Application of potato CP12.2 gene in regulation and control of cold resistance

Abstract

The invention provides an application of a potato CP12.2 gene in regulation and control of cold resistance. The method comprises the following steps: cloning a CP12.2 gene of a potato wild species Solanum acaule, constructing an over-expression vector and a gene editing vector, and transforming the over-expression vector and the gene editing vector into a potato material AC142 and Solanum tuberosum for functional verification. Experiments show that over-expression of the CP12.2 gene can significantly improve the cold resistance of the plant, the median lethal temperature of the plant is lower than that of a control group, and after freezing treatment at-2.5 DEG C, the damage score is reduced to 0.56-1.11 (the control group is 4.67); on the contrary, the LT50 of the gene editing strain is increased, and the damage score after treatment at-3.5 DEG C reaches 4.22-4.78 (the contrast is 0.56). The invention proves that the CP12.2 gene positively regulates the cold resistance of potatoes for the first time, and provides a new target for cold-resistant molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics and breeding, specifically relating to the application of the potato CP12.2 gene in regulating the cold resistance of potatoes. Background Technology

[0002] The potato (Solanum tuberosum L.) is an annual herbaceous plant belonging to the Solanaceae family and the Solanum genus. It is the world's fourth largest food crop after rice, wheat, and corn, playing a vital role in ensuring global food security. Low temperature is one of the major abiotic stresses faced by plants during growth, directly affecting their growth, development, and physiological metabolism. Plants can acquire stronger cold resistance after being treated with non-lethal temperatures; this is known as cold acclimatization. Cold acclimatization is a self-protective response of plants to low temperatures, a biochemical and physiological process related to improving plant cold resistance, mainly including cold acclimatization and freeze acclimatization. To cope with low temperature stress, plants have developed a set of mechanisms to resist low-temperature damage over a long period of evolution, including a series of changes at the physiological and biochemical levels and gene expression levels, such as plant hormones, transcription factors, kinases, and functional proteins. This indicates that the cold acclimatization ability of plants is a polygenic trait involving numerous gene interactions. Currently, the molecular mechanisms of plant cold resistance are mainly elucidated through two pathways: CBF-dependent signal transduction pathways and CBF-independent signal transduction pathways. The CBF regulatory pathway is the most clearly understood (Thomashow, 2010). CBF-like transcription factors are the core of cold acclimatization regulation, and the ICE-CBF-COR signaling pathway is considered to play a key role in cold acclimatization. The basic signal transduction pathway of cold acclimatization is the induction of intracellular Ca2+ by low temperature. 2+ Signal,

[0003] Stimulating the expression of multiple transcription factors, such as CBF / DREB, ICE, b-ZIP, and WRKY, regulates the responses of downstream proteins, including COR, aquaporins, and tubulin, leading to changes in intracellular metabolic processes and structural alterations, enabling cells to gradually adapt to low-temperature environments (Licausi et al., 2013). CBFs play a crucial role in signal transduction pathways, acting as key regulators of cold adaptation, photosynthetic changes, and plant architecture. CBF gene expression is regulated by both transcriptional activators and repressors. Three CBF genes exist in the Arabidopsis genome; overexpression of CBF1, CBF2, and CBF3 significantly enhances plant frost resistance. To date, CBF transcription factors with cold-inducible properties have been identified in crops such as rapeseed, wheat, rye, tomato, rice, and maize; furthermore, overexpression of Arabidopsis CBF in other species has also enhanced plant frost resistance (Liu et al., 2017). Potatoes contain two gene clusters: CBF3-CBF1-CBF2-CBF-2B and CBF5-CBF4. However, only CBF1 and CBF4 can be induced by low temperature (Liu et al., 2020). Besides CBF-dependent signal transduction pathways, many genes regulate low-temperature responses independently of CBF signaling; these regulatory mechanisms are collectively referred to as CBF-independent signaling pathways. Transcriptome analysis revealed that only about 12% of cold-response genes depend on CBF. For example, Vogel et al. (2005) found that constitutive expression of the transcription factor ZAT12 can induce the expression of low-temperature response genes, improving the cold resistance of Arabidopsis thaliana; the induced genes only partially overlap with those induced by CBF2. Furthermore, the regulation of plant responses to abiotic stresses such as low temperature also involves abscisic acid (ABA)-mediated signaling pathways. Kou et al. (2018) discovered in their study on the regulatory mechanisms of cold resistance in potatoes that low-temperature stress directly regulates the expression of StADC1 by inducing the expression of AREB3 and ABI5 in the ABA signaling pathway, and mediates putrescine synthesis to enhance the expression of related genes in the CBF pathway, thereby improving domestication cold resistance. These molecular mechanisms provide a theoretical basis and genetic resources for molecular breeding of cold-resistant potatoes.

