TN1 gene for regulating the number of potato tubers, encoded protein and application thereof

Abstract

The application provides a TN1 gene for regulating the number of potato tubers and application of the TN1 gene and a coded protein thereof in potato planting or potato breeding, and belongs to the technical field of genetic breeding. The potato TN1 gene provided by the application has a nucleotide sequence as shown in SEQ ID NO:1, and the coded amino acid sequence is as shown in SEQ ID NO:2. After the TN1 gene is transferred into a potato plant, the tuber weight and the number of tubers of the plant over-expressing the TN1 gene are obviously higher than those of a wild type plant, and the yield of the potato plant can be improved. The potato TN1 gene and the coded protein thereof provided by the application have important theoretical significance and practical value in potato planting or potato breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic breeding technology, specifically relating to a TN1 gene that regulates the number of potato tubers, its encoded protein, and its applications. Background Technology

[0002] Cultivated potatoes (Solanum tuberosum L.) were domesticated in the southern Andes Mountains of Peru. A characteristic of wild potatoes is their complex population structure, exhibiting immense morphological and genetic diversity, including numerous species adapted to a wide range of environments. These species possess desirable agricultural traits, such as disease resistance and environmental adaptability, making them valuable genetic resources for potato improvement.

[0003] As one of the world's most important non-cereal crops, potatoes play a vital role in meeting global demands for food, feed, and industrial uses. However, due to the self-incompatibility of most cultivated potatoes and their highly heterozygous genomes, improvements in potato yield and quality are severely hampered. Therefore, identifying key genes regulating tuber development is crucial for breeding high-yielding and high-quality potato varieties. Currently, research on specific genes involved in potato tuber formation and development remains in-depth, and there is an urgent need to discover new functional genes and clarify their application value. Summary of the Invention

[0004] To address the aforementioned problems, the purpose of this invention is to provide a TN1 gene that regulates the number of potato tubers, its encoded protein, and its applications, thereby providing new genetic resources and technical means for potato variety improvement.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] Mature potato plants were selected as materials. RNA was extracted from each tissue using the MiniBEST Plant Total RNA Extraction Kit (catalog number: 9769) from TaKaRa (Takara Bio Engineering Co., Ltd., Dalian) using Protocol I simple plant lysis method. An appropriate amount of RNA was then reverse transcribed according to the instructions of the TaKaRa PrimeScript™ RT reagent kit with gDNAEraser, including genomic DNA removal and reverse transcription, to obtain cDNA. The obtained cDNA was then used as a template, and specific primers 1 and 2 were designed according to homologous cloning methods for PCR amplification. The amplified fragment was ligated to the cloning vector pEASY-blunt zero (a product of Beijing TransGen Biotech Co., Ltd., catalog number CB501) to obtain the TN1 gene.

[0007] The nucleic acid sequence of the TN1 gene is shown in SEQ ID NO: 1.

[0008] The present invention also provides a protein encoded by the TN1 gene, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0009] The amplification primers for the cDNA are shown below:

[0010] Primer 1: 5'-ATGTATGCTGAGAAGC-3'

[0011] Primer 2: 5'-CCACCGTGCACAACTAG-3'

[0012] This invention also provides a tissue-specific expression pattern of the TN1 gene:

[0013] The cDNA of the TN1 gene was diluted 10-fold for subsequent qRT-PCR. Gene expression levels were measured using a QuantStudio 7 Flex system (Applied Biosystems, USA). Ef1a (elongation factor 1a) was used as an internal control (Chakravarthy et al., 2005). The relative expression levels of the gene in each tissue were analyzed using the 2–ΔΔCT method. Each sample was subjected to three biological replicates.

[0014] The present invention also provides a recombinant plasmid, which is obtained by homologous recombination of the above-mentioned TN1 gene with the vector pCAMBIA2301 (a product of Cambia Corporation), and the recombinant plasmid is named pCAMBIA2301-TN1.

[0015] Furthermore, when performing homologous recombination with the vector pCAMBIA2301, the TN1 gene requires amplification and recovery using primers 3 and 4 with homologous arms. The primers with homologous arms are as follows:

[0016] Primer 3: 5'- CAGGTCGACTCTAGAGGATCC ATGTATGCTGAGAAGC -3` (The sequence with a single underscore is the recombinant arm, and the sequence with a double underscore is the BamHI recognition sequence)

[0017] Primer 4: 5'- ATGGTCTTTGTAGTCGGTACC CCACCGTGCACAACTAG -3` (The underlined sequence is the recombination arm)

[0018] Preferably, the homologous recombination is performed by double digestion of the vector pCAMBIA2301 with restriction endonucleases BamHI and KpnI, and the vector backbone is recovered.

