A gene for regulating plant oil accumulation and its application

Abstract

The present invention discloses a gene for regulating the accumulation of plant oil and fat and its application, belonging to the field of biotechnology. The present invention discloses a biomaterial capable of regulating the synthesis and accumulation of oil and fat in plants, the biomaterial comprising an RFS1 gene and an RFS1 protein; the nucleotide sequence of the RFS1 gene is shown in SEQ ID NO.2. The present invention verifies that the RFS1 gene and its encoded protein regulate the accumulation of plant oil and fat by affecting the de novo synthesis of fatty acids. Compared with the wild type, the RFS1 gene mutant of the present invention exhibits significant enhancement of fatty acid synthase activity, increased fatty acid synthesis rate, increased total fatty acid content and changes in fatty acid composition. Its level of regulation of fatty acid content is comparable to that of the CTI1 gene for which articles have been published and patents have been authorized.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a gene for regulating plant oil accumulation and an application thereof. Background Art

[0002] Vegetable oils, especially triglycerides, are valuable agricultural products in the food, fuel and petrochemical industries. Soybean oil, rapeseed oil, palm oil and peanut oil are the four most mainstream edible vegetable oils. With the rapid economic development and the steady progress of urbanization, the consumption of edible vegetable oils by residents has gradually increased. Due to the slow growth of oil production capacity, the gap between supply and demand continues to widen. Cultivating high-oil and high-quality oilseed crop varieties through traditional breeding and molecular breeding is a key link in overcoming the shortcomings in this agricultural field. Improving high-oil varieties through genetic engineering and directed gene modification can greatly shorten the breeding process. Exploring genes that regulate the synthesis and accumulation of plant oils as targets for directed molecular breeding can provide a gene resource library for molecular breeding.

[0003] Acyl lipids are the main components of plant oils and fats and play a vital role in plant growth and development, stress response, signal transduction and energy storage. Increasing the level of de novo synthesis of acyl lipids in plant cells is one of the most effective ways to increase plant oil accumulation. Acyl lipids are synthesized by de novo fatty acid synthesis (FAS) in plant plastids and then further assembled in the endoplasmic reticulum. The initial step of the de novo fatty acid synthesis process is catalyzed by acetyl-CoA carboxylase (ACCase), which converts acetyl-CoA into propionyl-CoA. In most plants, such as dicots and non-gramineous monocots, ACCase exists in the plastids as a heterolog (hetACCase). Plastidial hetACCase consists of a protein complex encoded by four different genes: biotin carboxylase (BC), biotin carboxylation carrier protein (BCCP), α-carboxyltransferase (α-CT) and β-carboxyltransferase (β-CT). As the most important key rate-limiting enzyme in fatty acid synthesis, ACCase has always been a hot research object in oil anabolism. ACCase activity and plant fatty acid synthesis are subject to complex and fine regulation. Mining the key regulatory genes that regulate ACCase activity can provide promising molecular targets for molecular breeding of high-oil crops. The regulation of hetACCase activity is achieved through multiple mechanisms, and its influencing factors include light intensity, downstream metabolites, interacting proteins, etc.

[0004] In addition to the above factors, another mechanism for regulating ACCase activity is the association of the CT subcomplex with the plastid membrane. Specifically, compared with the BC / BCCP subunit, the subcellular localization of the CT subunit is more closely associated with the plastid membrane. α-CT is speculated to interact with membrane proteins through its non-catalytic domain at the C-terminus and bind to the inner membrane of the plastid. Recent studies have found that a group of carboxyl transferase interacting proteins (CTIs) located on the inner membrane of the plastid anchor ACCase to the plastid membrane by directly interacting with the non-catalytic domain of α-CT. More importantly, this interaction reduces the activity of ACCase, thereby negatively regulating the oil content of plants. Studies have shown that the leaves of Arabidopsis cti mutants have increased ACCase activity, increased fatty acid synthesis rate, and increased oil content by 5-8 times compared with wild-type leaves. In view of the importance of CTIs genes in the regulation of plant oil synthesis, the gene has been granted an international patent, and was subsequently purchased by a US biotechnology company and is undergoing further transformation of results. On this basis, the present invention further explores other key genes that regulate oil synthesis. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a gene for regulating plant oil accumulation and its application. The present invention discloses that the RFS1 gene and its encoded protein regulate plant oil accumulation by affecting the de novo synthesis of fatty acids.

