Application of StASG gene in potato variety improvement

Abstract

This invention belongs to the field of plant genetic engineering technology, and particularly relates to... StASG Application of genes in potato variety improvement. This application discovers and confirms the application of genes in potato variety improvement. StASG The mechanism of gene regulation of anthocyanin synthesis, reducing StASG Gene expression levels can significantly increase anthocyanin content in potato tubers, advance the first flowering time of potato plants, and significantly increase plant height. Therefore, this study provides a key target gene for simultaneously increasing anthocyanin content, shortening the first flowering time, and increasing plant height in potatoes, further refining the anthocyanin biosynthesis and metabolism regulatory network, and providing ideal materials for high-quality breeding of colored potatoes.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology. In particular, it relates to... StASG Application of genes in potato variety improvement. Background Technology

[0002] potato( Solanum tuberosum L. is an important dual-purpose crop for both food and vegetables, with advantages such as strong adaptability, short growth cycle, and high yield. Colored potatoes are a group of varieties in the genus *Potamogeton* of the Solanaceae family with rich skin / tuber colors. Based on color, they can be divided into purple, red, blue, and bicolor series. Research on this type of potato has increased the diversity of the potato gene pool and provided key gene resources for breeding work on disease resistance, cold resistance, and drought resistance (Zhang et al., 2024; Sun et al., 2024).

[0003] Anthocyanins are the main cause of the diverse colors of potatoes. They are important secondary metabolites synthesized in higher plants and belong to the flavonoid family. Anthocyanins are readily soluble in water and alcohol, but insoluble in organic solvents such as chloroform and ether. They are glycosylated derivatives formed by anthocyanins and various monosaccharides (glucose, rhamnose, galactose, xylose, and arabinose, etc.) through glycosidic bonds. They are mainly stored in vacuoles and can exhibit different colors depending on changes in intracellular pH, thus giving different plant tissues different colors, such as red, purple, or blue (Sunil et al., 2022). Anthocyanins are powerful antioxidants, with antioxidant capacity far exceeding that of vitamin E and vitamin C. This strong antioxidant property endows anthocyanins with a variety of physiological functions, including anti-inflammatory and antibacterial effects, anti-proliferative effects, prevention of cardiovascular disease, and reduction of capillary permeability (Tian et al., 2016; Vishnu et al., 2019; Tang et al., 2021).

[0004] The anthocyanin synthesis pathway is highly conserved in plants, and all key enzyme genes in the metabolic pathway have been cloned and identified (Takos et al., 2006). Studies have found that in addition to the influence of structural genes in the metabolic pathway, MYB, the bHLH transcription factor family, WD40 protein, the WRKY transcription factor family, and zinc finger proteins also participate in the regulation of anthocyanin synthesis (Wang et al., 2017; Liu et al., 2018). Furthermore, various factors such as light, temperature, sugars, and hormones can also indirectly affect this metabolic pathway (Ji et al., 2015; Liu et al., 2021; Sun et al., 2024).

[0005] Currently, the regulatory network of potato anthocyanin synthesis is not fully understood, and research mainly focuses on the MYB-bHLH-WD40 core complex. StASG is a key factor regulating potato anthocyanin synthesis, but no relevant reports have been found to date. Summary of the Invention

[0006] Based on existing technology, this application discovers and confirms the potato StASG (Anthocyanin synthesis gene) The mechanism by which this gene regulates anthocyanin synthesis is to reduce... StASG Gene expression levels can significantly increase anthocyanin content in potato tubers and significantly increase plant height. Additionally, overexpression... StASG Genes can significantly advance the first flowering time of potato plants. Therefore, this study provides a key target gene for simultaneously increasing anthocyanin content, shortening the first flowering time, and increasing plant height in potatoes, further refining the anthocyanin biosynthesis and metabolism regulatory network, and providing ideal materials for high-quality breeding of colored potatoes.