[0019] Preferably, when the TN1 gene undergoes homologous recombination with the vector pCAMBIA2301, the gene fragment is ligated to the vector backbone using a homologous recombination cloning enzyme (Hieff Clone Universal One Step Cloning Kit, YEASEN). The ligation is performed at 50°C for 20 min to obtain the recombinant plasmid pCAMBIA2301-TN1.

[0020] The present invention also provides a transgenic engineered bacterium containing the above-mentioned recombinant plasmid pCAMBIA2301-TN1 or the genome of the transgenic engineered bacterium having the above-mentioned TN1 gene integrated therein.

[0021] Preferably, the genetically engineered bacteria are Escherichia coli DH5α strain (purchased from Beijing TransGen Biotechnology Co., Ltd., product catalog number CD201-01) and Agrobacterium strain GV3101.

[0022] The constructed recombinant plasmid pCAMBIA2301-TN1 was transformed into Escherichia coli DH5α and cultured at 37℃ for 14 h. PCR analysis and enzyme digestion identification of the recombinant vector were performed, followed by sequencing verification. pCAMBIA2301-TN1 contains a TN1 gene expression cassette, in which the 35S promoter initiates TN1 gene transcription.

[0023] Another objective of this invention is to provide the application of the TN1 gene in potato cultivation or potato breeding.

[0024] This invention also provides a method for propagating potatoes using the recombinant plasmid pCAMBIA2301-TN1, comprising the following steps:

[0025] 1. Preparation of receptor materials

[0026] Select healthy potato tissue culture seedlings that are about 3-4 weeks old, cut stem segments of about 0.5-1 cm without axillary buds, place them on a pre-culture medium, and culture them at 22 ℃ in the dark for 2 days for genetic transformation.

[0027] 2. Activation of donor Agrobacterium

[0028] (1) After introducing the recombinant plasmid pCAMBIA2301-TN1 into Agrobacterium strain GV3101 by heat shock, it was stored at -80℃.

[0029] (2) Take out the frozen Agrobacterium culture from the -80℃ freezer and streak it on the Agrobacterium culture plate (LB+Kan 50 µg / L+Rif 50 µg / L). Incubate in the dark and upside down in a 28℃ incubator. Pick a single clone and put it into 500 μL liquid LB (Kan 50 µg / L+Rif 50 µg / L). Incubate at 28℃ and 200 rpm for about 14 h. At the same time, perform colony PCR to verify whether the single clone is positive.

[0030] (3) Expand the culture of positive bacterial culture: Take 40 µL of bacterial culture into a 50 mL centrifuge tube and add 20 mL of LB (Kan 50 µg / L + Rif 50 µg / L) culture medium. Shake and culture at 28℃ and 220 rpm until the OD600 is about 0.5-0.6.

[0031] (4) Centrifuge the 50mL centrifuge tube containing the bacterial solution at 5000 rpm for 10 min to collect the bacterial cells and discard the supernatant.

[0032] (5) Resuspend the bacterial cells in 20 mL of 3% sucrose MS solution containing 200 μmol / L acetylsuccinone (AS), let stand in the dark for 3 h, and let stand for later use.

[0033] 3. Agrobacterium infection and co-culture

[0034] Place the potato stem segments prepared in step 1 into the resuspension solution prepared in step 2, and gently shake at 28°C and 80 rpm for 15 minutes. Use tweezers to remove the stem segments from the centrifuge tube, place them on filter paper to absorb any remaining liquid, and then place the stem segments on a co-culture medium. Ensure they are spaced apart, wrap them in aluminum foil, and co-culture at 22°C for 2 days.

[0035] 4. Plant induction and resistance screening

[0036] Stem segments were transferred to CIM (callus induction) medium for callus induction, with approximately 20 segments per plate. After 2 weeks, they were transferred to SIM (bud induction) medium for bud induction culture, with the SIM medium being changed every 2 weeks until buds were induced. These buds were then cut and transferred to ordinary MS medium for rooting culture and propagation. During this process, transgenic lines were screened based on Kans resistance.

[0037] 5. Identification of transgenic potato plants

[0038] Since the overexpression vector pCAMBIA2301 carries a GUS tag, staining can be used to initially determine whether it is positive. As mentioned earlier, after the plants have rooted on MS medium with Kan resistance, leaves are aseptically cut and immersed in GUS staining solution. The solution is then incubated at 25-37°C for several hours or overnight. The immersed material is then transferred to 70% ethanol for decolorization 2-3 times until the negative control material turns white. If the staining solution turns blue or blue spots are observed on a white background under the naked eye or microscope after decolorization, it can be preliminarily determined that the plant is likely positive, and further in-depth testing can be performed.

