Application of CmoCK1 gene in creating low temperature tolerant rootstock of cucumber

Abstract

The application relates to the field of plant molecular breeding and low-temperature stress cultivation technology, and particularly relates to application of a CmoCK1 gene in creating a low-temperature-resistant rootstock of cucumber; through overexpression of the CmoCK1 gene and low-temperature treatment of a wild-type cucumber, the low-temperature resistance of the cucumber with overexpression of the CmoCK1 gene is significantly improved; the wild type and the overexpression plant are grafted as rootstocks to wild-type scions respectively, it is found that the cucumber with overexpression of the CmoCK1 gene as the rootstock can improve the low-temperature resistance of the scion cucumber, and the physiological indexes such as the relative conductivity and the malondialdehyde content of the above plants are determined, which shows that the new function of the CmoCK1 gene has good application potential, provides a gene resource and a new idea for reducing low-temperature stress, delaying the low-temperature response of the cucumber and further cultivating a low-temperature-resistant special rootstock of the cucumber in production, and has a wide application prospect and high use value.

Description

TECHNICAL FIELD

[0001] The application relates to the field of plant molecular low-temperature stress, and particularly relates to application of a CmoCK1 gene in creating a low-temperature-tolerant cucumber rootstock. BACKGROUND

[0002] Cucumber (Cucumis sativus L) is an important facility fruit and vegetable crop in China, and the planting area and yield of the cucumber are ranked first in the world. The cucumber is a typical warm vegetable, and has poor low-temperature tolerance. The wintering seedlings and the initial planting of the cucumber are prone to low-temperature stress (<10 DEG C), which leads to physiological disorders and fruit drop, and greatly reduces the yield and quality of the fruit. In severe cases, the yield is reduced by more than 30%, the income is reduced by about 100,000 yuan per hectare, and serious economic losses are caused. Grafting as an effective means to improve the low-temperature tolerance has become one of the matching measures for the cultivation of the cucumber in the sunlight greenhouse in winter and spring. The pumpkin rootstock not only maintains the excellent traits of the cucumber cultivars, but also improves the disease resistance and stress resistance, especially the low-temperature tolerance. However, the cucumber grafted with different rootstocks has different advantages. For example, the black-seeded pumpkin has strong stress resistance, but the grafted cucumber has poor quality; the white-seeded pumpkin has good quality, but the grafted cucumber has unstable stress resistance. Therefore, there is an urgent need for a rootstock with multiple traits (maintaining the quality and improving the stress resistance, and easy to breed) in the production, which can improve the low-temperature tolerance and maintain the original quality of the cucumber. Therefore, the improvement of the pumpkin rootstock and the mechanism of the stock-scion interaction have been the research focus in the field of plant grafting.

[0003] In the early stage, the group of the present application verifies and mines the key gene choline kinase CmoCK1 of the low-temperature tolerance of the pumpkin by using the omics and molecular biology means. It is found that the mRNA can be transported to the cucumber scion from the pumpkin stock in a long distance, and the low-temperature tolerance of the grafted cucumber is improved. However, whether the transgenic cucumber special low-temperature-tolerant rootstock can be created by overexpressing CmoCK1, and the low-temperature tolerance of the grafted cucumber is improved is still unknown. SUMMARY

[0004] In view of the defects that the rootstock material with multiple traits (maintaining the quality and improving the low-temperature tolerance, and easy to breed) is insufficient in the cucumber cultivation and production in the market, the present application aims to provide a method for creating a low-temperature-tolerant cucumber rootstock by overexpressing the CmoCK1 gene, and improving the low-temperature stress tolerance of the cucumber cultivars.

[0005] The present application constructs a set of genetic and stable genetic transformation methods of the cucumber hairy root of the vegetable crop, obtains the transgenic cucumber with overexpressed CmoCK1 gene, carries out physiological and gene expression analysis of the response to the low temperature, and finally grafts the transgenic cucumber and the wild type to explore the influence of the overexpressed CmoCK1 gene on the cold resistance of the cucumber.

[0006] To solve the above technical problems, the technical scheme provided by the present application is as follows:

[0007] Application of the CmoCK1 gene in cucumber cold tolerance

[0008] The nucleotide sequence of the CmoCK1 gene is shown in SEQ ID NO.1.

[0009] Application of the CmoCK1 gene in the creation of cucumber rootstocks resistant to low temperatures

[0010] The nucleotide sequence of the CmoCK1 gene is shown in SEQ ID NO.1.

[0011] Preferred,

[0012] The amino acid sequence of the protein encoded by the CmoCK1 gene is shown in SEQ ID NO.2.

[0013] Preferred,

[0014] The cold tolerance of cucumber plants can be improved by increasing the expression level of the CmoCK1 gene.

