Proteins and encoding genes thereof for regulating sugar content, thousand kernel weight and oil content of plant seeds and uses thereof

Abstract

This invention discloses proteins that regulate sugar content, thousand-seed weight, and oil content in plant seeds, along with their encoding genes and uses. Specifically, it discloses the use of the protein CoSWEET1a (SEQ ID NO:2) and its encoding gene (SEQ ID NO:1) in regulating sugar content, starch content, oil content, size, and / or weight in plant seeds. This invention obtains these proteins through bioinformatics analysis and screening. CoSWEET1a Genes, and for the first time, CoSWEET1a This gene is used to breed plants with increased sugar, starch, and / or oil content in their seeds. Upregulating the content of the protein CoSWEET1a can increase the sugar, starch, oil content, size, and / or weight of plant seeds. This invention provides a valuable gene resource for breeding high-oil-content plant varieties and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to proteins that regulate sugar content, thousand-seed weight, and oil content in plant seeds, their encoding genes, and their uses. Background Technology

[0002] Camellia oleifera ( Camellia oleifera Camellia oleifera is an evergreen shrub or tree belonging to the genus Camellia in the family Theaceae. It is one of the most widely cultivated economic forest tree species in my country and a major woody oil crop. Camellia oil, produced from camellia oleifera, is rich in nutrients, including oleic acid and linoleic acid, and has a mellow flavor, making it a high-quality edible oil. It is also an excellent industrial raw material, used in soap making, cosmetics, rubber, and other industries. Currently, camellia oil production still faces the problem of low yield, with an average yield of only about 14 kg of seed oil per mu (approximately 0.16 acres), severely restricting the development of the camellia oil industry. As the main source of camellia oil, seeds suffer from poor development and abortion due to imbalances in seed supply and insufficient nutrient supply, ultimately leading to reduced yields.

[0003] Therefore, identifying genes that regulate oil accumulation and using genetic engineering or molecular breeding techniques to improve plant germplasm and promote efficient oil accumulation in plant seeds is of great significance for increasing the oil yield of oil crops such as camellia oleifera and cultivating high-oil plant varieties. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to regulate the sugar content, starch content, oil content, size and / or weight of plant seeds, and / or how to cultivate plants with altered seed sugar content, starch content, oil content, size and / or weight.

[0005] To address the aforementioned technical problems, the present invention first provides the use of proteins in any of the following: A1) Uses in regulating the sugar, starch and / or oil content of plant seeds; A2) Uses in regulating plant seed size and / or weight; A3) Use in cultivating plants with increased seed sugar, starch and / or oil content; A4) Use in cultivating plants with increased seed size and / or weight; A5) Uses in molecular breeding or germplasm improvement related to sugar content, starch content, oil content, size and / or weight of plant seeds; The protein (named CoSWEET1a) may be a protein with the amino acid sequence of SEQ ID NO:2, or a fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:2.

[0006] Furthermore, the connection can be made directly via peptide bonds or via a connector.

[0007] Furthermore, the tags include, but are not limited to, GST (glutathione thioredoxin) tag protein, Trx (thioredoxin) tag protein, His tag protein (His-tag), Strep tag protein, MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO (small molecule ubiquitin-like modified protein) tag protein, Myc tag protein, LacZ tag protein, CBD (cellulose-binding domain) tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein, or combinations of the above tag proteins.

[0008] Furthermore, the regulation described in A1) or A2) can be positive regulation.

[0009] Furthermore, the intended use can be achieved by increasing or decreasing the content of the protein CoSWEET1a.

[0010] Furthermore, the use may include increasing the sugar content, starch content, oil content, size, and / or weight of plant seeds by upregulating the content of the protein CoSWEET1a.

[0011] The present invention also provides the use of biological materials in any of the following: B1) Uses in regulating the sugar, starch and / or oil content of plant seeds; B2) Uses in regulating plant seed size and / or weight; B3) Use in cultivating plants with increased seed sugar, starch and / or oil content; B4) Use in cultivating plants with increased seed size and / or weight; B5) Uses in molecular breeding or germplasm improvement related to sugar content, starch content, oil content, size and / or weight of plant seeds; The biomaterial may be any of the following: C1) The nucleic acid molecule encoding the protein CoSWEET1a; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3); C5) A recombinant host cell containing the nucleic acid molecule described in C1), or a recombinant host cell containing the expression cassette described in C2), or a recombinant host cell containing the recombinant vector described in C3); C6) Transgenic plant tissue containing the nucleic acid molecules described in C1), or transgenic plant tissue containing the expression cassette described in C2); C7) A transgenic plant organ containing the nucleic acid molecule described in C1), or a transgenic plant organ containing the expression cassette described in C2).

[0012] Furthermore, all of the biological materials can express the nucleic acid molecules described in C1).

[0013] The expression cassette can refer to a nucleic acid construct containing sufficient nucleic acid elements to express the target gene. Expression cassettes typically contain a promoter, multiple cloning site (MCS), and / or terminator. Expression cassettes may also contain the target gene, marker genes (such as TK, DHFR, and CAT genes), transcription factor binding sites (TFBS), ribosome recognition and binding sites (SD), introns, enhancers, silencers, repressors, poly(A) signal sequences, and / or mRNA splicing signal sequences, etc.

[0014] In the above-mentioned biological materials, the recombinant vector described in C3) can be either a cloning vector or an expression vector.

[0015] Constructing cloning vectors is typically for the purpose of large-scale amplification of the target gene, sequencing, restriction enzyme digestion, long-term preservation of the target gene, and immediate use. Common cloning vectors include the pGEM series and pUC series vectors. Those skilled in the art can choose according to their needs.

[0016] The recombinant vector can be constructed using expression vectors (such as prokaryotic or eukaryotic expression vectors). The structure of the expression vector is known to those skilled in the art. Expression vectors typically contain elements required for target gene expression, such as promoters, multiple cloning sites, terminators, and ribosome binding sites. They may also contain selection marker genes (such as neomycin resistance gene neo, kanamycin resistance gene kanr, hygromycin resistance gene hyg, chloramphenicol resistance gene cat, and streptomycin resistance gene str). Expression vectors can be constructed using any method known in the art (such as recombination technology or synthetic technology) or can be commercially available. Those skilled in the art can choose a suitable expression vector as needed.