[0039] Plants selected through GUS staining were propagated and preserved. DNA was extracted from the corresponding plants, specifically as follows:

[0040] ① Take about 100 mg of potato leaves that have rooted under antibiotic screening conditions, place them in a 2 mL centrifuge tube containing steel balls, freeze them quickly with liquid nitrogen, and then shake them thoroughly into powder on a grinder.

[0041] ② Place on ice, add 408 μL of DNA extraction buffer, mix thoroughly, and then place in a 65°C water bath for 5 min.

[0042] ③ Centrifuge at 12,000 rpm (-13.100xg) for 5 min, and transfer the supernatant to a new centrifuge tube. (4) Add 0.7 times the volume of isopropanol to the supernatant and mix thoroughly. After flocculent genomic DNA appears, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and keep the precipitate.

[0043] ④ Add 600ul of 70% alcohol, vortex, centrifuge at 12,000 rpm for 5 min, and discard the supernatant.

[0044] ⑤ Repeat step ④.

[0045] ⑥ Open the lid, invert it, and let it sit at room temperature for 10 minutes to completely dry any remaining alcohol.

[0046] ⑦ Add 30 μL of ddH2O, incubate at 65°C for 10 minutes to dissolve the DNA, inverting the container during the process to mix thoroughly, resulting in the final DNA solution.

[0047] ⑧ Using this DNA as a template, with untransformed Désirée DNA as the negative control and ddH2O as the blank control, detection was performed based on the kan resistance fragment on the vector. The detection primers were kan-3F: GCACAATCCCACTATCCTTCG and kan-3R: TCCCGCTTCAGTGACAACG.

[0048] 6. Select plants with increased TN1 gene expression levels compared to wild-type (WT) transgenic potato plants for propagation, hardening-off, and transplanting.

[0049] The specific cultivation conditions and methods are the same as the steps below:

[0050] (1) Cultivation of tissue culture materials

[0051] Under aseptic conditions, potato tissue culture seedlings were cut into 1 cm sections with one axillary bud and inserted into MS solid medium containing 3% carbon source at pH 5.8. Four to six stem segments were inserted into each medium. The tissue culture rooms were then placed under the following conditions: temperature 22±2 ℃, light intensity 2500 lx, and photoperiod 16 h·d. -1 The relative humidity was 60%. The culture was carried out for three weeks.

[0052] (2) Cultivation of planting materials

[0053] Potato seedlings need to be hardened off before planting. The specific method involves removing tissue culture seedlings, washing the roots of the culture medium with running water, planting them in seedling trays, and cultivating them in an incubator at a temperature of 22°C, humidity of 70%, and a light duration of 16 hours. -1 After two weeks of hardening off, the seedlings showed generally uniform growth. They were then transplanted into pots. After transplanting into large pots, both non-GMO and overexpressing GMO potato plants were managed under the same conditions, and the physiological phenotypic changes of the plants were observed and statistically analyzed.

[0054] The experimental steps are as follows:

[0055] Take potato tissue culture seedlings (approximately 6-7 cm long) with consistent growth in the incubator. First, harden them off for 2 weeks in an artificial climate chamber (16 hours light / 8 hours dark), with 12 seedlings per line. Transplant the plants into plastic pots (32 cm × 21 cm), then move the pots outdoors for 8 weeks of normal growth (i.e., normal irrigation with water or nutrient solution). After 8 weeks, harvest and count the number of tubers per plant and the yield per plant.

[0056] The present invention has the following beneficial effects:

[0057] A comparison of the changes in stolons per plant between wild-type (WT) and TN1-overexpressing potato plants revealed that TN1-overexpressing plants exhibited a significant increase in both the number of main stolons and their average length. Statistical analysis of the number of tubers per plant and tuber yield per plant in both wild-type and TN1-overexpressing potato plants showed that after 8 weeks of normal growth, the number of tubers in TN1-overexpressing potatoes was significantly higher than that in the wild-type control. There was also a significant increase in the weight per plant. This invention provides the first complete identification of the TN1 protein and its encoding gene, clarifies its specific expression pattern in potato tuber tissue, and confirms its regulatory role in potato tuber development through the construction of a recombinant vector. This provides new gene targets and technical support for elucidating the molecular mechanisms of potato tuber development and for breeding superior potato varieties, possessing significant theoretical value and application prospects in potato cultivation and breeding. Attached Figure Description

[0058] Figure 1 This is the cloning result of TN1 in potato. M is the DNA molecular marker, and the sequencing result is consistent with the size of the complete open reading frame (ORF) of TN1, which is 1458 bp.

[0059] Figure 2 Tissue-specific expression pattern of the TN1 gene. qRT-PCR analysis showed that the TN1 gene was constitutively expressed in all potato tissues, with the highest expression level in the stem, followed by axillary buds and aerial stolons. Expression was lower in roots, leaves, stolons, and young tubers, and lowest in mature tubers.