[0015] The primer pairs used to amplify the CmoCK1 gene have nucleotide sequences as shown in SEQ ID NO. 9-10.

[0016] A method for breeding low-temperature resistant cucumbers.

[0017] The low-temperature tolerance of cucumber plants can be improved by increasing the expression level of the CmoCK1 gene; the nucleotide sequence of the CmoCK1 gene is shown in SEQ ID NO.1.

[0018] Preferably, the steps include:

[0019] 1) Amplify the CmoCK1 gene to obtain the amplification product;

[0020] 2) The restriction sites were determined to be XbaI and KpnI. The amplification product was ligated into the vector pCambia1305-35S:C-GUS-NOSter-35S:C-GFP-NOSter to obtain the overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter. After the sequencing was confirmed to be correct, the plasmid was extracted.

[0021] 3) The overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter was transformed into Agrobacterium GV3101 competent cells, and the genetic transformation of cucumber was carried out by infecting cucumber cotyledons.

[0022] 4) The CmoCK1 gene overexpression line was obtained by amplifying the GFP sequence on the overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter and by detecting the CmoCK1 gene in plants using qRT-PCR.

[0023] A method for creating low-temperature resistant rootstocks for cucumbers.

[0024] Cucumbers overexpressing the CmoCK1 gene were obtained through genetic transformation. These overexpressing CmoCK1 gene-based rootstocks were then used for grafting to obtain cold-resistant rootstocks.

[0025] The nucleotide sequence of the CmoCK1 gene is shown in SEQ ID NO.1.

[0026] The beneficial effects of this invention are:

[0027] This invention is innovative in elucidating the molecular mechanism by which pumpkin rootstock regulates the low-temperature tolerance of cucumber, supplementing and improving the mechanism by which grafting enhances cold resistance, and providing new ideas for the creation of cold-resistant cucumber rootstock varieties in the future. It is a scientific research method that combines scientificity, innovation and practicality. Attached Figure Description

[0028] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0029] Figure 1 The figure shows the results of an experiment identifying the transport of CmoCK1 from cucumber hairy roots to new leaves.

[0030] Figure 2 The experimental results show the improvement of cucumber's low-temperature resistance by upward transport of CmoCK1.

[0031] Figure 3 Figure showing the experimental results of improving the low-temperature tolerance of cucumbers by overexpressing the CmoCK1 gene.

[0032] Figure 4 Figure showing the experimental results of improving the low-temperature tolerance of cucumber by influencing the rootstock-scion transport of CmoCK1 mRNA. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments.

[0034] Example 1: Cucumber cultivation, self-grafting, and low-temperature treatment methods

[0035] (1) Cucumber cultivation: "Xintai Mici" cucumbers were cultivated in a constant temperature incubator until they reached the two-leaf-one-heart stage. The light intensity of the incubator was 600 μmol / m². -2 s -1 Photoperiod: 16h light / 8h darkness; Temperature: 26℃ during the day / 18℃ at night.

[0036] (2) Cucumber self-grafting: After the cucumber cotyledons are flattened, the scion is obliquely cut 1 cm below the growing point of the scion. A similar oblique cut is made at the growing point of the rootstock, retaining one cotyledon. The cuts of the rootstock and scion are then joined together and secured with a grafting clip to obtain a self-grafted cucumber. During the grafting process, gently spray water to keep the scion moist. Cover the seedling tray with a transparent lid and wrap the edges with plastic wrap to maintain the moisture of the grafted seedling. Transfer the seedling tray to a low-light growing environment. After 4 days, gradually increase the light and decrease the humidity.

[0037] (3) Cucumber low temperature treatment method: Two-leaf and one-heart cucumber materials with the same growth for low temperature treatment were placed in a light incubator cooled to 4℃. The plant materials were measured or sampled at the set treatment time. At least 3 technical replicates were set for each experimental group.

[0038] Example 2: RNA extraction from self-grafted cucumber

[0039] Take an appropriate amount of self-grafted cucumber into a 2mL enzyme-free centrifuge tube, add a sterilized steel ball, and grind it into powder using a plant grinder.

[0040] RNA extraction using the TRizol method:

[0041] (1) Add 1 mL of TRizol extract to the sample tube, vortex to mix, and let stand at room temperature for 5 min.

[0042] (2) Pre-cool the centrifuge at 4°C, centrifuge at 12000 rpm for 5 min, and take 800 μL of the supernatant and add it to a 1.5 mL centrifuge tube without RNase enzyme;

[0043] (3) Add 160 μL of chloroform to a 1.5 mL centrifuge tube, invert to mix, and let stand at room temperature for 5 min;

[0044] (4) Centrifuge at 4℃ and 12000rpm for 15min, and take 300μL of the supernatant into a new 1.5mL centrifuge tube;

[0045] (5) Add 300 μL of isopropanol to the supernatant and mix thoroughly by inverting. Let stand at -20℃ for 20 min.