[0004] Problems with existing technologies: There are no reports on the application of potato CP12.2 gene regulation of cold resistance in existing technologies. Summary of the Invention

[0005] In view of the shortcomings of the prior art, this invention provides the application of the potato CP12.2 gene in regulating potato cold resistance. To achieve the above objective, this invention adopts the following technical solution:

[0006] 1. The CP12.2 gene: The CDS sequences of the CP12.2 gene from the highly cold-resistant wild potato material W3 (S. acaule) and the frost-sensitive wild potato material Cph12 (S. cardiophyllum) are shown in SEQ ID NO.1 of the sequence listing, and the sequences are completely identical. This is the same as the sequence (Soltu.DM.01G024860.1) published in the Potato Genome Database (http: / / spuddb.uga.edu / ). Using the genome of the moderately cold-resistant potato material Etuberosum as a template, the CP12.2 fragment SeCP12.2_Etuberosum_gDNA (SEQ ID NO.2) was amplified. Sequencing analysis revealed that SeCP12.2_Etuberosum_gDNA and SaCP12.2_W3_cDNA / SaCP12.2_W3_cDNA had 4 base variations and 1 amino acid site difference in the protein sequence (non-functional region, see SEQ ID NO.3 and SEQ ID NO.4).

[0007] 2. The method for cloning and functional verification of the CP12.2 gene includes the following steps: (1) primer design; (2) RNA extraction; (3) cDNA preparation; (4) PCR reaction and expression vector construction.

[0008] 3. Application of the potato CP12.2 gene to improve the cold resistance of potatoes, wherein the CDS sequence of the CP12.2 gene is shown in SEQ ID NO.1 or SEQ ID NO.2 of the sequence listing.

[0009] Furthermore, the application of the CP12.2 gene to enhance the cold resistance of potatoes is achieved through overexpression of the CP12.2 gene.

[0010] Furthermore, the application of the CP12.2 gene to enhance the cold resistance of potatoes is reflected in the fact that overexpression of the CP12.2 gene significantly reduces the plant's half-lethal temperature and freezing damage score.

[0011] Compared with existing technologies, the beneficial effects of this invention are as follows: Using sequences published in the potato genome database and cDNA from leaves of the wild species *Solanum acaule* (W3) as a template, the CP12.2 gene was cloned. Overexpression vectors and gene-editing vectors were constructed, and these were genetically transformed into wild species AC142 and *Solanum etuberosum*, with transgenic function verified. It was found that overexpression of the CP12.2 gene can improve the cold resistance of potato materials, while gene-edited CP12.2 gene can reduce the cold resistance of potato materials, mainly manifested in the median lethal temperature (LT) of potato plants overexpressing the CP12.2 gene.50 The frost damage scores of the control potato plant AC142 were lower than those of the control plant AC142. After treatment at -2.5℃ for 3 hours and recovery at 20℃ for 1 day, the frost damage scores of the control material AC142 were (4.67), while those of the potato plants 18# and 19# with overexpression of the CP12.2 gene were (0.56 and 1.11), respectively. The median lethal temperature (LT) of the gene-edited CP12.2 plants was lower than that of the control plant AC142. 50 The freezing damage scores of the control potato plant Solanume tuberosum were higher than those of the control plant. After treatment at -3.5℃ for 3 hours and recovery at 20℃ for 1 day, the freezing damage scores of the control material Solanume tuberosum were (0.56), while those of the gene-edited CP12.2 gene potato plants #4 and #8 were (4.78 and 4.22, respectively). Attached Figure Description

[0012] Figure 1 Phenotypic images of AC142 and its over-produced strain, Solanum etuberosum and its edited strain after freezing were taken before freezing and after being frozen at -2.5 °C ± 0.2 °C for 3 hours. AC142, CP12-2 OE-AC42-18, and CP12-2 OE-AC42-19 were then recovered under normal conditions for 1 day. Figure 1 A, 1C, and 1E represent the phenotypes of AC142, CP12-2 OE-AC42-18, and CP12-2 OE-AC42-19 before freezing, respectively. Figure 1 B, 1D, and 1F are the phenotypes of AC142, CP12-2 OE-AC42-18, and CP12-2 OE-AC42-19 after being frozen (-2.5 °C ± 0.2 °C for 3 hours) and then recovered for 1 day under normal conditions (20 °C, 12h / 12h light and dark).

[0013] Figure 2 Phenotypic images of Etuberosum, CP12-2 CC-Etuberosum-4, and CP12-2 CC-Etuberosum-8 were taken before freezing and after being thawed at -3.5 °C ± 0.2 °C for 3 hours, and then recovered under normal conditions for 1 day. Figure 2 A, 2C, and 2E are images of the phenotypes of Etuberosum, CP12-2 CC-Etuberosum-4, and CP12-2 CC-Etuberosum-8 before freezing, respectively. Figure 2B, 2D, and 2F show the phenotypes of Etuberosum, CP12-2 CC-Etuberosum-4, and CP12-2 CC-Etuberosum-8 after being frozen (-3.5 °C ± 0.2 °C for 3 hours) and then recovered for 1 day under normal conditions (20 °C, 12h / 12h light and dark).

[0014] Figure 3 This is a sequence diagram of the potato CP12.2 gene. The underlined nucleotides represent different nucleotides in different varieties.