[0007] Early flowering, increased plant height, and high anthocyanin content all have beneficial effects on potato production, quality, and / or economic benefits. Specifically, (1) Early flowering: Potato flowering is a crucial indicator of the transition from vegetative to reproductive growth. Earlier flowering allows for earlier maturity, increasing the land's multiple cropping index. After flowering, photosynthetic products are gradually transferred and accumulated in the tubers, and earlier flowering simultaneously advances the tuber enlargement initiation time, shortening the overall growth period. In areas with two or more crops per year (southern winter cropping areas and intercropping areas in the Central Plains), early-maturing potatoes can be followed by planting corn, vegetables, rapeseed, and other crops, significantly improving land utilization and increasing the annual total output value per unit area. Furthermore, it expands suitable planting areas. In cold regions with high altitudes and short frost-free periods, traditional potatoes often struggle to achieve normal tuber formation and high yields due to insufficient growth period. The early maturity resulting from earlier flowering allows these areas to successfully complete their growth cycle, expanding the suitable planting area for potatoes and revitalizing marginal arable land resources. To avoid the high temperatures and late blight outbreaks in the later stages of potato growth, which are often caused by high temperatures and concentrated rainfall in major potato-producing areas, this approach helps farmers avoid the high temperatures and late blight. Early flowering shifts the tuber formation, tuber enlargement, and maturity stages forward, allowing the plant to complete its main growth and development before the peak of high temperature and humidity, reducing pesticide use, lowering the risk of yield reduction, and significantly improving yield stability. Shorter growing seasons mean shorter periods of irrigation, cultivation, weeding, and pest and disease control, resulting in reduced inputs such as labor, water, electricity, pesticides, and fertilizers, and lowering the average cost per acre. Early-maturing potatoes can also capture a larger share of the market, filling the gap in fresh potato supply. The early spring fresh potato market is in high demand and sells for significantly higher prices than conventional potatoes harvested during peak seasons, allowing farmers to benefit from off-peak pricing and significantly improve their economic returns per acre.

[0008] (2) Increased plant height: This invention enhances photosynthesis, increases biomass and yield, and leads to increased plant height accompanied by greater foliage density and total leaf area. It significantly improves the field canopy's light-harvesting capacity and carbon dioxide fixation efficiency, and substantially increases the synthesis of photosynthetic products (carbohydrates). Potato tubers are nutrient storage organs; the increased translocation of photosynthetic products to tubers directly increases the number of tubers per plant and the weight of each tuber, resulting in increased yield per acre. It also synergistically enhances characteristic qualities (anthocyanins), with this invention reducing... StASGGenes can simultaneously increase the anthocyanin content of potato tubers. Sufficient photosynthetic products not only supply tuber enlargement but also provide the material and energy basis for the synthesis of secondary metabolites such as anthocyanins, thus consolidating the nutritional quality of colored potatoes while increasing yield, achieving a double improvement in both yield and quality. It enhances lodging and wind resistance (within a reasonable plant height range). Stronger, taller plants have thicker stems, higher mechanical strength, and a more coordinated root-to-above-ground ratio, making them more resistant to lodging from strong winds and heavy rains compared to shorter, weaker plants. When potatoes lodge, the stems and leaves lie flat on the ground, making them susceptible to disease, hindering photosynthesis, and causing tuber deformities. Increasing plant height appropriately can effectively avoid these problems and ensure normal growth. It improves tolerance to poor soil and low light. Plants with abundant stems and leaves have simultaneously enhanced root absorption capacity, resulting in higher efficiency in absorbing and utilizing nitrogen, phosphorus, potassium, and trace elements in the soil. They can still grow normally in moderately or poorly fertile soils, reducing the limitation of soil conditions on yield. In shady areas with insufficient sunlight, a larger canopy can also compensate for insufficient light. Increased plant height leads to greater canopy closure in potatoes, which blocks sunlight from reaching the ground, effectively suppressing the germination and growth of weeds in the field. This reduces labor input for weeding and lowers the amount of herbicide used, saving costs and meeting the requirements of green planting.