[0046] (6) Centrifuge at 4℃ and 12000rpm for 15min, then discard the supernatant;

[0047] (7) Wash the RNA precipitate with 1 mL of pre-cooled 75% ethanol, centrifuge at 12000 rpm at 4℃ for 5 min and discard the supernatant;

[0048] (8) Open the centrifuge tube and dry it at room temperature for about 5 minutes to allow the remaining ethanol to evaporate and dry.

[0049] (9) Add 40-60 μL of RNA-free water to the centrifuge tube to dissolve the RNA precipitate. Measure the RNA concentration using NanoDrop and store at -80°C. Obtain RNA from the self-grafted cucumber.

[0050] Example 3: Reverse transcription of RNA from self-grafted cucumber into cDNA

[0051] According to Novozymes The steps of the IIQ RT SuperMix for qPCR (+gDNA wiper) (R223-01) kit are as follows.

[0052] Genomic DNA removal:

[0053] The reaction solution was prepared in an RNase-free centrifuge tube as follows:

[0054]

[0055] At 42°C, gently pipette and mix for 2 minutes;

[0056] The reverse transcription reaction system was prepared as follows:

[0057] Add 5×HiScriptⅡqRT SuperMixⅡ directly to the reaction tube from the previous step;

[0058]

[0059] At 50°C, the RNA from the self-grafted cucumber was reverse transcribed into cDNA by pipetting and mixing for 15 min, followed by 5 s at 85°C.

[0060] Example 4: Expression detection of CmoCK1 gene and CBFs-COR cold pathway gene in CmoCK1 gene overexpression cucumber under normal temperature (26℃) and cold treatment (4℃) conditions.

[0061] The nucleotide sequence of the CmoCK1 gene in cucumber is shown in SEQ ID NO.1, and the sequence of its encoded protein is shown in SEQ ID NO.2.

[0062] The CBFs-COR cold pathway genes include CBF1 (C-repeat-Binding Factor 1) and COR (Cold-Regulated) genes, which are genes that mediate cold signal transduction and cold-regulated protein expression.

[0063] The CmoCK1 gene in cucumber consists of 3581 bases and is composed of 8 exons. The first exon consists of bases 1 to 110, the second exon consists of bases 519 to 1199, the third exon consists of bases 2122 to 2243, the fourth exon consists of bases 2329 to 2524, the fifth exon consists of bases 2724 to 2841, the sixth exon consists of bases 2930 to 2977, the seventh exon consists of bases 3288 to 3364, and the eighth exon consists of bases 3435 to 3581. The remaining exons are intron sequences.

[0064] The qRT-PCR primer sequences designed based on the CmoCK1 gene sequence are as follows:

[0065] CmoCK1 gene primer sequence:

[0066] qRT-CmoCK1-F 5'-TCATCCACCGCCAGGACGCTATCAGC-3'(SEQ ID NO.3)

[0067] qRT-CmoCK1-R 5'-CTTACCCAATTTCTCAATCTGTCCCA-3'(SEQ ID NO.4)

[0068] CsaCBF1 gene primer sequence:

[0069] qRT-CsaCBF1-F 5'-TACAGAGGAGTCAGGAGGA-3'(SEQ ID NO.5)

[0070] qRT-CsaCBF1-R 5'-AGAATCGGCGAAATTGA-3'(SEQ ID NO.6)

[0071] CsaCOR gene primer sequence:

[0072] qRT-CsaCOR-F 5'-ACTTTGAGAGGACATTTGATG-3'(SEQ ID NO.7)

[0073] qRT-CsaCOR-R 5'-GAAGCTCCAATTTTGACTTG-3'(SEQ ID NO.8)

[0074] The cDNA product from self-grafted cucumber was diluted 5-fold to prepare diluted cDNA samples. The qRT-PCR reaction was performed according to the steps outlined in the Novizan Bio-Fluorescence Quantitative Reaction Kit (Q711-02). The qRT-PCR reaction system is as follows:

[0075]

[0076] After thorough mixing, place the mixture into a QuantStudio™ 6Flex real-time PCR instrument for PCR amplification. The qPCR program is as follows:

[0077]

[0078] The measured CT values ​​were converted into relative copy numbers using the ΔΔCT method and compared with the copy number of the internal reference. For example... Figure 2 and Figure 3 As shown, overexpression of CmoCK1-GFP in cucumber roots or introduction of CmoCK1-GFP into cucumbers through stable transformation resulted in significantly higher levels of CmoCK1, CsaCBF1, and CsaCOR genes than the control after 12 h of cold treatment, demonstrating that the CmoCK1 gene responds to 4℃ low-temperature stress.