[0060] Figure 3 Genetic transformation of potato stem segments. Désirée tissue culture seedlings were propagated in long in vitro tubes (Figure A). Stem segments approximately 0.5-1 cm long with axillary buds were cut, and the axillary buds were removed. After pre-culturing for 2 days, the Désirée stem segments were infected with a prepared resuspension of bacteria. After 2 days of co-culturing (Figure B), the culture was transferred to CIM medium to induce callus (Figure C), and then buds were induced on SIM medium (Figure D), entering the rooting culture stage (Figure E).

[0061] Figure 4 The expression levels of the TN1 gene in overexpressing transgenic potato plants and wild-type plants (WT) are shown. In a total of 10 transformant lines, the expression level of the TN1 gene was significantly increased compared to the wild-type (WT).

[0062] Figure 5 shows the phenotypic statistics of potato plants overexpressing the TN1 gene and wild-type plants. The stolon length statistics of wild-type and transgenic potato plants are shown in [Figure 5]. Figure 5-1and Figure 5-2 The statistical analysis results of the number of tubers per plant and the yield per tuber of wild-type and transgenic potato plants are shown in [the table below]. Figure 5-3 . Detailed Implementation

[0063] The following description, in conjunction with specific embodiments of the present invention, provides further details. It should be noted that the description of the embodiments is intended to aid in understanding the present invention but does not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0064] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, P < 0.001 (***) indicates a highly significant difference, and P < 0.001 (****) indicates a highly significant difference.

[0065] Example 1

[0066] Acquisition of the TN1 gene and its encoded protein

[0067] Experimental materials: Stems and leaves of aseptic tissue culture seedlings of potato cultivar Désirée were removed, flash-frozen in liquid nitrogen, and stored at -80 °C.

[0068] The cloning vector pEASY-blunt zero is a product of Beijing TransGen Biotech Co., Ltd., catalog number CB501. The vector pCAMBIA2301 is a product of Cambia Biotech. The plant total RNA extraction kit is a product of TaKaRa (Takara Bio Engineering Co., Ltd., Dalian) – MiniBEST Plant Total RNA Extraction Kit (catalog number: 9769). The PrimeScript RT Kit is a product of TaKaRa (catalog number: RR047).

[0069] 1. Obtaining cDNA template

[0070] (1) RNA was extracted from each tissue using the TAKARA kit. Mature Désirée potato plants were used as materials. The tissues to be tested were placed in a pre-cooled mortar, liquid nitrogen was added, and the tissue materials were ground thoroughly with a pestle. Liquid nitrogen was added as needed until the materials were completely powdered. About 0.5 ug of powder was weighed and RNA was extracted from each tissue using the Protocol I simple plant lysis method of the TAKARA kit. Finally, the tissues were stored at -80℃ for long-term use.

[0071] (2) Total RNA integrity detection: Take the RNA solution obtained in the previous step, place it on ice to thaw, and then use an RNAase-free pipette tip to draw 5 μl. Quickly mix it with 1 μl of 6× Loading Buffer solution, and then add it to a 1% concentration agarose gel mixed with nucleic acid dye for electrophoresis detection. Irradiate the gel with ultraviolet light to observe the results. After electrophoresis, the sample bands are complete and not diffused, with two obvious bright bands and a band brightness ratio of about 2:1.

[0072] (3) Total RNA quantification: RNA concentration and A260 / A280 value were determined by spectrophotometer, with the ratio ranging from 1.8 to 2.0.

[0073] (4) cDNA synthesis: Take an appropriate amount of RNA for reverse transcription. The reverse transcription reaction system was prepared according to the instructions of the TaKaRa reverse transcription kit (PrimeScript™ RT reagent kit with gDNAEraser). This includes the removal of genomic DNA and the reverse transcription reaction. The cDNA was stored at -20 ℃ for later use.

[0074] 2. Using the cDNA obtained in step 1 as a template, specific primers 1 and 2 were designed according to the homologous cloning method for PCR amplification. The amplified fragment was ligated with the cloning vector pEASY-blunt zero to obtain the TN1 gene, the nucleic acid sequence of which is shown in SEQ ID NO: 1.

[0075] Primer 1: 5'-ATGTATGCTGAGAAGC-3'

[0076] Primer 2: 5'-CCACCGTGCACAACTAG-3'

[0077] 3. The protein encoded by the TN1 gene is named TN1. The amino acid sequence of this protein is shown in SEQ ID NO: 2, and it consists of 468 amino acid residues.