[0006] The present invention provides the following technical solutions:

[0007] Technical solution 1: A biological material that can regulate oil synthesis and accumulation in plants, the biological material includes RFS1 gene and RFS1 protein; the nucleotide sequence of the RFS1 gene is shown in SEQ ID NO.2; the amino acid sequence of the RFS1 protein is shown in SEQ ID NO.1.

[0008] Furthermore, the RFS1 protein is selected from at least one of the following:

[0009] (1) A protein whose sequence has one or more amino acids replaced, deleted or added and has the same or similar function as the RFS1 protein;

[0010] (2) a nucleic acid molecule encoding the RFS1 protein;

[0011] (3) a nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of the nucleic acid molecule described in (2) and which can encode a protein with the same or similar functions;

[0012] (4) A substance capable of regulating the level or activity of at least one of (1) to (3).

[0013] Furthermore, the nucleic acid molecule encoding the RFS1 protein includes genomic DNA, cDNA, recombinant DNA, mRNA or hnRNA encoding the RFS1 protein; or a nucleic acid molecule that is reverse complementary to the DNA, the cDNA, the recombinant DNA or the mRNA.

[0014] Furthermore, the nucleic acid molecule encoding the RFS1 protein includes the RFS1 gene, and the nucleotide sequence of the RFS1 gene is shown in SEQ ID NO.2.

[0015] Technical Solution 2: Application of the biomaterial in regulating oil synthesis in plants.

[0016] Technical solution three: Application of the biomaterial in regulating oil accumulation in plants.

[0017] Technical Solution 4: A method for increasing the oil content of plants, wherein the method is to knock out the RFS1 gene by gene editing to terminate the translation of the RFS1 protein prematurely and cause the loss of gene function, thereby increasing the oil content of plants.

[0018] Technical Solution 5: Application of gene knockout mutants in regulating oil synthesis and accumulation in plants, wherein the mutants are obtained by inserting a base A or G at position 181 from the start codon in the nucleotide sequence of the RFS1 gene.

[0019] Technical Solution 6: Application of a homozygous triple mutant in which RFS1 gene, RFS2 gene and CTI1 gene are all mutated in increasing plant oil accumulation, the nucleotide sequence of the RFS1 gene is shown in SEQ ID NO.2; the nucleotide sequence of the RFS2 gene is shown in SEQ ID NO.3.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention discloses that the RFS1 gene and its encoded protein regulate the accumulation of plant oils by affecting the de novo synthesis of fatty acids. Compared with the wild type, the RFS1 gene mutant exhibits significantly enhanced fatty acid synthase activity, increased fatty acid synthesis rate, increased total fatty acid content and changes in fatty acid composition. Its level of regulation of fatty acid content is comparable to that of the CTI1 gene, which has been published and patented. A triple mutant with simultaneous functional loss of RFS1, RFS1 homologous gene RFS2 and CTI1 gene can increase the increase in fatty acid content ( Figure 3-Figure 5 ).

[0022] The present invention screened out a gene AtRFS1 that can inhibit de novo fatty acid synthesis from the cruciferous plant Arabidopsis thaliana by co-expression analysis of isoacetyl-CoA carboxylase (hetACCase) and its known regulatory factors. The present invention successfully cloned the gene and constructed its deletion mutant by gene editing technology. The research results show that compared with the wild-type plant, the ACCase activity in the leaves of the RFS1 gene deletion mutant of the present invention is more than doubled, the fatty acid synthesis rate is nearly doubled, the total fatty acid content in the leaves is increased by nearly 16%, and the triglyceride accumulation level is significantly improved. In addition, the results of fatty acid composition analysis show that the fatty acid composition in the leaves of the RFS1 mutant has changed, among which the contents of C16:0, C16:1, C16:2, C18:0, C18:1 and C18:2 are increased, and the contents of C16:3 and C18:3 are decreased. The above RFS1 functional level related to improving oil accumulation is equivalent to the reported CTI1 gene, and the RFS1 gene function is more significant in terms of fatty acid synthesis rate and triglyceride accumulation level. A triple mutant with simultaneous functional loss of RFS1, RFS1 homologous gene RFS2 and CTI1 gene can promote fatty acid synthesis and increase oil accumulation to a greater extent. While the oil accumulation of the RFS1 gene mutant is improved, the biomass and other growth traits of the plant are not significantly affected, indicating that it will not affect other yield-related traits during application. RFS1 has homologous genes in oil crops, such as soybeans and rapeseed, and may have similar functions. Therefore, the present invention has application value in using genetic engineering methods to carry out molecular breeding to improve the oil level of higher plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 These are the mutant genotypes of four genes obtained by CRISPR / Cas9 gene editing;