[0079] Experimental Example 5: Method for Identifying the Transportability of Cucumbers

[0080] Using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), with the species set to Cucurbitaceae, the CDS sequence of CmoCK1 was entered in the PCR Template options, and the target product size was selected to be between 300-500 bp. Primers specific to 'Xintai Mici' were screened based on the website's design results. RT-PCR was used to verify the primer specificity, ensuring successful amplification of the specific CmoCK1 gene sequence from both above-ground and below-ground parts.

[0081] Experiment Example 6: CmoCK1 Protein Extraction and Western Blot Technique

[0082] Total protein was extracted from cucumber leaves using lysis buffer and protease inhibitors. A 12% SDS-PAGE separating gel and a 5% SDS-PAGE stacking gel were prepared. Protein electrophoresis was performed for 1 hour, followed by blocking the membrane with 5% skim milk in 1×TBST for 1 hour. The membrane was then incubated overnight at 4°C with monoclonal antibodies against CmoCK1 and GFP (green fluorescent protein, used as a reporter gene marker protein). After washing the membrane five times with 1×TBST, it was incubated in blocking buffer for 40 minutes with a 1:20 (v / v) dilution of anti-rabbit antibody. Protein bands were detected using a chemiluminescence analyzer.

[0083] Example 7: Full-length cloning of the CmoCK1 gene

[0084] Cloning primers were designed based on the CmoCK1 gene fragment.

[0085] The designed primer sequences are as follows:

[0086] CmoCK1-F 5'-ATGGCAATAAAGTCAAACGGATTCAC-3'(SEQ ID NO.9)

[0087] CmoCK1-R 5'-GGAGGCATTCATCATTAGACAAAAGTGA-3'(SEQ ID NO.10)

[0088] PCR reaction system: Max DNAPolymerase;

[0089]

[0090] The PCR reaction procedure using cucumber cDNA as a template is as follows:

[0091]

[0092] PCR product detection: Prepare a 1% agarose gel according to the target fragment size, add nucleic acid dye (1 / 10,000), 0.1% TAE electrophoresis buffer, electrophoresis at 130V for about 25 minutes, detect the size of PCR product fragments under UV light, and cut and recover the target band from the gel.

[0093] Example 8: Construction of 1305-CmoCK1 overexpression vector

[0094] The restriction enzyme sites were XbaI and BamHI. Then, the amplification product from Example 7 was ligated into the vector pCambia1305-35S:C-GUS-NOSter-35S:C-GFP-NOSter. After successful sequencing, the plasmid was extracted to obtain the overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter.

[0095] The expression vector was ligated according to the instructions of the NovoRec Plus One-Step PCR Cloning Kit (NR005) from Suzhou Nearshore Protein Technology Co., Ltd. The ligation system (10 μL) is as follows:

[0096]

[0097] Example 9: Genetic transformation of hairy roots

[0098] Sowing: Select plump cucumber seeds, soak them in 65℃ warm water for 30 minutes, then cool them at room temperature for several hours. Peel off the seed coat with tweezers. Shake in 70% alcohol for 30 seconds, then shake in 3% sodium hypochlorite (NaClO) for 8 minutes. Sow approximately 20 seeds per bottle in MS medium and culture in the dark for 4 days.

[0099] Explant acquisition: Remove the seedlings, cut off half of the cotyledons, and cut off the hypocotyl 0.5 cm below the growing point, using the part with the growing point as the explant for transformation.

[0100] Cucumber infection: Scrape the Agrobacterium rhizogenes inoculum plate coated one day in advance, wash with infection solution and readjust OD600 to between 0.15 and 0.20. After the adjusted bacterial solution is allowed to stand at 28℃ for 1-3 hours, place it into the obtained explants and incubate at 80-120 rpm for 20 minutes.

[0101] Co-culturing: After infection, remove excess bacterial culture from the explants and transfer them to a co-culture medium covered with sterile filter paper. Co-culture at 25°C in the dark for 3 days.

[0102] Induced rooting: After co-culture, one end of the hypocotyl of the explant was inserted into the rooting medium and placed in a light incubator to induce the formation of hairy roots.

[0103] Observe GFP under a fluorescent flashlight and select plants containing fluorescent roots.

[0104] Example 10: Identification of positive seedlings from genetic transformation of hairy roots with CmoCK1 gene overexpression

[0105] Hairy roots were transformed with *Agrobacterium rhizogenes* strain K599 containing either the empty vector (pCambia1300-35S::GFP) or pCambia1300-35S::CmoCK1-GFP. K599 was transformed alone as a control. Fourteen days after transformation, the transportability of *CmoCK1-GFP* or *GFP* was verified by RT-PCR using primers for *CmoCK1-GFP* or *GFP*.

