GhLACS4 gene for regulating oil content of plant seeds and application of GhLACS4 gene

Abstract

The invention discloses a GhLACS4 gene for adjusting the oil content of plant seeds and application of the GhLACS4 gene, and belongs to the technical field of molecular biology. The nucleotide sequence of the GhLACS4 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein of the GhLACS4 gene is as shown in SEQ ID NO.2. The gene mainly converts free fatty acid 18: 2 into 18: 2-CoA, further participates in synthesis of fatty acid to influence oil accumulation in seeds, and provides a gene resource for high-oil cotton breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a GhLACS4 gene for regulating the oil content of plant seeds and an application thereof. Background Art

[0002] Cotton is not only an important textile raw material, but its byproduct, cottonseed, is also a high-quality oil resource. As one of the main products of cotton production, cottonseed boasts significant yield advantages. Research shows that every 100 kilograms of cotton fiber harvested yields approximately 150 kilograms of cottonseed. In terms of yield per unit area, cottonseed significantly outperforms traditional oil crops. One acre of cottonseed can produce as much protein and oil as 1.5 acres of soybeans. More importantly, cottonseed possesses excellent oil properties, with the oil content of its shelled kernels reaching 25%-39%. Therefore, accelerating the functional analysis and molecular mechanism research of key genes involved in cottonseed oil synthesis and creating new high-oil cotton germplasm through modern biobreeding techniques are of strategic importance for improving oilseed self-sufficiency and ensuring the security of edible oil supply. Summary of the Invention

[0003] The purpose of the present invention is to provide a GhLACS4 gene for regulating the oil content of plant seeds and its application, so as to solve the problems existing in the above-mentioned prior art.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is a GhLACS4 gene for regulating the oil content of plant seeds, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The second technical solution of the present invention is that the amino acid sequence of the protein encoded by the GhLACS4 gene is shown in SEQ ID NO.2.

[0007] The third technical solution of the present invention is the use of the GhLACS4 gene, the protein or a recombinant vector containing the GhLACS4 gene in regulating the oil content of plant seeds.

[0008] A fourth technical solution of the present invention is a method for regulating the oil content of plant seeds, which overexpresses the GhLACS4 gene or upregulates the level of the protein encoded by it to increase the oil content of plant seeds.

[0009] A fifth technical solution of the present invention is the use of the GhLACS4 gene, the protein or a recombinant vector containing the GhLACS4 gene in cultivating transgenic plant lines with high oil yield.

[0010] The sixth technical solution of the present invention is a method for cultivating a transgenic plant line with high oil yield, comprising the following steps: introducing the GhLACS4 gene into the target plant.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] The present invention screened out a cottonseed oil phenotype regulatory gene, LACS4, which encodes long-chain acyl-CoA synthetase (LACS4). Heterologous transformation of Saccharomyces cerevisiae revealed a significant increase in the lipid droplet area of ​​yeast cells. A lacs4 mutation in Arabidopsis thaliana resulted in reduced seed oil content, while the oil content of seeds in revertant lines of the mutant returned to that of the wild type. Furthermore, heterologous expression of the GhLACS4 gene in Arabidopsis thaliana resulted in a significant increase in seed oil content compared to the wild type, with both C18:1 and C18:2 levels significantly elevated, with C18:2 showing the greatest increase. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0014] Figure 1 Identification of GhLACS4 transgenic Saccharomyces cerevisiae; (a) Semi-quantitative PCR analysis of transgenic Saccharomyces cerevisiae, (b) Triglyceride content in WT and transgenic Saccharomyces cerevisiae, (c) Nile red staining of WT and transgenic Saccharomyces cerevisiae. *: P < 0.05, ***: P < 0.001.

[0015] Figure 2 Identification and analysis of Arabidopsis mutants and revertant lines; (a) identification of homozygous Arabidopsis mutants, (b) screening of Arabidopsis mutant revertant lines, (c) semi-quantitative PCR analysis of Arabidopsis mutant revertant lines, (d) analysis of seed oil content differences between mutant lines, mutant revertant lines, and wild-type Arabidopsis, and (e) analysis of seed fatty acid content differences between mutant lines, mutant revertant lines, and wild-type Arabidopsis. ***: P < 0.001, ns: not significant.

