Plant oil content-related proteins and their related biomaterials and applications

Abstract

This invention relates to the field of biotechnology, and discloses the protein IpSTP5, which is related to the oil content of *Vernicia fordii* fruit, and its related biomaterials and applications. Specifically, the IpSTP5 protein can be one of the following: A1), A2), or A3): A1) a protein whose amino acid sequence is SEQ ID No. 2 in the sequence listing; A2) a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues to the protein of A1), possessing more than 90% identity and the same activity as the protein shown in A1); A3) a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2). The IpSTP5 protein and its related biomaterials can be used to positively regulate the oil content of *Vernicia fordii* fruit.

Description

Technical Field

[0001] This invention relates to plant oil content-related proteins and their related biomaterials and applications in the field of biotechnology. Background Art

[0002] Edible oil is a vital strategic resource for the national economy and people's livelihood, and an important component of national food security. The international grain and oil trade situation is complex and volatile. Especially under the current new situation of continuous international disputes, the international situation for edible oil is very severe. Globally, the area planted with oil crops remains unchanged, production does not increase, demand rises, and prices rise. The most obvious example is that prices in 2021 increased by 65.8% compared to 2020.

[0003] The Chinese tung tree, also known as the mountain fir, half-frost red, oil grape, chair tung, and water winter melon, originated in the Tertiary period of the Cenozoic era, making it an ancient tree species dating back 60 million years. It is a deciduous, light-loving, fast-growing tree, typically 10-20 meters tall, with the tallest known tree exceeding 20 meters. The flowers are unisexual, with some plants being dioecious, though monoecious species are occasionally found on the same tree. It boasts a beautiful tree shape, abundant flowers and foliage, and pearl-like fruit. It can withstand temperatures as low as -14℃ and as high as 40℃, and is tolerant of pH levels from 4.5 to 8.5. It is also tolerant of poor soil and highly adaptable, earning it the title of "China's Miracle Tree" from plant experts. The Chinese tung tree is cold-hardy, heat-tolerant, drought-tolerant, and tolerant of poor soil conditions; it can be planted almost anywhere trees can grow, even in rocky crevices, making it suitable for nationwide promotion and large-scale afforestation.

[0004] The fruit of the *Vernicia fordii* is rich in oil, earning it the nickname "oil depot on the tree." During peak production, a single tree yields an average of 10-30 kg of fresh fruit, with yields exceeding 50 kg per tree during the high-yield period, and sometimes reaching 150-200 kg. Based on 50 trees per mu (approximately 0.16 acres) and 50 kg per tree, the yield of fresh fruit per mu can conservatively be over 1 ton. Studies show that the oil yield of fresh *Vernicia fordii* fruit is 15%-23%, the oil yield of dried fruit is 22%-26%, and the oil content of the pulp is 43.6%, with an average oil content of 36.3%. Given the high oil content of fresh *Vernicia fordii* fruit (15-23%), identifying and characterizing its functional genes is increasingly important for further improving oil production. Currently, the transformation system for *Vernicia fordii* is still immature, making the verification of gene functions within the fruit itself difficult.

[0005] Virus-induced gene silencing (VIGS) technology involves inserting a target gene fragment into the viral genome. After infecting plant tissues, this leads to the degradation of host homologous RNA, resulting in post-transcriptional gene silencing and allowing for rapid verification of target gene function. The VIGS system offers numerous advantages, including ease of operation, short cycle time, and avoidance of plant transformation, and has been widely used in plant gene function identification research. Therefore, the VIGS method is proposed for rapid and efficient verification of *Vernicia fordii* gene function and its application in actual *Vernicia fordii* production. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention first provides a protein named IpSTP5, which is a protein as follows (A1), A2), or A3):

[0007] A1) The amino acid sequence of this protein is SEQ ID No. 2;

[0008] A2) A protein derived from A1) or having more than 80% identity with and the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1).

[0009] A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1), A2) or A3).

[0010] Of the proteins mentioned above, protein IpSTP5 may be derived from the fruit of the privet tree.

[0011] Of the proteins mentioned above, SEQ ID No. 2 in the sequence listing consists of 507 amino acid residues.

[0012] The term "more than one amino acid residue" as mentioned above can specifically refer to up to ten amino acid residues.

[0013] In the above applications, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity.