[0025] Figure 2 The growth phenotypes of Arabidopsis plants of different genotypes 4 weeks after germination;

[0026] Figure 3 is the activity of ACCase in leaves of different genotypes of Arabidopsis thaliana;

[0027] Figure 4 is the content of total fatty acids in leaves of different genotypes of Arabidopsis thaliana;

[0028] Figure 5 The composition of fatty acids in leaves of different genotypes of Arabidopsis thaliana. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1

[0035] 1. Identification and cloning of the RFS1 gene

[0036] In order to explore unknown genes involved in regulating fatty acid synthesis, the present invention uses Arabidopsis as experimental material, key enzymes and regulatory genes for de novo fatty acid synthesis as target groups, conducts co-expression analysis, and finally identifies the RFS1 gene in the present invention. The protein of the RFS1 gene is a transmembrane protein with a predicted chloroplast transit peptide structure and two coiled-coil domains at the C-terminus, and the amino acid sequence of the RFS1 protein is shown in SEQ ID NO.1. The CDS sequence of the RFS1 gene was cloned using Arabidopsis Col-0 as experimental material, and its nucleotide sequence is shown in SEQ ID NO.2. By searching and comparing the Arabidopsis genome, the homologous gene RFS2 of the RFS1 gene was identified, and the nucleotide sequence of the CDS of the RFS2 gene is shown in SEQ ID NO.3.

[0037] Amino acid sequence of RFS1 protein:

[0038] MLFSPSKLQSPQSLISWNPILHNGFIDKNLSFRRRVTLLASPISRLTKGSDSIASSTPSALYSNPQEPSISSQLTSSVGQPPLQLSQWTFTQKHFVLLNVVACVTAISASWLFFAAIPTLLAFKKAAES LEKLLDVTREELPDTMAAVRLSGMEISDLTMELSDLGQGITQGVKSSTRAIRVAEDRLRRLTNMNPVASMQEVMRQTKTDETEPMLAKQARSFREGVVKGRSLWQLFSTITRFSKITTSYLAKRAKQ(SEQ ID NO.1);

[0039] Nucleotide sequence of the RFS1 gene CDS:

[0040] ATGTTATTCTCGCCTTCGAAATTGCAATCACCCCAATCTCTAATCTCTTGGAATCCGATTCTCCATAACGGATTCATCGATAAAAATCTCTCCTTTCGCCGACGGGTCACTCTCCGGCTAGCTTCACCGATTTCACGATTGACCAAGGGTTCAGACTCAATCGCATCTTCTACTCCCTCTGCATTGTACTCGAATCCTCAGGAGCCATCGATCTCTTCCCAATTAACATCGTCCGTGGGACAGCCTCCTCTCCAACTCTCTCAATGGACATTCACACAGAAGCACTTCGTCTTGCTCAATGTCGTCGCTTGCGTGACAGCAATTTCAGCATCGTGGCTCTTCTTTGCTGCGATTCCTACTCTTCTGGCTTTCAAGAAAGCAGCTGAGTCTCTTGAAAAGCTTTTGGATGTAACTAGAGAAGAGCTACCAGATACAATGGCTGCTGTTCGTTTATCGGGAATGGAGATCAGTGACTTAACTATGGAGCTCAGTGATTTAGGCCAAGGTATCACTCAAGGTGTTAAAAGCTCGACACGGGCTATTCGTGTAGCTGAAGATAGACTAAGACGGTTAACAAACATGAATCCAGTAGCTTCAATGCAGGAGGTGATGCGTCAAACCAAGACAGATGAAACAGAACCAATGTTGGCTAAACAAGCGAGGAGCTTTAGAGAAGGTGTTGTTAAGGGACGCTCTTTATGGCAATTGTTCTCCACAATCACTCGTTTCTCTAAAATCACTACAAGTTATCTTGCAAAGCGTGCTAAGCAGTAG(SEQ ID NO.2);