[0106] Observe GFP under a fluorescent flashlight, and select plants containing fluorescent roots, such as... Figure 1 As shown in Figure A, under a fluorescence microscope, the root tips of wild cucumber hairy roots emit weak fluorescence, while the hairy roots of GFP and CmoCK1-GFP transgenic cucumbers emit strong and stable green fluorescence.

[0107] Total protein was extracted from leaves and roots of hairy roots transformed with (pCambia1300-35S::GFP) or pCambia1300-35S::CmoCK1-GFP(A). GFP fluorescence was identified using a Leica M205 FA UV microscope, and GFP density was analyzed using ImageJ GFP channels. At least 10 transformation-independent replicates were analyzed. Results are as follows: Figure 1 As shown in Figure B, fluorescent signals were observed in both the roots and leaves of the CmoCK1-GFP transgenic hairy-root cucumber. Strong fluorescent signals were observed in the roots of the GFP transgenic hairy-root cucumber, but almost no fluorescent signals were observed in the aboveground parts.

[0108] Western blot analysis was performed on the protein levels of leaves and roots of hairy roots transformed with (pCambia1300-35S::GFP) or pCambia1300-35S::CmoCK1-GFP. The WB analysis used three replicates, each containing 10 independent transgenic seedlings. R: Agrobacterium-transformed cucumber hairy roots; L: new leaves emerging 14 days after transformation. Figure 1 As shown in Figure C, CmoCK1 and GFP monoclonal antibodies were used to detect the fused CmoCK1-GFP protein (approximately 70 kDa) and GFP protein (approximately 27 kDa). Coomassie brilliant blue staining was used as a control.

[0109] DNA was extracted from cucumber leaves using the CTAB method. Primers were designed using the GFP sequence of the overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter as a template. The primer sequences are as follows:

[0110] GFP-F 5'-ACGTAAACGGCCACAAGTTCAGCG-3'(SEQ ID NO.11)

[0111] GFP-R 5'-CTCGTTGGGGTCTTTGCT-3'(SEQ ID NO.12)

[0112] Amplification system (20 μL):

[0113]

[0114] After mixing the mixture, centrifuge and perform PCR. The PCR procedure is as follows:

[0115]

[0116] The PCR products were detected by 1% agarose gel electrophoresis. Positive seedlings were obtained by amplifying the GFP sequence on the overexpression vector (the target band appeared, and the product length was 578 bp).

[0117] like Figure 1 Figure D shows the results of RT-PCR verification of the transportability of CmoCK1-GUS or GUS using primers fused to CmoCK1-GUS or GUS. Seeds 1 and 2 represent independent transgenic seedlings as biological replicates. Transport rates were analyzed as a percentage of mobile seedlings / total transgenic seedlings. CsaACTIN7 was used as an internal reference for mRNA abundance. HygR was used to exclude Agrobacterium rhizogenes contamination or mobility. The results show that CmoCK1-GUS mRNA can be transported from the roots to the shoots.

[0118] Example 11: Phenotypic and physiological analysis of low-temperature response in hairy-rooted plants overexpressing the CmoCK1 gene

[0119] Plants overexpressing the CmoCK1 gene in hairy roots and wild-type plants were simultaneously treated at 4°C for 12 hours. Their physiological phenotypes were observed and relevant physiological data were measured. Related phenotypes and physiological indicators are as follows: Figure 2 As shown, Figure 2 A shows the phenotypes of cucumber seedlings (control) with root overexpression of 35S::CmoCK1-GFP and 35S::GFP before and after 12 hours of low-temperature treatment. After low-temperature treatment, the leaves of the GFP transgenic hairy-root cucumber showed obvious wilting and curling phenotypes, while some leaves of the CmoCK1-GFP transgenic hairy-root cucumber drooped slightly. Figure 2 B represents NBT (nitroblue tetrazolium) staining of cucumber leaves. Compared with GFP transgenic hairy root cucumber leaves, the NBT staining area of ​​CmoCK1-GFP transgenic hairy root cucumber leaves was smaller and the staining degree was significantly reduced after low temperature treatment. Figure 2 C and 2D represent relative electrolyte permeability (REP) and MDA content; each treatment included 6-12 biological replicates, with 3-4 plants per replicate (mean ± standard deviation, two-way ANOVA followed by t-test, *p<0.05, **p<0.01, ***p<0.001). Under low temperature treatment, the relative electrolyte permeability (REP) and MDA content of leaves of CmoCK1-GFP transgenic hairy-root cucumber were significantly lower than those of GFP transgenic hairy-root cucumber. Figure 2 E represents the relative expression levels of CsaCBF1 and CsaCOR in the leaves of root-transformed 35S::CmoCK1-GFP and 35S::GFP cucumber seedlings before and after 12 hours of low-temperature treatment. Compared with 0 hours of low-temperature treatment in root-transformed 35S::GFP cucumbers, *p<0.05, **p<0.01, ***p<0.001, cucumber ACTIN7 was used as an internal control. Under low-temperature treatment, the relative expression levels of CsaCBF1 and CsaCOR genes in root-transformed 35S::CmoCK1-GFP were significantly increased.