[0015] Figure 4 This is a sequence diagram of the protein encoded by the potato CP12.2 gene. The underlined parts represent different amino acids in different varieties. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the invention clearer, the specific embodiments of the invention will be described in detail below with reference to the accompanying drawings. Unless otherwise specified, the reagents, instruments, and methods used in the embodiments are all conventional techniques.

[0017] Example 1

[0018] This embodiment provides the potato CP12.2 gene, the sequence of which is shown in SEQ ID NO.1 and... Figure 3 As shown. The sequence of the CP12.2 gene published in the Potato Genome Database (http: / / spuddb.uga.edu / ) is Soltu.DM.01G024860.1. Using the genome of the moderately cold-resistant potato material *Etuberosum* as a template, the CP12.2 fragment *SeCP12.2_Etuberosum_gDNA* (SEQ ID NO.2) was amplified. Figure 4 As shown, sequencing analysis revealed that SeCP12.2_Etuberosum_gDNA and SaCP12.2_W3_cDNA / SaCP12.2_W3_cDNA have 4 base variations and 1 amino acid site difference in the protein sequence (see SEQ ID NO.3, SEQ ID NO.4).

[0019] Example 2

[0020] This embodiment provides cold-resistant potatoes (LT). 50The method for determining the value specifically includes the following steps: The selected test material, detached leaf blades, is subjected to low-temperature treatment. Six temperature points are set up in the experiment (0℃, -1℃, -2℃, -3℃, -4℃, -5℃), with three replicates for each temperature point. The specific process is as follows: ① The leaf blades are rinsed multiple times with distilled water to absorb surface moisture; ② The leaf blades are placed at the bottom of glass test tubes (25cm×15cm), leaf surface facing down, one blade per tube; ③ The test tubes are placed in a low-temperature constant temperature bath (DC-120, Jintan Tianjing Test Instrument Factory, liquid medium is alcohol and distilled water). The initial temperature of the water bath was set to 0℃ and maintained for 30 min. Three tubes were taken out as controls. The temperature was then lowered to -0.5℃ and maintained for another 30 min. When the temperature dropped from -0.5℃ to -1℃ and maintained for 30 min, a small piece of ice was added to each tube, and the temperature was maintained for another 30 min before sampling. The temperature was then lowered from -1℃ to -1.5℃ and maintained for 1 h, then lowered to -2℃ and maintained for 30 min before sampling. This process was repeated until the target temperature was reached, and samples were taken at each temperature. The samples were then placed on ice and thawed overnight in a 4℃ freezer. After thawing overnight, 25 ml of deionized water was added and the mixture was shaken at 220 r / min at room temperature for 1 h. After standing, the conductivity R1 of the samples was measured using a conductivity meter (Leici DDS-307, Shanghai). After boiling in a water bath for 30 min and cooling to room temperature, the total conductivity R2 of the samples was measured. The electrolyte leakage rate (%) was calculated as (R1 / R2) × 100%. ⑤ By fitting the Logistic equation, the temperature at which the electrolyte leakage rate reaches 50% is the half-lethal temperature of the material.

[0021] Example 3

[0022] This embodiment provides a method for evaluating frost resistance, which includes the following steps: Nine plants of the same growth were selected and treated in a low-temperature artificial climate chamber (PAX-250C, Changzhou) at -2.5℃ and -3.5℃ in the dark for 3 hours, and then cultured at 20℃ for 1 day to score the frost damage. The scoring criteria adopted the method of Li Fei et al.

[16] , and the specific criteria were: 0 = no damage; 1 = slight damage to the top leaves; 2 = some top leaves were frozen to death; 3 = all top leaves were frozen to death; 4 = all leaves and petioles were frozen to death; 5 = leaves and stems were frozen to death. The damage index (DI) of each degree of damage was calculated. DI = (x0 + x1 + x2 + x3 + x4 + x5) / total number of plants, where x0 to x5 represent the number of plants of each damage level.

[0023] Example 4

[0024] This embodiment provides the potato CP12.2 gene, cloning method, method for obtaining and functionally verifying gene-modified lines, specifically including the following steps:

[0025] (1) Primer design

[0026] In this invention, primers were designed to amplify the full-length CDS sequence of the CP12.2 gene based on the sequence (Soltu.DM.01G024860.1) published in the Potato Genome Database (http: / / spuddb.uga.edu / ) (e.g., sequence SEQ No. 1); and primers for the gene editing vector were designed based on the previously amplified SeCP12.2_Etuberosum_gDNA sequence (e.g., sequence SEQ ID NO. 2). The primer sequences are as follows:

[0027] CP12.2 overload vector primers

[0028] pH7lic-CP12.2-F: 5'-ATTACGCCGAGGCCTATGGCAACAATTGCTGGT-3'

[0029] pH7lic-CP12.2-R: 5'-ATAGGGAAGAGGCCTACTATCATATGTACGGCACTCA-3'