[0009] (3) High anthocyanin content: The tubers have a bright and uniform color. Increased anthocyanin content gives the tubers a deep purple or bright red hue, with significantly better color uniformity and gloss than ordinary white / yellow-fleshed potatoes. In the sale of fresh potatoes, appearance is a crucial factor for consumers; high-quality tubers directly elevate the product's grade, distinguishing them from ordinary potatoes. Enhanced nutritional function and upgraded eating quality: Anthocyanins are natural, powerful antioxidants, rich in polyphenolic active ingredients. Compared to ordinary potatoes, their nutritional value is significantly improved. Anthocyanins have antioxidant, free radical scavenging, and metabolic regulation effects, transforming potatoes from a traditional staple food into a functional nutritional food, aligning with current demands for healthy eating and wellness consumption. High-anthocyanin plants exhibit vigorous photosynthetic metabolism, resulting in more abundant accumulation of starch, vitamin C, minerals, and other conventional nutrients in the tubers. While retaining the original taste and flavor of potatoes, they achieve the dual attributes of "staple food + functional food." Anthocyanin synthesis under normal regulation does not alter the original flavor and texture of potatoes, balancing flavor and nutrition, and is suitable for various consumption methods such as fresh eating, steaming, boiling, and salads. It enhances post-harvest storage and resistance to spoilage, improving storage and transportation quality. Freshly cut potatoes are highly susceptible to enzymatic browning, affecting sales and processing. Anthocyanins themselves have antioxidant properties, inhibiting polyphenol oxidase activity, significantly delaying browning in tubers, shreds, and mashed potatoes, and extending fresh shelf life. High-anthocyanin tuber cells have stronger antioxidant capacity, slower post-harvest respiration and aging rates, reducing the proportion of rot, water loss, and spoilage during long-distance transportation and cold storage, thus lowering post-harvest losses. Increased anthocyanin content is achieved through gene modification, while simultaneously optimizing field planting management and stress resistance, reducing production risks and costs. It also enhances the plant's resistance to biological stress. Anthocyanins are important plant defense substances, enhancing potato plants' resistance to some fungal, bacterial diseases, and pests, reducing the probability of disease in the field. Combining the early flowering and robust plant characteristics of this variety, it further reduces the risk of infection by common diseases such as late blight and leaf spot, thus reducing the frequency and amount of pesticide spraying. It enhances tolerance to abiotic stresses; anthocyanins can absorb ultraviolet radiation and eliminate reactive oxygen species generated under adverse conditions, increasing the plant's tolerance to environmental stresses such as strong light, drought, and low temperatures. In open fields, high-altitude areas, and areas with strong sunlight, plant growth is more stable, reducing yield reduction caused by extreme weather. Ordinary potatoes are mainly distributed as bulk, affordable grains and vegetables, resulting in meager profits; while high-anthocyanin colored potatoes are positioned as high-end fresh produce and specialty health foods, fetching prices 2-3 times higher than ordinary potatoes in supermarkets, boutique farmers' markets, and online fresh food platforms, significantly increasing the per-acre yield of fresh potatoes. High-anthocyanin potatoes are no longer limited to fresh consumption; they can undergo multi-level deep processing, extending the industrial chain and increasing product added value, which is the core direction for increasing industry income. Anthocyanins are safe and non-toxic natural food colorings that can replace artificial synthetic colorings and are widely used in the food, beverage, pastry, and daily chemical industries.Potatoes rich in anthocyanins are a high-quality raw material for extraction, with high utilization rate and low extraction cost. This allows for the creation of an integrated industry of "raw material planting + pigment deep processing" and the formation of a stable profit model.

[0010] In this regard, the technical solutions of the present invention include, but are not limited to, the following: In one aspect, the present invention provides StASG Application of genes in regulating anthocyanin content in potatoes, inactivation / reduction of the aforementioned StASG Gene expression.

[0011] In one aspect, the present invention provides StASG Application of the gene in regulating the flowering time of potato plants, overexpression of the above StASG Gene expression.

[0012] In one aspect, the present invention provides StASG Application of genes in regulating potato plant height, inactivation / reduction of the aforementioned StASG Gene expression.

[0013] In one aspect, the present invention also provides deactivation / reduction of the... StASG Application of recombinant vectors for gene expression or recombinant engineered bacteria containing said recombinant vectors in regulating anthocyanin content, flowering time and / or plant height in potatoes.

[0014] In one aspect, the recombinant vector of the present invention comprises StASG The recombinant pHELLSGATE8 vector containing gene-specific fragments, preferably, StASG The sequence of the gene-specific fragment is shown in SEQ ID NO: 3.