[0120] The results of low-temperature treatment showed that plants overexpressing the CmoCK1 gene through root transformation were more cold-resistant. The degree of NBT staining, relative conductivity, and malondialdehyde content (higher NBT staining, higher relative conductivity, and higher malondialdehyde content indicate greater cell membrane damage and more severe cold stress, indicating weaker cold resistance) were significantly lower than those of the control plants. The expression level of the CsaCOR gene was significantly increased. All of the above indicate that CmoCK1 gene overexpression through root transformation enhances the cold resistance of cucumbers.

[0121] Example 12: Stable genetic transformation with CmoCK1 gene overexpression

[0122] The full-length 1083bp sequence of the CmoCK1 gene was amplified by RT-PCR to obtain the amplification product (Example 7). The restriction sites were XbaI and KpnI. The amplification product was then ligated into the vector pCambia1300-Super:CmoCK1-GFP. After the sequencing was correct, the plasmid was extracted to obtain the overexpression vector pCambia1300-Super:C-GFP (same as Example 8).

[0123] The plasmid containing the CmoCK1 gene was chemically transformed into Agrobacterium GV3101 competent cells, and the genetic transformation of cucumber was carried out by infecting cucumber cotyledons.

[0124] Sowing: Select plump cucumber seeds, soak them in 65℃ warm water for 30 minutes, then cool them at room temperature for several hours. Peel off the seed coat with tweezers. Shake in 70% alcohol for 30 seconds, then shake in 3% sodium hypochlorite (NaClO) for 8 minutes. Sow approximately 20 seeds per bottle in MS medium and culture in the dark for 36 hours.

[0125] Explant acquisition: In a clean bench, use tweezers to remove the radicle of the previously prepared germinating cucumber seed, being careful not to damage the surrounding tissues. Then, cut off one-third of the cotyledon from the end away from the radicle, remove the inner seed coat, and completely separate the two cotyledons and place them in MS liquid medium for later use.

[0126] Cucumber infection: Scrape the Agrobacterium tumefaciens-coated bacterial plate prepared one day in advance, wash it with the infection solution, and readjust the OD600 to between 0.2 and 0.3. After the bacterial solution of the adjusted concentration has been allowed to stand at 28°C for 1-3 hours, it is then infected twice using a syringe to create a vacuum, each time for 1.5 minutes.

[0127] Co-culturing: After infection, remove excess bacterial culture from the explants and transfer them to a co-culture medium covered with sterile filter paper. Co-culture at 25°C in the dark for 4 days.

[0128] Inducing shoot formation: The explants were transferred to a shoot formation medium and cultured for three weeks.

[0129] Rooting induction: Cut off the newly formed buds of cucumber explants from the previous step, insert them into the rooting medium, and place them in a light incubator to induce root production.

[0130] Three CmoCK1 gene overexpression lines, OE#1, OE#2, and OE#4 (same as in Example 10), were obtained by amplifying the GFP sequence on the overexpression vector (GFP-F / R) and detecting the CmoCK1 gene in plants by qRT-PCR (qRT-CmoCK1-F / R).

[0131] Example 13: Phenotypic and physiological analysis of low temperature response in plants overexpressing the CmoCK1 gene

