Application of CmoCK1 gene in creating cucumber low-temperature-resistant rootstock

Abstract

The invention relates to the technical field of plant molecular breeding and low-temperature stress cultivation, in particular to application of a CmoCK1 gene in creating a low-temperature-resistant cucumber stock. According to the scheme, through low-temperature treatment of the overexpressed CmoCK1 gene and the wild cucumber, the low-temperature resistance of the obtained CmoCK1 gene overexpressed cucumber is remarkably improved, the wild plant and the overexpressed plant are used as rootstocks to be grafted with the wild scion, and it is found that the low-temperature resistance of the scion cucumber can be improved by using the overexpressed CmoCK1 gene cucumber as the rootstock, and the low-temperature resistance of the scion cucumber can be improved by using the overexpressed CmoCK1 gene. The new function of the CmoCK1 gene has good application potential, provides gene resources and new ideas for reducing low-temperature stress and delaying low-temperature response of cucumbers in production and further cultivating special low-temperature-resistant rootstocks for cucumbers, and has a wide application prospect in the field of low-temperature-resistant cucumber rootstocks and application of the CmoCK1 gene in low-temperature-resistant cucumber rootstocks and low-temperature-resistant cucumber rootstocks and low-temperature-resistant cucumber rootstocks and low-temperature-resistant cucumber rootstocks. And the method has a wide application prospect and a relatively high use value.

Description

Technical Field

[0001] The invention relates to the field of plant molecular low temperature stress, and in particular to application of CmoCK1 gene in creating low temperature resistant cucumber rootstock. Background Art

[0002] Cucumber (Cucumis sativus L) is an important greenhouse fruit and vegetable crop in my country, ranking first in the world in terms of planting area and yield. Cucumber is a typical thermophilic vegetable with poor tolerance to low temperatures. It is easily subjected to low temperature stress (<10℃) during the early stages of winter seedling raising and transplanting, resulting in physiological disorders and melon rot, and a significant reduction in fruit yield and quality. In severe cases, the yield can be reduced by more than 30%, with a reduction of about 100,000 yuan per hectare, causing serious economic losses. Grafting, as an effective means to improve low temperature tolerance, has become one of the supporting measures for winter and spring cultivation of cucumbers in solar greenhouses. Pumpkin rootstocks can not only maintain the excellent traits of cucumber cultivars, but also improve disease resistance and stress resistance, especially low temperature tolerance. However, cucumbers regulated by different rootstocks have different advantages. For example, black seed pumpkin has strong stress resistance but the quality of grafted cucumbers is poor, while white seed pumpkin grafted cucumbers have good quality but unstable stress resistance. In production, there is an urgent need for some rootstocks with multiple traits (maintaining quality, improving resistance, and easy reproduction) that can not only improve low temperature tolerance but also maintain the original fruit quality of cucumbers. Therefore, pumpkin rootstock improvement and the mechanism of rootstock-scion interaction have always been a research hotspot in the field of plant grafting.

[0003] In the early stage, the research team used omics and molecular biology to verify and discover the key gene of pumpkin cold tolerance, choline kinase CmoCK1. Analysis found that its mRNA can be transported to cucumber scions over long distances through pumpkin rootstocks and improve the cold tolerance of grafted cucumbers. However, it is still unclear whether it is possible to create a cucumber-specific cold-resistant rootstock by overexpressing CmoCK1 transgenes and improve the cold tolerance of cucumber varieties after grafting. Summary of the invention

[0004] In view of the shortage of rootstock materials with multiple traits (maintaining quality and improving low temperature resistance and easy reproduction) in cucumber cultivation and production in the existing market, the purpose of the present invention is to provide a method for creating low-temperature-resistant cucumber rootstock by overexpressing the CmoCK1 gene, thereby improving the resistance of cucumber varieties to low temperature stress.

[0005] The present invention constructs a set of methods for genetic and stable genetic transformation of hairy roots of vegetable crops cucumber, obtains transgenic cucumbers overexpressing the CmoCK1 gene, conducts physiological and gene expression analysis of their response to low temperature, and finally grafts the overexpressed cucumbers with the wild type to explore the effect of overexpressing the CmoCK1 gene on the cold resistance of cucumbers.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] Application of CmoCK1 gene in low temperature tolerance of cucumber,

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

[0009] Application of CmoCK1 gene in creating cucumber rootstocks resistant to low temperatures.

