Application of SiMYB113 protein and coding gene thereof in regulation and control of synthesis of plant tocopherol

Abstract

The invention discloses application of SiMYB113 protein and a coding gene thereof in regulation and control of synthesis of plant tocopherol, and belongs to the technical field of plant genetic engineering. The amino acid sequence of the SiMYB113 protein is as shown in SEQ ID NO. 1; according to the invention, the function and application of the sesame SiMYB113 gene in tocopherol synthesis are reported for the first time, a SiMYB113 gene overexpression vector is further constructed to be overexpressed in sesame hairy roots, it is verified that SiMYB113 in transgenic sesame hairy roots can induce synthesis of tocopherol, and it is verified that the SiMYB113 gene has the functions of positively regulating tocopherol synthesis, inducing tocopherol synthesis and inducing tocopherol synthesis. The method has important significance in analyzing the genetic regulation and control mechanism of the tocopherol content of the sesame, cultivating new varieties of the sesame with high tocopherol content and improving the nutritional quality of the sesame.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of the SiMYB113 protein and its encoding gene in regulating the synthesis of tocopherols in plants. Background Technology

[0002] Tocopherol, also known as vitamin E, was discovered in 1922. Tocopherol is a collective term for tocopherols and tocotrienols. Naturally occurring tocopherols include eight types: α, β, γ, δ-tocopherol and α, β, γ, δ-tocotrienol, differing mainly in the degree of hydrophobic tail saturation and the number and position of methyl groups on the aromatic ring. Tocopherol is an essential fat-soluble vitamin that humans and animals cannot synthesize independently, and it is widely known for its antioxidant properties. As a natural antioxidant, tocopherol can effectively reduce non-enzymatic oxidative damage to cells and lipids caused by free radicals or reactive oxygen species, inhibit lipid peroxidation, and protect biological membranes. Numerous studies have shown that tocopherol has various potential beneficial effects on human health, such as anti-cancer, anti-atherosclerosis, neuroprotection, and inhibition of chronic diseases such as Alzheimer's disease. Therefore, increasing the tocopherol content in plants, especially crops, is of great significance for promoting human health. The synthetic pathway of tocopherol in plants has gradually become clear, but reports on genes regulating tocopherol synthesis are relatively few.

[0003] Sesame( Sesamum indicum Sesame (L.) is an annual herbaceous oilseed crop belonging to the genus Sesame in the family Sesamaceae. It is widely cultivated in tropical and subtropical regions of Asia and Africa, with a global planting area exceeding 200 million mu (approximately 13.3 million hectares) and distributed in more than 70 countries. my country has a long history of sesame cultivation, with plantings throughout the country. Major production areas are concentrated in the Huang-Huai, Jiang-Huai, and Yangtze River basins (Henan, Hubei, Anhui, Jiangxi, etc.), ranking among the world's top in terms of planting area and yield. Sesame can be used for oil extraction or direct consumption, and can also be used as a spice, medicine, and chemical raw material, possessing a wide range of applications. The diverse uses of sesame are inseparable from its unique nutritional qualities. Sesame is not only rich in unsaturated fatty acids, various minerals, and proteins, but also rich in lignans, tocopherols, and other antioxidant components. Sesame seeds have a high tocopherol content, making them an ideal source of tocopherols.

[0004] But currently, for SiMYB113 The role of genes in regulating tocopherol synthesis in plants has not been reported. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the SiMYB113 protein and its encoding gene in regulating plant tocopherol synthesis. This addresses the problem in existing technologies of lacking an efficient SiMYB113 protein and its encoding gene for regulating plant tocopherol synthesis.

[0006] In a first aspect, the present invention provides the application of SiMYB113 protein in regulating plant tocopherol synthesis, wherein the SiMYB113 protein is selected from any of the following: A1) a protein with an amino acid sequence as shown in SEQ ID NO.1; A2) a protein with the same function obtained by substitution, deletion or addition of one or more amino acids compared with the amino acid sequence defined in A1); A3) a protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0007] The SiMYB113 protein provided by the present invention can be a natural, recombinant or synthetic active polypeptide. The active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants) using recombinant technology.