[0014] This invention also protects biological materials associated with the protein IpSTP5, said biological material being any one of B1) to B5) below:

[0015] B1) Nucleic acid molecules encoding IpSTP5;

[0016] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0017] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1);

[0018] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0019] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3).

[0020] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0021] In the above-mentioned biological materials, the nucleic acid molecule described in B1) is a cDNA molecule or a DNA molecule whose coding sequence is the nucleotide of SEQ ID No. 1.

[0022] Of these, SEQ ID No. 1 in the sequence listing consists of 1524 nucleotides and encodes the protein shown in SEQ ID No. 2.

[0023] In the aforementioned biological materials, the expression cassette (IpSTP5 gene expression cassette) containing the nucleic acid molecule described in B2) refers to a nucleic acid molecule capable of expressing IpSTP5 in host cells. This nucleic acid molecule may include not only a promoter to initiate IpSTP5 gene transcription but also a terminator to terminate IpSTP5 transcription. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; wound-inducible promoters from tomatoes, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology 120:979-992); chemically inducible promoters from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both can be induced by jasmonic acid methyl ester); heat shock promoter (US Patent 5,187,267); tetracycline-inducible promoters (US Patent 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 20071)). 0099169.7), seed storage protein-specific promoters (e.g., promoters of bean globular protein, napin, oleosin, and soybean beta conglycin (Beachy et al. (1985) EMBO J.4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator (see, e.g., Odell et al. (I 985Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res. 15:9627.

[0024] In the aforementioned biological materials, the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi.

[0025] The present invention also provides a substance for regulating the activity or content of the protein IpSTP5, or an application of a substance for regulating the expression of the gene encoding the protein IpSTP5, wherein the application is any one of the following:

[0026] P1. Application in regulating the oil content of *Vernicia fordii* fruit;

[0027] P2. Application in the preparation of products that regulate the oil content of *Vernicia fordii* fruit;

[0028] P3. Application in the breeding of *Vernicia fordii*.

[0029] In the above applications, the protein IpSTP5 can be derived from the fruit of the privet tree.

[0030] In the above applications, the gene encoding the protein IpSTP5 can be a DNA molecule as shown in a1), a2), or a3) below:

[0031] a1) The coding sequence is the DNA molecule shown in SEQ ID No. 1 of the sequence listing;

[0032] a2) has 90% or more identity with the nucleotide sequence defined in a1) and encodes a DNA molecule that encodes the protein IpSTP5 described above.

[0033] a3) hybridizes under strict conditions to the nucleotide sequence defined by a1) or a2) and encodes the DNA molecule containing the protein IpSTP5 described above.

[0034] In the above applications, the gene encoding the protein IpSTP5 can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0035] In the above applications, the regulation can be to promote or increase the expression of the gene encoding the protein IpSTP5 to increase the oil content of the fruit of *Vernicia fordii*, or it can be to inhibit or decrease the expression of the gene encoding the protein IpSTP5 to decrease the oil content of the fruit of *Vernicia fordii*.

[0036] In the above applications, the inhibition or reduction of the expression of the gene encoding the protein IpSTP5 may be a substance that knocks out the gene encoding the protein IpSTP5, and / or a substance that inhibits or reduces the expression of the gene encoding the protein IpSTP5.

[0037] In the above applications, the inhibition or reduction of the expression of the gene encoding the protein IpSTP5 can be achieved by gene knockout or gene silencing.

[0038] Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by altering its DNA sequence.

[0039] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0040] In the above applications, the inhibition or reduction of the expression of the gene encoding the protein IpSTP5 can be achieved by an agent that inhibits or reduces the expression of the gene. The agent that inhibits or reduces the expression of the gene can be a gene knockout agent, such as an agent that silences the gene via VIGS. The agent that inhibits or reduces the expression of the gene can contain a polynucleotide that targets the gene, such as sgRNA, shRNA, siRNA, miRNA, or antisense RNA.

[0041] In the above applications, inhibiting or reducing the expression of the gene encoding the protein IpSTP5 can be achieved by introducing any one of the following substances (c1)-c4) into the fruit of the *Vernicia fordii*:

[0042] c1) Nucleic acid molecules that inhibit or reduce the expression of the IpSTP5-encoding gene of the protein;

[0043] c2) Recombinant microorganisms containing the nucleic acid molecules described in c1).