[0016] Figure 3Identification and analysis of GhLACS4-overexpressing Arabidopsis plants; (a) Transgenic Arabidopsis screening, (b) Semi-quantitative PCR analysis of transgenic Arabidopsis, (c) Oil content of transgenic Arabidopsis, and (d) Fatty acid content of transgenic Arabidopsis. *: P < 0.05, ***: P < 0.001, ns: Not significant. DETAILED DESCRIPTION

[0017] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0020] 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 without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0023] An embodiment of the present invention provides a GhLACS4 gene for regulating the oil content of plant seeds, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0024] The present invention also provides a protein encoded by the GhLACS4 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0025] The embodiments of the present invention also provide the use of the GhLACS4 gene, the protein, or a recombinant vector containing the GhLACS4 gene in regulating the oil content of plant seeds.

[0026] In some specific embodiments, the plants include cotton and Arabidopsis thaliana.

[0027] The embodiments of the present invention also provide a method for regulating the oil content of plant seeds, which overexpresses the GhLACS4 gene or upregulates the level of the protein encoded by the gene to increase the oil content of plant seeds.

[0028] In some specific embodiments, the plants include cotton and Arabidopsis thaliana.

[0029] The embodiments of the present invention also provide the use of the GhLACS4 gene, the protein or a recombinant vector containing the GhLACS4 gene in cultivating high-oil-yielding transgenic plant lines.

[0030] In some specific embodiments, the plants include cotton and Arabidopsis thaliana.

[0031] The embodiment of the present invention also provides a method for cultivating a high-yield oil-producing plant transgenic line, comprising the following steps: introducing the GhLACS4 gene into a target plant to obtain a high-yield oil-producing plant transgenic line.

[0032] In some specific embodiments, the GhLACS4 gene is introduced into the target plant via the recombinant vector.

[0033] Example 1

[0034] 1. Materials and Methods

[0035] 1.1 Materials

[0036] 1.1.1 Bacterial strains and vectors

[0037] The Saccharomyces cerevisiae strain INVSc was purchased from Shanghai Weidi Biological Company, and the pYES2 vector was preserved in this laboratory.

[0038] 1.1.2 Plant materials

[0039] The wild-type Arabidopsis thaliana Col-0 (Columbia-0) was preserved in our laboratory, and the Arabidopsis thaliana lacs4 mutant (SALK_120357C) was purchased from Arashare (http: / / www.arashare.cn / ).

[0040] 1.1.3 Molecular biology reagents

[0041] 37 kinds of mixed fatty acid methyl ester standard solutions were purchased from SIGMA Company, USA, and Nile red was purchased from Beijing Solebow Technology Co., Ltd.

[0042] 1.2 Transgenic Saccharomyces cerevisiae experiment

[0043] 1.2.1 Construction of the expression vector GhLACS4-pYES2

[0044] Based on the reference sequences (TM-1, ZJUv2.1, GH_D03G0968) provided by the cottonfgd website (https: / / cottonfgd.org / ), a pair of specific primers was designed, encompassing the coding frame of the GhLACS4 gene. The primer sequences are shown in Table 1.

[0045] Table 1

[0046]

[0047] The cDNA template of upland cotton 901-001 was used for amplification.

[0048] The nucleotide sequence of the GhLACS4 gene is shown in SEQ ID NO.1:

[0049]

[0050] The amino acid sequence of the encoded protein is shown in SEQ ID NO.2:

[0051] SEQ ID NO. 2: MAGNNFVIEVEKGKDASDGQPSIGPVYRSSFAANGFPAPIPGME*.

[0052] The yeast expression vector pYES2 was digested with Hind III and Bam HI restriction endonucleases. The amplified fragment and digested vector were recovered by 1% agarose gel electrophoresis and ligated with the homologous recombinase at 50°C for 10 minutes. The ligation product was transformed into competent Escherichia coli DH5α, and positive clones were selected for sequencing. Following the instructions provided in the plasmid extraction kit, the plasmid of a single clone that was sequenced correctly was isolated and obtained as GhLACS4-pYES2. The plasmid was stored at -20°C until further use.