[0078] Sequence 1 (SEQ ID No. 1, 1458bp) is as follows:

[0079]

[0080] Sequence 2 (SEQ ID No. 2 458aa) is as follows:

[0081] MYAEKQAEEAIVSNFNGTDRDGEEVEKLGEEDQSIFSVKSLLWHGGSVWDAWFSCASNQVAQVLLTLPYSFSQLGMVSGIVFQVFYGLVGSWTAYLISVLYIEYRSRKEKEGVNFKNHVIQ WFEVLDGLLGPYWKAAGLAFNCTFLFGSVIQLIACASNIYYISDHLDKRTWTYIFGACCATTVFIPSFHNYRIWSFLGLGMTTYTAWYLTIAAVIHGQVENVQHTAPAKIVLYFTGATNI LYTFGGHAVTVEIMHAMWKPQKFKYIYLIATLYVFTLTIPSASAVYWAFGDQLLNHSNAFSLLPKDRWRDAAVILMLIHQFITFGFACTPLYFVWEKVIGMHDTKSICLRALVRLPVVIPI WFLAIIFPFFGPINSAVGALLVSFTVYIIPALAHMLTYRTTSARQNAAEKPPSFMPSWTIMYVINIFIVGWVLVVGFGFGGWASMSNFIKQVDTFGLFAKCYQCKPPAGQPPHPAPQATVHN

[0082] Example 2

[0083] Tissue-specific expression pattern of the TN1 gene

[0084] Potato tissues were collected, and total RNA was extracted and cDNA synthesized according to the steps described in Example 1. The cDNA was diluted 10-fold for subsequent qRT-PCR. Gene expression levels were measured using a QuantStudio 7 Flex system (Applied Biosystems, USA). Ef1a (elongation factor 1a) was used as an internal control (Chakravarthy et al., 2005). The relative expression levels of genes in each tissue were analyzed using the 2–ΔΔCT method. Each sample was subjected to three biological replicates.

[0085] Example 3

[0086] Application of TN1 gene in influencing potato tuber development

[0087] I. Construction of recombinant plasmid pCAMBIA2301-TN1

[0088] 1. The TN1 gene was amplified by PCR using primers 3 and 4, and double-stranded DNA molecules (fragment 1) containing recombinant arms at both ends were recovered.

[0089] Primer 3: 5'- CAGGTCGACTCTAGAGGATCC ATGTATGCTGAGAAGC -3` (The sequence with a single underscore is the recombinant arm, and the sequence with a double underscore is the BamHI recognition sequence)

[0090] Primer 4: 5'- ATGGTCTTTGTAGTCGGTACC CCACCGTGCACAACTAG -3` (The underlined sequence is the recombination arm)

[0091] 2. After completing step 1, digest the vector pCAMBIA2301 with the restriction endonucleases BamHI and KpnI, and recover the vector (backbone 2).

[0092] 3. Fragment 1 and vector backbone 2 were ligated using homologous recombination cloning enzyme (Hieff Clone Universal One StepCloning Kit, YEASEN) at 50℃ for 20 min to obtain recombinant plasmid pCAMBIA2301-TN1.

[0093] pCAMBIA2301-TN1 was transformed into Escherichia coli DH5α (purchased from Beijing TransGen Biotech Co., Ltd., product catalog number CD201-01). The transformation method was as follows:

[0094] 1. Remove competent cells from the -70℃ freezer and thaw them on ice. If aliquoting is required, aliquot the freshly thawed cell suspension into sterile, pre-cooled centrifuge tubes and place them in an ice bath.

[0095] 2. Add the target DNA (1-10 ng, volume <10 ml) to the competent cell suspension, gently rotate the centrifuge tube to mix the contents, and incubate on ice for 30 minutes.

[0096] 3. Place the centrifuge tubes in a 42°C water bath for 90 seconds, then quickly transfer them to ice for 3 minutes.

[0097] 4. Add 900 ml of sterile LB medium (antibiotic-free) to the centrifuge tube, mix well, and incubate at 37°C on a shaker for 1 hour (160-220 rpm).

[0098] 5. After centrifuging the transformed competent cells at low speed (5000 rpm, 4 minutes), discard part of the supernatant and retain 100-150 ml of culture medium. Gently pipette the suspended cells and add them all to LB solid agar medium containing the corresponding antibiotic. Spread the cells evenly using a sterile curved glass spreader.

[0099] 6. Place the plate at room temperature until the liquid is absorbed, then invert the plate and incubate at 37°C for 12-16 hours to see single colonies or blue-white spots.

[0100] After transformation, the recombinant vector was analyzed by PCR and identified by enzyme digestion, and then verified by sequencing. Sequencing results showed that pCAMBIA2301-TN1 is a recombinant vector obtained by replacing a small fragment between the BamHI and KpnI recognition sites of pCAMBIA2301 with a DNA molecule whose nucleotide sequence is SEQ ID NO: 1, while keeping the other nucleotide sequences of pCAMBIA2301 unchanged. pCAMBIA2301-TN1 contains a TN1 gene expression cassette, in which the 35S promoter initiates TN1 gene transcription.