[0132] Plants overexpressing the CmoCK1 gene and wild-type plants were simultaneously treated at 4°C for 12 hours. Their physiological phenotypes were observed and relevant physiological data were measured. Relevant phenotypes and physiological indicators are as follows: Figure 3 As shown, Figure 3 A shows the phenotypes of two-leaf-aged cucumber seedlings overexpressing 35S::CmoCK1-GFP (OE#1, #2 and #4) and wild-type cucumber seedlings before and after 12 hours of low-temperature treatment. Under low-temperature treatment, compared with wild-type, cucumber seedlings overexpressing the CmoCK1 gene showed lower wilting. Figure 3 B represents relative electrolyte permeability (REP) and MDA content; each treatment included 4 biological replicates, with 3-4 plants per replicate (mean ± standard deviation, two-way ANOVA followed by t-test, *p<0.05, **p<0.01, ***p<0.001). Under low temperature treatment, the relative electrolyte permeability (REP) and MDA content of the transgenic lines overexpressing 35S::CmoCK1-GFP (OE#1, #2, and #4) were significantly lower than those of wild-type cucumber seedlings. Figure 3 C represents the relative expression levels of CmoCK1, CsaCBF1, and CsaCOR in leaves of 35S::CmoCK1-GFP transgenic cucumber seedlings (OE#2 and #4) before and after 12 hours of low-temperature treatment. Compared with wild-type seedlings treated for 0 hours of low temperature, *p<0.05, **p<0.01, ***p<0.001. Cucumber ACTIN7 was used as an internal control. Under low-temperature treatment, the expression level of the CmoCK1 gene in the 35S::CmoCK1-GFP transgenic lines (OE#1, #2, and #4) was significantly higher than that in wild-type cucumber seedlings.

[0133] The results of low-temperature treatment showed that plants overexpressing the CmoCK1 gene were more cold-resistant. The relative conductivity and malondialdehyde content of the three overexpressing lines were significantly lower than those of the wild-type plants. These results indicate that overexpression of the CmoCK1 gene enhances the cold resistance of cucumbers.

[0134] Example 14: Phenotypic and physiological analysis of low-temperature response in wild-type plants and CmoCK1 gene-overexpressing plants, respectively, using rootstocks and wild-type scions.

[0135] Plants overexpressing the CmoCK1 gene and wild-type plants were grafted onto rootstocks and scions respectively, and simultaneously subjected to a 4℃ low-temperature treatment. Physiological phenotypes were observed and relevant physiological data were measured. Relevant phenotypes and physiological indicators are as follows: Figure 4 As shown, Figure 4 A represents the phenotypes of two-leaf wild-type cucumber seedlings grafted onto transgenic lines (OE#2 and #4) and wild-type cucumber rootstocks before and after 12 hours of cold treatment. The arrows indicate the grafting junctions. After low-temperature treatment, compared with the grafted WT / WT, the leaves of WT / OE#2 and WT / OE#4 cucumbers showed lower wilting. Figure 4 B represents the RT-PCR identification results of CmoCK1 in grafted WT / WT, WT / OE#2, and WT / OE#4 before and after 12 hours of cold treatment; R: rootstock, S: wild-type cucumber scion, H2O: negative control; asterisks indicate the transported mRNA of CmoCK1-GFP; each replicate includes 9 grafted plants, and the results show that CmoCK1-GFP mRNA can be transported from the rootstock to the scion. Figure 4 C represents Western blot analysis of protein levels in rootstocks and scions grafted from wild-type plants onto OE#2 / #4 rootstocks before and after 12 hours of cold treatment; the fused CmoCK1-GFP protein (approximately 70 kDa) was detected using a CmoCK1 monoclonal antibody; Coomassie brilliant blue staining was used as a control; each replicate included 9 independent grafted plants. R: rootstock, S: scion. The results showed that specific bands were detectable around 70 kDa in WT / OE#2 and WT / OE#4 cucumber scions. Figure 4 D represents the relative electrolyte permeability (REP) and MDA content of wild-type cucumber rootstock grafted onto OE#2 / #4 and wild-type cucumber rootstocks under 12-hour cold treatment. Each treatment included 6 biological replicates, with 3-4 plants per replicate (mean ± standard deviation, two-way ANOVA followed by t-test, *p<0.05, **p<0.01, ***p<0.001). Under cold treatment, the relative electrolyte permeability (REP) and MDA content of WT / OE#2 and WT / OE#4 were significantly lower than those of WT / WT. Figure 4E represents the relative expression levels of CsaCBF1 and CsaCOR on leaves grafted from wild-type cucumber rootstocks to OE#2 / #4 and wild-type cucumbers before and after 12 hours of cold treatment. Each condition included four replicates, with each replicate containing three plants (mean ± standard deviation, one-way ANOVA followed by t-test, *p<0.05, **p<0.01, ***p<0.001). Cucumber ACTIN7 was used as an internal control. Under low-temperature treatment, the relative expression levels of CsaCBF1 and CsaCOR genes in leaves grafted from wild-type cucumbers to OE#2 / #4 were significantly enhanced compared to wild-type and wild-type grafts. The PC / PE ratio of two-leaf-aged transgenic cucumbers overexpressing CmoCK1 and grafted from wild-type cucumbers was also observed before and after 12 hours of cold treatment. Each condition included three replicates, with each replicate containing 9 plants (mean ± standard deviation, one-way ANOVA, t-test, *p<0.05, **p<0.01, ***p<0.001). Under low temperature treatment, the PC / PE ratio of the leaves of grafted OE#2 and wild-type cucumbers was significantly higher than that of wild-type and wild-type grafted cucumbers.