[0015] In one aspect, the present invention also provides a method comprising... StASG The application of recombinant vectors of genes or recombinant engineered bacteria containing said recombinant vectors in regulating plant flowering time, and overexpression of said genes. StASG Gene.

[0016] Preferably, the genetically engineered bacteria of the present invention are genetically engineered Escherichia coli or Agrobacterium.

[0017] In one aspect, the genetically engineered bacteria of the present invention are genetically engineered Agrobacterium.

[0018] In another aspect, the present invention provides a method for obtaining potato varieties with improved traits, comprising the following processing steps: (1) Infecting callus tissue with genetically engineered bacteria; and (2) The infected callus tissue was cultured into complete plants; The genetically engineered bacteria described herein contain inactivation / reduction of the... StASG Recombinant vectors for gene expression or containing StASG Gene recombination vectors.

[0019] In another aspect, the present invention provides a potato with improved traits, having inactivation / reduction properties. StASG Gene expression or overexpression StASG Gene.

[0020] In one aspect, the improved traits described in this invention are increased anthocyanin content, earlier flowering time, and / or increased plant height.

[0021] In one aspect, the present invention describes StASG The gene encodes the amino acid sequence shown in SEQ ID NO: 2.

[0022] The present invention StASG The NCBI reference sequence number for the gene is: NM_001318553.1.

[0023] In one aspect, the present invention describes StASG The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0024] In one aspect, the potato of the present invention is preferably the 'Huacai No. 3' or 'Desiree' variety.

[0025] The beneficial effects of the present invention include at least the following: This application is the first report of intervention in potatoes. StASG This invention promotes anthocyanin synthesis, resulting in a darker potato skin color; significantly advances the first flowering time of potato plants; and significantly increases plant height. It provides a target gene for improving anthocyanin content and plant traits in potatoes, which can further enrich colored potato resources. Through interference... StASG Lowering the expression level of this gene or knocking it out using gene editing can promote anthocyanin synthesis, advance the first flowering time of potato plants, and increase plant height. Attached Figure Description

[0026] Figure 1 The electrophoresis results of transgenic positive lines are shown. In this case, A shows overexpression positive lines and B shows interference expression positive lines.

[0027] Figure 2 The results of protein expression detection in overexpression positive lines are shown.

[0028] Figure 3 Showing interference in transgenic plants StASG Expression level results.

[0029] Figure 4 Showing overexpression StASG The effect of anthocyanin content on potatoes is shown in the following figures: A shows the skin and flesh color of 'Huacai 3'; B shows the total anthocyanin content in 'Huacai 3' tubers; C shows the skin and flesh color of 'Desiree'; and D shows the total anthocyanin content in 'Desiree' tubers.

[0030] Figure 5 interference StASG The effect of gene expression on anthocyanin content in potatoes, where A represents potato skin color and B represents total anthocyanin content in potato tubers.

[0031] Figure 6 show StASG gene The effect on potato plant height, where A showed overexpression. StASG Transgenic plants; B shows interference StASG Expressing transgenic plants.

[0032] Figure 7 Showing overexpression StASG Effects on the timing of first flowering in potato plants. Wild-type and overexpression of the 'Desiree' variety. StASG Statistics on the first budding and flowering time of the plant tubers about 35 days after planting.

[0033] Figure 8 show StASG Representative phenotypic diagrams showing the effect of the effect on the first flowering time of potato plants. Wild type and overexpression of the 'Desiree' variety. StASG A visual phenotypic diagram of the plant in the early stage of flower bud formation and the opening of the flower buds. Detailed Implementation

[0034] Example 1: Identification and Cloning of the StASG Gene Using quantitative real-time PCR (qRT-PCR) technology, we discovered an ethylene response factor in potatoes (NCBI reference sequence number: NM_001318553.1), which we named... StASG The expression levels of this gene differed significantly among different colored potatoes, suggesting that the gene may be involved in anthocyanin synthesis.

[0035] StASG Gene cloning: Amplification primers were designed based on the StASG (672 bp) coding region sequence: attB1-StASG-F: AAAAAGCAGGCTTCACCATGGCGCCCAAGGAA; attB2-StASG-R: AGAAAGCTGGGTCCTATCACATATTTTCCGGTGGA.

