Gene for modulating oil accumulation in plants, and use thereof

Abstract

The present invention belongs to the field of biotechnology. Disclosed are a gene for modulating the oil accumulation in plants, and the use thereof. Disclosed is a biological material capable of modulating oil synthesis and accumulation in plants. The biological material comprises an RFS1 gene and an RFS1 protein, wherein the RFS1 gene has a nucleotide sequence as shown in SEQ ID NO. 2. It is verified that the RFS1 gene and a protein encoded thereby modulate the oil accumulation in plants by means of affecting de novo synthesis of fatty acids. Compared with a wild type, an RFS1 gene mutant exhibits a significantly increased fatty acid synthase activity, an increased fatty acid synthesis rate, an increased total fatty acid content, and changes in fatty acid composition. The ability of the mutant to modulate fatty acid content is comparable to that of the CTI1 gene that has been published in articles and authorized in patents.

Description

Gene for regulating plant oil accumulation and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a gene for regulating plant oil accumulation and application thereof. BACKGROUND

[0002] Plant oil, especially triacylglycerol, is a valuable agricultural product in food, fuel and petrochemical industries. Soybean oil, rapeseed oil, palm oil and peanut oil are the four most popular edible plant oil consumption varieties. With the rapid economic development, the urbanization process is steadily advancing, and the consumption of edible plant oil by residents is gradually increasing. Due to the slow growth of oil production capacity, the gap between supply and demand continues to widen. Through traditional breeding and molecular breeding, cultivating high-oil and high-quality oil crop varieties is a key link to overcome this shortcoming in the agricultural field. Genetic engineering and directional gene modification for high-oil variety improvement can greatly shorten the breeding process. Mining genes that regulate plant oil synthesis and accumulation as a target for directional molecular breeding can provide a gene resource library for molecular breeding.

[0003] Acyl lipids are the main components of plant oils, and play a crucial 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 synthesis of fatty acids (FAS) in plant plastids, followed by further assembly in the endoplasmic reticulum. The initial step of the fatty acid de novo synthesis process is catalyzed by acetyl-CoA carboxylase (ACCase), which converts acetyl-CoA to propionyl-CoA. In most plants, such as dicotyledons and non-grass monocotyledons, ACCase exists in the form of a heteromer (hetACCase) in plastids. Plastid hetACCase is composed of a protein complex encoded by four different genes: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), alpha-carboxyltransferase (alpha-CT) and beta-carboxyltransferase (beta-CT). ACCase, as the most important key rate-limiting enzyme in fatty acid synthesis, has always been a hot research object in oil synthesis metabolism. ACCase activity and plant fatty acid synthesis are regulated in a complex and delicate manner. Mining key regulatory genes that regulate ACCase activity can provide promising molecular targets for high-oil crop molecular breeding. The regulation of hetACCase activity is achieved through multiple mechanisms, and the influencing factors include light intensity, downstream metabolites, interacting proteins, etc.

[0004] In addition to the above factors, another mechanism to regulate ACCase activity is the association of the CT sub-complex with the plastid membrane. Specifically, the subcellular localization of the CT subunit is more closely associated with the plastid membrane than the BC / BCCP subunits. Alpha-CT is hypothesized to interact with membrane proteins through its non-catalytic domain at the C-terminus, binding to the inner membrane of the plastid. Recent studies have found that a group of carboxyltransferase interacting proteins (CTIs) localized on the inner membrane of the plastid anchor ACCase to the plastid membrane by directly interacting with the non-catalytic domain of alpha-CT. More importantly, this interaction reduces the activity of ACCase, thereby negatively regulating the content of plant oil. Studies have shown that the leaf of Arabidopsis thaliana cti mutant has increased ACCase activity, increased fatty acid synthesis rate and increased oil content by 5-8 times compared with wild type. Given the importance of CTIs genes in the regulation of plant oil synthesis, the gene has been granted an international patent, and has been purchased by a US biotechnology company and is being further transformed. The present application further excavates other key genes that regulate oil synthesis on this basis.

[0005] SUMMARY

[0006] To solve the above technical problems, the present application provides a gene for regulating plant oil accumulation and its application. The present application discloses that RFS1 gene and its encoded protein regulate plant oil accumulation by affecting de novo synthesis of fatty acids.

[0007] The present application provides the following technical solutions:

[0008] Technical solution one: a biological material capable of regulating oil synthesis and accumulation in plants, the biological material comprising 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.