[0053] 1.2.1 Transformation of INVSc1 Competent Cells with GhLACS4-pYES2 Recombinant Plasmid

[0054] (1) Pretreatment of carrier DNA: insert the carrier DNA into a 95°C metal bath for 5 min or into a 95°C water bath in a float for 3 min, then quickly insert into ice after heating;

[0055] (2) Take 100 μL of INVSc1 competent cells melted on ice, add 2-5 μg of pre-chilled target plasmid (GhLACS4-pYES2), 10 μL of pre-treated carrier DNA, and 500 μL of PEG / LiAc, mix well by pipetting, and incubate in a 30°C water bath for 30 min (invert 6-8 times every 15 min to mix well);

[0056] (3) Place the tube in a 42°C water bath for 15 min (invert 6-8 times at 7.5 min to mix thoroughly);

[0057] (4) Centrifuge at 5000 rpm for 40 seconds, discard the supernatant, resuspend in 400 μL ddH2O, centrifuge for 30 seconds, and discard the supernatant;

[0058] (5) Resuspend in 50 μL ddH2O, apply to plates (for screening, use SD-U medium plates), incubate at 29°C for 48-96 h, and select single clones for testing.

[0059] 1.2.2 Observation of transgenic yeast lipids

[0060] Transgenic Saccharomyces cerevisiae and WT controls were inoculated into 15 mL of SD / -Ura liquid medium, respectively, and cultured at 30°C, 200 rpm, for 48 h. 1 mL of bacterial culture was added to a 2 mL enzyme-free centrifuge tube, centrifuged at 5000 g for 10 min, and resuspended in sterile distilled water to an OD600 of 0.5. 10 μL of Nile red (5 mg / mL methanol), 50 μL of dimethyl sulfoxide, and 750 μL of distilled water were added, and 200 μL of yeast cells were added for staining in the dark for 15 s. The cells were observed using a laser confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 560–620 nm for Nile red.

[0061] 1.2.3 Determination of lipid content in transgenic Saccharomyces cerevisiae

[0062] Transgenic Saccharomyces cerevisiae and WT control were inoculated into 50 mL of SD / -Ura liquid medium and cultured at 30°C and 200 rpm for 48 h. The induced bacterial solution was centrifuged at 5000 rpm for 10 min. The bacterial solution was freeze-dried for 48 h and then ground into powder using an automatic sample grinder. 16 mg of powder was weighed from each sample for lipid content determination. The specific method was referenced to the tissue cell triglyceride enzymatic assay kit (E1013).

[0063] 1.3 Arabidopsis genetic transformation

[0064] 1.3.1 Arabidopsis thaliana cultivation

[0065] (1) Place an appropriate amount of Arabidopsis seeds in a 2 mL centrifuge tube, add 1 mL of sterile water, shake several times, and discard the supernatant and floating seeds;

[0066] (2) Add 1 mL of 75% ethanol to the centrifuge tube, shake for 10 seconds, and remove the supernatant;

[0067] (3) Add 1 mL of sterile water to the centrifuge tube, shake, and remove by aspiration. Repeat twice.

[0068] (4) Add 1 mL of 10% sodium hypochlorite solution to the centrifuge tube, shake vigorously on a vortex shaker for 8 minutes, and remove the supernatant;

[0069] (5) Add 1 mL of sterile water to the centrifuge tube, shake it, and remove the sterile water. Repeat this process 7-8 times or more.

[0070] (6) Add a small amount of sterile water to the centrifuge tube, use a pipette (1 mL) to draw up the seeds, and then plant them on 1 / 2 MS medium;

[0071] (7) Seal the culture dish with sealing film;

[0072] (8) Place the sealed plate in a refrigerator at 4°C for 2-3 days for vernalization to break the seed dormancy period;

[0073] (9) Place the vernalized plate in a tissue culture room (light at 22°C for 16 h, darkness at 20°C for 8 h) and culture for 3-4 days until the seeds germinate.

[0074] (10) After 7-10 days of cultivation in the culture room, when the seedlings have grown two true leaves, they can be transplanted in large quantities.

[0075] 1.3.2 Arabidopsis overexpression vector construction and genetic transformation

[0076] Based on the reference sequence (TM-1, ZJUv2.1, GH_D03G0968) provided by the cottonfgd website (https: / / cottonfgd.org / ), a pair of specific primers was designed, encompassing the coding frame of the GhLACS4 gene. The primer sequences are shown in Table 2.