[0044] In the above applications, the nucleic acid molecule described in c1) can be a silencing vector containing a DNA molecule whose nucleotide sequence is SEQ ID NO. 1, positions 621-1026 or SEQ ID NO. 1, positions 1123-1521.

[0045] To address the aforementioned technical problems, this invention also provides a method for regulating the oil content of *Vernicia fordii* fruit, comprising reducing the oil content of *Vernicia fordii* fruit by inhibiting or reducing the expression level of the gene encoding the protein IpSTP5 in the *Vernicia fordii* genome.

[0046] The above-mentioned inhibition or reduction of the expression level of the gene encoding the protein IpSTP5 in the genome of *Vernicia fordii* can be achieved by any method in the prior art, so as to induce deletion mutations, insertion mutations or base transformation mutations in the gene, thereby reducing or losing gene function. Specifically, this can be achieved by chemical mutagenesis, physical mutagenesis, RNAi, site-directed genome editing or homologous recombination, etc.

[0047] Among the aforementioned site-specific genome editing methods, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, clustered regularly interspaced short palindromic repeats / CRISPR-associated (CRISPR / Cas9 system) technology, and other technologies capable of site-specific genome editing can be employed. Regardless of the method used, the entire coding gene of the aforementioned proteins can be targeted, or individual elements regulating the expression of the coding gene can be targeted, as long as gene function loss or reduction is achieved. For example, exons or 5' UTRs of the coding genes of the aforementioned proteins can be used as targets.

[0048] In the above method, the inhibition or reduction of the expression level of the gene encoding the protein IpSTP5 in the genome of *Vernicia fordii* is achieved through virus-induced gene silencing. The virus-induced gene silencing includes introducing a VIGS gene silencing vector into the target *Vernicia fordii*, wherein the target site of the silencing vector is positions 621-1026 of SEQ ID NO. 1 or positions 1123-1521 of SEQ ID NO. 1.

[0049] In the above method, inhibiting or reducing the expression level of the gene encoding the protein IpSTP5 in the genome of *Vernicia fordii* can be achieved by introducing any of the following substances into *Vernicia fordii*:

[0050] c1) Inhibit or reduce the expression of the nucleic acid molecules encoding the IpSTP5 protein mentioned above;

[0051] c2) Recombinant microorganisms containing the nucleic acid molecules described in c1).

[0052] In the above method, c1) refers to a nucleic acid molecule that is a silencing vector containing a DNA molecule whose nucleotide sequence is SEQ ID NO. 1, positions 621-1026 or SEQ ID NO. 1, positions 1123-1521.

[0053] This invention discloses a method for silencing the IpSTP5 gene in *Vernicia fordii* to reduce the oil content of the fruit. Specifically, the VIGS method was used to silence the IpSTP5 gene in *Vernicia fordii*, demonstrating that silencing the IpSTP5 gene significantly reduced the oil content of the fruit, confirming that the IpSTP5 gene is related to oil content and providing new materials for the breeding of *Vernicia fordii* varieties. Attached Figure Description

[0054] Figure 1 This describes the process of obtaining the IpSTP5 gene in Example 1 of the present invention. Figure 1 A represents the association between oil content and resequencing in 42 *Vernicia fordii* samples analyzed by GWAS. Figure 1 B represents the expression of 20 genes in the candidate region for materials with high and low oil content.

[0055] Figure 2 This is a schematic diagram of the vector used in this invention. The pTRV2-IpSTP5_1 vector inserts IpSTP5_1, which is 621-1026 bp of the IpSTP5 sequence (SEQ ID NO.1); the pTRV2-IpSTP5_2 vector inserts IpSTP5_2, which is 1123-1521 bp of the IpSTP5 sequence (SEQ ID NO.1).

[0056] Figure 3 The results of the oil content detection of *Vernicia fordii* seeds after VIGS treatment with IpSTP5 in Example 1 of this invention are shown. The values ​​are mean ± standard error, with three biological replicates; p < 1e-2, indicating extremely significant differences. The statistical analysis method is one-way ANOVA.

[0057] Figure 4 The results of the detection of IpSTP5 expression level in *Vernicia fordii* after VIGS treatment in Example 1 of this invention are shown. The values ​​are mean ± standard error, with three biological replicates; p < 1e-2, indicating extremely significant differences. The statistical analysis method was one-way ANOVA. Detailed Implementation

[0058] 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.