[0030] CP12.2 gene editing vector primers

[0031] CR-SeCP12.2-F: '-CGGGTCTCCTGCATGTGGCTGATAGTGTTAAGGGTTTTAGAGCTAGAA-3'

[0032] CR-SeCP12.2-R: '-TAGGTCTCtAAACCTGCACTTGCCTCTCCACATGCACCAGCCGGGAA-3'

[0033] In addition, quantitative primers for CP12.2, quantitative primers for the internal reference gene β-tublin2, primers for the NPT II gene for positive detection in over-exploitation plants, primers for the Cas9 gene for positive detection in gene-edited plants, and CP12.2 HiTOM sequencing primers were designed. The sequences of each primer are as follows:

[0034] CP12.2 quantitative primers

[0035] CP12-2F: 5'-CTTTCTGACCTTGTGGCTGAT-3'

[0036] CP12-2R: 5'- CGCTGCACTTGCCTCTT-3'

[0037] Primers for the internal reference gene β-tublin2

[0038] β-tublin2-F: 5'-GATGTTGTGCCAAAGGATGT-3'

[0039] β-tublin2-R: 5'-AACTTGTGGTCAATGCGAGA-3'

[0040] NPT II primers

[0041] NPT II F: 5'-GCTATGACTGGGCACAACAG-3'

[0042] NPT II R: 5'-ATACCGTAAAGCACGAGGAA-3'

[0043] Cas9 primers

[0044] Cas9 F: 5'- GAGATACGACGAGCACCACC-3'

[0045] Cas9 R: 5'-CTTCACGGTCACTTTCCGGT-3'

[0046] CP12.2 HiTOM sequencing primers

[0047] SeCP12.2-HiTOM-F: 5'-GAGTACGTGTGCGTGCAGCAACACCAGATA-3'

[0048] SeCP12.2-HiTOM-R:5'-GGATGCTGGATGGTTCCTGCTTCTTCTTGTCC-3'

[0049] (2) RNA extraction

[0050] Leaf samples from wild potato materials (W3, Etuberosum) were ground into powder in liquid nitrogen, and RNA was extracted using the Plant Total RNA Kit from Zhuangmeng Biotechnology Co., Ltd. The specific method is as follows:

[0051] Take 60-100 mg of the above sample powder into a 1.5 mL centrifuge tube, add 1 mL of lysis buffer RL, shake immediately for 20 seconds to mix thoroughly and lyse completely, and incubate at 15-30℃ for 5 minutes to ensure complete lysis of ribosomes.

[0052] Add 0.2 mL of chloroform per 1 mL of RL. Tightly cap the sample tube, shake vigorously for 15 seconds, and incubate at room temperature for 3 minutes.

[0053] ③ Centrifuge at 12000 rpm at 4℃ for 10 minutes, and transfer the upper aqueous phase to a new 1.5 mL RNase-free centrifuge tube using a pipette.

[0054] Add half the volume of the aqueous phase (0.5 times) of anhydrous ethanol and mix thoroughly. Transfer the resulting solution and any possible precipitate into the adsorption column RA in the collection tube. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and reattach the adsorption column to the collection tube.

[0055] Add 500 µL of rinsing buffer RW, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid.

[0056] Add 500 µL of rinsing buffer RW, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid.

[0057] Place the adsorption column RA back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove as much of the wash solution as possible.

[0058] Place the adsorption column into a new 1.5 mL RNase-free centrifuge tube, add 50-80 uL of RNase-free water to the middle of the adsorption membrane, incubate at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute.

[0059] Take 4 µl of the obtained RNA solution and perform agarose gel electrophoresis (1% agarose gel) to detect the quality and concentration of RNA. The qualified RNA is used for subsequent reverse transcription cDNA preparation experiments.

[0060] (3) cDNA preparation

[0061] The extracted RNA was prepared into a reverse transcription system as shown in Table 1 using 200 µL eight-tube RNase-free centrifuge tubes. Based on the extracted RNA concentration, 12 µL of RNA was added as the minimum concentration, and the corresponding volumes of water were added to other RNA concentrations to ensure uniformity. Then, 1 µL of Oligo dT was added. 18 (50 µM), mix well and heat at 65°C for 5 min, then quickly place in an ice water bath to cool and let stand on ice for 2 min.

[0062] Table 1 RNA template denaturation system

[0063] Table 1 Reaction mixture of RNA template denaturation

[0064] Reaction components volume <![CDATA[OLigo dT 18 (50µM)]]> 1 µL Total RNA 10 pg-5µg <![CDATA[RNase free ddH2O]]> To 13 µL

[0065] Configure the above reaction products into the reaction system shown in Table 2, and synthesize the first strand of cDNA according to the following program: Reaction program: 1: 25℃ 5 min; 2: 50℃ 45 min; 3: 85℃ 5 min. The product can be used immediately for PCR reaction or stored at -20℃.

