytes, and CN104195169A uses the SAD gene of spinach to construct cold-resistant tobacco plants.

[0007] However, the existing technologies still have the following shortcomings: First, the FAD genes involved in the existing patents are mostly derived from materials such as cyanobacteria, halophytes, or spinach, and no genome-wide identification of the FAD family has been carried out for olive (Olea europaea L.), an important woody oilseed tree species. Second, the existing patents have not disclosed the specific nucleotide sequence of any FAD gene derived from olive and the amino acid sequence of its encoded protein, nor have they defined the range of homologous variants that have certain homology and maintain the same function, resulting in a gap in the utilization of olive FAD resources and intellectual property layout. Third, the existing technologies mainly focus on fatty acid composition regulation, and pay insufficient attention to the relationship between the subcellular localization of FAD genes (such as chloroplast localization) and low-temperature induced expression patterns. They have also not combined model plants and important crops (such as Arabidopsis thaliana, rapeseed, tomato, rice, wheat, and maize) to systematically evaluate the universality and stability of specific FAD genes in improving low-temperature resistance across species.

[0008] Therefore, it is urgent to identify and isolate a FAD gene in olive that has clear sequence information, is upregulated under low temperature stress, is located in chloroplasts, and can significantly improve plant low temperature resistance through overexpression. Furthermore, it is necessary to construct the corresponding expression vector, plasmid, and Agrobacterium competent cells, and establish a set of low temperature resistance improvement methods that can be used in a variety of plants to make up for the shortcomings of existing technologies in olive FAD gene resource development and plant cold resistance genetic improvement. Summary of the Invention

[0009] The technical objective of this invention is to identify and provide a fatty acid desaturase gene in olive trees that has a clear nucleotide and amino acid sequence, is significantly induced to express under low-temperature stress, and can effectively improve the plant's low-temperature resistance. The invention also aims to construct corresponding expression vectors, plasmids, and Agrobacterium competent cells, and establish a set of plant genetic transformation and low-temperature resistance improvement methods based on the fatty acid desaturase gene. This will be used to obtain transgenic plants that stably improve low-temperature resistance in various crops, thereby overcoming the shortcomings of existing technologies that lack olive-derived FAD genes and their application in cold-resistant breeding.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A type derived from olives ( Olea europaea L. The fatty acid desaturase gene, whose nucleotide coding sequence is as shown in SEQ ID NO:1;

[0012] Alternatively, a homologous variant sequence having at least 90% sequence homology with the nucleotide sequence shown and encoding an amino acid sequence that has the same function as the protein encoded by SEQ ID NO:1.

[0013] Furthermore, this application also provides a primer pair for amplifying the fatty acid desaturase gene shown in SEQ ID NO:1, wherein the primer pair includes an upstream primer and a downstream primer.

[0014] The nucleotide sequence of the upstream primer is as follows: ATGGCTTCGAAGCTGAGTCCAAATC;

[0015] The nucleotide sequence of the downstream primer is as follows: ATTTGACAGGGAAGTGAAGCTCTAA.

[0016] Furthermore, this application also provides the protein encoded by the fatty acid desaturase gene, the amino acid sequence of which is shown in SEQ ID NO:2.

[0017] Furthermore, this application also provides an expression vector containing the aforementioned fatty acid desaturase gene.

[0018] Furthermore, this application also provides a plasmid containing the aforementioned fatty acid desaturase gene.

[0019] Furthermore, this application also provides Agrobacterium competent cells containing the aforementioned fatty acid desaturase gene.

[0020] Furthermore, this application also provides the application of the fatty acid desaturase gene, the protein, the expression vector, the plasmid, or the Agrobacterium competent cells in improving plant low-temperature resistance.

[0021] As a preferred plant, Arabidopsis thaliana ( Arabidopsis thaliana ), Brassicaceae rapeseed ( Brassica napus ),tomato( Solanum lycopersicum ), rice Oryza sativa ),wheat( Triticum aestivum ) or corn ( Zea mays One of them.