[0041] Nucleotide sequence of the CDS of the RFS2 gene:

[0042] ATGAAATCTCTCTGCTTCATTTCTACAGCTCCATTGATTCGTTCGCCTCCATTTCTCGATCTTTCTCCAACAAGCTTGAATCGATTTACCCTTAAAATTCCAGCTTCTCTCTATGTATCTCATCCAACAAGATGCAGCATTTCAAACCCGTCGAGCTCTGAGGAATTGTTGAATTCCAATGGTGGAATGTCTAGGGCATCGATTAGTGTCTTCGGAGGTACTTCCCTTAACAATCTCAAGATGCAAGTGGGCAGTCCAATTTCTCTGCATTCAATCAATCCATTAGCAAAGCTGAGTTTGAGTGATCAGGCATTTCTTCTCTTGGCCTTCATCGTTTGCACGACTTCAGTGGCTTTTACAAGTCTTGTGATTACAGCTATTCCTACACTTGTGGCCATGGGTAGAGCTGCAACTTCCTTTGCAAAGTTAGCTGACACAGCTCGCAAAGAACTCCCTAGTACATTGGCAGCGGTAAGGCTCTCTGGCATGGAAATAAGCGATCTTACTCTTGAATTGAGCGATTTAAGCCAAGATATAACAGATGGGATAAACAAATCAGCTAAAGCGGTTCAAGCAGCTGAAGCTGGAATCAAACAGATTGGAACACTTGCACAGCAGCAAACTCTCTCAATGATTGAAGAGAGAGCAAATTTACCAGAAATATCACTACAACCTGTTGTGGCTGGTGCAGCCGAAAAGACTTCTCACGCAATCGGCAGTGCAACCAAGAGACTAATGAACATTATAACCGGAGGAAACAAGGATGAAGATTAG(SEQ ID NO.3).

[0043] Example 2

[0044] 1. Construction of Arabidopsis thaliana RFS gene deletion mutant

[0045] 1.1 Construction of CRISPR / Cas9 vector

[0046] The online CRISPR-P 2.0 software was used to design sgRNAs for the sequences of the RFS1 gene (SEQ ID NO.2) and the RFS2 gene sequence (SEQ ID NO.3). The sequences of the target sites of the RFS1 and RFS2 genes were designed as follows: GGATTCGAGTACAATGCAGA GGG (SEQ ID NO. 4), AATGGAGGCGAACGAATCAA TGG (SEQ ID NO.5) (The underlined part is the targeting sequence, and the bold sequence is PAM). Based on the target site sequence, sgRNA primers for editing the RFS1 gene and RFS2 gene were synthesized respectively:

[0047] RFS1-sgRNA-F: 5'-GATTGGATTCGAGTACAATGCAGA-3' (SEQ ID NO. 6);

[0048] RFS1-sgRNA-R: 5'-AAACTCTGCATTGTACTCGAATCC-3' (SEQ ID NO.7);

[0049] RFS2-sgRNA-F: 5'-GATTAATGGAGGCGAACGAATCAA-3' (SEQ ID NO. 8);

[0050] RFS2-sgRNA-R: 5'-AAACTTGATTCGTTCGGCCTCCATT-3' (SEQ ID NO. 9).