[0017] Expression vectors may include prokaryotic expression vectors and eukaryotic expression vectors. Prokaryotic expression vectors may be selected from Bacillus subtilis expression vectors (such as pHT01, pHT43), Escherichia coli expression vectors (such as pGEX series vectors, pET series vectors, pMAL series vectors), and Streptomyces expression vectors (such as pIJ702, pHJL197). Eukaryotic expression vectors may be selected from plant cell expression vectors, such as pCAMBIA series vectors, pBIN series vectors, pBI series vectors, pPZP series vectors, pGreen series vectors, and other plasmid vectors, as well as plant virus expression vectors. The plant virus expression vectors may be selected from tobacco mosaic virus (TMV) vectors, cowpea mosaic virus (CPMV) vectors, and cauliflower mosaic virus (CaMV) vectors.

[0018] Furthermore, the recombinant vector may be a recombinant expression vector obtained by cloning the coding gene of the protein CoSWEET1a described in this invention into an expression vector. Although the expression vector used in the embodiments provided in this invention is the pSuper1300 vector, this invention is not limited to this specific vector. Those skilled in the art can use other suitable expression vectors, as long as the vector can express the coding gene of the protein CoSWEET1a.

[0019] The microorganisms include bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasma, chlamydia, spirochetes, algae, etc. The bacteria may be derived from the genus *Bacillus* (…). Bacillus sp. (such as Bacillus), Escherichia coli ( Escherichia sp. (such as Escherichia coli), Erwinia spp. Erwinia sp. ), Agrobacterium ( Agrobacterium sp. (such as Agrobacterium tumefaciens), Pseudomonas spp. ( Pseudomonas sp. The viruses may include baculoviruses, rotaviruses, adenoviruses, adeno-associated viruses, retroviruses (such as lentiviruses), poxviruses, and herpes simplex viruses. The fungi may originate from the genus *Saccharomyces* (Yeast). Saccharomyces sp. (such as brewer's yeast, Pichia pastoris), Fusarium genus ( Fusarium sp. ), Rhizoctonia spp. Rhizoctonia sp. Verticillium ( Verticillium sp. ), Penicillium ( Penicillium sp. The actinomycetes may originate from the genus Streptomyces ( ). Streptomyces sp. (e.g., Streptomyces). The algae may originate from the phylum Cyanophyta (e.g., cyanobacteria), genus Aspergillus (e.g., *Cyanophyta*), or genus Aspergillus (e.g., *Cyanophyta*). Achnanthes sp. )wait.

[0020] The host cell, also known as the recipient cell, can refer to any type of cell that can be used to introduce the vector, such as microbial cells, plant cells, and animal cells. Suitable host cells are known in the art. The plant cell may be Arabidopsis thaliana (…). Arabidopsis thaliana ), camellia oil ( Camellia oleifera Abel), rapeseed ( Brassica napus ), rice ( Rice Plant cells, but not limited to these; animal cells may be mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), young hamster kidney cells (BHK cells)), insect cells (e.g., Sf21 cells, Sf-9 cells, or Hi-5 cells), avian cells (e.g., chicken or duck cells), fish cells (e.g., grass carp, carp, rainbow trout, or catfish cells), amphibian cells (e.g., African clawed frog cells), etc. Xenopus laevis ) cells or giant salamander ( Andrias davidianus (cells), etc.

[0021] In the above uses, the nucleic acid molecule described in C1) can be a DNA molecule whose coding sequence is SEQ ID NO:1.

[0022] The DNA molecule shown in SEQ ID NO:1 is CoSWEET1a The gene encodes DNA that encodes the protein CoSWEET1a, whose amino acid sequence is SEQ ID NO:2.

[0023] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID NO:1. Considering the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for expression in specific species as needed.

[0024] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as mRNA or hnRNA.

[0025] The sequence of the nucleic acid molecule encoding the protein CoSWEET1a can be either... CoSWEET1a The CDS sequence of a gene can also be CoSWEET1a The cDNA sequence of a gene can also be CoSWEET1a The genomic sequence of a gene, as long as that sequence can be transcribed and translated into the protein CoSWEET1a in an organism.

[0026] The present invention also provides a transgenic plant containing an inserted foreign gene, which may be the coding gene for the protein CoSWEET1a, and the transgenic plant having increased seed sugar content, starch content, oil content, size and / or weight compared to before the insertion of the foreign gene.

[0027] The gene encoding the protein CoSWEET1a described herein can be any gene capable of encoding the protein CoSWEET1a. In the art, those skilled in the art, knowing the amino acid sequence of a protein, can obtain the nucleotide sequence of the gene encoding that protein according to the rules of the genetic codon. Due to the degeneracy of the codon, the nucleotide sequence encoding the same amino acid sequence is not unique. For example, the nucleotide sequence of the gene encoding the protein CoSWEET1a may be as shown in SEQ ID NO:1.

[0028] The present invention also provides a method for cultivating plants with increased seed sugar content, starch content, oil content, size and / or weight, the method comprising increasing the content of the protein CoSWEET1a in the target plant to obtain plants with higher seed sugar content, starch content, oil content, size and / or weight than the target plant.

[0029] Increasing the level of protein CoSWEET1a (overexpression) can be achieved through regulation at the gene level (such as gene replication, transcription, translation, post-transcriptional modification, and / or post-translational modification) or by promoting or increasing the level of the target protein at the protein level. There are no particular limitations on the methods of overexpression, and many overexpression techniques are well-known to those skilled in the art. For example, the nucleic acid molecule encoding the protein to be overexpressed can be placed under the control of a strong promoter; the copy number of one or more genes encoding the protein can be increased; or the strength of the ribosome binding site or Kozak sequence can be increased, mRNA stability can be improved, codon usage can be altered, or repressive elements can be knocked out.

[0030] In the above method, increasing the content of the protein CoSWEET1a in the target plant can be achieved by increasing the expression level of the gene encoding the protein CoSWEET1a in the target plant.

[0031] In the above method, increasing the expression level of the gene encoding the protein CoSWEET1a in the target plant can be achieved by introducing the gene encoding the protein CoSWEET1a into the target plant.

[0032] In the above method, the nucleotide sequence of the gene encoding the protein CoSWEET1a can be as shown in SEQ ID NO:1.

[0033] The method described herein may include the following steps: S1) Construct a recombinant expression vector containing the coding gene of the protein CoSWEET1a; S2) Introduce the recombinant expression vector constructed in step S1) into the target plant; S3) Transgenic plants obtained through screening and identification are plants with increased seed sugar content, starch content, oil content, size and / or weight.