[0066] Table 2 First-strand cDNA synthesis reaction system

[0067] Table 2 Reaction mixture of synthesizing the first strand cDNA

[0068] Reaction components volume The mixture from the previous step 13 µL 5x HiScript II buffer 4 µL dNTP Mix (10mM each) 1 µL HiScript ⅡReverse Transcriptase (200U / µL) 1 µL RNase inhibitor (40 U / µL) 1 µL

[0069] (4) DNA extraction

[0070] DNA extraction was performed using the Omega Plant DNA Kit, as follows:

[0071] ① Grind the sample into powder using a mortar and pestle, and weigh 10-30 mg of sample powder into a 2 mL centrifuge tube. Add 600 μL of SP1 Buffer and 5 μL of RNase A, and vortex to mix. Incubate at 65 °C for 10 min. Add 210 μL of SP2 Buffer, and vortex to mix. Incubate on ice for 5 min. Centrifuge at 12,000 × g for 10 min at room temperature.

[0072] ② Insert the Homogenizer Columns into 2mL centrifuge tubes. Transfer the supernatant into the Homogenizer Columns. Centrifuge at 13,000×g for 2 min at room temperature.

[0073] ③ Transfer the supernatant to a new centrifuge tube. Add 1.5 times the volume of SP3 Buffer to the supernatant and vortex to mix. Insert the HiBind DNA Mini Columns into the collection tube.

[0074] ④ Transfer 650 μL of the mixture to the binding column, centrifuge at 10,000 × g for 1 min at room temperature, and discard the filtrate. Reinsert the HiBindDNA Mini Columns into the centrifuge tube.

[0075] ⑤ Connect the HiBindDNA Mini Columns to a new collection tube, add 65 μL of SPW Buffer to the binding column, centrifuge at 10,000 × g for 1 min, and discard the filtrate. Repeat the second SPW Buffer wash.

[0076] ⑥ Put the HiBindDNAMini Columns back into the collection tube and centrifuge at 12,000 o × g for 2 min.

[0077] ⑦ Insert the HiBind DNA Mini Columns into a new 1.5 mL centrifuge tube, add 50-100 μL of preheated 65°C Elution Buffer or sterile water to the binding column, incubate at room temperature for 3-5 min, then centrifuge at 10,000 × g for 1 min to elute the DNA. Repeat the elution process twice, and store the product at -20°C.

[0078] (5) Construction of overexpression vectors

[0079] Using the cDNA obtained in (3) above as a template, and with the synthesized amplification primer sequences, a PCR reaction system as shown in Table 3 was prepared (reagents from Novizan). The CDS sequence (375 bp) of CP12.2 was cloned. The reaction program was as follows: 1: 95°C, 3 min; 2: 95°C, 30 s; 3: 55°C, 30 s; 4: 72°C, 90 s; 5: 35 cycles of steps 2-4; 6: 72°C, 5 min; Store at 10°C.

[0080] Table 3 PCR reaction system of Phanta high-fidelity enzyme

[0081] Table 3 Reaction mixture of PCR with 2×Phanta ®Turbo Master Mix

[0082] Reaction components Volume (µL) template 1 2x Phanta Max Buffer 25 forward primer 2 reverse primer 2 <![CDATA[dNTP MixPhanta Max Super-Fidelity DNA PolymeraseddH2O]]> 1118

[0083] The PCR amplification products were detected by 1% agarose gel electrophoresis, and the target fragment was then excised and recovered. Agarose gel recovery was performed using a small-volume agarose gel DNA recovery kit (ZP202-3, Zhuangmeng), strictly following the manufacturer's instructions. The product was eluted with 30 μl of sterile ddH2O. 3 μl of the product was then analyzed by 1% agarose gel electrophoresis to determine product quality and concentration.

[0084] For plasmid vector digestion, the appropriate restriction endonuclease (Takara) and digestion system are selected according to the different inserted fragments. For vector linearization, taking the pH7-Lic-C-HA plasmid as an example, the digestion system is shown in Table 4. The reaction conditions are 37℃, digestion for 3-5 h, and after the reaction, it is placed at 65℃ for 15 min to inactivate the endonuclease.

[0085] Table 4 Enzyme digestion reaction system

[0086] Table 4 Reaction mixture of digestion

[0087] Reaction components Volume (µL) pH7-Lic-C-HA plasmid 30 Stu I 2 10 x QuickCut Green Buffer 2.5 <![CDATA[ddH2O]]> 13.5

[0088] The target fragment recovered from the gel was ligated into the pH7-Lic-C-HA vector and then transformed into *E. coli* DH5α. After bacterial growth, single colonies were picked and cultured in 400 µL of LB broth with the corresponding antibiotic at 37°C and 200 rpm for 4–5 h. PCR detection was performed using the appropriate primers. Sequencing was performed on positive colonies, and clones without mutations were selected. Plasmid extraction was carried out using a small-scale plasmid extraction kit (ZP101, Zhuangmeng), strictly following the manufacturer's instructions. The bacterial culture was preserved in sterile 50% glycerol (700 µL of bacterial culture was rapidly frozen in liquid nitrogen with 300 µL of glycerol and then stored at -80°C). The correctly sequenced target vector was named pH7-SaCP12.2.

