StTOE3 coding gene, StTOE3 gene, StTOE3 protein, recombinant vector and application thereof in improving anthocyanin content of plants

Abstract

This invention relates to the field of plant genetic engineering technology, specifically providing a StTOE3 encoding gene, the StTOE3 gene, the StTOE3 protein, a recombinant vector, and their applications. The base sequence of the encoding gene is shown in SEQ ID NO.1. The StTOE3 encoding gene determines the function of its encoded protein in plants. Recombining this encoding gene with the pBvVA(V)KS vector template yielded the recombinant vector pBvVA(V)KS-StTOE3, which was overexpressed in tobacco plants. This demonstrated that overexpression of the StTOE3 gene increased the anthocyanin content in plants, indicating that the function of the protein encoded by the StTOE3 encoding gene is to increase the anthocyanin content, laying the foundation for further research on the specific role of the StTOE3 gene in the anthocyanin synthesis pathway.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a potato StTOE3 encoding gene, the StTOE3 gene, the StTOE3 encoding gene-encoded protein, a recombinant vector containing the StTOE3 encoding gene, and its application in increasing the anthocyanin content of plants. Background Technology

[0002] Anthocyanins belong to the flavonoid family and are water-soluble pigments widely found in plants. They typically give plants their red, blue, or purple hues and significantly influence their overall appearance and nutritional value. Anthocyanins are currently recognized as the best natural antioxidants, possessing physiological functions such as anti-cancer, anti-aging, cardiovascular disease prevention, and cosmetic benefits. Therefore, cultivating crops high in anthocyanins not only meets daily dietary needs but also plays a vital role in maintaining human health, making it a primary goal for breeders. Currently, genetic engineering breeding has become one of the important methods for improving crop quality.

[0003] Potatoes are the world's fourth largest food crop and a specialty crop of Inner Mongolia. Potatoes can be divided into two main categories: common potatoes and colored potatoes. Common potatoes have white or yellow flesh, while colored potatoes have dark purple, purple, or red flesh. Colored potatoes not only contain all the nutrients found in common potatoes but are also rich in anthocyanins, giving them significant market value. In recent years, the mechanism of anthocyanin synthesis in colored potato tubers has become a research hotspot.

[0004] Using potato as the research material, some anthocyanin synthesis-related genes have been cloned and their functions verified, mainly focusing on MYB transcription factors (such as StMYB200, StMYB210, StMYB44-1, and StMYB44-2) and structural genes (such as StCHS, StF3H, StDFR, and StANS). StTOE3 is an important member of the AP2 / ERF superfamily. According to relevant studies, members of the AP2 / ERF superfamily can regulate anthocyanin synthesis by interacting with MYB-type transcription factor proteins, interacting with MBW complex members to form transcriptional regulatory circuits, affecting the promoter activity of anthocyanin synthesis structural genes, or directly activating structural gene promoters. However, the role of this family members in the regulation of potato anthocyanin synthesis has not yet been studied. Summary of the Invention

[0005] Therefore, this invention provides a StTOE3 encoding gene, a StTOE3 gene, a StTOE3 protein, a recombinant vector, and their application in increasing anthocyanin content in plants, laying the foundation for further research on the role mechanism of AP2 / ERF superfamily members in the regulation of anthocyanin synthesis in potatoes.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] A StTOE3 encoding gene, the base sequence of which is shown in SEQ ID NO.1.

[0008] The StTOE3 encoding gene of this invention is cDNA obtained by reverse transcription of total RNA extracted from potatoes using primers. This encoding gene (i.e., cDNA) can be transcribed and translated into the StTOE3 protein, which directly encodes the protein and determines the core functions of an organism, such as structure, metabolism, immunity, and development. It is the smallest and most necessary gene fragment for realizing protein function.

[0009] The second technical solution of the present invention is:

[0010] A StTOE3 gene, the base sequence of which is shown in SEQ ID NO.2.

[0011] In addition to containing the StTOE3 coding gene, the StTOE3 gene also contains a non-coding region, with a total length of 3695 bp.

[0012] The third technical solution of the present invention is:

[0013] A recombinant vector containing a base sequence as shown in SEQ ID NO.1.

[0014] To facilitate sequencing of cloned fragments of the StTOE3 coding gene or for transgenic expression in plants, it is usually necessary to recombine the StTOE3 coding gene with a corresponding empty vector to obtain a recombinant vector.

[0015] The fourth technical solution of the present invention is:

[0016] A StTOE3 protein, encoded by a gene and associated with anthocyanin synthesis in plants, the base sequence of which is shown in SEQ ID NO.1, or the gene is obtained by deleting one or more amino acid residues from a codon in the DNA sequence shown in SEQ ID NO.1, and / or by performing a missense mutation of one or more base pairs, and / or by attaching a tag to its 5' end and / or 3' end.