[0101] II. Obtaining Transgenic Potato Plants

[0102] 1. Preparation of receptor materials

[0103] Select healthy potato tissue culture seedlings that are about 3-4 weeks old, cut stem segments of about 0.5-1 cm without axillary buds, place them on a pre-culture medium, and culture them at 22 ℃ in the dark for 2 days for genetic transformation.

[0104] 2. Activation of donor Agrobacterium

[0105] (1) The plant expression vector pCAMBIA2301-TN1 was introduced into Agrobacterium strain GV3101 by heat shock method and then stored at -80℃.

[0106] (2) Take out the frozen Agrobacterium culture from the -80℃ freezer and streak it on the Agrobacterium culture plate (LB+Kan 50 µg / L+Rif 50 µg / L). Incubate in the dark and upside down in a 28℃ incubator. Pick a single clone and put it into 500 μL liquid LB (Kan 50 µg / L+Rif 50 µg / L). Incubate at 28℃ and 200 rpm for about 14 h. At the same time, perform colony PCR to verify whether the single clone is positive.

[0107] (3) Expand the culture of positive bacterial culture: Take 40 µL of bacterial culture into a 50 mL centrifuge tube and add 20 mL of LB (Kan 50 µg / L + Rif 50 µg / L) culture medium. Shake and culture at 28℃ and 220 rpm until the OD600 is about 0.5-0.6.

[0108] (4) Centrifuge the 50mL centrifuge tube containing the bacterial solution at 5000 rpm for 10 min to collect the bacterial cells and discard the supernatant.

[0109] (5) Resuspend the bacterial cells in 20 mL of 3% sucrose MS solution containing 200 μmol / L acetylsuccinone (AS), let stand in the dark for 3 h, and let stand for later use.

[0110] 3. Agrobacterium infection and co-culture

[0111] Place the potato stem segments prepared in step 1 into the resuspension solution prepared in step 2, and gently shake at 28°C and 80 rpm for 15 minutes. Use tweezers to remove the stem segments from the centrifuge tube, place them on filter paper to absorb any remaining liquid, and then place the stem segments on a co-culture medium. Ensure they are spaced apart, wrap them in aluminum foil, and co-culture at 22°C for 2 days.

[0112] 4. Plant induction and resistance screening

[0113] Stem segments were transferred to CIM (callus induction) medium for callus induction, with approximately 20 segments per plate. After 2 weeks, they were transferred to SIM (bud induction) medium for bud induction culture, with the SIM medium being changed every 2 weeks until buds were induced. These buds were then cut and transferred to ordinary MS medium for rooting culture and propagation. During this process, transgenic lines were screened based on Kans resistance.

[0114] III. Identification of Transgenic Potato Plants

[0115] 1. GUS staining detection

[0116] Since the overexpression vector pCAMBIA2301-TN1 carries a GUS tag, staining can be used to initially determine whether it is positive. As mentioned earlier, once the plants have rooted on MS medium with Kan resistance, leaves are aseptically cut and immersed in GUS staining solution. The solution is then incubated at 25-37°C for several hours or overnight. The immersed material is then transferred to 70% ethanol for decolorization 2-3 times until the negative control material turns white. If the staining solution turns blue or blue spots are observed on a white background under the naked eye or microscope after decolorization, it can be preliminarily determined that the plant is likely positive, and further in-depth testing can be performed.

[0117] 2. DNA testing

[0118] Plants selected through GUS staining were propagated and preserved. DNA was extracted from the corresponding plants, specifically as follows:

[0119] ① Take about 100 mg of potato leaves that have rooted under antibiotic screening conditions, place them in a 2 mL centrifuge tube containing steel balls, freeze them quickly with liquid nitrogen, and then shake them thoroughly into powder on a grinder.

[0120] ② Place on ice, add 408 μL of DNA extraction buffer, mix thoroughly, and then place in a 65°C water bath for 5 min.

[0121] ③ Centrifuge at 12,000 rpm (-13.100xg) for 5 min, and transfer the supernatant to a new centrifuge tube. (4) Add 0.7 times the volume of isopropanol to the supernatant and mix thoroughly. After flocculent genomic DNA appears, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and keep the precipitate.

[0122] ④ Add 600ul of 70% alcohol, vortex, centrifuge at 12,000 rpm for 5 min, and discard the supernatant.

[0123] ⑤ Repeat step ④.

[0124] ⑥ Open the lid, invert it, and let it sit at room temperature for 10 minutes to completely dry any remaining alcohol.

[0125] ⑦ Add 30 μL of ddH2O, incubate at 65°C for 10 minutes to dissolve the DNA, inverting the container during the process to mix thoroughly, resulting in the final DNA solution.

