Ghlacs4 gene for regulating oil content of plant seeds and application thereof

Abstract

This invention discloses a GhLACS4 gene that regulates the oil content of plant seeds and its applications, belonging to the field of molecular biology. The nucleotide sequence of the GhLACS4 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. This gene mainly participates in fatty acid synthesis and affects the accumulation of oil in seeds by converting free fatty acids 18:2 into 18:2-CoA, thus providing gene resources for breeding high-oil cotton.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a GhLACS4 gene that regulates the oil content of plant seeds and its applications. Background Technology

[0002] Cotton is not only an important textile raw material, but its by-product, cottonseed, is also a high-quality oilseed resource. As one of the main products of cotton production, cottonseed has a significant yield advantage. Studies have shown that approximately 150 kilograms of cottonseed can be produced simultaneously for every 100 kilograms of cotton fiber harvested. In terms of yield per unit area, cottonseed yield is significantly higher than that of traditional oilseed crops; the protein and oil produced from cottonseed per acre (0.067 hectares) are equivalent to the production capacity of 1.5 acres of soybeans. More importantly, cottonseed has excellent oil properties; the oil content of the hulled cotton kernels can reach 25%-39%. Therefore, accelerating the functional analysis and molecular mechanism research of key genes in cottonseed oil synthesis, and creating new high-oil cotton germplasm through modern bio-breeding technology, is of great strategic significance for improving oilseed self-sufficiency and ensuring the security of edible oil supply. Summary of the Invention

[0003] The purpose of this 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 prior art.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] One of the technical solutions of the present invention is a GhLACS4 gene that regulates 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 protein encoded by the GhLACS4 gene has the amino acid sequence shown in SEQ ID NO.2.

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

[0008] The fourth technical solution of the present invention is a method for regulating the oil content of plant seeds by overexpressing the GhLACS4 gene or upregulating the level of its encoded protein to increase the oil content of plant seeds.

[0009] The fifth technical solution of the present invention is the application of the GhLACS4 gene, the protein, or the recombinant vector containing the GhLACS4 gene in the cultivation of 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, thereby obtaining the desired result.

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

[0012] This invention screened a cottonseed oil content phenotypic regulatory gene, LACS4, which encodes long-chain acyl-CoA synthase 4 (LACS4). Heterologous transformation of *Saccharomyces cerevisiae* showed a significant increase in the lipid droplet area of ​​yeast cells. Mutations in *Arabidopsis thaliana* with lacs4 resulted in decreased seed oil content, while the oil content of the revertant mutant lines recovered to the wild-type state. Further heterologous expression of the GhLACS4 gene in *Arabidopsis thaliana* significantly increased the seed oil content compared to the wild type, with both C18:1 and C18:2 contents showing a marked increase, with C18:2 showing the largest increase. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 To identify GhLACS4 transgenic Saccharomyces cerevisiae; (a) semi-quantitative PCR analysis of transgenic Saccharomyces cerevisiae, (b) triglyceride content of WT and transgenic Saccharomyces cerevisiae, and (c) Nile red staining observation of WT and transgenic Saccharomyces cerevisiae. *: P<0.05, ***: P<0.001.

[0015] Figure 2 This study aimed to identify and analyze Arabidopsis thaliana mutants and their restorers; including (a) homozygous identification of Arabidopsis thaliana mutants, (b) screening of Arabidopsis thaliana mutant restorers, (c) semi-quantitative PCR analysis of Arabidopsis thaliana mutant restorers, (d) analysis of differences in seed oil content between mutant lines, mutant restorers, and wild-type Arabidopsis thaliana, and (e) analysis of differences in seed fatty acid content between mutant lines, mutant restorers, and wild-type Arabidopsis thaliana. ***: P < 0.001, ns: no significant difference.