[0022] Furthermore, this application also provides a method for improving the low-temperature resistance of plants, comprising the following steps:

[0023] S1. Provide the aforementioned fatty acid desaturase gene;

[0024] S2. Construct a plant expression vector, in which the coding sequence of the fatty acid desaturase gene is sequentially linked to the 3' terminator under the control of the plant expressible promoter, to form a recombinant plant expression vector containing the fatty acid desaturase gene expression cassette and a selection marker gene.

[0025] S3. The recombinant plant expression vector is introduced into the cells of the plant to be improved, and transgenic plants containing exogenous fatty acid desaturase genes are obtained through antibiotic screening and tissue culture regeneration, so that the transcription level of fatty acid desaturase genes in the leaves of the plants is at least 2 times higher than that of the control plants.

[0026] Preferably, the transgenic plants are subjected to low-temperature treatment. Plants grown at 24℃ for 3–5 weeks are cooled to 4℃ at a rate not exceeding 2℃ / h, treated at 4℃ for 24–72 hours, and then restored to normal culture at 20–24℃ for 5–10 days. Phenotypic evaluation is performed on the degree of leaf water loss / freezing damage, leaf yellowing rate, survival rate, and bolting rate of the transgenic plants and the untransformed control plants. It is determined that the survival rate of the transgenic plants after treatment at 4℃ for 48 hours and restoration culture for 7 days is at least 30% higher than that of the control plants, and the proportion of bolting plants is higher than that of the control plants.

[0027] This invention clones and functionally identifies a fatty acid desaturase gene derived from olive, demonstrating that this gene is significantly upregulated under low-temperature stress and located at the site of chloroplast membrane lipid synthesis. Constructing this gene into a plant expression vector and introducing it into plant cells significantly increases the proportion of unsaturated fatty acids in the leaves of transgenic plants, thereby lowering the membrane lipid phase transition temperature and mitigating low-temperature-induced membrane permeability damage and cell dehydration. In model plants such as Arabidopsis thaliana, transgenic lines overexpressing the fatty acid desaturase gene, after treatment at 4℃ for 48 h and recovery culture for 7 days, showed significantly reduced leaf wilting and frost damage, significantly lower leaf yellowing rate, increased survival rate by at least 30%, and bolting rate and growth vigor superior to the wild-type control. This indicates that the fatty acid desaturase gene can stably and significantly enhance the physiological adaptability of plants under low-temperature stress. Furthermore, the fatty acid desaturase gene, encoded protein, expression vector, plasmid, and Agrobacterium competent cells provided by this invention can be applied to various types of crops such as Arabidopsis thaliana, rapeseed, tomato, rice, wheat, and corn, enabling cross-species promotion and application. This provides new molecular tools and technical approaches for cold-resistant breeding of crops in cold or subarctic regions and for cold-resistant production in facility agriculture. Attached Figure Description

[0028] Figure 1 This represents the expression patterns of candidate genes.

[0029] Figure 2 This is a subcellular localization map of candidate genes.

[0030] Figure 3 The low-temperature treatment phenotype of the overexpressing transgenic lines was observed. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0032] I. Obtaining and Sequence Characteristics of Fatty Acid Desaturase Genes

[0033] In one embodiment, by processing olives ( Olea europaea L. The FAD gene family was identified genome-wide and its expression was induced by low temperature, screening for candidate fatty acid desaturase genes that were significantly upregulated under low temperature stress. Preferably, total RNA was extracted from olive leaves treated with low temperature, and a cDNA library was obtained using conventional reverse transcription. Primers were designed based on the obtained FAD gene family sequence information, and the amplified target fragment was purified, cloned, and sequenced to confirm its coding sequence as shown in SEQ ID NO:1, and the corresponding amino acid sequence as shown in SEQ ID NO:2. The primer pair included an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer was as follows: ATGGCTTCGAAGCTGAGTCCAAATC; the nucleotide sequence of the downstream primer was as follows: ATTGACAGGGAAGTGAAGCTCTAA.