[0017] Preferably, the tag is Poly-Arg, Poly-His, FLAG, Strep-tag II, or c-myc, wherein the amino acid sequence of the Poly-Arg tag is shown in SEQ ID NO.10, the amino acid sequence of the Poly-His tag is shown in SEQ ID NO.11, the amino acid sequence of the FLAG tag is shown in SEQ ID NO.12, the amino acid sequence of the Strep-tag II tag is shown in SEQ ID NO.13, and the amino acid sequence of the c-myc tag is shown in SEQ ID NO.14.

[0018] Preferably, the amino acid sequence of the StTOE3 protein is as shown in SEQ ID NO.3, or the StTOE3 protein is a derivative protein of the amino acid sequence shown in SEQ ID NO.3 with substitution and / or deletion and / or addition of one or more amino acid residues and related to the synthesis of plant anthocyanins.

[0019] The fifth technical solution of the present invention is:

[0020] A set of StTOE3 amplification primers for amplifying the base sequence shown in SEQ ID NO.1, and the base sequence shown in SEQ ID NO.4 and SEQ ID NO.5.

[0021] The sixth technical solution of the present invention is:

[0022] A set of StTOE3 detection primers for detecting the expression level of the base sequence shown in SEQ ID NO.1, whose base sequence is shown in SEQ ID NO.6 and SEQ ID NO.7.

[0023] The seventh technical solution of the present invention is:

[0024] A vector template, used to combine with the aforementioned gene to form a recombinant vector, wherein the vector template is pBvVA(V)KS, and its base sequence is shown in SEQ ID NO.17. To enable the StTOE3 encoding gene to undergo tobacco transgenic transformation and to express the gene in tobacco, this invention uses pBvVA(V)KS as a vector template to recombine with it to obtain a recombinant expression vector.

[0025] The eighth technical solution of the present invention is:

[0026] The StTOE3 encoding gene, StTOE3 gene, recombinant vector, and StTOE3 protein are used to increase the anthocyanin content in plants; the plants are monocotyledonous or dicotyledonous plants.

[0027] The technical solution of the present invention achieves the following beneficial technical effects:

[0028] 1. This invention obtained the potato StTOE3 gene through preliminary screening. By comparing the tuber formation period of purple-skinned, purple-fleshed potatoes, the expression abundance of this gene was significantly higher than that of yellow-skinned, white-fleshed potatoes, suggesting that this gene is involved in the regulation of anthocyanin synthesis.

[0029] 2. This invention demonstrates that overexpression of the StTOE3 gene can increase the anthocyanin content of tobacco plants by recombinant expression vector pBvVA(V)KS-StTOE3 and overexpressing it. It also shows that the function of the protein encoded by the StTOE3 gene is to increase the anthocyanin content of plants, laying the foundation for further research on the specific role of the StTOE3 gene in the anthocyanin synthesis pathway. Attached Figure Description

[0030] Figure 1 The results of phenotypic and anthocyanin content detection at different stages of tuber development of purple-skinned purple-fleshed potato (HS66) and yellow-skinned white-fleshed potato (DXY) in Example 1 of this invention are shown; where H1-H3: tuber formation, swelling, and maturity stages of 'HS66', and D1-D3: tuber formation, swelling, and maturity stages of 'DXY' (the same below); the anthocyanin content values ​​at each stage are shown in parentheses.

[0031] Figure 2 The results of the StTOE3 gene expression pattern detection in Example 1 of this invention are shown; (A) is a bar chart of relative expression levels detected by RT-qPCR, and (B) is a heatmap of RNA-seq sequencing results; H1 and D1 represent the tuber formation stage of HS66 and DXY, H2 and D2 represent the tuber enlargement stage of HS66 and DXY, and H3 and D3 represent the tuber maturation stage of HS66 and DXY.