[0034] Further, the nucleotide sequence of the gene encoding the protein CoSWEET1a described in step S1) may be as shown in SEQ ID NO:1.

[0035] Furthermore, the methods for introduction described in step S2) include, but are not limited to, Agrobacterium-mediated transformation, electroporation, liposome-mediated transformation, chemical stimulation, plant virus vector-mediated transformation, gene gun transformation, microinjection, laser microbeam transformation, ultrasound transformation, pollen tube channel transformation, air gun transformation, and eddy current transformation.

[0036] Furthermore, the method of introduction can be Agrobacterium-mediated transformation.

[0037] Furthermore, the Agrobacterium-mediated method may include the following steps: introducing the recombinant expression vector constructed in step S1) into Agrobacterium (e.g., by electroporation transformation, polyethylene glycol-mediated transformation, Ca ion-induced transformation, metal cation-mediated transformation, etc.) to obtain recombinant Agrobacterium; infecting the callus or explant of the target plant with the recombinant Agrobacterium; and inducing and culturing the obtained positive callus or explant to obtain regenerated plants after identification.

[0038] The explant can refer to a part of a plant used as in vitro culture material in plant tissue culture, which, after appropriate treatment and under suitable conditions, can regenerate into a whole plant. Those skilled in the art can select suitable explants for transformation based on different plants. The explants include, but are not limited to, seeds, leaves, petioles, cotyledons, cotyledonary petioles, roots, hypocotyls, stem segments, shoot apical meristems, epidermal parenchyma cells, tubers, stolons, embryogenic suspension cells, and protoplasts.

[0039] The term "callus" refers to the new tissue that forms on the surface of a wound after a localized injury to the original plant. It consists of living parenchyma cells and can originate from living cells in various tissues within any organ of the plant. In plant tissue culture, it refers to a cluster of disordered, rapidly dividing parenchyma cells formed from an explant. Cultivating callus on a suitable culture medium can induce the formation of a whole plant.

[0040] The screening and identification methods are well known to those skilled in the art, such as PCR detection, immunoblotting, Southern hybridization, Northern hybridization, enzyme-linked immunosorbent assay (ELISA), functional identification (testing for the presence of selectable marker genes and target genes) and / or in situ hybridization, etc., to identify transformed transgenic plants (including transgenic progeny materials).

[0041] In this article, the plant may be Camellia oleifera, Arabidopsis thaliana, or rapeseed.

[0042] Furthermore, the rapeseed may be Brassica napus.

[0043] The protein CoSWEET1a described herein, or the biological material described herein, is also within the scope of protection of this invention.

[0044] In this article, the protein CoSWEET1a may be derived from camellia oleifera ( Camellia oleifera Abel).

[0045] In this article, the regulation of plant seed sugar content, starch content and / or oil content can be achieved by increasing or decreasing the plant seed sugar content, starch content and / or oil content.

[0046] In this article, the regulation of plant seed size and / or weight may refer to increasing or decreasing plant seed size and / or weight.

[0047] In this article, the sugar content may refer to the soluble sugar content.

[0048] In this article, the seed weight may be the weight of 1000 seeds.

[0049] In this document, the term "transgenic plant" is understood to include not only the first-generation transgenic plants obtained by introducing the gene encoding the protein CoSWEET1a into the target plant, but also its progeny. The transgenic plant includes cells, seeds, callus tissue, and complete plants.

[0050] The term "comprising" in this document is not intended to be restrictive, but rather inclusive and implies the possible presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially consisting of". The terms "comprising" and "including" in this document have the same meaning and are used interchangeably.

[0051] The present invention has the following beneficial effects: This invention obtains through bioinformatics analysis and screening. CoSWEET1a The gene was found to be highly expressed in camellia seeds, suggesting that it may play an important role in seed development.

[0052] Agrobacterium-mediated genetic transformation technology was used in Arabidopsis thaliana. atsweet1 Constructing from mutants CoSWEET1a Heterogeneous Restoration System ( CoSWEET1a-R ), constructed from wild-type camellia oil CoSWEET1a Overexpression lines ( CoSWEET1a-OEThe study found that sugar and fatty acid content were significantly increased in seeds of Arabidopsis restorer lines and rapeseed overexpression lines, and that embryos in Camellia oleifera seeds showed significant enlargement and increased sugar content. Transient overexpression and silencing lines (IL60-CoSWEET1a, TRV2-CoSWEET1a) were constructed in Camellia oleifera seeds. The study found that the soluble sugar content in the overexpression lines was significantly increased. However, after specifically inhibiting gene expression using virus-induced gene silencing (VIGS) technology, the soluble sugar content was significantly lower than that in the empty vector control and overexpression lines. This demonstrates that the gene is a positive regulator in the process of sugar accumulation in plants, and its functional verification system provides a novel molecular target for improving the quality and increasing the yield of Camellia oleifera seeds.

[0053] This invention discloses for the first time the use of the protein CoSWEET1a (SEQ ID NO:2) and its encoding gene in regulating the sugar content, starch content, oil content, size, and / or weight of plant seeds. Upregulating the content of protein CoSWEET1a (e.g., overexpression) can increase the sugar content, starch content, oil content, size, and / or weight of plant seeds. Downregulating the content of protein CoSWEET1a (e.g., silencing or knocking out) can decrease the sugar content, starch content, oil content, size, and / or weight of plant seeds. This invention provides a valuable gene resource for molecular breeding (e.g., breeding high-oil-content plant varieties) or germplasm improvement related to the sugar content, starch content, oil content, size, and / or weight of plant seeds, and has broad application prospects. Attached Figure Description

[0054] Figure 1 for CoSWEET1a Gene expression pattern analysis and structure prediction. Among them, Figure 1 A in the middle is CoSWEET1a Analysis of expression patterns in different tissues of Camellia oleifera. Figure 1 B is CoSWEET1a Prediction of transmembrane structure of encoded proteins.

[0055] Figure 2 To evaluate the effects of different sugar signaling treatments on wild-type Arabidopsis thaliana, atsweet1 mutants and CoSWEET1a The impact on the growth of restorer line seedlings. Among them, Figure 2 Figure A shows the phenotypic observation of different Arabidopsis thaliana seedlings under sugar treatment. Figure 2 In section B, the fresh weight of Arabidopsis thaliana seedlings of different strains under sugar treatment was determined and analyzed.