[0034] Sequence analysis showed that the full-length CDS of the fatty acid desaturase gene is approximately 1200 bp, encoding about 399 amino acids. It possesses highly conserved catalytic and iron coordination sites characteristic of stearoyl-ACP desaturase, confirming that this protein belongs to the soluble fatty acid desaturase family. Figure 1 As shown, transcriptional analysis of FAD family members under different treatments of olive showed that the relative expression of fatty acid desaturase genes was significantly upregulated under low temperature conditions (e.g., 4℃ stress) compared to normal temperature treatment, with an increase of approximately 4.95-fold. However, most other FAD members did not show significant changes, suggesting that fatty acid desaturase genes have typical low temperature stress-induced expression characteristics.

[0035] The nucleotide coding sequence of the fatty acid desaturase gene:

[0036]

[0037] The amino acid sequence of the protein encoded by the fatty acid desaturase gene:

[0038] MASKLSPNPINCRAQKYSSFALPQMASLRSPKFFMASTLRSNSKEGETLKKKPFSPHREVHGQVTHSMPPQKIEIFKSLENWAEQNLLVHLKPVEKSWQPQDFLPDPASDGFHDQVKELRERTKEIPDDYFIVLVGDMVTEEALPTYQTVLNTLDGVQDETGASLTPWAIWTRAWTAEENRHGDLLNKYMYLSGRVDMKQ IEKTIQYLIGSGMYPRTENSPYLGFIYTSFQERATFICHGNTARLAKEHGDIKLAQICGTIASDEKRHETAYAKIVEKLFEIDPDGTVLAFSDMMRKKISMPACLMYDGQDDGLFEHFSAVAQRLGVYTTRDYADILEFLVCRWKVADLTGLSAEGRKAQDYVCGLPLRIRRLEERAQGRAKQAPIIPFSWIFDREVKL.

[0039] II. Localization of Fatty Acid Desaturase Genes and Construction of Expression Vectors

[0040] To clarify the subcellular localization of the fatty acid desaturase gene, in one embodiment, the coding sequence shown in SEQ ID NO:1, after removing the stop codon, was fused with the coding sequence of green fluorescent protein (GFP) and constructed into a transient expression vector under the control of a strong plant promoter (e.g., the CaMV35S promoter). The vector structure, from 5' to 3', sequentially includes: a plant promoter, a fatty acid desaturase gene ORF (without a stop codon), a GFP coding sequence, and a 3' stop codon sequence, along with a plant selection marker gene and a bacterial replication element.

[0041] The obtained fusion expression vector was introduced into Arabidopsis mesophyll protoplasts or other suitable plant cells, transformed using a conventional transient expression system, and the GFP fluorescence signal was observed using a laser confocal microscope after a certain period of culture. Figure 2 As shown, the fatty acid desaturase gene-GFP signal highly overlaps with the auto-red fluorescence of chloroplasts, while free GFP is diffusely distributed in the cytoplasm, indicating that the fatty acid desaturase protein is located in chloroplasts. This localization characteristic is consistent with its function in chloroplast membrane lipid synthesis and low-temperature response.

[0042] In another embodiment, to achieve stable overexpression of the fatty acid desaturase gene in plants, the full-length coding sequence shown in SEQ ID NO:1 (with or without the stop codon) is cloned into a binary plant expression vector, such as... Figure 3 As shown, the recombinant expression vector p35S::fatty acid desaturase gene was obtained. This expression vector includes at least: a plant-expressible promoter (such as a 35S promoter), a fatty acid desaturase gene coding sequence, a 3' terminator sequence, a plant selection marker gene (such as a hygromycin or kanamycin resistance gene), the left and right boundaries of T-DNA, and bacterial replication and selection elements.