[0008] In this invention (A3), the linkage can be achieved through direct peptide bonds or via adapters, using methods conventional in the art. The tags include, but are not limited to: GST (glutathione thioredoxin) tag protein, Trx (thioredoxin) tag protein, His tag protein (His-tag), Flag tag protein, LacZ tag protein, GFP (green fluorescent protein), sfGFP (hyperfolded green fluorescent protein), and HA tag (hemagglutinin tag). Those skilled in the art can select appropriate tag proteins according to actual needs. The use of tags does not alter the function of the target protein (SiMYB113 protein); its purpose is for separation, purification, detection, or tracing. The tags can be separated from the target protein (SiMYB113 protein) using chemical cleavage methods or enzymatic methods known in the art (e.g., introducing protease cleavage sites and using TEV protease to remove the tag).

[0009] In some implementations, the application is achieved by upregulating the content and / or activity of the SiMYB113 protein.

[0010] In some implementations, plants with increased tocopherol content are cultivated by upregulating the content and / or activity of the SiMYB113 protein.

[0011] In some preferred embodiments, upregulating the content and / or activity of the SiMYB113 protein can be achieved by overexpressing the gene for the SiMYB113 protein (…). SiMYB113 This is achieved through genes.

[0012] In a second aspect, the present invention provides the application of nucleic acid molecules encoding the aforementioned SiMYB113 protein in regulating plant tocopherol synthesis.

[0013] In some embodiments, the nucleic acid molecule is selected from any of the following: B1) a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.2; B2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in B1) under stringent conditions and encodes the SiMYB113 protein described above; B3) a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in B1) or B2) and encodes the SiMYB113 protein described above.

[0014] The nucleic acid molecules provided by this invention can be DNA, such as cDNA, genomic DNA, or recombinant DNA; or RNA, such as mRNA or hnRNA; and these nucleic acid molecules can usually be obtained by PCR amplification or artificial synthesis.

[0015] As used herein, the term "hybridization under stringent conditions" refers to the hybridization of two nucleic acid fragments under standard hybridization conditions as described in the section "Expression of Cloned Genes in E. coli" of *Molecular Cloning: A Laboratory Manual* (1989) (Cold Spring Lane Laboratory, New York, USA). Such conditions include hybridization in 6.0 × SSC at 45 °C, followed by washing in 2 × SSC at 50 °C. To select stringency, the salt concentration in the washing step can be chosen, for example, between 2.0 × SSC at 50 °C for low stringency and 2.0 × SSC at 50 °C for high stringency. Additionally, the temperature in the washing step can be varied between room temperature (approximately 22 °C) for low stringency and 65 °C for high stringency.

[0016] As used herein, the term “sequence identity” can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). Molecules are identical at that position when positions in the compared sequences are occupied by the same bases or amino acids. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. “Sequence identity” of a polynucleotide or amino acid sequence with another sequence having a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0017] In a third aspect, the present invention provides the application of recombinant vectors containing any of the above-mentioned nucleic acid molecules in regulating the synthesis of tocopherols in plants.

[0018] In a fourth aspect, the present invention provides the use of recombinant cells containing any of the above-mentioned nucleic acid molecules or recombinant cells containing the above-mentioned recombinant vectors in regulating plant tocopherol synthesis.

[0019] In a fifth aspect, the present invention provides a method for cultivating plants with increased tocopherol content, the method comprising increasing the content and / or activity of the aforementioned SiMYB113 protein in the target plant to obtain a plant with increased tocopherol content compared to the target plant.

[0020] In some implementations, increasing the content and / or activity of the SiMYB113 protein in the target plant is achieved by increasing the expression level of the gene encoding the SiMYB113 protein in the target plant.

[0021] In some implementations, increasing the expression level of the gene encoding the SiMYB113 protein in the target plant is achieved by at least one of the following methods: C1) increasing the copy number of the gene encoding the SiMYB113 protein; C2) expressing the gene encoding the SiMYB113 protein under the drive of a strong promoter; C3) overexpressing the gene encoding the SiMYB113 protein by adding regulatory elements, wherein the regulatory elements include enhancer elements, elements that improve mRNA stability, elements that improve translation efficiency, and / or elements that improve protein secretion; C4) codon optimization of the gene encoding the SiMYB113 protein.