[0016] Figure 3The study analyzed transgenic Arabidopsis thaliana overexpressing the GhLACS4 gene, including (a) screening of transgenic Arabidopsis thaliana, (b) semi-quantitative PCR analysis of transgenic Arabidopsis thaliana, (c) oil content of transgenic Arabidopsis thaliana, and (d) fatty acid composition of transgenic Arabidopsis thaliana. *: P < 0.05, ***: P < 0.001, ns: no significant difference. Detailed Implementation

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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 obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0021] 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.

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

[0023] This 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] This invention also provides a protein encoded by the GhLACS4 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0025] This invention also provides the application 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 implementations, the plants include cotton and Arabidopsis thaliana.

[0027] This invention also provides a method for regulating the oil content of plant seeds by overexpressing the GhLACS4 gene or upregulating the level of its encoded protein to increase the oil content of plant seeds.

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

[0029] This invention also provides the application of the GhLACS4 gene, the protein, or a recombinant vector containing the GhLACS4 gene in the cultivation of transgenic plant lines with high oil yield.

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

[0031] This invention also provides a method for cultivating transgenic plant lines with high oil yield, comprising the following steps: introducing the GhLACS4 gene into the target plant, thereby obtaining the desired result.

[0032] In some specific implementations, 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 Strains and Carriers

[0037] The Saccharomyces cerevisiae strain INVSC was purchased from Shanghai Weidi Biotechnology Co., Ltd., and the pYES2 vector was preserved in our laboratory.

[0038] 1.1.2 Plant materials

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

[0040] 1.1.3 Molecular Biology Reagents

[0041] The 37-component mixed fatty acid methyl ester standard solution was purchased from SIGMA Corporation, USA, and Nile Red was purchased from Beijing Solarbio Technology Co., Ltd.

[0042] 1.2 Experiment with transgenic Saccharomyces cerevisiae

[0043] 1.2.1 Construction of expression vector GhLACS4-pYES2

[0044] Based on the reference sequences (TM-1, ZJUv2.1, GH_D03G0968) provided on the cottonfgd website (https: / / cottonfgd.org / ), a pair of specific primers containing the coding frame of the GhLACS4 gene were designed. 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 its 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 the digested vector were recovered by 1% agarose gel electrophoresis and ligated with homologous recombinase at 50°C for 10 min. The ligation product was transformed into competent *E. coli* DH5α cells, and the selected positive clones were sent for sequencing. The plasmid of the correctly sequenced single-clone strain was extracted according to the instructions of the plasmid extraction kit; this plasmid was GhLACS4-pYES2 and stored at -20°C for later 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 float in a 95°C water bath for 3 min, and then quickly insert it into ice after heating;

[0055] (2) Take 100 μL of INVSC1 competent cells thawed on ice, add 2-5 μg of pre-cooled target plasmid (GhLACS4-pYES2), 10 μL of pretreated carrier DNA, and 500 μL of PEG / LiAc, and incubate at 30°C for 30 min (invert 6-8 times at 15 min to mix).

[0056] (3) Place the tube in a 42℃ water bath for 15 minutes (invert 6-8 times at 7.5 minutes to mix well);

[0057] (4) Centrifuge at 5000r / min for 40s and discard the supernatant. Resuspend in 400μL of ddH2O and centrifuge for 30s and discard the supernatant.

[0058] (5) Resuspend in 50 μL of ddH2O, spread on a plate (for screening plates: SD-U medium plate), incubate at 29℃ for 48-96 h, and select single clones for testing.

[0059] 1.2.2 Observation of transgenic yeast oil

[0060] Transgenic Saccharomyces cerevisiae and the WT control were inoculated into 15 mL of SD / -Ura liquid medium and cultured at 30℃ and 200 r / min for 48 h. 1 mL of the 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 with 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, along with 200 μL of yeast cells, and the mixture was stained in the dark for 15 s. The excitation wavelength of Nile red was 488 nm, and the emission wavelength was 560–620 nm. The staining was performed using a laser confocal microscope.