[0043] III. Preparation of Agrobacterium and transgenic plants containing fatty acid desaturase genes

[0044] In one embodiment, the recombinant expression vector p35S::fatty acid desaturase gene was introduced into Agrobacterium competent cells using conventional methods to obtain an Agrobacterium strain containing the fatty acid desaturase gene expression cassette, as a specific implementation method. The structure of the fatty acid desaturase utilization system can be summarized as follows:

[0045] 1) Fatty acid desaturase gene module: containing the coding sequence shown in SEQ ID NO:1 and its homologous variants;

[0046] 2) Plant expression vector module: carrying promoter, terminator, selection marker and T-DNA boundary;

[0047] 3) Agrobacterium-mediated transformation module: The recombinant vector enters Agrobacterium competent cells;

[0048] 4) Plant receptor module: Transgenic plants are obtained by Agrobacterium-mediated transformation using Arabidopsis thaliana, rapeseed, tomato, rice, wheat or corn as receptors.

[0049] Taking Arabidopsis thaliana Col-0 as an example, T1 generation transgenic plants were obtained using conventional Agrobacterium-mediated transformation. Positive lines integrating the fatty acid desaturase gene expression cassette were selected through resistance screening and PCR identification. Further self-pollination and segregation yielded genetically stable T3 generation homozygous transgenic lines, designated OE-1, OE-2, and OE-3, respectively. qRT-PCR analysis showed that, under normal temperature conditions, the transcriptional level of the fatty acid desaturase gene in these three transgenic lines was significantly higher than that in wild-type Col-0, with relative fold increases of approximately 5.2, 6.0, and 5.5, respectively. The expression level was further upregulated after low-temperature treatment, indicating successful overexpression of the fatty acid desaturase gene in the transgenic plants.

[0050] IV. Technical Approaches and Experimental Design for Improving Plant Low Temperature Resistance

[0051] The method for improving plant low-temperature resistance based on fatty acid desaturase genes in this invention can be summarized into steps S1 to S3, including:

[0052] S1: Provide the fatty acid desaturase gene and its encoded protein sequence, and complete sequence function prediction and subcellular localization verification;

[0053] S2: Construct a plant-expressible fatty acid desaturase gene expression vector and introduce it into Agrobacterium to form a transformation system carrying the fatty acid desaturase gene;

[0054] S3: Using Agrobacterium-mediated transformation of the plant to be improved, transgenic plants that stably express fatty acid desaturase genes were obtained, and their low-temperature resistance was systematically evaluated.

[0055] 1. Low-temperature stress treatment conditions

[0056] In one specific implementation, wild-type Col-0 and three T3 generation homozygous transgenic lines OE-1, OE-2, and OE-3 were simultaneously sown and cultured under suitable greenhouse or artificial climate chamber conditions. The plants were used as experimental materials when they reached approximately 4 weeks of age and had several true leaves. The low-temperature treatment followed these principles:

[0057] Control group: The entire process was maintained at room temperature (e.g., around 24°C);

[0058] Low temperature treatment group: After pre-culturing at room temperature, the temperature was slowly reduced to 4℃ at a rate not exceeding 2℃ / h, and then treated at 4℃ for 48h.

[0059] Recovery culture: After the low temperature treatment, the plants were restored to normal temperature conditions and cultured for another 7 days. The recovery and growth of the plants were observed and recorded.

[0060] The above treatment conditions can fully simulate the chilling or freezing damage processes that may occur in the natural environment.

[0061] 2. Phenotypic observation and physiological index measurement

[0062] To objectively evaluate the low-temperature resistance of transgenic plants, phenotypic and physiological indicators were measured before low-temperature treatment, 48 hours after treatment, and 7 days after recovery culture, including but not limited to:

[0063] Degree of leaf wilting and frost damage (grading score);

[0064] Leaf yellowing rate (the proportion of yellowed leaves to the total number of leaves).

[0065] Plant survival rate (the proportion of plants that can continue to grow out of the total number of plants).