[0036] RNA was extracted from Desiree potato seedlings in vitro. RNA purity and concentration were assessed by electrophoresis. The RNA was reverse transcribed into single-stranded cDNA, which was then used as a template for in vitro amplification of the StASG coding region. The amplification system was as follows: 25 μl of 2×PhantaMax Buffer, 1 μl each of forward and reverse primers (10 μM), 1 μl of dNTP Mix (10 mM), 2 μl (100 ng) of Desiree cDNA template, 1 μl of PhantaMax Super-Fidelity DNA Polymerase, and sterile water to a final volume of 50 μl. The reaction program was: 95℃ for 5 min; 95℃ for 20 s, 56℃ for 20 s, 72℃ for 60 s, 35 cycles; 72℃ for 10 min.

[0037] Using the above PCR products as templates, primers attB1-F: GGGGACAAGTTTGTACAAAAAAGCAGGCT and attB2-R: GGGGACCACTTTGTACAAGAAAGCTGGGT were used for in vitro amplification. The target band in the gel was recovered using the Magen HiPure GelPure DNA Mini Kit.

[0038] Construction of the pK7FGW2.0 expression vector: The recovered gene fragment was inserted into the entry vector pDOR201 using BP recombinase (Gateway BP Clonase™ II Enzyme mix, Invitrogen®), constructing the pDOR201-StASG intermediate entry vector. Subsequently, the above entry vector was subjected to LR recombination with the target expression vector pK7FGW2.2 (Gateway LR Clonase™ II Enzyme mix, Invitrogen®). Successful insertion of the target gene was confirmed by sequencing. The gene sequence and encoding amino acid information are shown in SEQ ID NO: 1 and 2.

[0039] Example 2 StASG Plant expression vector construction 1) Vector used for plant overexpression: pH7.0 LIC-HA. The amplification primers used to construct the vector are as follows: LIC-StASG-F: 5'- ATTACGCCGAGGTCatggcgcccaaggaaaaaat; LIC-StASG-R: 5'- TagggaagaggTCACATATTTTCCGGTGGAGGAAA.

[0040] After amplifying the gene using the primers described above, the gene fragment was recovered and inserted into the Agrobacterium plant overexpression vector pH7.0LIC-HA using homologous recombinase (ClonExpress II One Step Cloning Kit, Vazyme®). This vector was then transformed into *E. coli* DH5α. PCR and sequencing confirmed successful insertion of the gene into the expression vector, yielding pH7.0LIC-HA-StASG. The fusion vector plasmid was then transformed into *Agrobacterium* GV3101 for later use.

[0041] 2) Vector used for plant interference expression: pHELLSGATE8. Analysis was performed using the NCBI website. StASG Gene-specific fragments of 200-300 bp were used to construct StASG-RNAi. The two sets of primers used were: StASG-RNAi-XhoI-F: 5'-TTTGGAGAGGACACGCTCGAGGTTGAATCGTCGAGTCCGGT; StASG-RNAi-XhoI-R: 5'-TGGGGTACCGAATTCCTCGAGGTCACCACCACCGAGGAAAT.

[0042] StASG-RNAi-XbaI-F: 5'-TCATTAAAGCAGGACTCTAGAGTCACCACCACCGAGGAAAT; StASG-RNAi-XbaI-R: 5'-GATAAGCTTGGATCCTCTAGAGTTGAATCGTCGAGTCCGT.

[0043] Amplification using the above primers StASG A specific 237 bp fragment of the gene was obtained and inserted into the Agrobacterium plant interference expression vector pHELLSGATE8 via two homologous recombinations using the ClonExpressII One Step Cloning Kit (Vazyme®). This vector was then transformed into E. coli DH5α, and the pHELLSGATE8-StASG vector was obtained by PCR detection and sequencing. This vector plasmid was then transformed into Agrobacterium GV3101 for later use.