[0032] Figure 3 This is a schematic diagram of the structure of the recombinant plasmid pBvVA(V)KS-StTOE3 constructed in Example 3 of the present invention; wherein, the brown MCS fragment in the lower right corner is the T-DNA region of plasmid pBvVA(V)KS, and the target gene fragment is inserted and amplified in this region; upstream is the CaMV35S promoter fragment, and downstream is connected to the eGFP fluorescent label and NOS terminator;

[0033] Figure 4This is an electrophoresis diagram of PCR identification of transgenic tobacco plants in Example 4 of the present invention; where M is the molecular weight standard (fragment sizes from top to bottom are 2000, 1000, 750, 500, 250 and 100 bp respectively), CK+ is the positive control of plasmid pBvVA(V)KS-StTOE3, CK- is the control of empty plasmid pBvVA(V)KS, WT is the negative control of wild-type tobacco, and the remaining lanes numbered 1-12 are the transgenic tobacco plants to be identified, and those containing the target fragment are transgenic positive plants;

[0034] Figure 5 The image shows the RT-qPCR detection results of the T3 generation transgenic tobacco plants in Example 4 of this invention; where WT is the negative control of wild-type tobacco, and OE-STOE3-1, OE-STOE3-2, and OE-STOE3-3 are three different positive plants; lowercase letters a and b indicate significant differences at the P<0.05 level.

[0035] Figure 6 The results of detecting the petal phenotype and anthocyanin content of transgenic tobacco lines in Example 5 of this invention are shown. Among them, CK-: petals of control plants transgenic with empty plasmid pBvVA(V)KS, OE-STOE3: petals of positive tobacco plants transgenic with pBvVA(V)KS-StTOE3, OE-STOE3-1, OE-STOE3-2, and OE-STOE3-3 are petals of three different positive plants; lowercase letters a and b indicate significant differences at the P<0.05 level. Detailed Implementation

[0036] This invention involves planting purple-skinned, purple-fleshed potatoes (HS66) and yellow-skinned, white-fleshed potatoes (DXY) in an experimental field at the Inner Mongolia Agricultural University Science and Technology Park, under conventional field management. Samples were collected at the tuber formation, tuber enlargement, and maturity stages to detect anthocyanin content and StTOE3 gene expression levels. The study found that the expression abundance of the StTOE3 gene was significantly higher in purple-skinned, purple-fleshed potatoes during tuber formation than in yellow-skinned, white-fleshed potatoes, suggesting that this gene participates in the regulation of anthocyanin synthesis. Using Agrobacterium-mediated genetic transformation, the overexpression vector pBvVA(V)KS-StTOE3 was transformed into tobacco to obtain transgenic StTOE3-positive plants. Molecular identification and anthocyanin content detection results showed that the anthocyanin content in the petals of transgenic StTOE3 plants was significantly increased, indicating that this gene plays a positive regulatory role in plant anthocyanin synthesis. The invention will be described in detail below with reference to specific embodiments.

[0037] The anthocyanin synthesis-related protein provided by this invention is derived from potato (Solanum tuberosum L.), named StTOE3, and is a protein as follows a) or b):

[0038] a) A protein consisting of SEQ ID NO.3;

[0039] b) Proteins derived from (a) whose amino acid sequence shown in SEQ ID NO.3 has been modified by substitution and / or deletion and / or addition of one or more amino acid residues and are associated with anthocyanin synthesis in plants;

[0040] SEQ ID NO.3 consists of 507 amino acid residues.

[0041] To facilitate the purification of the protein in (a) above, a tag as shown in Table 1 may be attached to the amino or carboxyl terminus of the protein composed of SEQ ID NO.3.

[0042] Table 1. Sequence of Labels

[0043]

[0044] The proteins mentioned in (b) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0045] The gene encoding the protein in (b) above can be obtained by deleting one or more amino acid residues from the codons of the DNA sequence shown in SEQ ID NO.1, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5' end and / or 3' end.

[0046] The gene encoding the protein is any one of the following genes 1)-6):

[0047] 1) Its base sequence is the DNA molecule shown in positions 1 to 1524 of SEQ ID NO.1;

[0048] 2) Its base sequence is the DNA molecule shown in positions 1 to 3695 of SEQ ID NO.2;

[0049] 3) Its base sequence is the DNA molecule shown in SEQ ID NO.1;

[0050] 4) Its base sequence is the DNA molecule shown in SEQ ID NO.2;

[0051] 5) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA sequence defined in 1), 2), 3), or 4) and encodes the protein therein;

[0052] 6) A DNA molecule that hybridizes under stringent conditions to the DNA sequence defined in 1) or 2) or 3) or 4) or 5) and encodes the protein therein.

[0053] SEQ ID NO.1 consists of 1524 deoxynucleotides and is the full-length CDS sequence of the potato (Solanum tuberosum L.) protein StTOE3; positions 1 to 1524 are open reading frames. SEQ ID NO.2 is the potato genomic DNA corresponding to the StTOE3 gene, with a full length of 3695 bp, consisting of 5 exons (positions 309-857, 1457-1886, 2172-2331, 2479-2684, and 3130-3312) and 4 introns.