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

[0011] Preferably,

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

[0013] Preferably,

[0014] By increasing the expression level of the CmoCK1 gene in cucumber plants, the low temperature resistance of cucumber plants is improved.

[0015] A primer pair for amplifying the CmoCK1 gene, the nucleotide sequence of the primer pair is shown in SEQ ID NO.9-10.

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

[0017] By increasing the expression level of the CmoCK1 gene in the cucumber plant, the low temperature resistance of the cucumber plant is improved; the nucleotide sequence of the CmoCK1 gene is shown in SEQ ID NO.1.

[0018] Preferably, the method comprises the following steps:

[0019] 1) Amplifying the CmoCK1 gene to obtain an amplified product;

[0020] 2) Determine the restriction sites as XbaI and KpnI, connect the amplified product to 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, and extract the plasmid after sequencing is correct;

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

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

[0023] A method for creating a low-temperature resistant cucumber rootstock,

[0024] Cucumbers that overexpress the CmoCK1 gene are obtained through genetic transformation, and the rootstocks that overexpress the CmoCK1 gene are used for grafting to obtain low-temperature resistant rootstocks;

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

[0026] The beneficial effects of the present invention are:

[0027] The present invention has the innovation of analyzing the molecular mechanism of pumpkin rootstock regulating the low temperature tolerance of cucumber, supplements and improves the mechanism of grafting to improve cold resistance, and provides new ideas for the creation of cold-resistant rootstock varieties of cucumber in the future. It is a set of scientific research methods that are both scientific, innovative and practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0029] Figure 1 This is a diagram showing the results of an identification experiment on the transport of CmoCK1 from cucumber hairy roots to new leaves.

[0030] Figure 2 A diagram showing the experimental results of improving the low temperature tolerance of cucumber by transporting CmoCK1 upward.

[0031] Figure 3 The experimental results of overexpressing the CmoCK1 gene to improve the low temperature tolerance of cucumber.

[0032] Figure 4 The experimental results of improving the cold tolerance of cucumber by the stock-scion transport of CmoCK1 mRNA. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific embodiments.

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

[0035] (1) Cucumber cultivation: The “Xintai Mici” cucumber was cultured in a constant temperature incubator until it reached the stage of two leaves and one heart. The light intensity of the incubator was 600 μmol / m -2 s -1 , photoperiod: 16h light / 8h dark, temperature: 26℃ day / 18℃ night.

[0036] (2) Self-grafting of cucumbers: Flatten the cotyledons of cucumbers and graft them. Use a blade to cut the hypocotyl obliquely 1 cm below the growth point of the scion, and cut it obliquely at the growth point of the rootstock, leaving one cotyledon. Fit the cuts of the rootstock and scion together and fix them with a grafting clamp to produce a self-grafted cucumber. Spray water gently during the grafting process to keep the scion moist. Cover the plug tray with a transparent cover and wrap the edges with plastic wrap to keep the grafted seedlings moist. Transfer the plug tray to low-light growth conditions, and gradually increase the light and reduce the humidity after 4 days.

[0037] (3) Cucumber low temperature treatment method: Place the two-leaf, one-heart cucumber materials with the same growth for low temperature treatment in a light incubator cooled to 4°C. Test or sample the plant materials at the set treatment time. Set at least 3 technical replicates for each test group.

[0038] Example 2: RNA extraction from grafted cucumber

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

[0040] TRizol method for RNA extraction:

[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) Precool the centrifuge at 4°C, centrifuge at 12,000 rpm for 5 min, 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°C and 12,000 rpm for 15 min and take 300 μL of the supernatant into a new 1.5 mL centrifuge tube;

[0045] (5) Add 300 μL of isopropanol to the supernatant, mix thoroughly by inversion, and let stand at -20°C for 20 min;

[0046] (6) Centrifuge at 12,000 rpm for 15 min at 4°C and discard the supernatant;

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

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

[0049] (9) Add 40-60 μL RNA-free water to the centrifuge tube to dissolve the RNA precipitate, measure the RNA concentration using NanoDrop, and store in a -80°C refrigerator. RNA from the grafted cucumber was obtained.