[0044] The specific fragment 237bp is: GTTGAATCGTCGAGTCCGGTTATGGTTGATTCATCATCGCCGTTAGATCTAAGACTTTGTGGATCAATCGGCGGGTTTAACCATAATACTGTTAGATTTCCGATCTCCGGTGGAGGTTTTACCGGTGCTGTTCCGGCGTGAATCATATGTACTATTTAGATGCACTTGAACGCGCCGGAGTTATAAATTTAGAAACAAATCGGAAGAAGACGGTGGATTTCCTCGGTGGTGGTGAC (SEQ ID NO: 3) Example 3: Potato genetic transformation and detection of transgenic plants 1) Potato genetic transformation: Potato genetic transformation used in vitro plantlets of the red-skinned, red-fleshed potato variety 'Huacai 3' and the red-skinned, yellow-fleshed potato variety 'Desiree' as transformation recipients. The specific steps are as follows: Potato plantlets were grown in 3% MS solid medium and cultured in a tissue culture room (22℃, 16 h light / 8 h dark) for 3 weeks before genetic transformation. Agrobacterium tumefaciens (GV3101) carrying the target gene vector was activated on YEB solid medium and cultured in the dark at 28℃ for 2 days. Single colonies were picked and added to 5 mL of YEB liquid medium containing kanamycin (kan), and cultured overnight at 200 rpm at 28℃ (log phase). After centrifugation at 4000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in 30 mL of YEB. The leaves were immersed in the infection solution and cultured in the dark for 2 days, then blotted dry with sterile paper and cultured on P1 medium (callus induction medium) for 7 days. Subsequently, resistant shoots were induced on P2 medium (selection medium). After the resistant shoots emerged, they were cut and transferred to rooting selection medium P3 for rooting. Positive selection of rooted single plants was then carried out.

[0045] Table 1. Potato transgenic culture medium

[0046] 2) Detection of transgenic positive lines: For plant lines exhibiting overexpression and interference expression, DNA was extracted using the CTAB method. This DNA was then used as a template for PCR detection. Primers used for detecting overexpression positive lines: F-35S:CAAAGGGCTATTGAGACTTTTCAAC, R: gene-specific primer. Primers used for detecting interference expression positive lines: F-35S:CAAAGGGCTATTGAGACTTTTCAAC, R: gene-specific primer.

[0047] Regardless of whether it is overexpression or interference expression, the vector transformed into the strain contains the 35S sequence fragment, while the wild-type strain does not. Therefore, in the positive test, only the PCR results of the positive strain show a band, while the wild-type strain does not contain the 35S sequence and therefore does not show a band. Figure 1 Electrophoresis results showed that no positive bands were detected in the WT control group, while the constructed overexpression lines 2, 5, and 11 showed clear bands. Figure 1 The A) indicates that these strains already contain overexpression. StASG The vector of genes; similarly, clear bands were observed in the positive tests of interference expression lines 1, 2, 4, 6, and 9. Figure 1 (B) indicates that these strains already contain interference. StASG Vectors for gene expression.

[0048] 3) Detection and analysis of protein and expression levels of transgenic positive strains: The positive test-tube seedlings obtained by genetic transformation were planted in a greenhouse and samples were taken after 30-40 days of growth for later use.

[0049] Detection of protein expression in overexpression strains: Tag-specific antibodies, specifically HA tag-specific antibodies, are used to detect protein expression. Overexpression transgenic lines contain a vector with the target fragment (the vector carries a tag sequence for detection), while wild-type lines do not contain a tag. Therefore, no bands are detected in the extracted proteins from wild-type lines during protein analysis.

[0050] Specific steps: Add the sample to a grinding ball, shake at 45 Hz for 90 s, then add 200 µL of protein extraction buffer and place on ice for 0.5 h. Shake for a few seconds every 10 min and immediately return to ice. After the sample is fully dissolved, centrifuge at 4°C and 12,000 rpm for 10 min. Transfer the supernatant to a pre-chilled 1.5 mL centrifuge tube on ice and centrifuge again at 4°C and 12,000 rpm for 10 min. Carefully transfer the supernatant to a new centrifuge tube, add 2 × SDS loading buffer, mix well, and denature at 95°C for 10 min. Store the extracted protein at -80°C for subsequent experiments. Western blot detection of protein: After thawing on ice, the extracted protein is spotted onto an SDS denaturing protein gel. After electrophoresis, transfer, antibody hybridization, and chemical ECL development, no protein bands were found in the WT control group, while lines 2, 5, and 11 showed clear protein bands, proving the exogenous transfer. StASG Normal expression in potatoes ( Figure 2 ).