[0056] Figure 3 For wild-type Arabidopsis thaliana, atsweet1 mutants and CoSWEET1a Phenotypic observation and internal solubility analysis of restorer line seeds. Figure 3 A represents the observation of seed phenotype. Figure 3In section B, the determination of the thousand-seed weight, sugar content, starch content, and oil content of different strains is presented.

[0057] Figure 4 Camellia oleifera seeds CoSWEET1a Phenotypic and related trait analysis of transient overexpression and silencing. Among them, Figure 4 Figure A shows the phenotypic observation of the seed embryo. Figure 4 B is CoSWEET1a Gene expression level analysis. Figure 4 C represents the determination of seed sugar content.

[0058] Figure 5 Wild type of rapeseed and Brassica napus CoSWEET1a Seed phenotype observation of overexpression lines ( Figure 5 (A) Thousand-grain weight ( Figure 5 (B) and oil content ( Figure 5 (C) determination. Detailed Implementation

[0059] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0061] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0062] The pSuper1300 vector used in the following examples is described in the following literature: Zhou Jing, Du Bingshuai, Cao Yibo, Liu Kui, Ye Zhihua, Huang Yiming and Zhang Lingyun. Genome-wide identification of sucrose transporter genes in Camellia oleifera and characterization of CoSUT4. Journal of Integrative Agriculture. 2024, 7, 1-31. It is publicly available from the applicant for replication of the experiments described in this application.

[0063] The IL60-1, IL60-2, pTRV1, and pTRV2 vectors used in the following examples were kindly provided by Professor Hao Yujin of Shandong Agricultural University and are described in the following literature: An JP, Wang XF, Li Y Yet al. EIN3-LIKE1, MYB1, and ETHYLENE RESPONSE FACTOR3 act in a regulatory loop that synergistically modulates ethylene biosynthesis and anthocyanin accumulation[J]. PlantPhysiology, 2018, 178: 808-823. These vectors are available to the public from the applicant for replication of the experiments described in this application.

[0064] The wild-type Arabidopsis thaliana in the following examples ( Arabidopsis thaliana Col-0) and atsweet1 The Arabidopsis mutant seeds were purchased from the ABRC (Arabidopsis Biological Resource Center) website. atsweet1 The mutant seeds were sown, and after germination, seedlings were cultivated. DNA was then extracted from each seedling as a template for subsequent identification. Arabidopsis thaliana was found on the TAIR website. atsweet1 The primer sequences corresponding to the mutants, LP (5'-GTATGGACTTCCCTTTGTGTCA -3'), RP (5'-TTAAACTTGAAGGTCTTGCTTTCC -3'), and LB1.3 (5'-ATTTTGCCGATTTCGGAAC -3'), were used to perform PCR amplification using the extracted mutant DNA as a template via a three-primer method. Based on the PCR amplification results, homozygous mutant plants were selected for further cultivation. Only the seeds of the harvested homozygous mutant lines could be used for subsequent experiments.

[0065] The experimental results of the following examples are expressed as mean ± standard deviation. One-way ANOVA and t-test methods were used. P < 0.05 (*) indicates statistical difference, P < 0.01 (**) indicates statistically significant difference, and P < 0.001 (***) indicates extremely significant difference.

[0066] Example 1 CoSWEET1a Gene expression pattern analysis Using different tissues (flowers, stems, leaves, roots, and seeds) of Camellia oleifera 'Huashuo' at different developmental stages as materials, genes potentially involved in sugar transport in Camellia oleifera seeds were screened and identified. A gene involved in influencing sugar transport in Camellia oleifera seeds was discovered, named [gene name missing]. CoSWEET1a Analysis by real-time quantitative PCR CoSWEET1a Gene expression in different tissues includes the following steps: Prepare the following primer pairs: CoSWEET1a-qF: 5'-GGTGAAGCCAAGAAACCTCCT-3'; CoSWEET1a-qR: 5'-CTACTTGCTCGATCGCTTCTCT-3'.

[0067] CoGAPDH-qF: 5'- GGTGCCAAGAAGGTGGTAATA -3'; CoGAPDH-qR: 5'-GTTGTGCAGCTTGCATTAGAG-3'.

[0068] RNA was extracted from the flowers, stems, leaves, roots, and seeds of Camellia oleifera, and cDNA was synthesized using a reverse transcription kit. Then, using cDNA from fruits at different developmental stages as templates, PCR amplification was performed using the aforementioned RT-qPCR primers. CoGAPDH Genes are used as internal reference genes for detection. CoSWEET1a Gene expression in different tissues of Camellia oleifera. CoSWEET1a The expression level in the stem was normalized to 1.

[0069] result( Figure 1 The display shows that, based on the trend of expression level changes, CoSWEET1a The gene expression level was highest in seeds, 3.43 times that in flowers; followed by roots, 2.72 times that in flowers; and lowest in stems. This indicates that... CoSWEET1a Genes may play an important role in sugar absorption and transport in camellia seeds.

[0070] Example 2 CoSWEET1a Gene cloning and its sequence Using 5'-ATGGGTAATACTGCGCATTTCG-3' and 5'-CTACTTGCTCGATCGCTTCTCT-3' as primers and cDNA extracted from Camellia oleifera seeds as a template, the cDNA was cloned. CoSWEET1a A sequence of open reading frames. CoSWEET1aThe open reading frame sequence was ligated to the unique insertion site (DNA insert region) of the pTOPO-Blunt vector (purchased from Beijing Adley Biotechnology Co., Ltd., catalog number: CV16) to obtain the recombinant vector pTOPO-Blunt-CoSWEET1a, which was then sequenced. Sequencing results showed that *Camellia oleifera*... CoSWEET1a The coding sequence of the gene in the camellia oleifera variety 'Asus' is shown below: 5'--3' (SEQ ID NO:1).

[0071] CoSWEET1aThe gene encodes a protein named CoSWEET1a, and the amino acid sequence of protein CoSWEET1a is shown below: MGNTAHFVFGVFGNANGLLLFLAPTITFKRIIMSKSTEQFSGIPYVMTLLNCLLLSAWYGMPFVSPHNMLVSTINGTGAAIEAIYVFIFIIFAPKKEKGKILGLLIFVLSVFTAVALISMFALHGKNR KIFCGLAASIFSIIMYASPLSIMRMVIKTKSVEFMPFFLSLFVFLCGTSWFVFGLLGKDPFVAVPNGFGSGLGVIQLILYVIYRNNKGEAKKPPNADGSLEIGLEKSQPHEEKRSSYEEKRSSK (SEQ ID NO:2).