[0059] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0061] The pTRV1 and pTRV2 vectors in the following examples are described in the non-patent literature “Liu et al., 2002. Tobacco Rar1, EDS1 and NPR1 / NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J. 30(4): 415-429”, which are available to the public from the applicant for replication of this experiment.

[0062] The competent Agrobacterium in the following examples is GV3101, a product of Beijing Jinsha Biotechnology Co., Ltd.

[0063] The LB solid culture medium in the following examples is prepared as follows (taking 1L as an example): 10g peptone, 5g yeast extract, 10g sodium chloride, 15g agar powder, distilled water to a final volume of 1L, pH adjusted to 7.2 with 5mol / L NaOH, and sterilized at 121℃ for 30min.

[0064] The LB liquid culture medium in the following examples is prepared as follows (taking 1L as an example): 10g peptone, 5g yeast extract, 10g sodium chloride, distilled water to a final volume of 1L, pH adjusted to 7.2 with 5mol / L NaOH, and sterilized at 121℃ for 30min.

[0065] In the examples below, the concentration of the antibiotic kana sulfate (Kana) was 50 mg / L, and the concentration of the antibiotic rifampin (Rif) was 50 mg / L.

[0066] The inoculum solution was formulated as follows: 10 mM MgCl2, 10 mM MES (pH 5.7), and 100 μm AS.

[0067] Example 1

[0068] I. Discovery of IpSTP5 and its encoding gene The IpSTP5 gene

[0069] Candidate genes were obtained by combining genome-wide association analysis (GWAS) with transcriptome (RNA-seq) analysis.

[0070] To analyze the differences in oil content of *Vernicia fordii*, this study collected 42 *Vernicia fordii* accessions from different regions and performed resequencing. GWAS analysis was performed on the oil content of these 42 accessions, identifying a candidate region in Chr4. Based on the linkage disequilibrium distance of 150 kb, the candidate region was narrowed down to Chr4:13.99-14.35. Simultaneously, using genome annotation files, 20 candidate genes were identified within this region. Further analysis of transcriptome data from high- and low-oil-content materials revealed significant differences in IpSTP5 expression between the two types of materials (see...). Figure 1 Therefore, IpSTP5 is considered as a subsequent gene related to oil content.

[0071] The coding sequence of the IpSTP5 gene is shown in SEQ ID NO.1; the protein encoded by this gene is named IpSTP5, and its amino acid sequence is shown in SEQ ID NO.2.

[0072] SEQ ID NO.1

[0073]

[0074] SEQ ID NO.2MAGGGFVADGPASGFNGKITVSVVITCIVAASSGLIFGYDIGISGGVTTMAPFLIKFFPEVFRKASEVKTNMYCQFDSQVLTAFTSSLYIAGLASSLVASRLTAAMGLKNTMVLGGCTFLAGAA INGGAANIAMLLLGRILLGFGVGFTNQVATPVYLSEVAPPKWRGAFGTGFQFFIGIGVVAANCINFGMAKHSWGWRFSLGLAVVPAALMTIGALFISDTPSSLVERGKVKQARQSLTKVRGINSNVEA ELADLLKFNEMSKDAQKEPFLTIFERQHRPHLVMSIAIPFFQQLTGINIIAFYAPVIFQSVGFGSNSALIAAIVLGLVNLGSILVSTGMVDRHGRRFLFIIGGIQMFICQVAVTIMLVVTTGISGTKQ VSKGYGILLLVLMCIYASGFGWSWGPLSWLVPSEIFPMKIRPTGQSINVAVNFATTFVLSQTFLTMLCQFKFGTFLFYAGWIALMTIFVMLFLPETKGIPLDSMYAVWERHWYWGRFVRGSGRQILV*

[0075] II. Construction of pTRV2 vector of IpSTP5 from *Vernicia fordii*

[0076] 1. Obtaining cDNA template

[0077] The *Vernicia fordii* used was a female *Vernicia fordii* plant grown at the Institute of Botany, Chinese Academy of Sciences, in Xiangshan, Beijing. Other female *Vernicia fordii* plants can be used to reproduce this embodiment and are available for purchase by the public.

[0078] RNA was extracted from the fruit of the *Vernicia fordii* and reverse transcribed into cDNA.

[0079] 2. Using the cDNA obtained in step 1 as a template, amplification was performed in the following manner using primer pairs consisting of primers IpSTP5_BamHⅠ_F1 and IpSTP5_XhoⅠ_R1, and primer pairs consisting of primers IpSTP5_BamHⅠ_F2 and IpSTP5_XhoⅠ_R2.