[0009] Further, the RFS1 protein is selected from at least one of the following:

[0010] (1) a protein having one or more amino acids substituted, deleted or added in the sequence and having the same or similar function as the RFS1 protein;

[0011] (2) a nucleic acid molecule encoding the RFS1 protein;

[0012] (3) a nucleic acid molecule having one or more nucleotides substituted, deleted or added in the nucleotide sequence of the nucleic acid molecule of (2) and capable of encoding a protein having the same or similar function;

[0013] (4) a substance capable of regulating the level or activity of at least one of (1)-(3).

[0014] Further, 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 reverse complementary to the DNA, the cDNA, the recombinant DNA or the mRNA.

[0015] Further, the nucleic acid molecule encoding the RFS1 protein includes an RFS1 gene, and a nucleotide sequence of the RFS1 gene is shown as SEQ ID NO. 2.

[0016] The biological material is used for regulating oil synthesis in plants.

[0017] The biological material is used for regulating oil accumulation in plants.

[0018] The biological material is used for regulating oil synthesis in plants.

[0019] The biological material is used for regulating oil synthesis in plants.

[0020] The biological material is used for regulating oil synthesis in plants.

[0021] Compared with the prior art, the present application has the following advantages and technical effects:

[0022] The present application discloses that RFS1 gene and its encoded protein regulate plant oil accumulation by affecting de novo synthesis of fatty acids. Compared with wild type, RFS1 gene mutant shows significant increase in fatty acid synthase activity, fatty acid synthesis rate, total fatty acid content and change in fatty acid composition. The regulation level of fatty acid content is equivalent to that of CTI1 gene published in articles and authorized in patents. The three mutants of RFS1, RFS1 homologous gene RFS2 and CTI1 gene simultaneously lacking of function can increase the amplitude of fatty acid content (Fig. 3-5).

[0023] The present application screens a gene AtRFS1 which can inhibit de novo synthesis of fatty acids from co-expression analysis of heteromeric acetyl-CoA carboxylase (hetACCase) and its known regulatory factors in Brassica rapa, the present application successfully clones the gene, and constructs its deletion mutant by gene editing technology. The research results show that, compared with wild type plants, ACCase activity in leaf of RFS1 gene deletion mutant of the present application is increased by more than one time, fatty acid synthesis rate is increased by nearly one time, total fatty acid content in leaf is increased by nearly 16%, and triacylglycerol accumulation level is significantly increased. In addition, the results of fatty acid composition analysis show that, fatty acid composition in leaf of RFS1 mutant changes, wherein 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 function level of RFS1 for improving oil accumulation is equivalent to that of reported CTI1 gene, and the function of RFS1 gene is more significant in terms of fatty acid synthesis rate and triacylglycerol accumulation level. The triple mutant with simultaneous functional deletion of RFS1, RFS1 homologous gene RFS2 and CTI1 gene can promote fatty acid synthesis and increase oil accumulation to a higher extent. The RFS1 gene mutant improves oil accumulation without obviously affecting plant biomass and other growth traits, indicating that it will not affect other yield-related traits in application process. RFS1 has homologous genes in oil crops such as soybean and rapeseed, and may have similar functions. Therefore, the present application has application value in improving oil level of higher plants by using genetic engineering means for molecular breeding. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0025] Fig. 1 is the genotype of four genes obtained by CRISPR / Cas9 gene editing method;

[0026] Fig. 2 is the growth phenotype of different genotypes of Arabidopsis plants 4 weeks after germination;

[0027] Fig. 3 is the activity of ACCase in leaves of different genotypes of Arabidopsis;

[0028] Fig. 4 is the content of total fatty acid in leaves of different genotypes of Arabidopsis;

[0029] Fig. 5 is the composition of fatty acid in leaves of different genotypes of Arabidopsis. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] Example 1

[0036] 1. Identification and cloning of the RFS1 gene

[0037] In order to mine unknown genes involved in regulating fatty acid synthesis, the present application takes Arabidopsis thaliana as experimental material, takes key enzymes and regulatory genes for de novo synthesis of fatty acids as target groups, and performs co-expression analysis, and finally identifies the RFS1 gene in the present application. The protein of the RFS1 gene is a transmembrane protein, has a predicted chloroplast transit peptide structure and two coiled coil domains at the C terminal, and the amino acid sequence of the RFS1 protein is shown as SEQ ID NO. 1. The CDS sequence of the RFS1 gene is cloned by taking Arabidopsis thaliana Col-0 as experimental material, and the nucleotide sequence is shown as SEQ ID NO. 2. By searching and aligning the Arabidopsis thaliana genome, the homologous gene RFS2 of the RFS1 gene is identified, and the nucleotide sequence of the CDS of the RFS2 gene is shown as SEQ ID NO. 3.