[0072] CoSWEET1a The genome sequence of the gene is shown in SEQ ID NO:3.

[0073] Example 3 CoSWEET1a The role of genes in regulating plant sugar accumulation This embodiment uses camellia fruit, Arabidopsis thaliana, and rapeseed as materials to... CoSWEET1a The function of genes is analyzed and verified.

[0074] I. Over-expression and silence CoSWEET1a Construction of recombinant vectors 1. Obtaining overexpression vectors Prepare the following primer pairs: CoSWEET1a-pSuper1300-F: 5'-GGGGCCCGGGGTCGACATGGGTGGCGTTGCAC-3'; CoSWEET1a-pSuper1300-R: 5'-CCATGGTACCGGATCCGCTAGACATGCCCGTTT-3'.

[0075] PCR amplification was performed using the recombinant vector pTOPO-Blunt-CoSWEET1a as a template to obtain PCR products. The PCR products and pSuper1300 vector were digested with XbaI and SmaI restriction enzymes, respectively, and then recovered from the gel. The two were ligated using DNA ligase, and the ligation product was transformed into *E. coli* DH5α competent cells and cultured overnight at 37°C. Positive single clones were detected by PCR and sequenced. After verification, the results were obtained. CoSWEET1a The gene overexpression vector was named pSuper1300-CoSWEET1a.

[0076] The recombinant vector pSuper1300-CoSWEET1a is the vector shown in SEQ ID No:1. CoSWEET1a The gene was inserted between the XbaI and SmaI restriction sites of the expression vector pSuper1300, while keeping the other sequences of the vector pSuper1300 unchanged, to obtain a recombinant vector. The pSuper1300-CoSWEET1a vector can express the protein CoSWEET1a with the amino acid sequence SEQ ID No:2.

[0077] 2. Obtaining the IL60-2 recombinant viral vector Prepare the following primer pairs: IL60-CoSWEET1a-F: 5'-GTCGACAAGCTTCTCGAGATGGGTAATACTGCGC-3'; IL60-CoSWEET1a-R: 5'-CCACACGTGTGGTCTAGACTACTTGCTCGATCGC-3'.

[0078] PCR amplification was performed using the recombinant vector pTOPO-Blunt-CoSWEET1a as a template to obtain the PCR product. The PCR product was then ligated with the recovered product of the IL60-2 vector, which had been digested with the same restriction enzyme. The ligation product was transformed into *E. coli* DH5α competent cells and cultured overnight at 37°C. Positive single clones were detected by PCR and sequenced. The recombinant viral vector was subsequently named IL60-CoSWEET1a.

[0079] The recombinant viral vector IL60-CoSWEET1a is the vector shown in SEQ ID No:1. CoSWEET1a The recombinant vector was obtained by inserting the gene between the XhoI and XbaI restriction sites of the viral vector IL60-2 while keeping other sequences of the vector IL60-2 unchanged.

[0080] Recombinant vector IL60-CoSWEET1a is used for CoSWEET1a Transient overexpression of genes.

[0081] 3. Obtaining the pTRV2 recombinant viral vector Prepare the following primer pairs: pTRV2-CoSWEET1a-F: 5'- GGTTACCGAATTCTATAGTGTTGCTGTTTTTGGCAC -3'; pTRV2-CoSWEET1a-R: 5'-TAGAGACGCGTGAGCTCGACGCTGAGCACAAAGATG-3'.

[0082] PCR amplification was performed using the recombinant vector pTOPO-Blunt-CoSWEET1a as a template to obtain the PCR product. The PCR product was then ligated with the recovered product of the pTRV2 vector, which had been digested with the same restriction enzyme. The ligation product was transformed into *E. coli* DH5α competent cells and cultured overnight at 37°C. Positive single clones were detected by PCR and sequenced. The recombinant viral vector obtained after verification was named pTRV2-CoSWEET1a.

[0083] The recombinant viral vector pTRV2-CoSWEET1a is a recombinant vector obtained by inserting a 266 bp DNA positive-sense fragment with gene specificity from position 187 to position 452 of SEQ ID No:1 between the XbaI and KpnI restriction sites of the viral vector pTRV2, while keeping the other sequences of the vector pTRV2 unchanged.

[0084] Recombinant vector pTRV2-CoSWEET1a is used for CoSWEET1a Gene silencing.

[0085] two, CoSWEET1a Determination of inclusions in heterologous replenished Arabidopsis seeds 1) CoSWEET1a Obtaining heterologous Arabidopsis thaliana lines The recombinant vector pSuper1300-CoSWEET1a was transformed into Agrobacterium GV3101. The recombinant bacteria that was verified by bacterial culture PCR was named GV3101 / pSuper1300-CoSWEET1a.

[0086] Recombinant bacteria GV3101 / pSuper1300-CoSWEET1a were inoculated into YEB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, respectively, and cultured overnight at 28°C with shaking. The bacteria were collected by centrifugation at 6000 rpm for one minute. The resulting Agrobacterium pellet was thoroughly mixed with transformation buffer and resuspended at OD200. 600 = Around 1.0.

[0087] Arabidopsis thaliana was infected using the Agrobacterium-mediated inflorescence immersion method. atsweet1 The mutant strain was obtained using the recombinant strain GV3101 / pSuper1300-CoSWEET1a. It was then inoculated onto MS medium containing 25 mg / L hygromycin for selection until stably transformed plants were obtained.

[0088] Wild-type strains (WT, wild-type Arabidopsis thaliana) and atsweet1 The mutant was used as a control; the line stably transformed with pSuper1300-CoSWEET1a was used as a control. CoSWEET1aHeterogeneous replacement lines, whose test lines are marked as CoSWEET1a- R Seeds from contemporary transgenic Arabidopsis plants (T1 generation) were harvested and screened on MS medium containing kanamycin (50 ug / mL). The plants were then passaged until homozygous T3 generation transgenic plants were obtained. CoSWEET1a Arabidopsis thaliana plant seeds. Three independent, stable, homozygous plants were selected for the experiment. CoSWEET1a -R2 and CoSWEET1a -R6 T3 generation lines were used for the following phenotypic experiments, wild-type lines (WT) and atsweet1 The mutant was used as a control.