[0054] The above stringent conditions can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; alternatively: hybridization at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; alternatively: hybridization at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; alternatively: hybridization at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS; alternatively: hybridization at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS; alternatively: hybridization at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50°C in 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA. Hybridization was performed in a mixed solution of Na3PO4 and 1 mM EDTA, followed by rinsing at 65°C with 0.1×SSC and 0.1% SDS. Alternatively, hybridization was performed in a solution of 6×SSC and 0.5% SDS at 65°C, followed by rinsing once with 2×SSC and 0.1% SDS and once with 1×SSC and 0.1% SDS.

[0055] Recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria, or recombinant viruses containing the above-mentioned genes are also within the scope of protection of this invention.

[0056] Recombinant expression vectors containing the above-mentioned genes can be constructed using existing plant expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBvVA(V)KS, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3' end of the mRNA precursor; similar functions exist in the untranslated regions transcribed at the 3' end of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthase) and plant genes (such as the soybean storage protein gene). When constructing recombinant plant expression vectors using the genes described above, any type of enhancing promoter (such as the cauliflower mosaic virus (CAMV) 35S promoter, the maize ubiquitin promoter), constitutive promoter, or tissue-specific expression promoter (such as a seed-specific expression promoter) can be added before the transcription initiation nucleotide. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants that encode enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinic acid, the hph gene that confers resistance to the antibiotic hygromycin, the dhfr gene that confers resistance to methatrexate, and the EPSPS gene that confers resistance to glyphosate), or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose.

[0057] The aforementioned recombinant vector can be a recombinant expression vector obtained by inserting the gene into the vector pBvVA(V)KS, specifically, it can be a recombinant expression vector obtained by inserting the coding gene into the vector pBvVA(V)KS; the vector pBvVA(V)KS is prepared by a method including the following steps: amplifying the target gene with specific primers containing adapter sequences, recovering the 1.5 kb target fragment, and linking the target gene to the pBvVA(V)KS backbone using homologous recombination to obtain the recombinant vector pBvVA(V)KS-StTOE3.

[0058] This invention protects the application of the StTOE3 protein or the StTOE3 gene in regulating anthocyanin synthesis in target plants.

[0059] Another objective of this invention is to provide a method for cultivating transgenic plants, which involves introducing the StTOE3 gene into a target plant to obtain a transgenic plant with a higher anthocyanin content than the target plant.

[0060] The present invention also provides a method for increasing the anthocyanin content of a target plant, comprising the step of introducing the gene into the target plant.

[0061] In both of the above methods, the importation is achieved through the recombinant vector.

[0062] In both of the above methods, the anthocyanin content is detected using the pH method.

[0063] In the above applications and methods, the target plant can be a monocotyledonous plant or a dicotyledonous plant, and the dicotyledonous plant can specifically be tobacco.

[0064] Experiments have shown that transforming tobacco with the recombinant vector pBvVA(V)KS-StTOE3 containing the DNA molecule shown in SEQ ID NO.1 yields T3 generation homozygous transgenic lines. After the seeds germinate, they are cultured under conventional conditions. After the plants flower, the anthocyanin content of the petals of the transgenic plants and the control plants is detected. It is found that the anthocyanin content of the petals of the transgenic plants is significantly higher than that of the control.

[0065] Example 1: Expression analysis of the StTOE3 gene in potato tubers of different colors

[0066] This embodiment analyzes the expression of the StTOE3 gene in potato tubers. The experimental method is as follows:

[0067] (1) Sampling of potato tubers

[0068] The tested potato varieties were yellow-skinned, white-fleshed potato DXY and purple-skinned, purple-fleshed potato HS66, which were provided and stored in our laboratory. Plump, disease- and pest-free seed potatoes were selected and planted in the experimental field of the Inner Mongolia Agricultural University Science and Technology Park, under routine field management. Samples were collected at the tuber formation stage (flowering stage, June 30, 2023), the tuber enlargement stage (July 15, 2023), and the maturity stage (September 25, 2023). Anthocyanin content was determined using the pH method (described below), and the samples were flash-frozen in liquid nitrogen and stored at -80℃ for RNA extraction.

[0069] Among them, "HS66" represents "Huasong 66," bred by Huasong Seed Industry, with variety registration certificate number GPD Potato (2018) 110089. It is an antioxidant variety rich in anthocyanins, a mid-to-late maturing variety with a growth period of about 95 days. It has an upright plant type, strong growth, long oval tubers, purple skin, smooth surface, few and light-colored eyes, and particularly purple flesh. "DXY" represents "Atlantic," a variety introduced from the United States. It is a mid-to-late maturing variety with a growth period of about 90 days. It has an upright plant type, thick stems, a moderate number of branches, and strong growth. The tubers are oval or round with shallow eyes, pale yellow skin, and white flesh. The anthocyanin content of the tubers is extremely low.