[0051] The intermediate vector P1P2 of the Gateway system was digested with restriction endonuclease BbsI using the following digestion system: 5μL P1P2 vector, 3μL digestion buffer, 1.5μL BbsⅠ, Add ddH2O to 30μL. React at 37℃ for 30min, and gel-cut and recover DNA after electrophoresis. Phosphorylate and anneal the sgRNA primer pair according to the following reaction system: 1μL Primer F (100uM), 1μL Primer R (100uM), 0.5μL PNKbuffer, 0.5μL T4 DNAligasebuffer 0.5μL T4 PNK, Add ddH2O to 10μL. Perform annealing reaction in a PCR instrument with the following program: 37℃ 30min, 95℃ 5min, and then gradually cool to 25℃ at a rate of 5℃ / min.

[0052] The annealed oligonucleotide fragments were connected to the digested P1P2 vector using the following reaction system: XμL BbsIdigestedP1P2 vector (50ng), 1μL phosphorylated and annealed oligo primers (1:200 dilution), 1μL T4 DNA ligase buffer, 1μL T4 DNA ligase, Add ddH2O to 10μL. After reacting at room temperature for 30 minutes, E. coli DH5a was transformed and positive clones were identified by PCR. After extracting the plasmid, sequencing was performed to confirm that the vector construction was correct.

[0053] The pU6-RFS1sgRNA cassette in the intermediate vector P1P2 was cloned into the pYAO::Cas9 final vector site by LR reaction (for details of the pYAO::Cas9 vector, see Yan L, Wei S, Wu Y, Hu R, Li H, Yang W, Xie Q. High-Efficiency Genome Editing in Arabidopsis Using YAO Promoter-Driven CRISPR / Cas9 System. Mol Plant. 2015: 8(12): 1820-3. https: / / doi.org / 10.1016 / j.molp.2015.10.004): 1μL P1P2, 1μL YAO::Cas9, 0.5μL Gateway LR Clonase II EnzymeMix. After reacting at room temperature for 2 hours, the reaction system was transformed into Escherichia coli DH5a. Positive clones were identified by PCR. Plasmids were extracted and sequenced to confirm that the vector construction was correct.

[0054] 1.2. Acquisition, screening and identification of mutant Arabidopsis

[0055] The wild-type Arabidopsis Col-0 in full flowering period was transformed using the inflorescence infection method to obtain T1 generation transgenic Arabidopsis. The specific operations are as follows: The pYAO::Cas9-pU6::RFS1sgRNA and pYAO::Cas9-pU6::RFS2sgRNA vectors constructed above were respectively transformed into the Agrobacterium tumefaciens strain GV3101, and the positive clones were identified by colony PCR. Pick the positive single clone and culture it in 2mL of the corresponding resistance LB medium at 28°C overnight, then inoculate it into 200mL LB medium and shake it overnight. Centrifuge the bacterial solution at 5000g for 20min, then resuspend the bacteria in 120ml of infiltration solution (10% sucrose + 400μl / L Silwet-77, mix well before dipping the flowers, OD 600=0.8-1.0). At the same time, cut off the fruit pods and fully opened flowers on the plants. Immerse the above-ground parts of the plants in the bacterial solution for 90 seconds and shake gently. Cover the infected plants with plastic wrap to maintain humidity, culture in the dark for 1 day, and then place them in normal culture conditions. Continue to infect several times regularly. Continue to culture until the seeds are mature, collect the seeds and dry them at 37℃ for about 3 days, and then you can screen the transformants.

[0056] After sowing, the obtained seeds were screened on hygromycin-resistant 1 / 2MS plates and continued to be planted to harvest T2 generation seeds. DNA was extracted and the T2 generation plants were genotyped and mutants were identified using the designed identification PCR primers. The primers used were as follows:

[0057] RFS1-CF: 5'-CACCATGTTATTCTCGCCTTCGAAA-3' (SEQ ID NO. 10);

[0058] RFS1-CR: 5'-CTGCTTAGCACGCTTTGCAAG-3' (SEQ ID NO. 11).

[0059] RFS2-CF: 5'-CGGTATAATCCCGGTTTGCAG-3' (SEQ ID NO. 12);

[0060] RFS2-CR: 5'-TGCAAACGATGAAGGCCAAG-3' (SEQ ID NO. 13).