[0089] 2) CoSWEET1a Molecular detection of transgenic Arabidopsis thaliana Extract separately CoSWEET1a Transgenic Arabidopsis thaliana T3 generation plants ( CoSWEET1a -R2、 CoSWEET1a -R6, wild-type strains (WT) and atsweet1 The mutant RNA was reverse transcribed into cDNA. Then, using the cDNA from each of the aforementioned strains as templates, PCR amplification was performed with 5'-GGTGAAGCCAAGAAACCTCCT-3' and 5'-CTACTTGCTCGATCGCTTCTCT-3' RT primers. Detection was then performed. CoSWEET1a The expression of [a specific substance] in transgenic plants.

[0090] 3) CoSWEET1a Determination of fresh weight of transgenic Arabidopsis seedlings Seed sterilization: Place Arabidopsis seeds in a 1.5 mL centrifuge tube, add an appropriate amount of 75% ethanol, and shake the centrifuge tube up and down for 5 minutes; then aspirate the 75% ethanol, add an appropriate amount of 95% sodium hypochlorite, and shake the centrifuge tube up and down for 20 minutes.

[0091] Seed washing: In a clean bench, aspirate 95% sodium hypochlorite, add an appropriate amount of high-temperature and high-pressure sterilized distilled water, and shake the centrifuge tube to wash the seeds; aspirate the distilled water, add an appropriate amount of distilled water again, and shake to wash the seeds; repeat the distilled water washing step 7 times.

[0092] Seed sowing: Aspirate seeds into a 1 mL pipette tip and gently sow them onto MS solid medium containing different types of sugars (sucrose, glucose, fructose). Clearly label the seed type on the culture dish, seal the dish with sealing film, remove it from the laminar flow hood, wrap it in newspaper (to create a dark environment), and place it upright in a 4°C refrigerator (simulating vernalization). After 3 days, remove it and individually place the culture dishes into a constant temperature and light incubator to allow seeds to germinate. The incubator temperature is 22°C, with a light duration of 16 hours, a dark duration of 8 hours, and a relative humidity of 65%. After 2 weeks of culture, observe the phenotypic characteristics and photograph the seeds. Fresh weight detection: Use tweezers to separate the wild-type Arabidopsis thaliana seeds... atsweet1 mutants and CoSWEET1a The seedlings of the replanted line were completely removed along with their roots, laid flat on weighing paper, and their weight was accurately read using a balance with a precision of 0.01%. Each group was repeated ten times, and the average value was recorded and calculated. One-way ANOVA was performed using Duncan (p=0.05), and the graphs were plotted using Sigmaplot 10.0.

[0093] The results are as follows Figure 2 As shown, there were no significant differences in growth and fresh weight among different strains on sugar-free medium. After growth on MS medium with 1.5% sucrose, CoSWEET1a_R6 showed an increase in fresh weight compared to other strains. atsweet1 The growth rate increased by 8.41%; however, after growing on a medium with a high concentration of sucrose, the leaves of R2 and R6 darkened, the plants became smaller, and their fresh weight decreased by 10.99% and 14.91% respectively compared to the mutants. After growth on MS medium with 1.5% glucose, the leaf area and growth vigor of the restorer lines were significantly better than those of the wild type and mutants. Fresh weight analysis showed that the fresh weight of the restorer lines grown on low-concentration glucose medium was not only higher than that of the wild type and mutants, but also higher than that of the restorer lines grown on low-concentration sucrose and fructose media. Specifically, the fresh weight of R2 was 1.28 times that of the mutant under the same treatment, and the fresh weight of R6 was 1.32 times that of the mutant under the same treatment. However, after growth on high-concentration glucose medium, the restorer lines were most significantly inhibited, with reduced leaf area and inhibited germination of some seeds. The fresh weight of R2 seedlings was 37.42% lower than that of the mutant under the same treatment, and the fresh weight of R6 seedlings was 41.54% lower than that of the mutant under the same treatment. After growth on MS medium with 1.5% fructose, the growth of the restorer lines was slightly better than that of the wild type and mutants, with their fresh weight increasing by 14.13% and 9.65% respectively compared to the mutants. Compared with sucrose and glucose, high concentrations of fructose had a more significant inhibitory effect on all lines. Among the various lines, the restorer lines were the most inhibited, and the fresh weight of the two restorer lines was significantly lower than that of the wild type and mutants.

[0094] 4) CoSWEET1a Determination of Inclusion Content in Seeds of Heterogeneous Recombinant Lines Seeds were collected and dried at the maturity stage of various Arabidopsis lines for photographic observation. Observation results under a stereomicroscope showed ( Figure 3 (A) CoSWEET1a The seeds of the two restorer lines were significantly larger than those of the wild-type and mutant lines. Based on these observations, further studies were conducted on the wild-type lines (WT), atsweet1 mutants and CoSWEET1a-R The contents of the seeds were determined.

[0095] Soluble sugar content determination: The sugar content extraction kit (visible spectrophotometry) (Beijing Solarbio Science & Technology Co., Ltd., catalog number BC2710) was used for determination. The specific steps were as follows: 0.1 g of sample was thoroughly ground with liquid nitrogen and placed in a pre-chilled 2 mL centrifuge tube. 1 mL of 80% ethanol was added, and the mixture was placed in an 80℃ water bath for 10 min. After centrifugation, the supernatant was collected into a new centrifuge tube. A small amount of carbon powder was added to the centrifuge tube, and the mixture was placed in an 80℃ water bath for decolorization for 30 min. The tube was then centrifuged at 5000 rpm for 10 min at room temperature, and the supernatant was collected for sugar content determination. Sample addition and calculations were performed according to the corresponding kit instructions. All assays were performed in triplicate.

[0096] Starch content determination: The starch content extraction kit (visible spectrophotometry) (Beijing Solarbio Science & Technology Co., Ltd., catalog number BC0700) was used for determination. The specific steps were as follows: 0.03 g of sample was thoroughly ground with liquid nitrogen and placed in a pre-cooled 2 mL centrifuge tube. 0.6 mL of reagent 1 was added and thoroughly mixed, then the mixture was placed in an 80℃ water bath for 30 min. After centrifugation, double-distilled water was added to the precipitate for high-temperature gelatinization, followed by the addition of 0.6 mL of reagent 2, and the mixture was reacted again at high temperature for 15 min. After high-speed centrifugation, the supernatant was collected for determination. Sample addition and calculations were performed according to the kit instructions, and absorbance values ​​were measured at 620 nm. All assays were performed in triplicate.