[0070] (2) Determination of anthocyanin content by pH method

[0071] Take 1 g of fresh potato tuber sample, grind it, and place it in a test tube. Add 15 mL of extraction solution (a mixture of 0.1 mol / L HCl and 95% ethanol at a volume ratio of 1:1). Incubate in a water bath at 60℃ for 1.5 h, filter, and collect the supernatant. Add another 15 mL of extraction solution to the filter residue and repeat the extraction once. Mix the supernatants from the two extractions and adjust the pH to 1.0 and 4.5. Measure the absorbance at 530 nm and 700 nm using a visible spectrophotometer (Beijing, TU-1810SPC). Calculate the anthocyanin content using the pH difference method. Based on Beer-Lambert's law, the difference in absorbance of the anthocyanin solution is proportional to the anthocyanin content. Absorbance value A = (A... 530 nm -A 700 nm pH 1.0-(A) 530 nm -A 700 nm pH 4.5, anthocyanin content (mg / kg FW) = [(A / Ɛ· L)·V / m]·MW·100. Extinction coefficient Ɛ = 31600 L·mol -1 ·cm -1 L is the optical path length, 1 cm; V is the extraction volume, 30 mL; m is the fresh weight of the tested potato; MW is the molecular weight of anthocyanin, 449.2 g·mol⁻¹ -1 .

[0072] (3) Extraction of total RNA and acquisition of cDNA

[0073] The improved CTAB method (Zhang Xi, Analysis of early response genes to salt stress in cotton and cloning of salt tolerance-related genes: [Doctoral dissertation], China Agricultural University, 2010) was used to extract total RNA from the samples collected in step (1), and cDNA was obtained by reverse transcription using M-MLV RTaseRNase H- (Promega, M5301).

[0074] (4) RT-PCR and Real-time PCR

[0075] The cDNA obtained in step (3) was diluted 5-fold and used as a template for RT-PCR and real-time quantitative PCR. Specific primers StTOE3F and StTOE3R were designed at the 3′ end of the StTOE3 gene sequence, with StActin as the internal reference gene. The results are as follows: Figure 1 and Figure 2 As shown. Figure 1 The results showed that the anthocyanin content of purple-skinned, purple-fleshed potatoes was significantly higher than that of yellow-skinned, white-fleshed potatoes at all stages of tuber development. Figure 2 The results showed that the expression abundance of the StTOE3 gene during the tuber formation stage of purple-skinned, purple-fleshed potatoes was significantly higher than that during the tuber formation stage of yellow-skinned, white-fleshed potatoes.

[0076] Real-time quantitative PCR was performed on an ABI 7500 real-time quantitative PCR instrument, with each parallel experiment having three replicates. The method reported by Livak KJ and Schmittgen TD (2001) was used, i.e., 2... -∆∆CT Calculate the relative expression level. The method for calculating ∆∆CT is as follows:

[0077] ∆∆CT=(C T.Target -C T.Actin ) Time x - (C T.Target -C T.Actin ) Time 0

[0078] Where Time x represents any time point, and Time 0 represents the target gene expression at 1-fold after actin correction.

[0079] The base sequences of primers StTOE3F and StTOE3R are as follows:

[0080] The base sequence of the StTOE3F (SEQ ID NO.6) primer is: 5′- GGTCGAGGCTCAAGCAACATAGTC-3′ (corresponding to positions 1123-1146 of SEQ ID NO.1);

[0081] The base sequence of the StTOE3R (SEQ ID NO.7) primer is: 5′-ATGTAGGTGCGTTTGGCTCAAGAG-3′ (corresponding to positions 1206-1229 of SEQ ID NO.1).

[0082] The primer sequences for the internal reference gene StActin are as follows:

[0083] The base sequence of the StActinF (SEQ ID NO.8) primer is: 5′-ATTGTGAGCAACTGGGATGA-3′

[0084] The base sequence of the StActinR (SEQ ID NO.9) primer is: 5′-GTAGATGGGGACGGTGTGAG-3′

[0085] Example 2: Cloning of the potato StTOE3 gene

[0086] The specific method for cloning the potato StTOE3 gene and constructing the recombinant vector in this embodiment is as follows:

[0087] (1) Total RNA was extracted from the tetraploid material Atlantic potato (Solanum tuberosum L.) tuber material (Reference: Wang Yanjun. Characteristics and high-yield cultivation techniques of Atlantic potato. Shanxi Agricultural Economy. 2019, 21. Available to the public from Inner Mongolia Agricultural University) and cDNA was obtained by reverse transcription. The cDNA was used as a template and E26386F and E26386R were used as primers for PCR amplification. The PCR amplification reaction system consisted of: 2 μL cDNA, 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 5 μL 10×LA buffer, 2.5 μL dNTPs (2.5 mM each), 0.5 μL LA-Taq enzyme, and 38 μL ddH2O. The PCR amplification reaction program was: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 2 min, for 30 cycles; and 72℃ extension for 10 min.