[0022] In some implementation methods, the method for cultivating plants with increased tocopherol content specifically includes the following steps: 1) constructing a recombinant vector containing a nucleic acid molecule encoding the SiMYB113 protein; 2) introducing the recombinant vector into the target plant; 3) obtaining plants with increased tocopherol content through screening and identification.

[0023] In some preferred embodiments, in step 1), the recombinant vector includes the recombinant vector pKR203S-SiMYB113, and the recombinant vector pKR203S-SiMYB113 is a material containing pKR203S-SiMYB113. SiMYB113 The gene was constructed by ligating it into the plant expression vector pKR203S.

[0024] In some more preferred embodiments, SiMYB113 The genes were obtained by amplification using primers with nucleotide sequences as shown in SEQ ID NO. 3-4.

[0025] In some preferred embodiments, in step 2), the introduction can be carried out by Agrobacterium-mediated transformation, specifically including the following steps: introducing the recombinant vector into Agrobacterium to obtain recombinant Agrobacterium; infecting sesame callus or explants with the recombinant Agrobacterium; and inducing and culturing the obtained positive callus or explants to obtain hairy roots and / or regenerated plants after identification.

[0026] It is understood that in step 3), the screening and identification methods are conventional methods in the field, such as screening and identification by PCR detection.

[0027] In some implementations, the plant includes sesame.

[0028] The beneficial effects of this invention are: unlike the prior art, this invention is the first to report sesame seed processing technology. SiMYB113 The function and application of genes in tocopherol synthesis, further explored through the construction of... SiMYB113 Gene overexpression vectors were used to overexpress the gene in sesame hairy roots, verifying the efficacy of transgenic sesame hairy roots. SiMYB113 It can induce the synthesis of tocopherol, confirming that... SiMYB113 The gene has the function of positively regulating tocopherol synthesis, thereby increasing the tocopherol content in sesame. This is of great significance for elucidating the genetic regulation mechanism of tocopherol content in sesame, cultivating new sesame varieties with high tocopherol content, and improving the nutritional quality of sesame. Attached Figure Description

[0029] Figure 1 This is a Manhattan plot of genome-wide association analysis of tocopherol content in sesame seeds in Example 1 of the present invention, where WH and LQ are association results of two different environmental phenotypes;

[0030] Figure 2 As in Embodiment 1 of the present invention SiMYB113 Expression map of the plant expression vector pKR203S-SiMYB113; Figure 3 The images show the induction and culture of transgenic sesame hairy roots in Example 2 of this invention. (A) shows the hairy roots produced 8 days after explant infection, and (B) shows the hairy roots after 2 weeks of subculture. Figure 4 In the transgenic sesame hairy root line of Example 2 of this invention SiMYB113 The results of quantitative real-time PCR detection of the gene, where VC is the empty vector control. SiMYB113 -OE#1 and -OE#2 are two transgenic root systems.