[0097] Oil content determination: The oil content of mature seeds was determined using Soxhlet extraction. Seed samples were dried to constant weight, ground into powder, and an appropriate amount was weighed. Petroleum ether was used as the extraction solvent, and extraction was performed by continuous reflux in a Soxhlet extractor until constant weight. The oil content was calculated based on the mass difference of the sample before and after extraction. The samples were tested by Beijing Yangou Technology Co., Ltd. All tests were performed in triplicate.

[0098] Experimental results are as follows Figure 3 As shown in B: Compared to WT, CoSWEET1a-R The soluble sugar content was significantly higher than that of the wild type and mutants, compared to atsweet1 The mutant strain showed a 28.96% higher level of starch content; further analysis of the starch content in seeds from different strains revealed...CoSWEET1a-R The starch content in it was also significantly higher than that in the wild type and the mutant, exceeding the mutant by 35.21%. Additionally, CoSWEET1a After restoration of expression in a homologous Arabidopsis mutant, the oil content of mature seeds also increased significantly. In summary, CoSWEET1a It has a positive effect on increasing plant yield.

[0099] III. Transient Overexpression and Silence CoSWEET1a Phenotypic observation of camellia seeds 1) Virus-induced transient overexpression and silencing CoSWEET1a Obtaining Camellia oleifera fruit Vectors pTRV1, pTRV2, and recombinant vector pTRV2-CoSWEET1a were transformed into competent Agrobacterium GV3101 cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.) to obtain recombinant bacteria GV3101 / pTRV1, GV3101 / pTRV2, and GV3101 / pTRV2-CoSWEET1a. The recombinant bacteria were mixed with 50% glycerol at a 1:1 ratio, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0100] Single clones of GV3101 / pTRV1, GV3101 / pTRV2, and GV3101 / pTRV2-CoSWEET1a were inoculated into YEB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, respectively, and cultured overnight at 28°C with shaking. The bacteria were collected by centrifugation at 6000 rpm for one minute. The resulting Agrobacterium pellet was thoroughly mixed with transformation buffer and resuspended at OD200. 600 =Approximately 1.0. The resuspended GV3101 / pTRV1 bacterial solution was mixed with an equal volume of GV3101 / pTRV2 bacterial solution or recombinant bacteria GV3101 / pTRV2-CoSWEET1a, and injected into Camellia oleifera seeds respectively, and cultured in the dark for 10 days.

[0101] The plasmids of vector IL60-1 and IL60-2 or the recombinant vector IL60-CoSWEET1a were mixed in equal volumes and added to transformation buffer. The mixture was then injected into Camellia oleifera seeds and cultured in the dark for 10 days.

[0102] The strain that transiently transforms IL60-1+IL60-CoSWEET1a is CoSWEET1aThe overexpression lines were designated as IL60-CoSWEET1a (also denoted as pIR-CoSWEET1a); the lines transiently transformed with GV3101 / pTRV1+GV3101 / pTRV2-CoSWEET1a were designated as CoSWEET1a silencing lines, and their test lines were designated as TRV-CoSWEET1a; the lines transiently transformed with IL60-1+IL60-2 and GV3101 / pTRV1+GV3101 / pTRV2 were designated as control lines, and their designations were pIR and TRV, respectively.

[0103] CoSWEET1a Phenotypic observation results of Camellia oleifera seed embryos with transient gene overexpression and silencing are as follows: Figure 4 As shown in Figure A, the results indicate that the embryo volume in the overexpression line was significantly larger than that in the wild-type and silent lines, while the transiently silent line showed the opposite, with a significantly smaller embryo volume than the wild-type. This suggests that... CoSWEET1a Genes positively regulate the embryonic development of Camellia oleifera seeds.

[0104] 2) CoSWEET1a The expression of genes in various lines of transiently transformed Camellia oleifera seeds is as follows: Figure 4 As shown in Figure B. The results indicate that in the pIR-CoSWEET1a line... CoSWEET1a Gene expression was significantly higher in TRV-CoSWEET1a than in the pIR empty vector control line; while in TRV-CoSWEET1a... CoSWEET1a Gene expression was suppressed, significantly lower than in the TRV empty vector control line. Therefore, this transgenic material can be used for subsequent functional verification.

[0105] 3) CoSWEET1a promotes the accumulation of soluble sugars in transgenic camellia seeds. Ten days after injection, seeds of pIR, pIR-CoSWEET1a, TRV, and TRV-CoSWEET1a that were in good growth condition and did not wilt were selected for soluble sugar content determination (the determination method is the same as that for Arabidopsis seeds).

[0106] Experimental results are as follows Figure 4 As shown in C, overexpression CoSWEET1a Following gene administration, the soluble sugar content in seeds showed an increasing trend, increasing by 22.35% compared to the empty control. Conversely, silencing... CoSWEET1a After gene therapy, the soluble sugar content decreased by 33.16%, and the differences were statistically significant. In summary, CoSWEET1a Genes can significantly promote the accumulation of soluble sugars in camellia seeds.

[0107] IV. Overexpression CoSWEET1a Phenotypic observation of Brassica napus seeds 1) Stable overexpression CoSWEET1aObtaining Brassica napus plants Single clones of recombinant bacteria GV3101 / pSuper1300-CoSWEET1a were inoculated into YEB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, respectively, and cultured overnight at 28°C with shaking. The bacteria were collected by centrifugation at 6000 rpm for one minute. The resulting Agrobacterium pellet was thoroughly mixed with transformation buffer and resuspended to OD. 600 = Around 1.0.

[0108] Rapeseed was transformed using Agrobacterium-mediated hypocotyl infection, with the recombinant strain GV3101 / pSuper1300-CoSWEET1a used. The prepared Agrobacterium culture was centrifuged at 5000 rpm for 8 min, and the supernatant was discarded. The bacterial cells were resuspended in sterile DM resuspension (containing 100 mM AS) in a clean bench, and the OD of the culture was adjusted. 600 The value is approximately 0.1. Pour about 20 mL of DM liquid medium into a petri dish for later use. Using scissors, cut the hypocotyl 1 cm above the root and 1 cm below the cotyledon. Cut the obtained hypocotyl into 0.8-1 cm segments at an angle with a scalpel and place them in the DM liquid medium to prevent dehydration and wilting. Filter the DM liquid through sterilized gauze. Pour Agrobacterium resuspension into an empty petri dish and incubate for 15 min in the dark, shaking the dish every 1 min. Then, aspirate the inoculum with a pipette tip and use tweezers to evenly place the hypocotyls in M1 solid medium, 20-30 hypocotyls per dish, and incubate in the dark for 48 h. After two days of total incubation, transfer the uncontaminated Agrobacterium hypocotyls from M1 to M2 selection medium in a clean bench at 25℃, with 16 h of light and 8 h of darkness per day, changing the medium every 20 days. After 21 days, the cells were transferred to M3 medium with added hygromycin resistance, and the subculture cycle was 21 days until green leaves appeared.