[0061] The following PCR reaction system and program were used: 2×Es Taq Master Mix 7.5μL, Primer F 0.75μL, Primer R 0.75μL, gDNA 1μL, ddH2O 5μL. PCR identification reaction program: initial denaturation, 94℃2min; denaturation, 94℃30s; annealing, 55℃30s; extension, 72℃30s (35 cycles); final extension, 72℃2min.

[0062] The purified PCR fragments were sent to Sangon Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared with the CDS sequences of the RFS1 gene and the RFS2 gene. The homozygous mutants rfs1-1, rfs1-2, rfs2-1, and rfs2-2 were identified and obtained. The mutation forms are shown in Figure 1 .like Figure 1As shown, in the mutants rfs1-1 and rfs1-2, a base A and a base G were inserted at the 181st position from the start codon of RFS1, respectively; in the mutants rfs2-1 and rfs2-2, a base A and a base G were inserted at the 37th position from the start codon of RFS2, respectively. The double mutants rfs1rfs2, RFS1, RFS2 of the RFS1 gene and its homologous gene RFS2, and the triple mutant rfs1rfs2cti1 of the CTI1 gene were constructed by conventional artificial hybridization methods (for details of the mutant cti1, see YeY, Nikovics K, To A, Lepiniec L, Fedosejevs ET, et al. Docking of acetyl-CoA carboxylase to the plastid envelope membrane attenuates fatty acid production in plants. Nat Commun. 2020: 11(1): 6191. https: / / doi.org / 10.1038 / s41467-020-20014-5). The specific operation steps are as follows: the homozygous rfs1 single mutant was selected as the female parent and rfs2 as the male parent. Before hybridization, remove the fruit pods and the petals and stamens of the mother plant that have grown and those that have bloomed or have not bloomed, and spread the pollen of the father plant on the stigma of the mother plant that has not been stained with pollen. The stigma hairs wilt 10 hours after pollination, and fruit pods are formed and continue to elongate after 3 days, indicating that the hybridization is successful. The obtained F1 generation of hybridization is harvested to obtain F2 generation seeds. After sowing, the genotype is identified by PCR to obtain homozygous double mutants. Using the same method, the double mutant rfs1rfs2 is used as the mother parent and cti1 is used as the father parent for hybridization. After identification in the F2 generation, a homozygous triple mutant is obtained. The situation of the above Arabidopsis mutant plants is as follows. Figure 2 The results showed that they grew and developed normally, and no obvious growth defect phenotype was found.

[0063] Example 3

[0064] 1. Analysis of ACCase activity in RFS1 gene deletion mutants

[0065] About 5 g of leaves from 4-week-old plants of wild-type Col-0, homozygous mutants rfs1-1, rfs1-2, and other related mutants were taken to extract chloroplasts. 300 μL of protein extraction buffer (50 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 1% (v / v) Triton X-100, 10% (v / v) glycerol and plant protease inhibitors) was added to each sample, incubated on ice for 10 min, centrifuged at 12.000 × g for 10 min, and the supernatant was collected and desalted using a spin column (ThermoFisher, catalog number 89882). The insoluble precipitate was resuspended with extraction buffer. 40 μL of protein extract was added to 10 μL of reaction buffer (100 mM Tricine pH 8.2, 100 mM KCl, 15 mM ATP, 5 mM MgCl2, 1 mM DTT, 2.5 mM acetyl-CoA, 50 μM halpyralid and 1 μCi 14 C-NaHCO3 (PerkinElmer; catalog number NEC 086H001MC). The reaction without acetyl-CoA was used as a control. After 30 minutes of reaction, 50 μL of 12N HCl was added to quench the reaction. The mixed solution was transferred to a scintillation vial with filter paper. The vial was heated at 80°C for 1 hour to evaporate the free 14 Then 1 mL of scintillation mixture was added to each vial, and the ACCase activity was quantitatively determined by liquid scintillation method.

[0066] The results show that ( Figure 3 ) Compared with wild-type Col-0, the ACCase activity in RFS1 mutants rfs1-1 and rfs1-2 increased by more than 1.5 times, which was comparable to that of cti1. The ACCase activity in its homologous gene RFS2 mutants (rfs2-1 and rfs2-2) also increased, but the increase was smaller than that of RFS1 mutants. The triple mutants rfs1-1 rfs2-1 cti1 showed stronger ACCase activity.