[0088] The primer sequences for the above PCR amplification are as follows:

[0089] Primer E26386F (SEQ ID NO.4): 5′-CATatgcatcaccatcaccatcacatgtggaatctaaacaactctccag-3′

[0090] Primer E26386R (SEQ ID NO.5): 5′-TCCTCGCCCTTGCTCACCATgttggagggtctggtgagagag-3′

[0091] (2) The obtained PCR amplification products were subjected to 1% agarose gel electrophoresis, and a fragment of about 1.5 kb was recovered, ligated into the pMD18-T vector (TAKARA, D101A), and transformed into Escherichia coli DH5α strain. Positive clones were screened using the blue-white screening method, and the positive clones were sequenced. The sequencing results showed that the positive clone vector (named pMD18-T-StTOE3) had the DNA fragment shown in SEQ ID NO.1 inserted into the pMD18-T vector, indicating that the recombinant vector was successfully constructed.

[0092] It is worth noting that the sequence of SEQ ID NO.1 is the CDS sequence of the StTOE3 gene, which is 1524 bp long and is the coding sequence of the potato StTOE3 gene. The full-length potato genomic DNA corresponding to the StTOE3 gene is 3695 bp long, as shown in the sequence listing SEQ ID NO.3. It consists of 5 exons (positions 309-857, 1457-1886, 2172-2331, 2479-2684, and 3130-3312) and 4 introns. The amino acid sequence of the protein StTOE3 encoded by the coding sequence is shown in SEQ ID NO.2. This protein consists of 507 amino acid residues.

[0093] Example 3 Construction of recombinant plant expression vectors

[0094] This embodiment constructs a recombinant plant expression vector using homologous recombination, as detailed below:

[0095] 1. Obtaining the target gene StTOE3

[0096] Using pMD18-T-StTOE3 obtained in Example 2 as a template, and primers E26386F and E26386R as specific primers, PCR amplification was performed. The PCR amplification product was detected by 1% agarose gel electrophoresis, and the PCR product of approximately 1.5 kb was recovered and purified. Sequencing results showed that the PCR product contained a 1524 bp DNA fragment as shown in positions 1 to 1524 of SEQ ID NO. 1.

[0097] 2. Construction of plant expression vectors using homologous recombination method

[0098] (1) Preparation of linearized vector: A linearized vector was obtained by enzyme digestion to obtain a fragment whose ends precisely correspond to the homologous arms of the inserted target gene fragment. The enzyme digestion system was as follows: 20 μL plasmid, 1.25 μL Nco I, 1.25 μL Lpe I, 5 μL 10×Buffer, 0.3 μL 100×BSA, 22.2 μL ddH2O; digestion for 2-3 h. The amplification system and primers for this target gene fragment were the same as in step (1) of Example 2.

[0099] (2) Purify all DNA fragments using gel recovery or PCR purification kits and accurately determine the concentration using a micro-nucleic acid quantification instrument.

[0100] (3) According to the recommended ratio of the commercial kit (NEB Gibson Assembly HiFi Mix), the linearized vector and the inserted target gene fragment were mixed in a microcentrifuge tube, and a recombinase premix was prepared. The reaction system was incubated in a 50°C water bath for 30 min to obtain the recombinant reaction product.

[0101] (4) The recombinant reaction product was directly added to competent DH5α cells, and after a 30-minute ice bath, it was heat-shocked in a 42°C water bath for 90 seconds. It was then immediately removed and ice-bathed again for 5 minutes. The transformed bacterial culture was spread onto solid LB plates containing 50 μg / mL kanamycin antibiotic and incubated overnight at 37°C. Rapid screening was performed using universal vector primers or gene-specific primers. Clones that amplified the expected size were considered preliminary positive clones, indicating successful construction of the recombinant plant expression vector pBvVA(V)KS-StTOE3, which can be used for subsequent transgenic function verification. A schematic diagram of the structure of the recombinant plant expression vector pBvVA(V)KS-StTOE3 is shown below. Figure 3 As shown.

[0102] The pBvVA(V)KS vector was modified from the pCAMBIA1300 vector, and its base sequence is shown in SEQ ID NO.17.