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

[0062] Transgenic Saccharomyces cerevisiae and the WT control were inoculated into 50 mL of SD / -Ura liquid medium and cultured at 30℃ and 200 r / min for 48 h. The induced bacterial culture was centrifuged at 5000 r / min for 10 min. The bacterial culture was freeze-dried for 48 h. Then, the freeze-dried yeast was ground into powder using a fully automated sample grinder. 16 mg of powder was weighed from each sample for the determination of lipid content. The specific method was based on the tissue cell triglyceride enzyme assay kit (E1013).

[0063] 1.3 Arabidopsis genetic transformation

[0064] 1.3.1 Arabidopsis thaliana cultivation

[0065] (1) Take an appropriate amount of Arabidopsis seeds and put them into a 2mL centrifuge tube. Add 1mL 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 aspirate, repeat twice;

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

[0069] (5) Add 1 mL of sterile water to the centrifuge tube, shake and remove the sterile water, repeat 7-8 times or more;

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

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

[0072] (8) Place the sealed plate in a refrigerator at 4℃ for 2-3 days to carry out vernalization treatment and break the seed dormancy period;

[0073] (9) Place the vernalization-treated plates into a tissue culture chamber (light 22℃, 16h, darkness 20℃, 8h) and culture for 3-4 days until the seeds germinate.

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

[0075] 1.3.2 Construction and genetic transformation of Arabidopsis thaliana overexpression vectors

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

[0077] Table 2

[0078]

[0079] The CDS sequence was amplified using the *Upland cotton* 901-001 cDNA template. The *Arabidopsis thaliana* overexpression vector pCAMBIA3301 was digested with Nco I and BstE II restriction endonucleases. The amplified fragment and the digested vector were recovered by 1% agarose gel electrophoresis and ligated with homologous recombinase at 50°C for 10 min. The ligation product was transformed into competent *E. coli* DH5α cells, and the selected positive clones were sent for sequencing. The plasmid of the correctly sequenced single-clone strain was extracted according to the plasmid extraction kit instructions; this plasmid was GhLACS4-pCAMBIA3301 and stored at -20°C for later use.

[0080] The recombinant vector was transformed into Agrobacterium GV3101, and Arabidopsis thaliana was infected using the flower-dip method: when Arabidopsis thaliana reached its full flowering stage, the pods and open flowers were removed, and the plant was thoroughly watered 24 hours before infection; the Agrobacterium containing the target gene vector was activated by adding 100 μL to 1 mL of LB liquid medium (containing the corresponding antibiotics), and incubated overnight at 28℃ and 200 rpm until the OD600 reached 1.2-1.5 (orange juice color); for scale-up culture, 1 mL of the above bacterial solution was added to 50 mL of... LB liquid medium (containing the corresponding antibiotic) was cultured overnight at 28℃ and 200 r / min. The culture was stopped when the OD reached 600 to 0.8-1.2. The supernatant was discarded and the bacterial cells were collected after 5000 r / min for 10 min. The resuspension was prepared according to Table 3, and the bacterial cells were resuspended and allowed to stand for 4-5 h. AS was added and mixed before infection. The resuspension was poured into a petri dish, and Arabidopsis flower buds were immersed in the resuspension for about 45 s. The infected Arabidopsis plants were placed flat on a tray and treated in the dark for 24-36 h before being allowed to grow normally. One week later, the plants were infected again and cultured normally until the seeds matured.

[0081] Table 3

[0082]

[0083] After infection, mature Arabidopsis seeds were harvested and placed at 4°C for 15 days. The seeds were then evenly sown into nutrient soil and covered with plastic wrap until germination (approximately 3-4 days). A herbicide solution (750 μL of 10% Basta in 500 mL of water) was sprayed twice daily for one week, followed by 5-6 days of continued cultivation. Positive transgenic Arabidopsis showed green cotyledons and true leaves with larger leaves, while negative transgenic Arabidopsis showed yellowing and wilting cotyledons and true leaves. Healthy Arabidopsis seedlings were transplanted. After 3-4 weeks of normal growth, rosette leaves were harvested, DNA was extracted, and PCR identification was performed using specific primers, with wild-type Arabidopsis serving as a negative control. Based on the PCR 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 herbicides were continuously sprayed for screening. Leaf DNA was extracted for PCR testing, and homozygous lines were retained. Seeds were collected after normal growth.