[0109] The callus tissue was removed from the germinated hypocotyls, and surgical incisions were made at the stem base. The seedlings were transferred from M3 to M4 rooting medium. After rooting, the culture bottles were opened for 6 hours to harden the seedlings. The culture medium was then removed from the roots in warm water, and excess water was removed before transplanting into a 1:2 mixture of potting soil and vermiculite. Newly transplanted seedlings were covered with plastic wrap for 3 days to allow them to recover. The plants were then placed in an artificial climate chamber at 24°C, 80% humidity, with 16 hours of light and 8 hours of darkness. After harvesting mature seeds, they were screened and subcultured on MS medium containing kanamycin (50 ug / mL) until homozygous transgenic seeds were obtained. CoSWEET1aRapeseed seeds were then compared with wild-type lines (WT) as controls; the line stably transformed with pSuper1300-CoSWEET1a was... CoSWEET1a The overexpression lines, whose test line markers are: CoSWEET1a-OE , used for the following phenotypic experiments.

[0110] 2) Overexpression CoSWEET1a Phenotypic observation and thousand-seed weight determination of rapeseed seeds Select wild-type cells in good condition and overexpressing them. CoSWEET1a 1000 seeds of each homozygous rapeseed line were spread evenly on a petri dish with a diameter of 20 cm, and the plumpness of the seeds was determined by the area occupied. Then, 1000 seeds were spread evenly on weighing paper, and the weight of the seeds was accurately read using a balance with a precision of 0.01%. Each group was repeated 5 times, and the average value was recorded and calculated. One-way ANOVA was performed using Duncan (p=0.05), and Sigmaplot 10.0 was used for plotting.

[0111] 3) Overexpression CoSWEET1a Promotes the determination of fatty acid content in rapeseed seeds For further testing CoSWEET1a Regarding the effect on seed yield in plants, this embodiment harvested mature seeds from overexpression line OE-8 / 9 and wild-type (WT) plants after the rapeseed pods were fully mature. After natural air drying and removal of impurities, the thousand-seed weight and oil content of wild-type WT and overexpression lines were measured (the measurement method is the same as that used for measuring oil content in Arabidopsis seeds).

[0112] Experimental results are as follows Figure 5 As shown, the seeds of the overexpression lines are more plump ( Figure 5 In the overexpression strain (A), the cumulative coverage area of ​​1000 seeds was greater than that of the wild type (approximately 2.86 g per thousand seeds), while the average weight of the overexpression lines was 3.48 g per thousand seeds. There was no significant difference in the weight of the thousand seeds among the overexpression lines. CoSWEET1a It can increase the thousand-seed weight of mature rapeseed by 21%. Figure 5 (B). Simultaneously, the oil content of mature seeds was measured, revealing a significant increase in oil content in the overexpression lines. The oil content of the WT lines was approximately 37.21%, while the oil content of mature seeds from the OE-8 / 9 overexpression lines was 41.41% and 44.31%, respectively, representing increases of 11.3% and 19%, respectively. Figure 5 (C) In summary, CoSWEET1a Genes influence the soluble sugar and starch content of rapeseed seeds and play a positive regulatory role in seed weight and oil synthesis.

[0113] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The uses of proteins in any of the following: A1) Uses in regulating the sugar, starch and / or oil content of plant seeds; A2) Uses in regulating plant seed size and / or weight; A3) Use in cultivating plants with increased seed sugar, starch and / or oil content; A4) Use in cultivating plants with increased seed size and / or weight; A5) Uses in molecular breeding or germplasm improvement related to sugar content, starch content, oil content, size and / or weight of plant seeds; The protein is a protein with the amino acid sequence of SEQ ID NO:2, or a fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:

2.

2. Uses of biomaterials in any of the following: B1) Uses in regulating the sugar, starch and / or oil content of plant seeds; B2) Uses in regulating plant seed size and / or weight; B3) Use in cultivating plants with increased seed sugar, starch and / or oil content; B4) Use in cultivating plants with increased seed size and / or weight; B5) Uses in molecular breeding or germplasm improvement related to sugar content, starch content, oil content, size and / or weight of plant seeds; The biomaterial is any one of the following: C1) A nucleic acid molecule encoding the protein described in claim 1; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3); C5) A recombinant host cell containing the nucleic acid molecule described in C1), or a recombinant host cell containing the expression cassette described in C2), or a recombinant host cell containing the recombinant vector described in C3); C6) Transgenic plant tissue containing the nucleic acid molecules described in C1), or transgenic plant tissue containing the expression cassette described in C2); C7) A transgenic plant organ containing the nucleic acid molecule described in C1), or a transgenic plant organ containing the expression cassette described in C2).

3. The use according to claim 2, characterized in that, C1) The nucleic acid molecule described is a DNA molecule whose coding sequence is SEQ ID NO:

1.

4. A genetically modified plant, characterized in that, The transgenic plant contains an inserted foreign gene, which is the gene encoding the protein described in claim 1. Compared with the transgenic plant before the insertion of the foreign gene, the seed sugar content, starch content, oil content, size and / or weight of the transgenic plant are increased.

5. A method for cultivating plants with increased seed sugar content, starch content, oil content, size, and / or weight, characterized in that, The method includes increasing the content of the protein described in claim 1 in the target plant to obtain a plant with higher seed sugar content, starch content, oil content, size and / or weight than the target plant.

6. The method according to claim 5, characterized in that, The increase in the protein content of the target plant as described in claim 1 is achieved by increasing the expression level of the gene encoding the protein in the target plant.

7. The method according to claim 6, characterized in that, The improvement in the expression level of the protein-coding gene in the target plant is achieved by introducing the protein-coding gene of claim 1 into the target plant.

8. The method according to claim 7, characterized in that, The nucleotide sequence of the gene encoding the protein is shown in SEQ ID NO:

1.

9. The use according to any one of claims 1-3, or the transgenic plant according to claim 4, or the method according to any one of claims 5-8, characterized in that, The plant in question is Camellia oleifera, Arabidopsis thaliana, or rapeseed.

10. The protein of claim 1, or the biomaterial of claim 2 or 3.

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