[0126] ⑧ Using this DNA as a template, with untransformed Désirée DNA as the negative control and ddH2O as the blank control, detection was performed based on the kan resistance fragment on the vector. The detection primers were kan-3F: GCACAATCCCACTATCCTTCG, and kan-3R: TCCCGCTTCAGTGACAACG.

[0127] 3. Analysis of the relative expression level of the TN1 gene in transgenic materials

[0128] Following the method in Example 1, RNA was extracted, reverse transcribed into cDNA as a template, and its concentration was measured and then diluted to the same concentration. qRT-PCR was then performed to analyze the relative expression level of the TNI gene in positive plants.

[0129] Results after fluorescence quantification are as follows Figure 4 The results showed that among the 11 transformed materials obtained, the expression level of 10 lines increased significantly, indicating that 10 plants overexpressing the TN1 gene were obtained in this process.

[0130] III. Phenotypic observation of potato plants overexpressing the TN1 gene

[0131] After qRT-PCR, plants with increased expression levels compared to WT were selected for propagation, hardening, and transplanting.

[0132] The specific cultivation conditions and methods are the same as the steps below:

[0133] 1. Cultivation of tissue culture materials

[0134] Under aseptic conditions, potato tissue culture seedlings were cut into 1 cm sections with one axillary bud and inserted into MS solid medium containing 3% carbon source at pH 5.8. Four to six stem segments were inserted into each medium. The tissue culture rooms were then placed under the following conditions: temperature 22±2 ℃, light intensity 2500 lx, and photoperiod 16 h·d. -1 The relative humidity was 60%. The culture was carried out for three weeks.

[0135] 2. Cultivation of planting materials

[0136] Potato seedlings need to be hardened off before planting. The specific method involves removing tissue culture seedlings, washing the roots of the culture medium with running water, planting them in seedling trays, and cultivating them in an incubator at a temperature of 22°C, humidity of 70%, and a light duration of 16 hours. -1 After two weeks of hardening off, the seedlings showed generally uniform growth. They were then transplanted into pots. After transplanting into larger pots, both non-transgenic and overexpressing Désirée plants were managed under the same conditions, and physiological phenotypic changes were observed and statistically analyzed.

[0137] The experimental steps are as follows:

[0138] (1) Take potato tissue culture seedlings (about 6-7 cm long) with the same growth status in the incubator. First, harden the seedlings in an artificial climate chamber (16h light / 8h dark) for 2 weeks, with 12 seedlings for each line.

[0139] (2) After completing step (1), transplant the plant into a plastic pot (32 cm × 21 cm).

[0140] (3) After completing step (2), move the plastic pot outdoors for 8 weeks of normal growth (i.e., normal irrigation with water or nutrient solution). After 8 weeks, harvest and count the number of tubers per plant and the yield of tubers per plant.

[0141] The changes in individual runners of wild-type and TN1-overexpressing potato plants are shown in the figure. Figure 5-15-2, the results showed that the number of main stolons and the average length of stolons in overexpressing plants increased significantly, with the highest increase in average length per plant being 1.49 times (OE-31) and the lowest being 1 time (OE-39). Statistical analysis results of the number of tubers per plant and tuber yield per plant in wild-type potato and TN1 gene overexpressing potato plants are shown in [the table below]. Figure 5-3 The results showed that after 8 weeks of normal growth, the number of tubers in potatoes overexpressing the TN1 gene was significantly higher than that in the wild-type control, with the highest increase being 1.28 times (OE-31) and the lowest being 0.89 times (OE-10). In terms of single-plant weight, the highest increase was 38% (OE-45).

[0142] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A TN1 gene that regulates the number of potato tubers, characterized in that, Cloning this gene involves the following steps: using mature potato plants as material, total RNA is extracted from the material; using a reverse transcription kit, genomic DNA removal and reverse transcription reactions are performed sequentially to obtain cDNA; using the cDNA as a template, PCR amplification is performed using specific primers 1 and 2, wherein the nucleotide sequence of primer 1 is 5'-ATGTATGCTGAGAAGC-3' and the nucleotide sequence of primer 2 is 5'-CCACCGTGCACAACTAG-3'; the PCR amplified fragment is ligated to the cloning vector pEASY-blunt zero to obtain the TN1 gene, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. A protein encoded by the TN1 gene as described in claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