[0080] Table 1 Primers used to construct the pTRV2 vector for IpSTP5.

[0081]

[0082] The PCR amplification reaction system and PCR reaction procedure are as follows:

[0083] Table 2. Conventional PCR reaction system

[0084] Reaction components Dosage 2×Taq MasterMix 10μL IpSTP5_BamHⅠ_F 1μL IpSTP5_XhoⅠ_R 1μL <![CDATA[ddH2O]]> 7μL cDNA 1μL Total Volume 20μL

[0085] After instantaneous centrifugation to ensure homogeneity, the PCR reaction was performed using a touch-down annealing program, with the annealing temperature decreasing by 0.5°C for the first 10 cycles. The program settings were as follows:

[0086] Table 3. Conventional PCR Amplification Procedure

[0087] step Temperature (°C) time Remark Pre-variation 95 5min transsexual 95 30s annealing 62 30s -0.5℃ extend 72 30s 10 cycles transsexual 95 30s annealing 57 30s extend 72 30s 24 cycles extend 72 5min save 4 ∞

[0088] 4 μL of PCR product was taken and detected by 1% agarose gel electrophoresis. The target band of the reaction product was approximately 400 bp.

[0089] DNA was recovered by gel cutting using the Novozymes standard agarose gel DNA recovery kit, following the instructions in the kit's manual.

[0090] The specific fragment amplified by the primer pair consisting of primers IpSTP5_BamHⅠ_F1 and primer IpSTP5_XhoⅠ_R1 is called IpSTP5_1.

[0091] The specific fragment amplified by the primer pair consisting of primers IpSTP5_BamHⅠ_F2 and primer IpSTP5_XhoⅠ_R2 is called IpSTP5_2.

[0092] pTRV2 was double-digested using the restriction endonucleases BamHI and XhoI produced by NEB. The large fragment was then recovered to obtain the digested pTRV2.

[0093] Using the one-step recombination kit manufactured by Novizan, the above-mentioned IpSTP5_1 was recombined into the enzyme-digested pTRV2, and the operation method was followed according to the kit instructions to obtain ligation product 1.

[0094] Using the one-step recombination kit manufactured by Novizan, the above-mentioned IpSTP5_2 was recombined into the enzyme-digested pTRV2, and the operation method was followed according to the kit instructions to obtain ligation product 2.

[0095] 5 μL of ligation product 1 was transformed into *E. coli* DH5α and cultured on solid LB medium containing kanamycin (Kana) for 12 h. Single colonies were picked and cultured in liquid LB medium containing Kana. After positive PCR identification of the bacterial culture, the plasmid was extracted and verified by double enzyme digestion. The successfully verified plasmid was sent to Qingke Biotechnology for sequencing. After correct sequencing, the successfully constructed silencing vector pTRV2-IpSTP5_1 was obtained (see...). Figure 2 The structure of the silencing vector pTRV2-IpSTP5_1 is described as follows: The small fragment between the recognition sequences of the restriction endonucleases BamHI and XhoI in pTRV2 is replaced with the double-stranded DNA molecule shown in positions 621-1026 from the 5' end of SEQ ID NO.1, while keeping the other sequences of the vector pTRV2 unchanged, resulting in the silencing vector pTRV2-IpSTP5_1.

[0096] 5 μL of ligation product 2 was transformed into *E. coli* DH5α and cultured on solid LB medium containing kanamycin (Kana) for 12 h. Single colonies were picked and cultured in liquid LB medium containing Kana. After positive PCR identification of the bacterial culture, the plasmid was extracted and verified by double enzyme digestion. The successfully verified plasmid was sent to Qingke Biotechnology for sequencing. After correct sequencing, the successfully constructed silencing vector pTRV2-IpSTP5_2 was obtained (see...). Figure 2 The structure of the silencing vector pTRV2-IpSTP5_2 is described as follows: The small fragment between the recognition sequences of the restriction endonucleases BamHI and XhoI in pTRV2 is replaced with the double-stranded DNA molecule shown in positions 1123-1521 from the 5' end of SEQ ID NO.1, while keeping the other sequences of the vector pTRV2 unchanged, resulting in the silencing vector pTRV2-IpSTP5_1.