[0089] (6) Construction of gene editing vector

[0090] Using the gDNA obtained in (4) above as a template, and with the synthesized amplification primer sequences, a PCR reaction system as shown in Table 3 was prepared (reagents from Novizan). The gene-editing sequence of CP12.2 (196 bp) was cloned. The reaction program was as follows: 1: 95°C, 3 min; 2: 95°C, 30 s; 3: 55°C, 30 s; 4: 72°C, 90 s; 5: 35 cycles of steps 2-4; 6: 72°C, 5 min; stored at 10°C.

[0091] The PCR amplification products were detected by 1% agarose gel electrophoresis, and the target fragment was then excised and recovered. Agarose gel recovery was performed using a small-volume agarose gel DNA recovery kit (ZP202-3, Zhuangmeng), strictly following the manufacturer's instructions. The product was eluted with 30 μl of sterile ddH2O. 3 μl of the product was then analyzed by 1% agarose gel electrophoresis to determine product quality and concentration.

[0092] For plasmid vector digestion, the appropriate restriction endonuclease (Takara) and digestion system are selected according to the different inserted fragments. For vector linearization, taking pJCV55 plasmid as an example, the digestion system is shown in Table 4. The reaction conditions are 37℃, digestion for 3-5 h, and after the reaction, it is placed at 65℃ for 15 min to inactivate the endonuclease.

[0093] Table 5 Enzyme digestion reaction system

[0094] Table 5 Reaction mixture of digestion

[0095] Reaction components Volume (µL) pJCV55 plasmid 30 Alu I 2 10 x QuickCut Green Buffer 2.5 <![CDATA[ddH2O]]> 13.5

[0096] The target fragment recovered from the gel was ligated into the pJCV55 vector and then transformed into *E. coli* DH5α. After colony growth, single clones were picked and cultured in 400 µL of LB broth with the corresponding antibiotic at 37°C and 200 rpm for 4–5 h. PCR detection was performed using the appropriate primers. Sequencing was performed on positive clones, and mutation-free clones were selected. Plasmid extraction was carried out using a small-scale plasmid extraction kit (ZP101, Zhuangmeng), strictly following the manufacturer's instructions. The bacterial culture was preserved in sterile 50% glycerol (700 µL of bacterial culture was rapidly frozen with liquid nitrogen using 300 µL of glycerol) and then stored at -80°C. The correctly sequenced target vector was named pJCV55-SaCP12.2.

[0097] (7) Carrier transformation

[0098] Following the vector construction method described above, pH7-SaCP12.2 and pJCV55-SaCP12.2 were constructed, and the plasmids were electroporated into Agrobacterium GV3101. The specific procedures are as follows:

[0099] Clean the electroporation cup with pure anhydrous ethanol beforehand to kill any bacteria remaining from the previous use. Place it upside down on absorbent paper and put it into the laminar flow hood along with the electroporation apparatus. Set the apparatus to the "bacteria" setting and check if it is working properly. Then, place a sterile pipette tip and a pipette with the appropriate volume. Sterilize with UV light for about 10 minutes.

[0100] ② During this period, place the plasmid to be transformed and the Agrobacterium GV3101 to be activated on ice. After 10 min, place the electroporation cup on ice to pre-cool it to avoid directly heat shocking the Agrobacterium.

[0101] ③ Take 2 µL of plasmid into Agrobacterium, repeatedly aspirate and mix well for electroporation, wash the bacterial solution with 400 µL YEB into the original Agrobacterium centrifuge tube, and revive at 28℃ and 250 rpm for 2 h or more;

[0102] ④ Centrifuge at 4000 rpm for 4 minutes. According to the experimental requirements, aspirate different volumes of transformed competent cells and add them to YEB agar medium containing the corresponding antibiotics, spreading the cells evenly. After the liquid is absorbed, seal the plate, invert it, and incubate at 28°C for 2-3 days.

[0103] ⑤ Select a single colony for PCR colony detection to obtain a positive clone.

[0104] (8) Potato genetic transformation

[0105] Agrobacterium GV3101 with the above-mentioned overexpression vector and gene editing vector were streaked onto YEB solid medium for activation. Single clones were picked and cultured in 20 ml of YEB liquid medium at 28°C and 200 rpm for 24 hours. 2 ml of bacterial culture was then transferred to 40 ml of YEB liquid medium and subcultured at 28°C and 200 rpm until the OD600 was approximately 0.5 (approximately 6 hours). The culture was then centrifuged at 5000 rpm for 6 minutes, the supernatant was discarded, and the culture was resuspended in 10 ml of 3% MS liquid medium.