[0038] The amino acid sequence of the RFS1 protein is:

[0039] The nucleotide sequence of the CDS of the RFS1 gene is:

[0040] The nucleotide sequence of the CDS of the RFS2 gene is:

[0041] Example 2

[0042] 1. Construction of Arabidopsis thaliana RFS gene deletion mutant

[0043] 1.1 Construction of CRISPR / Cas9 vector

[0044] The sgRNA is designed for the sequence of the RFS1 gene (SEQ ID NO. 2) and the sequence of the RFS2 gene (SEQ ID NO. 3) using the online CRISPR-P 2.0 software, and the sequences of the target sites of the RFS1 and RFS2 genes are (SEQ ID NO. 4) and (SEQ ID NO. 5) (the underlined sequence is the target sequence, and the bold sequence is PAM). The sgRNA primers for editing the RFS1 gene and the RFS2 gene are synthesized based on the target site sequences:

[0045] RFS1-sgRNA-F: 5'-GATTGGATTCGAGTACAATGCAGA-3' (SEQ ID NO. 6);

[0046] RFS1-sgRNA-R: 5'-AAACTCTGCATTGTACTCGAATCC-3' (SEQ ID NO. 7);

[0047] RFS2-sgRNA-F: 5'-GATTAATGGAGGCGAACGAATCAA-3' (SEQ ID NO. 8);

[0048] RFS2-sgRNA-R: 5'-AAACTTGATTCGTTCGGCCTCCATT-3' (SEQ ID NO. 9).

[0049] The Gateway system's intermediate vector P1P2 was digested with the restriction endonuclease BbsI using the following digestion regimen: 5 μL P1P2 vector, 3 μL digestion buffer, and 1.5 μL BbsI. Add ddH2O to 30 μL. Incubate at 37°C for 30 min, and then recover the DNA by gel extraction after electrophoresis. Phosphorylate and anneal the sgRNA primer pair according to the following reaction system: 1 μL Primer F (100 μM), 1 μL Primer R (100 μM), 0.5 μL PNK buffer, 0.5 μL T4 DNA ligase buffer, 0.5 μL T4 PNK, and add ddH2O to 10 μL. Perform the annealing reaction in the PCR instrument according to the following program: 37°C for 30 min, 95°C for 5 min, and then gradually decrease the temperature to 25°C at a rate of 5°C / min.

[0050] The annealed oligonucleotide fragments were ligated into the enzyme-digested P1P2 vector using the following reaction system: X μL BBSI digested P1P2 vector (50 ng), 1 μL phosphorylated and annealed oligo primers (1:200 dilution), 1 μL T4 DNA ligase buffer, 1 μL T4 DNA ligase, and ddH2O added to 10 μL. After reacting at room temperature for 30 min, *E. coli* DH5α was transformed, and positive clones were identified by PCR. Plasmid extraction and sequencing confirmed successful vector construction.

[0051] Cloning of pU6-RFS1 sgRNA cassette in intermediate vector P1P2 into pYAO::Cas9 final vector site by LR reaction (pYAO::Cas9 vector information 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): 1 μL P1P2, 1 μL YAO::Cas9, 0.5 μL Gateway LR Clonase II Enzyme Mix. After 2 h reaction at room temperature, the reaction system was transformed into E. coli DH5a. Positive clone identification was performed by PCR. After plasmid extraction, sequencing was performed to confirm that the vector construction was correct.

[0052] 1.2. Obtaining, screening and identifying mutant Arabidopsis

[0053] Wild-type Arabidopsis Col-0 at flowering stage was transformed using inflorescence infection method to obtain T1 generation transgenic Arabidopsis. The specific operation is as follows: the above constructed pYAO::Cas9-pU6::RFS1 sgRNA and pYAO::Cas9-pU6::RFS2 sgRNA vectors were transformed into Agrobacterium tumefaciens strain GV3101, and positive clones were identified by colony PCR. The positive single clone was picked into 2 mL of corresponding resistance LB medium and cultured at 28°C overnight, and then inoculated into 200 mL of LB medium and shaken overnight. The bacterial solution was centrifuged at 5000g for 20 min, and then resuspended in 120 ml of infiltration solution (10% sucrose + 400 μl / L Silwet-77, mixed well before dipping, OD 600 = 0.8-1.0). At the same time, the pods and fully opened flowers on the plants were cut off. The aerial part of the plant was immersed in the bacterial solution for 90 s with slight shaking. The infected plants were covered with plastic wrap to maintain humidity and dark cultured for 1 d, and then placed in normal culture conditions. Regularly continue to infect several times. Continue to culture until seed maturity, dry the seeds at 37°C for about 3 days, and then screen the transformants.