[0097] III. VIGS Verification of the Function of the IpSTP5 Gene in *Vernicia fordii*

[0098] 1. Infection of Mangosteen fruit

[0099] The *Vernicia fordii* used was a female *Vernicia fordii* plant grown at the Institute of Botany, Chinese Academy of Sciences, Xiangshan, Beijing. Other female *Vernicia fordii* plants can be used to reproduce this embodiment and are available for purchase by the public.

[0100] pTRV1, pTRV2, and the obtained plasmids pTRV2-IpSTP5_1 and pTRV2-IpSTP5_2 were transformed into Agrobacterium GV3101 and grown at 28°C for 48 h on solid LB medium containing 50 mg / L Kana and 50 mg / L Rif. Single colonies were picked and placed in liquid LB medium containing Kana and Rif and cultured overnight at 150 rpm at 28°C. For colonies that were positive by PCR, a portion was added to 50% glycerol to preserve the bacterial culture, while the rest were expanded in liquid LB medium (containing Kana + Rif + 10 mM MES + 20 μM AS) to obtain the shaken Agrobacterium GV3101 / pTRV1, Agrobacterium GV3101 / pTRV2, Agrobacterium GV3101 / pTRV2-IpSTP5_1, and Agrobacterium GV3101 / pTRV2-IpSTP5_2 cultures.

[0101] The shaken Agrobacterium tumefaciens suspensions GV3101 / pTRV1, GV3101 / pTRV2, GV3101 / pTRV2-IpSTP5_1, and GV3101 / pTRV2-IpSTP5_2 were poured into 50 mL centrifuge tubes, centrifuged at 4000 rpm for 10 min at room temperature, the supernatant was discarded, the cells were resuspended in the infection solution, the OD was adjusted to 0.5, vortexed to mix, and then allowed to stand at room temperature for 3 h to obtain the Agrobacterium tumefaciens resuspensions GV3101 / pTRV1, GV3101 / pTRV2, GV3101 / pTRV2-IpSTP5_1, and GV3101 / pTRV2-IpSTP5_2.

[0102] The control group and two experimental groups were set up as follows:

[0103] Equal volumes of pTRV1 Agrobacterium resuspension and pTRV2 Agrobacterium resuspension were thoroughly mixed to obtain the control group infection solution. Using a disposable 1mL syringe, the prepared control group infection solution was drawn up, and the needle was inserted into a *Vernicia fordii* fruit growing on a tree. The solution was then injected directly into the fruit under slight pressure, allowing it to spread throughout the fruit.

[0104] Equal volumes of pTRV1 Agrobacterium resuspension and pTRV2-IpSTP5_1 Agrobacterium resuspension were thoroughly mixed to obtain the infection solution for experimental group 1. Using a disposable 1mL syringe, the prepared infection solution for experimental group 1 was drawn up, and the needle was inserted into a *Vernicia fordii* fruit growing on a tree. The bacterial solution was then injected directly into the fruit under slight pressure, allowing it to diffuse within the fruit.

[0105] Equal volumes of pTRV1 Agrobacterium resuspension and pTRV2-IpSTP5_2 Agrobacterium resuspension were thoroughly mixed to obtain the infection solution for experimental group 2. Using a disposable 1mL syringe, the prepared infection solution for experimental group 2 was drawn up, and the needle was inserted into a *Vernicia fordii* fruit growing on a tree. The bacterial solution was then injected directly into the fruit under slight pressure, allowing it to diffuse within the fruit.

[0106] After injecting the infected bacterial solution, the fruit of the *Vernicia fordii* was covered with a bag and then allowed to grow under normal natural conditions for 12 hours.

[0107] 2. Determination of oil content in *Vernicia fordii* seeds

[0108] Two weeks after the fruit was infected with the bacterial solution, fruits from experimental groups 1, 2, and the control group were collected, with three replicates for each group. The fruit was blanched at 120℃ for 30 minutes, dried at 65℃, and the oil content was measured using nuclear magnetic resonance (NMR). The experiment was performed in three biological replicates, and the average of the three results was taken. Figure 3 As shown in the figure. The results showed that after IpSTP5 was silenced, the oil content of the fruit of *Vernicia fordii* decreased significantly, especially in experimental group 2, where the oil content of the fruit introduced with the pTRV2-IpSTP5_2 vector decreased more significantly.