[0077] Table 2

[0078]

[0079] The CDS sequence was amplified using the G. hirsutum 901-001 cDNA template. The Arabidopsis thaliana overexpression vector pCAMBIA3301 was digested with Nco I and BstE II restriction endonucleases. The amplified fragment and digested vector were recovered by 1% agarose gel electrophoresis and ligated with the homologous recombinase at 50°C for 10 minutes. The ligation product was transformed into competent Escherichia coli DH5α, and positive clones were selected for sequencing. Following the instructions provided in the plasmid extraction kit, the single clone plasmid, GhLACS4-pCAMBIA3301, was isolated and sequenced correctly. The plasmid was stored at -20°C until further use.

[0080] The recombinant vector was transformed into Agrobacterium GV3101 and infected with Arabidopsis thaliana by the floral dip method: when Arabidopsis thaliana reached the flowering stage, the fruit pods and opened flowers were removed and the plants were watered 24 hours before infection; the Agrobacterium containing the target gene vector was activated, 100 μL was added to 1 mL of LB liquid medium (containing the corresponding antibiotics), and cultured at 28°C and 200 rpm overnight. When the OD600 reached 1.2-1.5 (orange juice color), the culture was expanded and the above 1 mL of bacterial solution was added to 50 mL LB liquid medium (containing the corresponding antibiotics) was cultured overnight at 28°C at 200 rpm, and the culture was stopped when the OD600 reached 0.8-1.2; the above liquid was cultured at 5000 rpm for 10 minutes, the supernatant was discarded, and the bacteria were collected; the resuspension solution was prepared according to Table 3, the above bacteria were resuspended, and the cells were allowed to stand for 4-5 hours; AS was added and mixed before infection, and the resuspension was poured into a culture dish, and Arabidopsis thaliana flower buds were immersed in the resuspension solution for about 45 seconds. The infected Arabidopsis plants were placed flat on a tray and kept in the dark for 24-36 hours to allow normal growth. One week later, the plants were infected again and cultured under normal management until the seeds matured.

[0081] Table 3

[0082]

[0083] After infection, the Arabidopsis thaliana harvested normal mature seeds, placed at 4 ℃ for 15 days, then the seeds were evenly spread in the nutrient soil, and covered with plastic wrap until the Arabidopsis thaliana germinated (about 3-4 days), sprayed with herbicide (750 μL 10% Basta plus 500mL water), once in the morning and evening, sprayed continuously for one week, and continued to cultivate for 5-6 days. The cotyledons and true leaves of the positive transgenic Arabidopsis thaliana were green and larger, while the cotyledons and true leaves of the negative transgenic Arabidopsis thaliana turned yellow and withered. The Arabidopsis thaliana seedlings with normal growth were transplanted, and after 3-4 weeks of normal growth, rosette leaves were taken, DNA was extracted, and specific primers were used for PCR identification, while the wild-type Arabidopsis thaliana was used as a negative control. According to the PCR test results, false positive plants were removed and positive plants were retained.

[0084] Stable transgenic Arabidopsis lines were obtained. Harvested seeds were planted and cultured, and herbicide spraying was continued for screening. Leaf DNA was extracted for PCR testing. Homozygous lines were retained and seeds were collected after normal growth.

[0085] 1.3.3 Construction of Arabidopsis mutant reversion vector and genetic transformation

[0086] Grow Arabidopsis lacs4 mutants according to the method in 1.3.1. After leaves emerge, remove rosette leaves and extract DNA. Using the sequence (TM-1, ZJU v2.1, GH_D03G0968) as a reference, design primers using the website http: / / signal.salk.edu / tdnaprimers.2.html. Identify homozygous mutants using the three-primer method. Primer sequences are shown in Table 4.

[0087] Table 4

[0088]

[0089] The GhLACS4 promoter sequence (TM-1, ZJU v2.1, GH_D03G0968) was downloaded from the CottonFGD website, and specific primers were designed to amplify the CDS sequence and promoter sequence of the GhLACS4 gene. The primer sequences are shown in Table 5.