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

[0086] Arabidopsis thaliana lacs4 mutants were planted according to method 1.3.1. After leaves emerged, rosette leaves were harvested and DNA was extracted. Primers were designed using the (TM-1, ZJU v2.1, GH_D03G0968) sequence as a reference, through the website http: / / signal.salk.edu / tdnaprimers.2.html. Homozygous mutants were identified using the three-primer method. Primer sequences are shown in Table 4.

[0087] Table 4

[0088]

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

[0090] Table 5

[0091]

[0092] The promoter sequence of the GhLACS4 gene (At-P-LACS4-F1 / R1) was amplified using the genome of *Gossypium uplandum* 901-001 as a template, and the CDS sequence (At-P-LACS4-F2 / R2) was amplified using *Gossypium uplandum* 901-001 cDNA as a template. The *Arabidopsis thaliana* complement vector WMV067 was digested with Sal I and BamHI restriction endonucleases (the promoter of the GhLACS4 gene replaced the 35S of the WMV067 vector). The amplified fragments and the digested vector were recovered by 1% agarose gel electrophoresis and ligated with homologous recombinase at 50℃ for 15 min. The ligation product was transformed into competent *E. coli* DH5α cells, and the selected positive clones were sent for sequencing. The plasmid of the correctly sequenced single-clone strain was extracted according to the procedures provided in the plasmid extraction kit; this plasmid was GhLACS4-WMV067 and stored at -20℃ for later use.

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

[0094] Obtaining stable transgenic Arabidopsis thaliana lines: The harvested seeds were planted and cultured, screened, and homozygous lines were retained. After normal planting and growth, the seeds were collected.

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

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

[0097] 2. Experimental Results and Analysis

[0098] 2.1GhLACS4 increases the content of yeast lipids.

[0099] To verify the function of the GhLACS4 gene in a fungal system, this 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 (a) Three positive clones were randomly selected for propagation and induction culture. Figure 1 (a)

[0100] Lipid content was determined in empty vector and recombinant yeast strains after induction culture. The results showed that the total lipid content of the transgenic yeast strain was significantly higher than that of the empty vector control group (P<0.001). Figure 1 (b) To further verify this result, Nile red fluorescence staining was used to visualize the lipids within yeast cells. Fluorescence microscopy showed that the area of ​​the fluorescently stained region in the transgenic yeast cells was significantly increased ( Figure 1 (c)

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

[0102] To verify the regulatory effect of GhLACS4 on seed oil content, a mutant reversion 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 for Arabidopsis thaliana lacs4 mutant replacement experiment. Figure 2 (a) The harvested seeds were sown on 1 / 2 MS medium containing spectinomycin for preliminary screening. Figure 2 (b) Further positive transgenic plants were obtained through PCR detection. Figure 2 (c) Positive plants were repeatedly self-crossed to obtain stable genetic lines for subsequent phenotypic identification.

[0103] Under the same environmental conditions, homozygous Arabidopsis thaliana lacs4 mutant lines, wild-type Arabidopsis thaliana, and Arabidopsis thaliana lacs4 mutant restorer lines were planted, and the oil content and fatty acid composition of Arabidopsis thaliana seeds were measured.

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

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

[0106] To study the function of the GhLACS gene, the GhLACS4 gene was ligated into an overexpression vector for Arabidopsis genetic transformation. Harvested seeds were evenly sown in nutrient soil, and green shoots emerged after 2-4 days. Subsequently, Basta herbicide was sprayed for selection. First, plants that could grow normally were selected. Figure 3 (a) Further PCR technology was used to identify positive seedlings, and transgenic positive seedlings were screened out. Figure 3 (b) After normal seed harvesting, continue generation culture to obtain stable transgenic Arabidopsis lines.