[0103] Example 4: Transformation of Tobacco with Potato StTOE3 Gene

[0104] In this embodiment, transgenic tobacco plants overexpressing the StTOE3 gene were obtained using the Agrobacterium-mediated transformation method. The specific method is as follows:

[0105] (1) Transformation of Agrobacterium tumefaciens with recombinant expression vector: The recombinant plant expression vector pBvVA(V)KS-StTOE3 prepared in Example 3 was used to transform competent cells of Agrobacterium tumefaciens GV3101 (purchased from Beijing Tianenze Gene Technology Co., Ltd.) by freeze-thaw method. The cells were screened and cultured at 28°C in YEP solid culture containing 50 μg / mL kanamycin sulfate and 50 μg / mL rifampin. Positive monoclonal colonies were identified by colony PCR (using primers E26386F and E26386R from Example 2). The Agrobacterium tumefaciens that were correctly identified, i.e. the recombinant Agrobacterium tumefaciens containing the recombinant plant expression vector pBvVA(V)KS-StTOE3, was named GV3101 / pBvVA(V)KS-StTOE3.

[0106] (2) Preparation of Agrobacterium infection solution: Take the recombinant Agrobacterium tumefaciens GV3101 / pBvVA(V)KS-StTOE3 obtained in step (1) and inoculate it into 5 mL of YEP liquid medium (containing 50 μg / mL kanamycin sulfate and 50 μg / mL rifampin). Shake overnight. The next day, transfer the culture to 500 mL of YEP liquid medium and incubate at 28℃ until OD. 600 The bacterial cells were collected by centrifugation to a concentration of 1.6-2.0. The precipitate was resuspended in MS liquid medium, and then MS liquid medium was added again to fully suspend the bacterial cells, ensuring the OD of the suspension was maintained. 600 It is approximately 0.2.

[0107] (3) Obtaining genetically modified tobacco: Cut the leaves of sterile tobacco seedlings into 1 cm pieces 2 The leaves were sized, the main veins and edges were removed, and they were immersed in the suspension for 15 min, shaking constantly. The suspension on the leaf surface was blotted dry with sterile filter paper, and the leaves were placed face down on a co-culture medium lined with filter paper and incubated in the dark at 28°C for 2 days. The leaves were then washed three times with sterile water containing 200 mg / L Carb (carbenicillin), and then once with liquid MS containing the same concentration of Carb. After the washing solution was blotted dry with filter paper, the leaves were placed on selection medium (containing 200 mg / L Carb and 10 mg / L PPT) and incubated under light at 28°C.

[0108] (4) Transplanting and screening of resistant seedlings: When the adventitious buds on the screening medium in step (3) grow to 2-3 cm, they are cut off and placed on the rooting medium for rooting culture. When the root system is relatively robust, the seedlings are transplanted into water for hardening culture. After 5-7 days, the seedlings are transplanted into the soil and placed in the greenhouse to continue growing until T1 generation seeds are obtained. After disinfecting the T1 generation seeds, they are spread on MS solid medium (containing 50 μg / mL kanamycin sulfate) and cultured at 21℃ for 10 days. Then, resistant plants are selected and transplanted into the soil. After 30 days, T2 generation seeds are harvested per plant. T2 generation seeds are planted and screened in the same way as T1 generation seeds. Three T2 generation lines with a resistance segregation ratio of 3:1 are transplanted, and T3 generation seeds produced on each individual plant in the T2 generation line are harvested per plant. Sixteen T3 generation line seeds are randomly selected and subjected to resistance screening in the same way to obtain 12 homozygous transgenic lines of T3 generation that no longer produce resistance segregation. Plants or seeds from T3 generation homozygous transgenic lines were used for PCR identification and phenotypic identification. The tobacco rooting medium consisted of 40g MS + 0.5mg / L IAA + 300mg / L Timentin + 50mg / L Kan.

[0109] Meanwhile, empty vector pBvVA(V)KS was transformed using the same method as an empty vector control, resulting in three homozygous empty vector control lines in the T3 generation.

[0110] Wherein, T1 generation represents the seeds produced by self-pollination of the current generation of plants and the plants that grow from them, T2 generation represents the seeds produced by self-pollination of T1 generation and the plants that grow from them, and T3 generation represents the seeds produced by self-pollination of T2 generation and the plants that grow from them.