[0054] The obtained seeds were screened by hygromycin resistance 1 / 2MS plate after sowing, and the T2 generation seeds were planted and harvested. DNA was extracted, and the T2 generation plants were genotyped and identified mutants using designed identification PCR primers. The primers used are as follows:

[0055] RFS1-CF: 5'-CACCATGTTATTCTCGCCTTCGAAA-3' (SEQ ID NO. 10);

[0056] RFS1-CR: 5'-CTGCTTAGCACGCTTTGCAAG-3' (SEQ ID NO. 11).

[0057] RFS2-CF: 5'-CGGTATAATCCCGGTTTGCAG-3' (SEQ ID NO. 12);

[0058] RFS2-CR: 5'-TGCAAACGATGAAGGCCAAG-3' (SEQ ID NO. 13).

[0059] The following PCR reaction system and procedure were used: 2 x Es Taq Master Mix 7.5 μΐ, Primer F 0.75 μΐ, Primer R 0.75 μΐ, gDNA 1 μΐ, ddH2O 5 μΐ. The PCR identification reaction procedure was as follows: pre-denaturation, 94 °C for 2 min; denaturation, 94 °C for 30 s; annealing, 55 °C for 30 s; extension, 72 °C for 30 s (35 cycles); final extension, 72 °C for 2 min.

[0060] The purified PCR fragments were sent to Shenguo Company for sequencing, and the sequencing results were compared with the CDS sequences of RFS1 gene and RFS2 gene. The homozygous mutants rfs1-1, rfs1-2, rfs2-1 and rfs2-2 were identified, and the mutant forms are shown in Figure 1. As shown in Figure 1, in mutants rfs1-1 and rfs1-2, one base A and one base G were inserted at the 181st position from the start codon of RFS1, respectively; in mutants rfs2-1 and rfs2-2, one base A and one base G were inserted at the 37th position from the start codon of RFS2, respectively. The double mutant rfs1rfs2 of RFS1 gene and its homologous gene RFS2 gene, the triple mutant rfs1rfs2cti1 of RFS1 gene, RFS2 gene and CTI1 gene (mutant cti1 information is detailed in Ye Y, 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) were constructed by conventional artificial hybridization method. The specific operation steps are as follows: select homozygous rfs1 single mutant as female parent, and rfs2 as male parent. Remove the mother's long pod and the flower that has bloomed and the petals and stamens that have not bloomed before hybridization, and apply pollen of the male parent to the stigma of the female parent that has not been pollinated. The stigma hair wilts 10 hours after pollination, and the fruit pod forms and continues to elongate 3 days after pollination, indicating successful hybridization. The F2 generation seeds were obtained after hybridization of the F1 generation, and the genotype was identified by PCR after sowing to obtain homozygous double mutants. Using the same method, the double mutant rfs1rfs2 was used as the female parent, and the cti1 was used as the male parent for hybridization, and the homozygous triple mutant was obtained by F2 generation identification. The above Arabidopsis thaliana mutant plants are shown in Figure 2, and the results show that they grow normally and do not show obvious growth defect phenotype.

[0061] Example 3

[0062] 1. ACCase activity analysis in RFS1 gene deletion mutant

[0063] About 5 g of leaves from 4-week-old wild type Col-0, homozygous mutants rfs1-1, rfs1-2 and other related mutants were taken, and chloroplasts were extracted. 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, and incubated on ice for 10 min, centrifuged at 12.000 x g for 10 min, and the supernatant was collected and desalted using a spin column (Thermo Fisher, Cat. No. 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 fluazifop-butyl and 1 μCi of [14C] fluazifop-butyl (PerkinElmer; Cat. No. NEC 086H001MC)) in a 96-well plate. Reactions without acetyl-CoA were used as controls. After 30 min 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 h to evaporate free 14 C-NaHCO3. Then 1 mL of scintillation cocktail was added to each vial, and the ACCase activity was quantitatively determined by liquid scintillation. 14 C-NaHCO3. Then 1 mL of scintillation cocktail was added to each vial, and the ACCase activity was quantitatively determined by liquid scintillation.

[0064] The results (Figure 3) show that the ACCase activity in RFS1 mutants rfs1-1 and rfs1-2 increased by more than 1.5-fold compared to wild type Col-0, with a similar increase as cti1. The ACCase activity in RFS2 mutants (rfs2-1 and rfs2-2) also increased, but to a lesser extent than RFS1 mutants. The triple mutant rfs1-1 rfs2-1 cti1 showed even stronger ACCase activity.