[0031] Figure 5 The results of tocopherol content determination in transgenic sesame hairy root lines in Example 2 of this invention are shown, where VC is an empty vector control. SiMYB113 -OE#1 and -OE#2 are two separate terms. SiMYB113 Overexpression of transgenic roots. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0034] Example 1: Sesame SiMYB113 Gene acquisition and overexpression vector construction 1.1 Sesame SiMYB113 Acquisition of genes 1) Based on the geographical origin and genetic diversity detection results, 400 sesame materials were selected from 7,910 domestic and foreign resources preserved in the National Sesame Mid-term Bank and resequencing analysis was performed using a stepwise sampling strategy, resulting in 20-fold coverage of genome sequences. 2) Genome-wide association analysis (GWA) of tocopherol content in sesame seeds in two environments was performed using the rMVP software package. A single SNP located at 20151495 bp on chromosome 9 was detected in both environments and was significantly associated with seed tocopherol content (P < 10). -10 (Results as follows) Figure 1 (as shown) 3) Through comparison with the sesame reference genome and gene annotation analysis, a MYB transcription factor MYB113 (accession number: LOC105177882) was screened in the candidate region where the peak SNP site is located, and it is speculated that it may be related to the variation in tocopherol content; 4) According to SiMYB113 The gene's CDS sequence was used as the basis for primer design using Primer 5.0 to amplify its complete CDS sequence. The primer sequences (including modified bases) are as follows: SiMYB113-F:5′-aggatcccgggtcgaactagtGTGAACATAAATTGGAGTAGG-3′ (SEQ IDNO.3); SiMYB113-R:5′-atcttcatcttcataactagtCAGACACTGTACCATCGC-3′ (SEQ IDNO.4); 5) Take seeds of sesame germplasm Zhongzhi 13 10 days after flowering, extract total RNA, reverse transcribe it into cDNA, use the reverse transcribed cDNA as a template, and perform RT-PCR amplification using primers SiMYB113-F / R from step 5). Sequencing the amplified fragment yields... SiMYB113 The gene sequence, the nucleotide sequence of which is shown in SEQ ID NO.2, and the amino acid sequence of the protein it encodes, are shown in SEQ ID NO.1.

[0035] The amino acid sequence of the SiMYB113 protein is shown below: MRSADEDDGGIAAALLNQETTTQASNHLHSITLHLRPNVKGLHSITLHLRPAEKLVFLRDGLMENKASGLRKGAWTKEEDILLTKCIDKYGEGKWHQVPIRAGLNRCRKSCRLRWLNYLRPNIKRGLFTKDEVDLIVRLHKLLGNRWSLIAGRLPGRTANDVKNF WNTHMHRKLSGVGGCREKAMQKDITKSNIIRPRPRTFSKLQLTAPVAWSSETSKTNRENPENKKLMMSTSPSSPLKDDDANLIKSTQPPSSSSPDECIQWWSNLLEMSENRRDGLNTLSLPDDGQHQMEPLLSSNSYANQRGLSSLSGDVDVWELVNFQNL(SEQ ID NO.1); SiMYB113 The nucleotide sequence of the gene is shown below: ATGAGATCAGCAGATGAAGATGATGGAGGGATCGCAGCAGCACTCCTCAACCAGGAAACAACAACACAGGCCAGCAACCACCTCCACAGTATTACGCTGCACCTCCGCCCCAATGTCAAGGGCCTCCACAGTATTACGCTGCACCTCCGCCCCGCAGAGAAGCTGGTTTTCTTGAGGGATGGATTAATGGAAAACAAAGCATCAGGGTTGAGAAAAGGGGCGTGGACGAAAGAAGAAGATATTCTGCTAACCAAGTGCATTGACAAGTATGGAGAAGGGAAATGGCACCAAGTCCCCATCAGAGCTGGGCTGAACAGATGCAGGAAGAGTTGCAGGTTGAGATGGTTGAACTATCTCCGACCGAATATTAAAAGAGGTTTATTTACGAAAGATGAGGTGGATCTCATTGTAAGGCTTCACAAGTTATTAGGCAACAGATGGTCACTGATAGCTGGTAGACTTCCGGGAAGAACTGCAAACGATGTGAAGAACTTTTGGAACACCCACATGCACAGGAAATTATCCGGTGTTGGAGGTTGCAGAGAAAAAGCCATGCAGAAAGATATCACTAAGAGTAACATCATTAGACCCCGACCTCGGACCTTCTCCAAATTACAATTGACAGCACCAGTTGCTTGGTCCAGTGAAACAAGCAAAACAAACCGTGAAAATCCCGAGAACAAGAAGCTGATGATGTCTACATCTCCATCATCACCGTTGAAAGACGATGATGCAAATCTGATCAAGAGCACGCAACCGCCATCTTCATCATCACCAGATGAATGCATTCAGTGGTGGAGCAACTTACTCGAAATGAGTGAAAATCGTAGGGATGGATTAAACACGCTTTCGCTTCCCGACGATGGCCAGCACCAGATGGAACCATTGTTGTCGTCGAATAGTTACGCCAACCAACGAGGGTTGAGTAGTTTGTCGGGAGATGTCGACGTCTGGGAACTTGTAAACTTTCAAAATCTATGA(SEQ ID NO.2)。