[0111] (5) PCR identification of homozygous transgenic tobacco plants: Genomic DNA was extracted from leaves of the T3 generation homozygous transgenic plants that no longer produced resistance segregation obtained in step (4). PCR amplification was performed using primer 35seq across the promoter and primer E26386(514C) for the target gene. The predicted product size was 711 bp, and all results were positive. Electrophoresis images of PCR products from some plants are shown below. Figure 4 As shown. The T3 generation homozygous transgenic empty vector control lines and wild-type tobacco plants obtained according to step (4) of this method all showed negative results. Furthermore, RT-qPCR detection of the T3 generation homozygous transgenic lines and wild-type tobacco plants revealed (e.g.) Figure 5 As shown in the figure, the expression level of the StTOE3 gene in the T3 generation homozygous transgenic line was significantly higher than that in the wild type, indicating that it was a transgenic tobacco plant that overexpressed the StTOE3 gene.

[0112] The base sequences of primers 35seq and E26386(514C) are as follows:

[0113] The base sequence of 35seq (SEQ ID NO.15) is: 5′- tTCATTTGGAGAGAACACGGGggac-3′

[0114] The base sequence of E26386(514C) (SEQ ID NO.16) is: 5′-CACGAGCAGCAGCATGTGC-3′

[0115] Example 5: Determination of anthocyanin content in petals of StTOE3 transgenic tobacco plants

[0116] This embodiment identifies the anthocyanin content of the T3 generation homozygous transgenic lines obtained in Example 4, and the results are as follows: Figure 6 As shown. By Figure 6 It can be seen that the anthocyanin content in StTOE3 transgenic tobacco plants is significantly increased, indicating that overexpression of the StTOE3 gene can increase the anthocyanin content in tobacco petals.

[0117] The identification method was as follows: seeds of T3 generation homozygous transgenic lines (TL1-TL12), T3 generation homozygous empty vector control lines (CK-), and wild-type tobacco (WT) were sown on the surface of nutrient soil (soil:vermiculite = 3:1), sprayed with moisture, and then placed at 28℃ for 16 h / 8 h light-dark cycle culture. Each line was replicated three times. After the plants reached the flowering stage, the petal phenotype was observed and the anthocyanin content of the petals was detected.

[0118] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A StTOE3-encoding gene, characterized in that, The base sequence is shown in SEQ ID NO.

1.

2. A StTOE3 gene, characterized in that, The base sequence is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, It contains a base sequence as shown in SEQ ID NO.

1.

4. A StTOE3 protein, characterized in that, The protein is encoded by a gene and is related to the synthesis of anthocyanins in plants. The base sequence of the gene is shown in SEQ ID NO.

1. Alternatively, the gene may be obtained by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID NO.1, and / or by performing a missense mutation on one or more base pairs, and / or by attaching a tag to its 5' end and / or 3' end.

5. The StTOE3 protein according to claim 4, characterized in that, The tag is Poly-Arg, Poly-His, FLAG, Strep-tag II, or c-myc, wherein the amino acid sequence of the Poly-Arg tag is shown in SEQ ID NO.10, the amino acid sequence of the Poly-His tag is shown in SEQ ID NO.11, the amino acid sequence of the FLAG tag is shown in SEQ ID NO.12, the amino acid sequence of the Strep-tag II tag is shown in SEQ ID NO.13, and the amino acid sequence of the c-myc tag is shown in SEQ ID NO.

14.

6. The StTOE3 protein according to claim 4 or 5, characterized in that, The amino acid sequence of the StTOE3 protein is as shown in SEQ ID NO.3, or the StTOE3 protein is a derivative protein of the amino acid sequence shown in SEQ ID NO.3 with substitution and / or deletion and / or addition of one or more amino acid residues and related to the synthesis of plant anthocyanins.

7. A set of StTOE3 amplification primers, characterized in that, Used to amplify the base sequence shown in SEQ ID NO.1, whose base sequence is shown in SEQ ID NO.4 and SEQ ID NO.

5.

8. A set of primers for detecting StTOE3, characterized in that, Used to detect the expression level of the base sequence shown in SEQ ID NO.1, whose base sequence is shown in SEQ ID NO.6 and SEQ ID NO.

7.

9. A carrier template, characterized in that, The vector is used to form a recombinant vector by combining with the gene of claim 1 or 2, wherein the vector template is pBvVA(V)KS, and its base sequence is shown in SEQ ID NO.

17.

10. The application of the StTOE3 encoding gene, the StTOE3 gene, the recombinant vector, and the StTOE3 protein, characterized in that... The StTOE3 encoding gene of claim 1, or the StTOE3 gene of claim 2, or the recombinant vector of claim 3, or the StTOE3 protein of any one of claims 4-6, is used to increase the anthocyanin content in plants; the plant is a monocotyledonous plant or a dicotyledonous plant.

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