[0106] The process of infecting AC142 potato material with pH7-SaCP12.2 bacterial suspension is as follows: AC142 test tube potatoes grown for 6-8 weeks are cross-cut into thin slices with a thickness of about 1-2 mm; then, they are inoculated in the above resuspension for 10 minutes, with a gentle shake halfway through; the bacterial suspension is discarded, the surface bacterial suspension is blotted dry with sterile filter paper, and then transferred to P1 co-culture medium (3% MS solid medium + 0.2 mg / L GA3 + 0.2 mg / L IAA + 0.5 mg / L 6-BA + 2 mg / L ZT, pH 5.8), and incubated in the dark at 23°C for 48 hours. The samples were then transferred to P2 differentiation medium (3% MS solid medium + 0.2 mg / L IAA + 0.2 mg / L GA3 + 2 mg / L ZT + 0.5 mg / L 6-BA + 75 mg / L Kan + 400 mg / L Cef) and cultured at 23°C (16 hours light / 8 hours dark cycle). When the resistant shoots grew to 0.5-1 cm, they were inoculated into P3 rooting medium (3% MS + 50 mg / L Kan + 400 mg / L Cef). After the resistant shoots grew, they underwent secondary rooting, followed by positive detection using NPT II gene primers. The positive lines were propagated in 4% MS solid medium, RNA was extracted and reverse transcribed into cDNA (method as above), and the relative expression level of the CP12.2 gene was detected using quantitative primers (CP12-2F, CP12-2R) (with β-tublin2 as an internal control). The expression levels of the target gene in each strain are shown in Table 6. The strains CP12.2-OE-AC142-18 and CP12.2-OE-AC142-19 were selected for cold resistance identification.

[0107] Table 6. Detection of relative expression levels of CP12.2 in the CP12.2-OE-AC142 strain.

[0108] Material No. relative expression level Material No. relative expression level Material No. relative expression level Material No. relative expression level AC142 1.00 6 2.25 11 1.65 16 1.06 1 0.35 7 1.05 12 8.83 17 2.83 2 2.17 8 5.54 13 1.10 18 7.65 3 3.17 9 2.90 14 5.61 19 31.58 4 1.17 10 0.93 15 1.61 20 0.74 5 1.93

[0109] The inoculation of potato material *Etuberosum* with pJCV55-SaCP12.2 bacterial suspension was performed as follows: Leaflets (including part of the petiole) / stem segments approximately 1.5-2 cm long from sterile test-tube seedlings cultured in 2% MS for 3-4 weeks with fully expanded leaves were placed on MS plates containing 3% sucrose + 2 mg / L 6-BA + 0.2 mg / L NAA + 0.05 mg / L GA3. 20-25 explants were inoculated per plate. When inoculating leaves, the upper surface should be facing down, adhering to the entire culture medium. The leaves were incubated in the dark at 22±1℃ for 2 days. After culturing Agrobacterium at 28℃ and 200 rpm for 14-20 h in a shaker, 1-2 mL of bacterial suspension was transferred to 150 mL LB medium containing 50 mg / L Rif + 50 mg / L Kan / Spe; culturing at 28℃ and 200 rpm for 4-5 h, the culture was transferred to a 50 mL centrifuge tube and centrifuged at 4500 rpm for 8 min. The bacterial cells were collected on a clean bench and resuspended in 35-40 mL MS liquid medium, adjusting the concentration to an OD value of approximately 0.3. The culture was then used to infect pretreated explants for 8-10 min. Infected leaves and stem segments were removed and the bacterial suspension was completely blotted dry with filter paper. They were then placed on co-culture medium and incubated in the dark at 22±1℃ for 2 days. The culture medium was the same as the pretreatment medium. Leaves and stem segments co-cultured for 2 days were transferred to callus induction medium (3% MS + 2 mg / L 6-BA + 0.2 mg / L NAA + 0.05 mg / L GA3 + 50 mg / L Kan + 200 mg / L Cef + 200 mg / L Tim). Explants transferred to callus induction differentiation medium should be changed every 10-12 days. After approximately 3-4 weeks, regenerated shoots differentiated to over 1 cm were cut off and inserted into rooting medium (MS + 50 mg / L Kan + 200 mg / L Cef + 200 mg / L Tim) for root selection; rooted plantlets were then cut off for secondary rooting. The two-rooted lines were propagated in 4% MS solid medium, and DNA was extracted (using the same method as above). Positive lines were detected using Cas9 primers and HiTOM sequencing was performed at Wuhan Boyuan Biotechnology Co., Ltd. Gene editing vector lines CP12.2-CC-etuber-4 and CP12.2-CC-etuber-8 were selected for cold resistance identification.

[0110] Example 5

[0111] This embodiment provides the application of the potato CP2.2 gene, as detailed below:

[0112] The cold resistance of CP12.2 gene-modified plants was assessed as described in Examples 1 and 2. The selected positive gene-modified lines and wild-type materials were potted, and after 40 days, materials with consistent growth were selected for LT50 testing and cold resistance assessment. As shown in Tables 7 and 8, overexpression of the CP12.2 gene improved the cold resistance of potato materials, while gene-edited CP12.2 genes reduced the cold resistance. This was mainly reflected in the median lethal temperature (LT50) of potato plants overexpressing the CP12.2 gene. 50 The frost damage scores of the control potato plant AC142 were lower than those of the control plant AC142. After treatment at -2.5℃ for 3 hours and recovery at 20℃ for 1 day, the frost damage scores of the control material AC142 were (4.67), while those of the potato plants 18# and 19# with overexpression of the CP12.2 gene were (0.56 and 1.11), respectively. The median lethal temperature (LT) of the gene-edited CP12.2 plants was lower than that of the control plant AC142. 50 The freezing damage scores of the control potato plants Solanum etuberosum were higher than those of the control plants. After treatment at -3.5℃ for 3 hours and recovery at 20℃ for 1 day, the freezing damage scores of the control material Solanum etuberosum were (0.56), while those of the potato plants 4# and 8# with the gene-edited CP12.2 gene were (4.78 and 4.22).