[0136] The above low-temperature treatment results show that the relative conductivity and malondialdehyde content of the two overexpression lines as rootstocks are significantly lower than those of the wild-type plants, and the CmoCK1 gene overexpression lines as rootstocks are more cold-resistant.

[0137] Example 15: NBT staining

[0138] (1) Dissolve 0.1g NBT in 50ml of 50mM phosphate solution to prepare 0.2% NBT staining solution, and wrap the centrifuge tube with aluminum foil;

[0139] (2) Take the first true leaf of the cucumber plant, soak the plant material in the centrifuge tube with the prepared NBT staining solution in the dark, vacuum for half an hour, and place it in the dark for 4 hours after vacuuming.

[0140] (3) After 4 hours, discard the NBT staining solution, soak in anhydrous ethanol and boil in a water bath for 10 minutes, shaking several times during the process;

[0141] (4) Carefully remove the plant material and take photos for observation.

[0142] Example 16: Detection of relative conductivity

[0143] (1) Use a punch to take the first true leaf of the cucumber plant. Ten leaves are taken as a biological replicate (avoid the main vein when taking leaf rounds). Set up more than three biological replicates for each experimental group and place them in centrifuge tubes containing 20ml of deionized water.

[0144] (2) The conductivity of deionized water without blades was measured and recorded as S0. The centrifuge tube with blades was shaken on a horizontal shaker for 2 hours, and the conductivity of the solution was measured and recorded as S1.

[0145] (3) Heat the centrifuge tube containing the blades in a boiling water bath for 15 minutes, cool it to room temperature, and measure the conductivity of the solution, denoted as S2. Calculate REL according to the following formula:

[0146]

[0147] Example 17: Detection of Malondialdehyde (MDA) Content

[0148] (1) Take 0.5g of the first true leaf of cucumber plant into a mortar, add quartz sand and 2mL of 10% TCA solution, grind into a homogenate, add 8mL of 10% TCA to make a 10mL system, centrifuge the homogenate at 4000rpm for 10min and take the supernatant.

[0149] (2) Transfer 2 mL of supernatant to a new 10 mL centrifuge tube. Add 2 mL of distilled water to the control group, and then add 2 mL of 0.6% TBA to each tube. Boil in a water bath for 15 min, cool rapidly, and centrifuge at 4000 rpm for 10 min.

[0150] (3) Take 200 μL of the supernatant and measure the absorbance at wavelengths of 450 nm, 532 nm, and 600 nm. Calculate the MDA content (μmol / g) using the following formula:

[0151]

[0152] In the above formula, V is the total volume of the reaction system, Vs is the amount of extract during the determination, S is the total amount of extract, and W is the mass of the material.

[0153] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0154] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. CmoCK1 The application of genes in cucumber cold resistance is characterized by, The CmoCK1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; By increasing in cucumber plants CmoCK1 Increasing gene expression levels enhances the cucumber plant's resistance to low temperatures.

2. CmoCK1 The application of genes in creating low-temperature resistant rootstocks for cucumbers is characterized by, The CmoCK1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; By increasing in cucumber plants CmoCK1 Increasing gene expression levels enhances the cucumber plant's resistance to low temperatures.

3. The application according to claim 1 or 2, characterized in that, The CmoCK1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

4. A method for breeding low-temperature resistant cucumbers, characterized in that, By increasing the concentration in cucumber plants CmoCK1 The expression level of genes improves the low-temperature tolerance of cucumber plants; CmoCK1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The method for breeding low-temperature resistant cucumbers according to claim 4, characterized in that, Includes the following steps: 1) Amplification CmoCK1 Genes are amplified to obtain amplification products; 2) The restriction sites were determined to be XbaI and KpnI. The amplification product was ligated into the vector pCambia1305-35S:C-GUS-NOSter-35S:C-GFP-NOSter to obtain the overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter. After confirming that the sequencing was correct, the plasmid was extracted. 3) The overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter was transformed into Agrobacterium GV3101 competent cells, and the genetic transformation of cucumber was carried out by infecting cucumber cotyledons. 4) By amplifying the GFP sequence on the overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter and by analyzing the plants... CmoCK1 qRT-PCR detection obtained CmoCK1 Overexpression of genes in strains.

6. A method for creating a low-temperature resistant rootstock for cucumber, characterized in that, Overexpression was obtained through genetic transformation. CmoCK1 Cucumbers with overexpressed genes will CmoCK1 By grafting onto rootstocks with genetically modified genes, cold-resistant rootstocks can be obtained. The CmoCK1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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