[0066] Bolting rate (the proportion of plants that bolt within a specified time to the total number of plants);

[0067] Optionally, physiological and biochemical indicators such as relative conductivity, malondialdehyde (MDA) content, and unsaturated fatty acid ratio can also be measured.

[0068] V. Results and Data of Low Temperature Resistance Test

[0069] Taking wild-type Col-0 (WT) and three transgenic lines OE-1, OE-2, and OE-3 as examples, the typical results obtained under the above-mentioned low-temperature treatment scheme are as follows. All data are averages of multiple repeated measurements; fluctuations in specific values ​​do not affect the achievement of the technical effect of this invention.

[0070] 1. Survival rate and yellowing rate

[0071] After treatment at 4℃ for 48 hours and recovery culture for 7 days, the survival rate and leaf yellowing of plants in each treatment were recorded. The results are shown in Table 1.

[0072] Table 1. Survival rate and etiolation rate of Arabidopsis thaliana plants after low-temperature treatment

[0073]

[0074] As shown in Table 1, under the same low-temperature treatment conditions, the survival rates of the three fatty acid desaturase gene overexpression lines were significantly higher than those of the wild type, with the survival rate of OE-2 increasing by about 34.6 percentage points. Meanwhile, the leaf yellowing rate of the transgenic lines was significantly lower than that of the control, while the proportion of bolting plants was significantly higher than that of the control, indicating that the transgenic plants could still maintain good growth vigor and reproductive development capacity after experiencing low-temperature stress.

[0075] 2. Leaf damage index and relative conductivity

[0076] The degree of cell membrane damage can be further reflected by scoring the damage index (0-5 points, with higher scores indicating more severe damage) and measuring the relative conductivity of leaves after low-temperature treatment. The results are shown in Table 2:

[0077] Table 2 Leaf damage and relative conductivity after low-temperature treatment

[0078]

[0079] The results showed that the leaf damage index of the fatty acid desaturase gene overexpression lines was significantly lower than that of WT by 1.6 to 2.1 points, and the relative conductivity was reduced by about 20% to 27%, indicating that the transgenic leaves had better cell membrane integrity and lower permeability damage under low temperature stress.

[0080] 3. Changes in fatty acid composition

[0081] In one embodiment, the total fatty acids in the leaves of WT and OE-2 lines before and after low-temperature treatment were subjected to methyl esterification, and the results were analyzed using conventional gas chromatography. The changes in the main fatty acid composition are shown in Table 3 (data after 48 hours of low-temperature treatment).

[0082] Table 3. Fatty acid composition of leaves after low-temperature treatment (molar percentage)

[0083]

[0084] As can be seen, compared with WT, the proportion of saturated fatty acids (C16:0, C18:0) was significantly reduced in the fatty acid desaturase gene overexpression lines, while the proportions of linoleic acid (C18:2) and α-linolenic acid (C18:3) were significantly increased, resulting in an increase in the proportion of unsaturated fatty acids in total fatty acids from approximately 70.7% to approximately 80.0%. This result is consistent with the function of the fatty acid desaturase gene as a FAB2 / SAD type fatty acid desaturase, indicating that overexpression of this gene can increase the unsaturation of membrane lipids, thereby improving membrane lipid fluidity and stability under low-temperature conditions.

[0085] VI. Application in Other Crops In another embodiment, using Brassica rapa (a cruciferous vegetable) as the recipient plant, an Agrobacterium-mediated transformation process similar to that used in Arabidopsis thaliana was employed. The p35S:: fatty acid desaturase gene expression vector was introduced into rapeseed explants. Several rapeseed lines overexpressing the fatty acid desaturase gene were obtained through resistance screening, PCR, and qRT-PCR identification. Similar low-temperature treatment and recovery culture were then applied to these transgenic rapeseed plants. The results showed that the transgenic rapeseed plants exhibited significantly better performance than the control in terms of leaf frost damage, survival rate, and biomass. This indicates that the fatty acid desaturase gene of this invention and its low-temperature resistance improvement method have good cross-species applicability.