[0090] Table 5

[0091]

[0092] The GhLACS4 gene promoter sequence (At-P-LACS4-F1 / R1) was amplified using the Gossypium hirsutum 901-001 genome as a template, and the CDS sequence (At-P-LACS4-F2 / R2) was amplified using the Gossypium hirsutum 901-001 cDNA template. The Arabidopsis thaliana complementation vector WMV067 (replacing the 35S region of the WMV067 vector with the GhLACS4 gene promoter) was digested with Sal I and BamH I restriction endonucleases. The amplified fragments and digested vectors were recovered by 1% agarose gel electrophoresis and ligated with homologous recombinases at 50°C for 15 minutes. The ligation products were transformed into competent Escherichia coli DH5α, and positive clones were selected for sequencing. Following the instructions provided in the plasmid extraction kit, the single clone plasmid containing the sequenced plasmid, GhLACS4-WMV067, was extracted and stored at -20°C until further use.

[0093] The recombinant plasmid vector was transformed into Agrobacterium GV3101 and transformed into the Atlacs4 homozygous mutant using the inflorescence infection method (see 1.3.2). The harvested seeds were planted in Arabidopsis according to the method of 1.3.1. Spectinomycin sulfate was added to 1 / 2MS to screen positive plants. Transgenic positive Arabidopsis grew normally on the culture medium, while the wild type grew abnormally. Normally growing Arabidopsis seedlings were transplanted. After 3-4 weeks of normal growth, rosette leaves were taken, DNA was extracted, and PCR identification was performed using specific primers. The mutant was also used as a negative control. Based on the PCR test results, false positive plants were removed and positive plants were retained.

[0094] Obtaining stable transgenic Arabidopsis lines: Harvested seeds are planted and cultured, screened, and homozygous lines are retained. After normal planting and growth, seeds are collected.

[0095] 1.4 Detection of cottonseed oil content and fatty acids in Arabidopsis seeds

[0096] The cottonseed oil content was determined using infrared grain analyzer DA7200, and the cottonseed fatty acid content was measured using GC-MS.

[0097] 2. Experimental Results and Analysis

[0098] 2.1 GhLACS4 increases yeast lipid content

[0099] To verify the function of the GhLACS4 gene in a fungal system, the present invention constructed the GhLACS4 gene into the pYES2 yeast expression vector and regulated its expression using the galactokinase gene promoter. Positive transformants were screened using SC-Ura-deficient medium, and single clones were randomly selected for PCR verification ( Figure 1 In a), three positive clones were randomly selected for expansion and induction culture ( Figure 1 (a)

[0100] The lipid content of the empty and recombinant yeasts after induction culture was determined. The results showed that the total lipid content of the transgenic yeast was significantly higher than that of the empty control group (P<0.001) ( Figure 1 To further verify this result, Nile Red fluorescent staining was used to visualize lipids within yeast cells. Fluorescence microscopy revealed a significant increase in the fluorescent staining area of ​​transgenic yeast cells (1, c).

[0101] 2.2 Phenotypic identification of Arabidopsis lacs4 mutants and revertant lines

[0102] To verify the regulatory effect of GhLACS4 on seed oil content, a mutant restoration vector of the GhLACS4 gene was constructed. The 35S constitutive promoter of the WMV067 vector was replaced with the promoter sequence of GhLACS4 itself, and the full-length CDS of GhLACS4 was constructed into the WMV067 expression vector to perform Arabidopsis thaliana lacs4 mutant restoration experiments ( Figure 2 The harvested seeds were sown on 1 / 2MS medium containing spectinomycin for preliminary screening ( Figure 2 b), and further PCR detection was performed to obtain positive transgenic plants ( Figure 2 (c) The positive plants were self-pollinated continuously to obtain stable genetic lines for subsequent phenotypic identification.

[0103] Homozygous Arabidopsis lacs4 mutant lines, wild-type Arabidopsis thaliana, and Arabidopsis lacs4 mutant revertant lines were grown under the same environmental conditions, and the oil content and fatty acid composition of Arabidopsis seeds were determined.

[0104] The results showed that the oil content of lacs4 mutant seeds was significantly reduced by 12.81% compared with wild-type Arabidopsis, while the oil content of mutant revertant seeds was not significantly different from that of wild-type seeds ( Figure 2 In addition, the total fatty acid content of lacs4 mutant seeds was significantly reduced by 15.46% compared with the wild type ( Figure 2 (e)

[0105] 2.3 Overexpression of GhLACS4 can increase the oil content of Arabidopsis seeds

[0106] To investigate the function of the GhLACS gene, the GhLACS4 gene was linked to an overexpression vector and genetically transformed into Arabidopsis. The harvested seeds were evenly sown in nutrient soil. After 2-4 days, green shoots grew and were subsequently sprayed with Basta herbicide for screening. First, plants that could grow normally were screened ( Figure 3 a), and further use PCR technology to identify positive seedlings and screen out transgenic positive seedlings ( Figure 3(b) After harvesting the seeds normally, they were cultured for additional generations to obtain stable transgenic Arabidopsis lines.