[0036] 1.2 Contains sesame seeds SiMYB113 Construction of gene overexpression vectors The sesame seeds obtained from the above cloning SiMYB113 The gene was ligated to the pKR203S plasmid (provided by our laboratory, a genetic transformation vector carrying the CaMV35S promoter of cauliflower mosaic virus with constitutive and overexpression characteristics) using homologous recombination to construct a plant expression vector, named pKR203S-SiMYB113 (vector map shown). Figure 2 As shown in the image, the specific steps are as follows: 1) First, use single enzyme digestion ( Spe I) The linearized vector was obtained by method I, and then purified to obtain high purity linearized vector by agarose gel electrophoresis and gel recovery kit (Tiangen Biotech Co., Ltd.)

[0037] 2) The sesame seeds obtained from the above cloning SiMYB113 The target DNA fragment and linearized vector were added to a 1.5 mL centrifuge tube at a molar ratio of 3:1 for recombination reaction. After mixing, the mixture was incubated at 37 °C for about 30 min. 10 μL of the reaction solution was added to 50 μL of DH5α competent cells, and the mixture was gently mixed with a pipette. The mixture was incubated on ice for 20 min, heat-shocked in a 42 °C water bath for 45 seconds, and then rapidly cooled on ice for 2 min.

[0038] 3) Add 700 μL of LB liquid medium and incubate at 37°C for 45-60 min. Centrifuge at 5000 rpm for 2 min to collect the bacterial cells, discard part of the supernatant, resuspend the bacterial cells with the remaining medium, and gently spread them evenly on LB solid medium containing Kan resistance using a sterile spreader. Incubate upside down in a 37°C incubator for 16-24 h.

[0039] 4) Select several clones from the recombinant reaction transformation plate for colony PCR identification. For colonies that are identified as positive, select the corresponding single colonies and incubate them overnight in liquid LB medium containing Kan antibiotic at 37°C and 200 rpm. Extract plasmids or directly sequence the bacterial culture and identify the accuracy of the vector by enzyme digestion electrophoresis to obtain the correctly sequenced recombinant vector pKR203S-SiMYB113.

[0040] Example 2 Sesame SiMYB113 Functional verification of genes in sesame hairy roots In this embodiment, the recombinant vector pKR203S-SiMYB113 prepared in Example 1 was transformed into Agrobacterium rhizogenes K599, and then used to infect sesame cotyledon explants to induce the formation of transgenic hairy roots. The genetic hairy roots were then tested for the presence of transgenic hairy roots. SiMYB113 Gene expression and tocopherol levels. The specific procedures are as follows: 2.1 Turn SiMYB113 Induction and culture of sesame hairy roots 1) Recombinant vector transformed into Agrobacterium K599 a) Add 2 μg of plasmid DNA to every 100 μL of K599 Agrobacterium competent cells, mix by hand by tapping the bottom of the tube, and incubate on ice for 5 min, liquid nitrogen for 5 min, water bath at 37°C for 5 min, and ice bath for 5 min in sequence.

[0041] b) Add 700 μL of antibiotic-free TY liquid medium and incubate at 28°C with shaking for 2 h. Centrifuge at 6000 rpm for 1 min to collect the bacterial cells, and keep about 100 μL of supernatant. Gently pipette and resuspend the bacterial cells, and spread them evenly on TY solid medium containing Kan. Invert the medium and incubate at 28°C for 2 days. Pick a number of positive clones and verify the results using colony PCR.

[0042] 2) Induction and culture of sesame hairy roots a) Pre-sterilize sesame seeds using the following method: rinse with sterile water for 1 min; treat with 70-75% ethanol for 30 s; rinse once with sterile water; treat with 3% sodium hypochlorite for 8-10 min; rinse once with sterile water; rinse 3-5 times with sterile water, 1 min each time; then place the seeds on sterile filter paper and air dry naturally. Inoculate the sterilized sesame seeds onto MS medium containing 30 g / L sucrose, 8 g / L agar, and 0.075% PPM, pH 5.8. Culture conditions are: temperature 28±2℃, light intensity 1500-2000 lux, photoperiod 16 h / 8 h. Sterile sesame seedlings can be obtained in 7-10 days.