[0051] Interference efficiency detection of interference expression lines: RNA was extracted from the above samples and analyzed using qPCR. StASGExpression levels in the transgenic lines were detected. Total RNA was extracted from wild-type and transgenic plant lines using a plant total RNA rapid extraction kit (Truly Gold), and single-stranded cDNA was generated by reverse transcription using RT MasterMix with AccuRT (abm). Real-time quantitative PCR was performed using BlasTaq™ 2X qPCR MasterMix (abm). The primers used for qPCR are as follows: Stactin As an internal reference gene (actin-F 5'-ATTGGAAACGGATATGCTCCA-3', actin-R: 5'-TCCTTACCTGAACGCCTGTCA-3'); StASG Specific primers (qRT-StASG-F: 5'-GTTGAATCGTCGAGTCCGGT-3', qRT-StASG-R: 5'-GTCACCACCACCGAGGAAAT-3'). Using 2... -△△Ct Data calculation and analysis were performed using [a specific method]. The results showed that among the five interfering transgenic plants... StASG Expression levels decreased significantly ( Figure 3 ).

[0052] Example 4: Identification of color phenotype in transgenic potatoes Wild-type and positive transgenic in vitro plantlets were transplanted into plastic pots in a greenhouse. During the growth period, appropriate thinning was performed, and plant growth was observed under the same water, fertilizer, and light management conditions. Potatoes were harvested 90 days after planting. Phenotypic observation showed that overexpression... StASG This resulted in a lighter skin and flesh color in 'Huacai 3', while the wild-type 'Huacai 3' exhibited a red skin and red flesh phenotype. Subsequently, the total anthocyanin content in the 'Huacai 3' tuber materials was determined using the pH differential method. The absorbance difference of the same sample at 510–530 nm was measured, and combined with the anthocyanin molar absorptivity, the total anthocyanin content of the wild-type and transgenic lines was calculated. The results showed overexpression. StASG The anthocyanin content in the potato flesh was significantly reduced ( Figure 4 A and B); at the same time, we observed the same results in the 'Desiree' material, where the potato peel changed from red to yellow and the anthocyanin content in the potato peel was significantly reduced (A and B); Figure 4 (C and D). Interference in 'Huacai 3' and 'Desiree' materials. StASG Gene expression was clearly observed, and the tubers of the transgenic lines were significantly darker in color compared to wild-type tubers. Figure 5 A), with a higher content of anthocyanins ( Figure 5 (B). The above results indicate that... StASG It inhibits the synthesis and accumulation of anthocyanins.

[0053] Example 5 StASG Effects on potato plant height Wild-type and transgenic potato tubers of similar size and with good germination were selected and planted in greenhouse pots. After seedling emergence, appropriate thinning was carried out, and plant growth was observed under consistent management conditions. Significant differences in plant growth were observed 30 days after planting. Measurement of the above-ground height of the plants revealed that overexpression... StASG The transgenic plants were significantly shorter than the wild-type lines. Figure 6 (A), while the height of the StASG-RNAi interference lines was significantly increased ( Figure 6 (B). The above experimental results show that... StASG It participates in regulating the height of potato plant lines.

[0054] Example 6: Effect of StASG on the first flowering time of potato plants Wild-type and transgenic potato tubers of similar size and with good germination were selected and planted in greenhouse pots. After seedling emergence, appropriate thinning was carried out, and the flower bud development was observed under the same water, fertilizer, and light management conditions. The first flowering was determined by the appearance of the first flower bud, full opening, and normal opening. The corresponding times of the first appearance of each stage of the flower bud were recorded. After about 35 days of planting, significant differences in flower bud development were observed. The overexpression lines of the 'Huacai 3' and 'Desiree' varieties flowered significantly earlier than the wild type. Earlier flowering can shorten the overall growth cycle of potatoes, promote earlier tuber maturity, and effectively increase the land multiple cropping index. After potatoes enter the flowering stage, photosynthetic products are gradually transported to and stored in underground tubers. Earlier flowering can simultaneously initiate the tuber enlargement process, further shortening the entire growth period. Figure 7 and 8 ).