[0107] Stable transgenic Arabidopsis lines were grown under the same environmental conditions as wild-type Arabidopsis and their phenotypes were observed. Subsequently, the oil content and fatty acid composition of the transgenic Arabidopsis seeds were measured. The results showed that the oil content of the transgenic lines' seeds increased significantly by 3.99% ( Figure 3 In addition, the contents of C18:1 (oleic acid), C18:2 (linoleic acid) and total fatty acids in the transgenic lines increased significantly by 6.52%, 9.79% and 6.12%, respectively ( Figure 3 (d)

[0108] Long-chain fatty acyl-CoA synthetase, a homolog of LACS4, is responsible for converting free fatty acids into acyl-CoA and participates in multiple pathways of lipid metabolism. The primary function of LACS is fatty acid transport. In Escherichia coli, the single LACS enzyme, FadD, works together with the fatty acid transporter FadL to transport and activate long-chain fatty acids. In yeast, short- and medium-chain free fatty acids can directly enter the peroxisome, where they are reactivated to CoA thioesters by acyl-CoA synthetases. In Arabidopsis, LACS1 and LACS2 preferentially modify very-long-chain fatty acids (VLCFAs) for wax synthesis and long-chain fatty acids for cutin synthesis. Meanwhile, LACS6 and LACS7 in peroxisomes play overlapping roles in fatty acid β-oxidation, crucial for seedling growth and development. Studies in Arabidopsis thaliana found that 18:2 released from PC on the endoplasmic reticulum membrane was converted into 18:2-CoA by LACS4 and LACS9, and then 18:2-CoA was immediately used as a substrate to resynthesize PC on the plastid membrane.

[0109] The present invention expresses the GhLACS4 gene in Saccharomyces cerevisiae, and the results show that lipid droplets are significantly enlarged and TAG content is significantly increased. In Arabidopsis lacs4 mutant strains, seed oil content is significantly reduced, while the oil content of the complemented strains returns to normal levels. In addition, experiments on transgenic Arabidopsis found that GhLACS4 can significantly increase seed oil content and specifically increase the content of C18:2 ( Figure 3(d) In Arabidopsis, C18:2, C18:3, C20:1, C18:1, C18:0, and C16:0 are the preferred substrates for fatty acid synthesis, while C20:1 is more efficiently utilized in Arabidopsis. This is likely due to the high activity of LACS6 and LACS7 towards C20:1. The substrate preferences of different fatty acid enzymes lead to differences in seed fatty acid composition. This study demonstrates that GhLACS4 primarily influences seed oil accumulation by converting the free fatty acid 18:2 into 18:2-CoA, which in turn participates in fatty acid synthesis.

[0110] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A GhLACS4 gene for regulating the oil content of plant seeds, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the GhLACS4 gene according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

3. Use of the GhLACS4 gene according to claim 1, the protein according to claim 2, or a recombinant vector containing the GhLACS4 gene according to claim 1 in regulating the oil content of plant seeds.

4. The use according to claim 3, characterized in that Such plants include cotton and Arabidopsis thaliana.

5. A method for regulating the oil content of plant seeds, characterized in that: Overexpressing the GhLACS4 gene or upregulating the level of the protein it encodes can increase the oil content of plant seeds.

6. The method according to claim 5, characterized in that Such plants include cotton and Arabidopsis thaliana.

7. Use of the GhLACS4 gene according to claim 1, the protein according to claim 2, or a recombinant vector containing the GhLACS4 gene according to claim 1 in cultivating a high-oil-yielding transgenic plant strain.

8. The use according to claim 7, characterized in that Such plants include cotton and Arabidopsis thaliana.

9. A method for cultivating a high-yield oil and fat transgenic plant strain, characterized in that: The method comprises the following steps: introducing the GhLACS4 gene according to claim 1 into a target plant.

10. The method according to claim 6, characterized in that The GhLACS4 gene described in claim 1 is introduced into the target plant via the recombinant vector described in claim 3.

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