[0043] b) Separate the cotyledons from the sterile sesame seedlings, and make 2-3 small incisions (approximately 5 mm) on each cotyledon using a scalpel. Place the treated cotyledons on MS medium (NH4NO3 1650 mg / L + KNO3 1900 mg / L + CaCl2·2H2O 440 mg / L + MgSO4·7H2O 370 mg / L + KH2PO4 170 mg / L + KI 0.83 mg / L + H3BO3 6.2 mg / L + MnSO4·4H2O 22.3 mg / L + ZnSO4·7H2O 8.6 mg / L + Na2MoO4·2H2O 0.25 mg / L + CuSO4·5H2O 0.025 mg / L + CoCl2·6H2O 0.025 mg / L + Na2EDTA 37.25 mg / L + FeSO4·7H2O). 27.85 mg / L + inositol 100 mg / L + glycine 2 mg / L + thiamine hydrochloride 0.1 mg / L + pyridoxine hydrochloride 0.5 mg / L + nicotinic acid 0.5 mg / L + sucrose 30 g / L + agar 7-8 g / L (pH 5.8) was kept moist and ready for Agrobacterium infection.

[0044] c) Incubate strain K599 containing the pKR203S-SiMYB113 on TY solid medium (5 g / L tryptone + 3 g / L yeast extract + 10 mmol / L calcium chloride + 15 g / L agar) at 28°C for 2-3 days. Then, pick a single colony and inoculate it into 5 mL of TY liquid medium, shaking at 200 rpm and 28°C for 24 hours. Take 1 mL of the bacterial culture and inoculate it into 200 mL of TY liquid medium, shaking at 200 rpm and 28°C until the bacterial culture reaches OD. 600 When the value is 0.6-0.8, the cotyledon explants are infected for 10 minutes.

[0045] d) Place the infected cotyledons on sterile filter paper until there is no excess bacterial solution on the surface of the cotyledons, and then place them on a co-culture medium of 1 / 10 MS (NH4NO3 165mg / L + KNO3 190mg / L + CaCl2·2H2O 44mg / L + MgSO4·7H2O 37mg / L + KH2PO4 17mg / L + KI 0.83mg / L + H3BO3 6.2mg / L + MnSO4·4H2O 22.3mg / L + ZnSO4·7H2O 8.6mg / L + Na2MoO4·2H2O 0.25mg / L + CuSO4·5H2O 0.025mg / L + CoCl2·6H2O 0.025mg / L + Na2EDTA 37.25mg / L + FeSO4·7H2O). 27.85 mg / L + inositol 100 mg / L + glycine 2 mg / L + thiamine hydrochloride 0.1 mg / L + pyridoxine hydrochloride 0.5 mg / L + nicotinic acid 0.5 mg / L + sucrose 30 g / L + MES 3.9 g / L + agar 8 g / L + DTT 150 mg / L + AS 0.02 g / L, pH 5.4, co-cultured for 3 days.