[0113] Table 7. Logistic regression equations and half-lethal temperatures for conductivity of AC142, excess strains, Solanum etuberosum, and edited strains.

[0114] Material Name Regression equation Correlation coefficient <![CDATA[LT 50 / ℃]]> AC142 <![CDATA[y=57.974 / [1+e (2.335-4.65x) ]]]> 0.9984** -0.897℃ CP12.2-OE-AC142-18 <![CDATA[y=75.576 / [1+e (2.714-2.381x) ]]]> 0.9996** -1.42℃ CP12.2-OE-AC142-19 <![CDATA[y=71.368 / [1+e (2.305-2.158x) ]]]> 0.9946** -1.46℃ Solanum etuberosum <![CDATA[y=93.183 / [1+e (1.6869-0.65422x) ]]]> 0.9413** -2.8℃ CP12.2-CC-etuber-4 <![CDATA[y=89.613 / [1+e (2.00378-1.09129x) ]]]> 0.9709** -2.05℃ CP12.2-CC-etuber-8 <![CDATA[y=76.358 / [1+e (1.225-0.794x) ]]]> 0.9853** -2.35℃

[0115] Table 8 Evaluation of freezing damage to AC142 and its over-exploitation strain, Solanum etuberosum and its edited strain.

[0116] Material Name Artificial cryo-injury scoring AC142 (CK) 4.67 CP12.2-OE-AC142-18 0.56 CP12.2-OE-AC142-19 1.11 Solanum etuberosum (CK) 0.56 CP12.2-CC-etuber-4 4.78 CP12.2-CC-etuber-8 4.22

[0117] The above description is only a specific embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

[0118] References:

[0119] Thomashow MF. Molecular basis of plant cold acclimation: insights gained from studying the CBF cold response pathway[J]. Plant Physiology, 2010, 154(2):571-577.

[0120] Licausi F, Ohme-Takagi M, Perata P. APETALA / Ethylene ResponsiveFactor (AP2 / ERF) transcription factors: mediators of stress responses and developmental programs[J]. New Phytologist, 2013, 199(3):639-649.

[0121] Liu Jingyan, Shi Yiting, Yang Shuhua. CBF: Key to Balancing Plant Low Temperature Response and Growth Development [J]. Acta Botanica Sinica, 2017, 52(6): 689-698.

[0122] Liu Jitao, Suo Haicui, Li Chengchen, Wang Li, Shan Jianwei, An Kang, Li Xiaobo. Research progress on cold resistance of potato [J]. Guangdong Agricultural Sciences, 2020, 47(12): 73-81.

[0123] Vogel JT, Zarka DG, Van Buskirk HA, Fowler SG, Thomashow MF. Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis[J]. The Plant Journal, 2005, 41(2):195-211.

[0124] Kou S, Chen L, Tu W, Scossa F, Wang Y, Liu J, Fernie AR, Song BT, XieCH. The arginine decarboxylase gene ADC1, associated to the putrescinepathway, plays an important role in potato coldacclimated freezing toleranceas revealed by transcriptome and metabolome analyses[J]. Plant Journal, 2018,96(6): 1283-1298.

Claims

1. The potato CP12.2 gene, characterized by... The CDS sequences of the CP12.2 gene in the highly cold-resistant wild potato material W3 and the frost-sensitive wild potato material Cph12 are shown in SEQ ID NO.1, and the CDS sequence in the moderately cold-resistant potato material Etuberosum is shown in SEQ ID NO.

2.

2. A method for cloning and functional verification of the potato CP12.2 gene, characterized in that... The method includes the following steps: (1) primer design; (2) RNA extraction; (3) cDNA preparation; (4) PCR reaction and expression vector construction.

3. Application of the potato CP12.2 gene to enhance cold resistance in potatoes, characterized by... The CDS sequence of the CP12.2 gene is shown in SEQ ID NO.1 or SEQ ID NO.2 of the sequence listing.

4. The application of the potato CP12.2 gene to enhance cold resistance in potatoes according to claim 3, characterized in that... The application of the CP12.2 gene to enhance the cold resistance of potatoes is achieved through overexpression of the CP12.2 gene.

5. The application of the potato CP12.2 gene to enhance cold resistance in potatoes according to claim 3, characterized in that... The application of the CP12.2 gene in improving the cold resistance of potatoes is reflected in the fact that CP12.2 gene overexpression significantly reduces the plant's half-lethal temperature and freezing damage score.

Images