[0107] Stable transgenic Arabidopsis thaliana lines were planted alongside wild-type Arabidopsis thaliana under the same environmental conditions, and phenotypic observations were conducted. Subsequently, the oil content and fatty acid composition of the transgenic Arabidopsis seeds were determined. The results showed that, compared with wild-type Arabidopsis thaliana, the seed oil content of the transgenic lines was significantly increased by 3.99% (…). Figure 3 (c). Furthermore, 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] The long-chain fatty acid acyl-CoA synthase, a homolog of LACS4, is responsible for converting free fatty acids into acyl-CoA, participating in multiple pathways of lipid metabolism. The primary function of LACS is fatty acid transport. In *E. coli*, the single LACS enzyme FadD, along with the fatty acid transporter FadL, participates in the transport and activation of long-chain fatty acids. In yeast, short-chain and medium-chain free fatty acids can directly enter peroxisomes; these fatty acids cross the membrane partly because acyl-CoA synthase reactivates them into CoA thioesters. In *Arabidopsis thaliana*, LACS1 and LACS2 preferentially modify very long-chain fatty acids (VLCFAs) for wax synthesis, and long-chain fatty acids for keratin synthesis. On the other hand, LACS6 and LACS7 in peroxisomes play overlapping roles in fatty acid β-oxidation, which are crucial for seedling growth and development. Studies in Arabidopsis thaliana have shown that 18:2 released from PC on the endoplasmic reticulum membrane is converted into 18:2-CoA by LACS4 and LACS9, and then 18:2-CoA is immediately used as a substrate to resynthesize PC on the plastid membrane.

[0109] This invention expresses the GhLACS4 gene in *Saccharomyces cerevisiae*, resulting in significantly increased lipid droplets and a marked increase in TAG content. In *Arabidopsis thaliana* lacs4 mutant lines, seed oil content was significantly reduced, while the oil content in the replacement lines returned to normal levels. Furthermore, experiments with transgenic *Arabidopsis thaliana* showed that GhLACS4 significantly increased seed oil content and specifically increased the content of C18:2 (…). Figure 3(d). In Arabidopsis thaliana, 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 utilized more efficiently. This may be due to the higher activity of LACS6 and LACS7 for C20:1. The substrate preferences of different fatty acid enzymes lead to differences in the fatty acid composition of seeds. This invention shows that GhLACS4 mainly participates in fatty acid synthesis and affects oil accumulation in seeds by converting free fatty acid 18:2 into 18:2-CoA.

[0110] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of overexpression of the GhLACS4 gene or recombinant vectors containing the GhLACS4 gene in increasing the oil content of plant seeds, characterized in that, The nucleotide sequence of the GhLACS4 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; The plant in question is Arabidopsis thaliana.

2. A method for regulating the oil content of plant seeds, characterized in that, Overexpression of the GhLACS4 gene increases the oil content of plant seeds; The nucleotide sequence of the GhLACS4 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; The plant in question is Arabidopsis thaliana.

3. The application of the GhLACS4 gene or a recombinant vector containing the GhLACS4 gene in cultivating transgenic plant lines with increased seed oil content, characterized in that... The GhLACS4 gene is overexpressed in plants, the nucleotide sequence of which is shown in SEQ ID NO.1 and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2. The plant in question is Arabidopsis thaliana.

4. A method for cultivating transgenic plant lines with increased seed oil content, characterized in that, Includes the following steps: The GhLACS4 gene is introduced into the target plant to obtain the desired result. The nucleotide sequence of the GhLACS4 gene is shown in SEQ ID NO.1; The GhLACS4 gene was introduced into the target plant via a recombinant vector containing the GhLACS4 gene; the plant was Arabidopsis thaliana.

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