3. The application of a TN1 gene that regulates the number of potato tubers and its encoded protein in potato breeding.

4. The application according to claim 3, characterized in that, The steps include: homologous recombination of the TN1 gene with the vector pCAMBIA2301 to obtain the recombinant plasmid pCAMBIA2301-TN1; transformation of the constructed recombinant plasmid pCAMBIA2301-TN1 into Escherichia coli DH5α, cultured at 37℃ for 14 h, PCR analysis and enzyme digestion identification of the recombinant vector, and sequencing verification; activation of the recombinant plasmid pCAMBIA2301-TN1 by introducing Agrobacterium strain GV3101 via heat shock; cutting stem segments without axillary buds from potato tissue culture seedlings and placing them on pre-culture medium, incubating at 22℃. After culturing at ℃ in the dark for 2 days, Agrobacterium infection and co-culture were carried out; the stem segments were transferred to callus induction medium for callus induction; after 2 weeks, they were transferred to bud induction medium for bud induction culture, and the bud induction medium was changed every 2 weeks until buds were induced. The buds were cut off and transferred to ordinary MS for rooting culture and propagation; transgenic plants were screened according to Kans resistance; the transgenic plants were propagated, hardened off and transplanted.

5. The application according to claim 4, characterized in that, During homologous recombination, the TN1 gene requires amplification and recovery using primers 3 and 4 with homologous arms. The nucleotide sequence of primer 3 is shown in SEQ ID NO: 3, and the nucleotide sequence of primer 4 is shown in SEQ ID NO:

4. During homologous recombination, the vector pCAMBIA2301 is digested with restriction endonucleases BamHI and KpnI, and the vector backbone is recovered. When the TN1 gene and the vector pCAMBIA2301 undergo homologous recombination, the gene fragment is ligated to the vector backbone using a homologous recombination cloning enzyme at 50°C for 20 min to obtain the recombinant plasmid pCAMBIA2301-TN1.

6. The application according to claim 4, characterized in that, The activation step is as follows: (1) The recombinant plasmid pCAMBIA2301-TN1 was introduced into Agrobacterium strain GV3101 by heat shock and then stored at -80℃; (2) Take out the frozen Agrobacterium culture from the -80℃ freezer, streak it on the LB Agrobacterium culture plate containing 50 µg / L Kan and 50 µg / L Rif, and incubate it upside down in the dark at 28℃; pick a single colony into 500 μl of liquid LB containing 50 µg / L Kan and 50 µg / L Rif, and incubate it at 28℃ and 200 rpm for 12-16 hours, and at the same time perform colony PCR to verify whether the single colony is positive; (3) Expand the culture of positive bacterial culture: Take 40 µL of bacterial culture into a 50 mL centrifuge tube, add 20 mL of LB culture medium containing 50 µg / L Kan and 50 µg / L Rif, and shake at 28℃ and 220 rpm until the OD600 is 0.5-0.6; (4) Centrifuge the above-mentioned 50mL centrifuge tube containing bacterial solution at 5000 rpm for 10 min to collect the bacterial cells and discard the supernatant; (5) Resuspend the bacterial cells in 20 mL of 3% sucrose MS solution containing 200 μmol / L acetylsalicylic acid, let stand in the dark for 3 h, and let stand for later use.

7. The application according to claim 4, characterized in that, The steps for screening transgenic plants are as follows: when potato plants have rooted on MS medium with Kan resistance, plant leaves are cut off under aseptic conditions, soaked in GUS staining solution, kept at 25-37℃ overnight, the soaked material is transferred to 70% ethanol for decolorization 2-3 times, and observed.

8. The application according to claim 4, characterized in that, The steps for propagating, hardening off, and transplanting the transgenic plants are as follows: (1) Under aseptic conditions, potato tissue culture seedlings were cut into 1cm sections with one axillary bud and inserted into MS solid medium containing 3% carbon source at pH 5.

8. Four to six stem segments were inserted into each medium. The seedlings were placed in a plant tissue culture room under the following conditions: temperature 22±2℃, light intensity 2500 lx, and photoperiod 16 h·d. -1 Incubate at a relative humidity of 60% for three weeks; (2) Potato seedlings need to be hardened off before planting. The specific method is to take out the tissue culture seedlings, wash the culture medium off the roots with running water, plant them in seedling trays, and cultivate them in an incubator. The temperature of the incubator is 22 ℃, the humidity is 70%, and the light time is 16 h·d. -1 After two weeks of hardening off, when the growth was basically the same, the seedlings were transplanted into pots. After transplanting into large flower pots, the non-GMO potato plants and the overexpressing GMO potato plants were managed under the same conditions, and the physiological phenotypic changes of the plants were observed and statistically analyzed. (3) Take potato tissue culture seedlings with the same growth status in the incubator and harden them for 2 weeks in an artificial climate chamber with 16h light / 8h darkness, with 12 seedlings in each line; transplant the plants into plastic pots and then move the plastic pots outdoors for normal growth for 8 weeks; after 8 weeks, harvest and count the number of tubers per plant and the yield of tubers per plant.

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