[0109] 3. Detection of IpSTP5 expression in *Vernicia fordii* seeds

[0110] Two weeks after the fruit was infected with the bacterial solution, fruits from experimental group 1, experimental group 2 and control group were collected respectively. RNA was extracted using TRIzol reagent and reverse transcribed into cDNA, according to the kit instructions.

[0111] To detect the silencing effect of the viral vector, IpSTP5 real-time quantitative primers were designed, and the expression level in the fruit was detected by real-time quantitative PCR. IpPP2A was used as the internal control. The primer sequences are shown in Table 4.

[0112] Table 4. RT-qPCR primers for detecting relative expression of the IpSTP5 gene.

[0113]

[0114]

[0115] The reaction mixture for real-time quantitative PCR amplification is as follows:

[0116] Table 5 RT-qPCR reaction system

[0117] Reaction components Dosage 2×SuperRealPreMixPlus 7.5μL IpSTP5_qF 0.3μL IpSTP5_qR 0.3μL <![CDATA[ddH2O]]> 3.9μL cDNA 3μL Total Volume 15μL

[0118] After instantaneous centrifugation to ensure homogeneity, the real-time quantitative PCR amplification reaction program was as follows: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 15 s, for 40 cycles. After the program, the data were exported for analysis, and the expression level of the target gene was analyzed using a relative quantification method.

[0119] Experimental results are as follows Figure 4 As shown, the expression level of IpSTP5 decreased significantly, especially in the experimental group 2 fruits in which the pTRV2-IpSTP5_2 vector was introduced, indicating that the VIGS vector constructed from the specific fragment provided in this invention can successfully infect the fruit of *Vernicia fordii*, effectively reducing the expression level of the IpSTP5 gene and decreasing the oil content. This allows for a simple, efficient, and low-cost identification of the function of the IpSTP5 gene in *Vernicia fordii*.

[0120] The present invention has been described in detail above. For those skilled in the art, 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. Although specific embodiments have been given, 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. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A protein, characterized by: The protein is either A1) or A2) of the following: A1) The amino acid sequence of this protein is SEQ ID No. 2; A2) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1).

2. The biomaterial related to the protein of claim 1, characterized in that: The biological material is any one of B1) to B4) below: B1) A nucleic acid molecule encoding the protein of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that: B1) The nucleic acid molecule is a cDNA molecule or DNA molecule whose coding sequence is SEQ ID No. 1 nucleotide.

4. The application of a substance that regulates the activity or content of the protein of claim 1, or a substance that regulates the expression of the gene encoding the protein of claim 1, characterized in that: The application is any one of the following: P1. Application in regulating the oil content of *Vernicia fordii* fruit; P2. Application in the preparation of products that regulate the oil content of *Vernicia fordii* fruit; The regulation is to suppress or reduce the expression of the gene encoding the protein of claim 1 in order to reduce the oil content of the fruit of *Vernicia fordii*.

5. The application according to claim 4, characterized in that: The inhibition or reduction of the expression of the gene encoding the protein of claim 1 is achieved by introducing any one of the following substances into the fruit of the *Vernicia fordii*: c1) Nucleic acid molecules that inhibit or reduce the expression of the gene encoding the protein of claim 1; c2) Recombinant microorganisms containing the nucleic acid molecules described in c1).

6. The application according to claim 5, characterized in that: c1) The nucleic acid molecule described is a silencing vector containing a DNA molecule whose nucleotide sequence is SEQ ID NO. 1, positions 621-1026 or SEQ ID NO. 1, positions 1123-1521.

7. A method for regulating the oil content of *Vernicia fordii* fruit, characterized in that, This includes reducing the oil content of *Vernicia fordii* fruit by inhibiting or reducing the expression level of the gene encoding the protein described in claim 1 in the *Vernicia fordii* genome.

8. The method according to claim 7, characterized in that: The suppression or reduction of the expression level of the gene encoding the protein of claim 1 in the genome of *Vernicia fordii* is achieved by silencing the gene encoding the protein of claim 1 in *Vernicia fordii*.

9. The method according to claim 7, characterized in that: The inhibition or reduction of the expression level of the gene encoding the protein of claim 1 in the genome of *Vernicia fordii* is achieved by introducing any one of the following substances into *Vernicia fordii*: c1) Nucleic acid molecules that inhibit or reduce the expression of the protein-coding gene as described in claim 1; c2) Recombinant microorganisms containing the nucleic acid molecules described in c1).

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