[0065] Example 4

[0066] Fatty acid content and composition analysis in RFS1 gene deletion mutants

[0067] The method of fatty acid content and composition analysis refers to the method 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 are labeled with serial numbers, weighed with a precision balance, and 4-week-old plant leaves are placed in the corresponding test tubes. After drying in an oven at 65°C for 2-3 days, the dried leaves are weighed again, and the weight of the dried leaves is calculated. Add 200 μL of toluene solution containing 20 μg of 17:0 as an internal standard, and then add 1 mL of methanol solution containing 5% hydrochloric acid. Heat in a metal bath at 85-90°C for 1.5 h. Observe the boiling phenomenon of the liquid in the test tube during heating. After cooling to room temperature, add 0.2 mL of n-hexane, and then add 1.5 mL of 0.9% NaCl solution. Shake the test tube vigorously with a vortex for 30 s, and centrifuge at 1000 rpm for 1 min. Take about 200 μL of supernatant to a GC sample bottle for gas chromatograph analysis. Gas chromatograph (Fuli, GC9790plus) analysis program: temperature program 100°C, 4 min, 230°C, 20 min, total 50 min.

[0068] The total fatty acid content of different genotypes of Arabidopsis thaliana leaves was analyzed, and the results showed that compared with the wild type, the fatty acid content in the rfs1 mutant unit mass dry weight leaf increased by nearly 16%, and the increase level was comparable to the CTI1 gene reported and authorized by the United States patent (Figure 4). In the rfs1 rfs2 cti1 triple mutant, the increase reached 35%, almost twice the increase of the cti1 single mutant. The composition analysis results of fatty acids showed (Figure 5) that 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.

[0069] The above results show that RFS1 regulates oil accumulation in Arabidopsis by negatively regulating ACCase activity. Disabling the function of RFS1 by gene editing or reducing the expression of RFS1 by other genetic engineering methods or reducing the activity of RFS1 protein can increase the oil content in plants. Simultaneous knockout of RFS1, RFS2 and CTI1 can obtain plants with greater oil accumulation, which has great potential in agricultural breeding practice.

[0070] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. A biological material capable of regulating oil synthesis and accumulation in a plant, characterized in that, The biomaterial comprises an RFS1 gene and an RFS1 protein; the nucleotide sequence of the RFS1 gene is shown as SEQ ID NO. 2; and the amino acid sequence of the RFS1 protein is shown as SEQ ID NO.

1.

2. The biomaterial of claim 1, wherein, The RFS1 protein is selected from at least one of the following: (1) a protein having one or several amino acids substituted, deleted or added in the sequence and having the same or similar function as the RFS1 protein; (2) a nucleic acid molecule encoding the RFS1 protein; (3) a nucleic acid molecule having one or several nucleotides substituted, deleted or added in the nucleotide sequence of the nucleic acid molecule of (2) and capable of encoding a protein having the same or similar function; (4) a substance capable of regulating the level or activity of at least one of (1) to (3).

3. The biomaterial of claim 2, wherein, The nucleic acid molecule encoding the RFS1 protein comprises genomic DNA, cDNA, recombinant DNA, mRNA or hnRNA encoding the RFS1 protein, or a nucleic acid molecule reverse complementary to the DNA, the cDNA, the recombinant DNA or the mRNA.

4. The biomaterial of claim 2, wherein, The amino acid sequence of the RFS1 protein is shown as SEQ ID NO.

1.

5. The biomaterial of claim 2, wherein, The nucleic acid molecule encoding the RFS1 protein comprises an RFS1 gene, and the nucleotide sequence of the RFS1 gene is shown as SEQ ID NO.

2.

6. Use of the biomaterial of any one of claims 1 to 5 in regulating oil synthesis in a plant.

7. Use of the biomaterial of any one of claims 1 to 5 in regulating oil accumulation in a plant.

8. A method for increasing the lipid content of a plant, comprising, The method is to terminate the translation of the RFS1 protein in advance by knocking out the RFS1 gene through gene editing, so as to delete the gene function and improve the oil content of the plant.

9. Use of a gene knockout mutant in modulating oil synthesis and accumulation in a plant, 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 shown in claim 1.

10. Application of homozygous triple mutants of RFS1 gene, RFS2 gene and CTI1 gene, all of which are mutated, in increasing the oil accumulation of plants, characterized in that, The nucleotide sequence of the RFS1 gene is shown as SEQ ID NO. 2; and the nucleotide sequence of the RFS2 gene is shown as SEQ ID NO. 3.

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