[0086] Similarly, the fatty acid desaturase gene, encoded protein, expression vector, and Agrobacterium competent cells provided by this invention can also be applied to crops such as tomatoes, rice, wheat, and corn through conventional genetic transformation pathways in the art to molecularly improve their low-temperature resistance. Those skilled in the art can flexibly adjust the specific implementation details according to the technical route disclosed in this invention, combined with conventional transformation systems for different crops, all of which fall within the protection scope of this invention.

[0087] In summary, as can be seen from the above embodiments, the olive-derived fatty acid desaturase gene provided by the present invention has clear sequence characteristics and chloroplast localization characteristics. Its overexpression can significantly improve the survival rate of plants under low temperature stress, reduce leaf damage, and achieve adaptation to low temperature environment by increasing membrane lipid unsaturation. This fully demonstrates that the technical effect of the present invention in improving plant low temperature resistance is significant and stable.

Claims

1. A type derived from olive oil ( Olea europaeaL. The fatty acid desaturase gene plays a role in improving fatty acid desaturase levels in Arabidopsis thaliana. Arabidopsis thaliana ) or cruciferous rapeseed ( Brassica napus The application of this gene in low-temperature resistance is shown in SEQ ID NO:

1.

2. The protein encoded by the fatty acid desaturase gene of claim 1 improves the fatty acid desaturase activity in Arabidopsis thaliana (…). Arabidopsis thaliana ) or cruciferous rapeseed ( Brassica napus Applications in low-temperature resistance; the amino acid sequence of this protein is shown in SEQ ID NO:

2.

3. An expression vector containing the fatty acid desaturase gene as described in claim 1 can improve the expression of Arabidopsis thaliana (…). Arabidopsis thaliana ) or cruciferous rapeseed ( Brassica napus Applications in low-temperature resistance.

4. Plasmids containing the fatty acid desaturase gene as described in claim 1 can improve the fatty acid desaturase activity in Arabidopsis thaliana (…). Arabidopsis thaliana ) or cruciferous rapeseed ( Brassica napus Applications in low-temperature resistance.

5. Agrobacterium competent cells containing the fatty acid desaturase gene as described in claim 1 improve the fatty acid desaturase activity of Arabidopsis thaliana (… Arabidopsis thaliana ) or cruciferous rapeseed ( Brassica napus Applications in low-temperature resistance.

6. A method to improve Arabidopsis thaliana ( Arabidopsis thaliana ) or cruciferous rapeseed ( Brassica napus The method for low-temperature resistance is characterized by, Includes the following steps: S1. Provide the fatty acid desaturase gene according to claim 1; S2. Construct a plant expression vector, so that the coding sequence of the fatty acid desaturase gene is sequentially linked to the 3' terminator under the control of the plant expressible promoter, to form a recombinant plant expression vector containing the fatty acid desaturase gene expression cassette and the selection marker gene. S3. The recombinant plant expression vector is introduced into the cells of the plant to be improved, and transgenic plants containing exogenous fatty acid desaturase genes are obtained through antibiotic screening and tissue culture regeneration, so that the transcription level of fatty acid desaturase genes in the leaves of the plants is at least 2 times higher than that of the control plants.

7. The method according to claim 6, characterized in that, The transgenic plants were subjected to low-temperature treatment. Plants grown at 24℃ for 3–5 weeks were cooled to 4℃ at a rate not exceeding 2℃ / h, and treated at 4℃ for 24–72 h. Then, they were restored to 20–24℃ and cultured normally for 5–10 days. Phenotypic evaluations were performed on the degree of leaf water loss / freezing damage, leaf yellowing rate, survival rate, and bolting rate of the transgenic plants and the untransformed control plants. It was determined that the survival rate of the transgenic plants treated at 4℃ for 48 h and then restored to culture for 7 days was at least 30% higher than that of the control plants, and the proportion of bolting plants was also higher than that of the control plants.

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