[0046] e) After co-culture, the explants were transferred to washing medium MS (NH4NO3 1650 mg / L + KNO3 1900 mg / L + CaCl2·2H2O 440 mg / L + MgSO4·7H2O 370 mg / L + KH2PO4 170 mg / L + KI 0.83 mg / L + H3BO3 6.2 mg / L + MnSO4·4H2O 22.3 mg / L + ZnSO4·7H2O 8.6 mg / L + Na2MoO4·2H2O 0.25 mg / L + CuSO4·5H2O 0.025 mg / L + CoCl2·6H2O 0.025 mg / L + Na2EDTA 37.25 mg / L + FeSO4·7H2O). 27.85 mg / L + inositol 100 mg / L + glycine 2 mg / L + thiamine hydrochloride 0.1 mg / L + pyridoxine hydrochloride 0.5 mg / L + nicotinic acid 0.5 mg / L) + sucrose 30 g / L + thiazolycin 200-250 mg / L + carbenicillin 200-250 mg / L, pH 5.8. Wash three times, 1 min each time, until the medium is clear. Then place on sterile filter paper and air dry naturally until there is no obvious moisture on the cotyledon surface. Then place on 1 / 2 MS rooting and subculture medium (NH4NO3 825 mg / L + KNO3 950 mg / L + CaCl2·2H2O 220 mg / L + MgSO4·7H2O 185 mg / L + KH2PO4 85 mg / L + KI 0.83 mg / L + H3BO3 6.2 mg / L + MnSO4·4H2O) 22.3 mg / L + ZnSO4·7H2O 8.6 mg / L + Na2MoO4·2H2O 0.25 mg / L + CuSO4·5H2O 0.025 mg / L + CoCl2·6H2O 0.025 mg / L + Na2EDTA 37.25 mg / L + FeSO4·7H2O 27.85 mg / L + Inositol 100 mg / L + Glycine 2 mg / L + Thiamine Hydrochloride 0.1 mg / L + Pyridoxine Hydrochloride 0.5 mg / L + Nicotinic Acid 0.5 mg / L + Sucrose 30 g / L + MES 0.6 g / L + 200 mg / L Termedin + Agar 7 g / L, pH 5.8 or higher. After 7-8 days, hairy roots were observed to develop from wounds on the cotyledons and petioles, as well as at the wound sites on the cotyledons. Figure 3 As shown in Figure A.

[0047] f) Two weeks later, the rapidly growing hairy roots were cut from the explants and inoculated onto new rooting and subculture media for further expansion. The hairy roots after subculture were as follows: Figure 3 As shown in B, the generation will be repeated every two weeks thereafter.

[0048] The culture media used in the above steps were all treated with conventional sterilization methods, and were used after high temperature and high pressure (121℃, 1.5 atmospheres) sterilization for 20 minutes.

[0049] The culture chambers used in the above steps were in a conventional sesame tissue culture chamber (temperature around 28℃, light intensity 1000-1500 lux, light / dark cycle 16h / 8h, humidity around 70%).

[0050] 2.2 Detection of transgenic hairy roots by real-time quantitative PCR Total RNA was extracted from the transgenic hairy roots obtained above using an RNA extraction kit (Beijing Adley Biotechnology Co., Ltd.), and then cDNA was obtained using a reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd.). Using the respective cDNA as templates, sesame seeds were... Histone H3.3 The gene (LOC105159325) was used as an internal control. qRT-PCR expression validation of the target gene was performed (qRT-PCR Mix: Nanjing Novizan Biotechnology Co., Ltd.; Instrument: Roche LightCycler® 480). The detection... SiMYB113 The primers for the gene are SiMYB113QRT-F / R. Histone H3.3 The primers for the gene are shown below: SiMYB113QRT-F:5′-CCACCTCCACAGTATTACGC-3′ (SEQ ID NO.5); SiMYB113QRT-R:5′-TCCATCCCTCAAGAAAACCAG-3′ (SEQ ID NO.6); H3.3F: 5′-GTTGGTCTCTTTGAGGAC-3′ (SEQ ID NO.7); H3.3R: 5′-CAGCTGGATGTCTTTTGG-3′ (SEQ ID NO.8); qRT-PCR test results are as follows Figure 4 As shown.

[0051] from Figure 4 As can be seen, converting empty vector (VC) into a control, SiMYB113 The gene was detected in two transgenic hairy root lines ( SiMYB113 The expression levels in both -OE#1 and -OE#2 were significantly increased.

[0052] 2.3 Determination of tocopherol content in transgenic hairy roots Collect the above SiMYB113 The tocopherol content in overexpressed transgenic hairy root families and empty vector-transformed materials was detected by UPLC.