[0055] sequence list SEQ ID NO: 1 StASG gene sequence Atggcgcccaaggaaaaaattggtgcagtgacggcggcagcggtgatggcggtggggaaattgaatggaatttcgaaagaggtgcattatagaggtgtaaggaagaggccatgggggagatacgcggcggagataagagatcccggtaaaaaaagccgggtttggcttggtacttttgatactgcggaggaagcggccaaagcttatgataacgctgctagggaattccgtggagctaaagcgaaaactaattttcctcaattactgaaggaggaagatctgaaattccctgttaaaaatgaaatcaatcggagtccgagtcagactagtactgttgaatcgtcgagtccggttatggttgattcatcatcgccgttagatctaagactttgtggatcaatcggcgggtttaaccataatactgttagatttccgatctccggtggaggttttaccggtgctgttccggcggtgaatcatatgtactatttagatgcacttgaacgcgccggagttataaatttagaaacaaatcggaagaagacggtggatttcctcggtggtggtgactccgatacatcaacggtgattgattttatgcgtgttgacgtgaaaccaaccatcgccggtctaaatctggaccttaactttcctccaccggaaaatatgtga; StASG Amino acid sequence of the gene: MAPKEKIGAVTAAAVMAVGKLNGISKEVHYRGVRKRPWGRYAAEIRDPGKKSRVWLGTFDTAEEAAKAYDNAAREFRGAKAKTNFPQLLKEEDLKFPVKNEINRSPSQTSTVESSSPVMVDSSSPLDLRLCGSIGGFNHNTVRFPISGGGFTGAVPAVNHMYYLDALERAGVINLETNRKKTVDFLGGGDSDTSTVIDFMRVDVKPTIAGLNLDLNFPPPENM [[ID=​7]].

Claims

1. StASG The application of genes in regulating anthocyanin content in potatoes is characterized by, Inactivation / degradation of the aforementioned StASG Gene expression, the StASG The gene encodes the amino acid sequence shown in SEQ ID NO:

2.

2. StASG The application of genes in regulating the flowering time of potato plants is characterized by, Overexpression StASG Genes, the ones mentioned StASG The gene encodes the amino acid sequence shown in SEQ ID NO:

2.

3. StASG The application of genes in regulating the height of potato plants is characterized by, Inactivation / degradation of the aforementioned StASG Gene expression, the StASG The gene encodes the amino acid sequence shown in SEQ ID NO:

2.

4. Inactivation / Degradation StASG The application of recombinant gene expression vectors or recombinant engineered bacteria containing said recombinant vectors in regulating anthocyanin content and / or plant height in potatoes, characterized in that... The StASG The gene encodes the amino acid sequence shown in SEQ ID NO:

2.

5. Includes StASG The application of a recombinant gene vector or a recombinant engineered bacterium containing said recombinant vector in regulating plant flowering time, characterized in that... Overexpression StASG Genes, the ones mentioned StASG The gene encodes the amino acid sequence shown in SEQ ID NO:

2.

6. A method for obtaining potato varieties with improved traits, characterized in that, It includes the following processing steps: (1) Infecting callus tissue with genetically engineered bacteria; and (2) The infected callus tissue was cultured into complete plants; The genetically engineered bacteria contain inactivation / reduction StASG Recombinant vectors for gene expression or containing StASG Recombinant vectors of genes, the StASG The gene encodes the amino acid sequence shown in SEQ ID NO:

2.

7. A potato with improved traits, characterized in that, With inactivation / reduction StASG Gene expression or overexpression StASG Genes, the ones mentioned StASG The gene encodes the amino acid sequence shown in SEQ ID NO:

2.

8. The method according to claim 6 or the potato according to claim 7, characterized in that, The improved traits are increased anthocyanin content, earlier flowering time, and / or increased plant height.

9. The application according to any one of claims 1-5, the method according to claim 6, or the potato according to claim 7 or 8, characterized in that, The StASG The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

Images