[0067] Example 4

[0068] Analysis of fatty acid content and composition in RFS1 gene deletion mutants

[0069] The fatty acid content and composition analysis methods refer to the methods reported in the previous literature (Li Y, Beisson F, Pollard M, Ohlrogge J. Oil content of Arabidopsis seeds: the influence of seed anatomy, light and plant-to-plant variation. Phytochemistry. 2006: 67 (9): 904-15. https: / / doi.org / 10.1016 / j.phytochem.2006.02.015). 10 mL glass test tubes were marked with serial numbers and weighed with a precision balance. Four-week-old plant leaves were placed in the corresponding test tubes and placed in a 65 ° C oven. After drying for 2-3 days, they were taken out and weighed again to calculate the weight of the dried leaves. 200 μL of toluene solution containing 20 μg 17:0 was added to the test tube as an internal standard, and then 1 mL of methanol solution containing 5% hydrochloric acid was added. The metal bath was heated at 85-90 ° C for 1.5 h. During the heating period, the liquid in the test tube was observed to see if it was boiling. After cooling to room temperature, add 0.2 mL of n-hexane, then add 1.5 mL of 0.9% NaCl solution, vortex the test tube vigorously for 30 seconds, centrifuge at 1000 rpm for 1 minute, and take about 200 μL of supernatant into a GC sample bottle for gas chromatography analysis. Gas chromatograph (Fuli, GC9790plus) analysis program: temperature program 100 ° C, 4 minutes, 230 ° C, 20 minutes, a total of 50 minutes.

[0070] The total fatty acid content of leaves of Arabidopsis thaliana plants of different genotypes was analyzed. The results showed that the fatty acid content per unit dry weight of leaves in rfs1 mutants increased by nearly 16% compared with the wild type, which is comparable to the CTI1 gene that has been reported and granted US patents ( Figure 4 The increase in the rfs1 rfs2 cti1 triple mutant was as high as 35%, almost twice that of the cti1 single mutant. The analysis of fatty acid composition showed that ( Figure 5 ), in the rfs1, rfs2 and cti1 mutants, the contents of C16:0, C16:1, C16:2, C18:0, C18:1 and C18:2 increased, while the contents of two polyunsaturated fatty acids C16:3 and C18:3 decreased.

[0071] The above results show that RFS1 regulates oil accumulation in Arabidopsis by negatively regulating ACCase activity. By making it functionally ineffective through gene editing or by downregulating gene expression or reducing protein activity through other genetic engineering methods, the oil content in plants can be increased. Knocking out the three genes RFS1, RFS2 and CTI1 simultaneously can achieve a greater increase in plant oil accumulation, which has great potential in agricultural breeding practice.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An application of a biomaterial in regulating oil synthesis in plants, characterized in that: The biological material includes RFS1 gene and RFS1 protein; the nucleotide sequence of the RFS1 gene is shown as SEQ ID NO.2; the amino acid sequence of the RFS1 protein is shown as SEQ ID NO.1; and the plant is Arabidopsis thaliana.

2. An application of a biomaterial in regulating oil accumulation in plants, characterized in that: The biological material includes RFS1 gene and RFS1 protein; the nucleotide sequence of the RFS1 gene is shown as SEQ ID NO.2; the amino acid sequence of the RFS1 protein is shown as SEQ ID NO.1; and the plant is Arabidopsis thaliana.

3. A method for increasing the content of vegetable oil, characterized in that: The method is to knock out the RFS1 gene by gene editing so that the translation of the RFS1 protein is terminated prematurely and the gene function is lost, thereby increasing the oil content of the plant; the nucleotide sequence of the RFS1 gene is shown in SEQ ID NO.2; and the plant is Arabidopsis thaliana.

4. Application of gene knockout mutants in regulating oil synthesis and accumulation in plants, characterized in that: The mutant is obtained by inserting a base A or G at the 181st position from the start codon in the nucleotide sequence of the RFS1 gene; the nucleotide sequence of the RFS1 gene is shown in SEQ ID NO.2; and the plant is Arabidopsis thaliana.

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