[0053] Specifically, after drying, the sample was ground into powder, and 0.1 ± 0.001 g was weighed into a centrifuge tube. 1.5 mL of n-hexane was added, the centrifuge tube was sealed, and the mixture was shaken on a shaker for 2-3 hours, then placed in a 4℃ refrigerator for 16-24 hours of extraction. After centrifugation (10000 g, 4℃, 5 min), the supernatant was collected, filtered through a 0.22 μm organic membrane, and then used for chromatography. The chromatographic conditions were: an Agilent ZORBAXRX-SIL Analytical 4.6 × 250 mm 5-Micron column; a mobile phase of 99% n-hexane: 1% isopropanol (premixed); a column temperature of 30℃; an injection volume of 5 μL; and a flow rate of 1 mL / min. The detection wavelength was 292 nm. The detection results are as follows: Figure 5 As shown.

[0054] from Figure 5 As can be seen, in the empty vector control, the tocopherol content ranged from 91.609 to 94.847 mg / kg, while in the two SiMYB113 gene overexpression lines, the tocopherol content reached 136.961 to 162.649 mg / kg. The results indicate that... SiMYB113 The gene has the function of positively regulating tocopherol synthesis.

[0055] In summary, this invention provides sesame seeds. SiMYB113 The application of genes in regulating tocopherol synthesis, further through SiMYB113 The overexpression experiment of the gene in the hairy roots of sesame confirmed that sesame... SiMYB113 Genes have the function of positively regulating tocopherol synthesis, thereby increasing the tocopherol content in sesame. This is of great significance for elucidating the genetic regulation mechanism of tocopherol content in sesame, cultivating new sesame varieties with high tocopherol content, and improving the nutritional quality of sesame.

[0056] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of SiMYB113 protein in regulating plant tocopherol synthesis, characterized in that, The SiMYB113 protein is selected from any of the following: A1) A protein with the amino acid sequence shown in SEQ ID NO.1; A2) Proteins with the same function obtained by substituting, deleting or adding one or more amino acids compared to the amino acid sequence defined in A1); A3) A protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

2. The application of the nucleic acid molecule encoding the SiMYB113 protein of claim 1 in regulating plant tocopherol synthesis.

3. The application according to claim 2, characterized in that, The nucleic acid molecule is selected from any of the following: B1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; B2) A nucleic acid molecule that hybridizes with the nucleic acid molecule defined in B1) and encodes the SiMYB113 protein of claim 1; B3) is a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in B1) or B2) and encodes the SiMYB113 protein of claim 1.

4. The use of a recombinant vector containing any one of the nucleic acid molecules described in claims 2-3 in regulating the synthesis of plant tocopherols.

5. The use of recombinant cells comprising any one of the nucleic acid molecules of claims 2-3 or recombinant cells comprising the recombinant vector of claim 4 in regulating plant tocopherol synthesis.

6. A method for cultivating plants with increased tocopherol content, characterized in that, The method includes increasing the content and / or activity of the SiMYB113 protein of claim 1 in the target plant to obtain a plant with increased tocopherol content compared to the target plant.

7. The method according to claim 6, characterized in that, The improvement of the content and / or activity of the SiMYB113 protein of claim 1 in the target plant is achieved by increasing the expression level of the gene encoding the SiMYB113 protein in the target plant.

8. The method according to claim 7, characterized in that, The enhancement of the expression level of the gene encoding the SiMYB113 protein in the target plant is achieved through at least one of the following methods: C1) Increase the copy number of the gene encoding the SiMYB113 protein; C2) The gene encoding the SiMYB113 protein is expressed under the drive of a strong promoter; C3) Increase the regulatory elements of the gene encoding the SiMYB113 protein to overexpress it, wherein the regulatory elements include enhancer elements, elements that improve mRNA stability, elements that enhance translation efficiency, and / or elements that enhance protein secretion. C4) Codon optimization was performed on the gene encoding the SiMYB113 protein.

9. The method according to claim 6, characterized in that, The method for cultivating plants with increased tocopherol content specifically includes the following steps: 1) Construct a recombinant vector containing a nucleic acid molecule encoding the SiMYB113 protein; 2) The recombinant vector is introduced into the target plant; 3) Through screening and identification, plants with increased tocopherol content were obtained.

10. The application according to any one of claims 1-5 or the method according to any one of claims 6-9, characterized in that, The plant mentioned includes sesame.

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