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

[0051] Novozymes IIQ RT SuperMix for qPCR (+gDNA wiper) (R223-01) kit steps.

[0052] Genomic DNA Removal:

[0053] Prepare the reaction solution in an RNase-free centrifuge tube as follows:

[0054]

[0055] Mix by gently pipetting at 42°C for 2 min.

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

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

[0058]

[0059] Mix well by pipetting at 50°C for 15 min, and reverse transcribe the RNA from the grafted cucumber into cDNA at 85°C for 5 s.

[0060] Example 4: Expression detection of CmoCK1 gene and CBFs-COR cold pathway genes in CmoCK1 gene overexpressed cucumbers at room temperature (26°C) and cold treatment (4°C)

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

[0062] 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, which are composed of 8 exons. The first exon is from bases 1 to 110, the second exon is from bases 519 to 1199, the third exon is from bases 2122 to 2243, the fourth exon is from bases 2329 to 2524, the fifth exon is from bases 2724 to 2841, the sixth exon is from bases 2930 to 2977, the seventh exon is from bases 3288 to 3364, the eighth exon is from bases 3435 to 3581, and the rest are intron sequences.

[0064] The qRT-PCR primer sequences designed according to 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 sequences:

[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 sequences:

[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 the grafted cucumber was diluted 5 times to prepare a diluted cDNA sample, and the qRT-PCR reaction system was as follows according to the steps of the Novozymes Bioluminescence Quantification Kit (Q711-02):

[0075]

[0076] After thorough mixing, place in QuantStudio TM 6Flex real-time PCR instrument for PCR amplification. The qPCR procedure is as follows:

[0077]

[0078] The measured CT values ​​were converted to relative copy numbers using the ΔΔCT method and compared with the copy number of the internal reference. Figure 2 and Figure 3 As shown, CmoCK1-GFP was overexpressed in cucumber roots or introduced into cucumber by stable transformation. The results showed that after 12 hours of cold treatment, the levels of CmoCK1, CsaCBF1 and CsaCOR genes were significantly higher than the control, proving that the CmoCK1 gene responded to 4°C low temperature stress.

[0079] Experimental Example 5: Cucumber transportability identification method

[0080] Use the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), set the species to Cucurbitaceae, enter the CDS sequence of CmoCK1 in the PCR Template option, and select the target product size between 300-500bp. Screen the primers specific to 'Xintai Mi Ci' based on the design results of the website. RT-PCR verifies the specificity of the primers to ensure that the specific sequence of the CmoCK1 gene can be successfully amplified in the aboveground and underground parts.

[0081] Experimental Example 6: CmoCK1 protein extraction and Western blot technology

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

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

[0084] Cloning primers were designed according to 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 DNA Polymerase;

[0089]

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

[0091]

[0092] PCR product detection: Prepare 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 ultraviolet light, and cut the gel for recovery of the target band.

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

[0094] The restriction sites were XbaI and BamHI, and the amplified product in Example 7 was then connected to the vector pCambia1305-35S:C-GUS-NOSter-35S:C-GFP-NOSter. After correct sequencing, the plasmid was extracted to obtain the overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter.

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

[0096]

[0097] Example 9: Hairy Root Genetic Transformation

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

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

[0100] Cucumber infection: scrape the Agrobacterium rhizogenes plate that was coated one day in advance, wash it with infection solution and readjust the OD600 to between 0.15-0.20. After the adjusted concentration of the bacterial solution is placed at 28°C for 1-3 hours, put the obtained explants in and rotate at 80-120rpm for 20 minutes.

[0101] Co-cultivation: After infection, remove excess bacterial liquid from the explants and transfer them to co-cultivation medium covered with sterile filter paper. Co-cultivate at 25℃ in the dark for 3 days.

[0102] Root induction: After the co-cultivation is completed, one end of the hypocotyl of the explant is inserted into the rooting medium and placed in a light incubator to induce the production of hairy roots.

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

[0104] Example 10: Identification of positive seedlings of hairy root genetic transformation by overexpression of CmoCK1 gene

[0105] Hairy roots were transformed with Rhizobium rhizogenes strain K599 containing an empty vector (pCambia1300-35S::GFP) or pCambia1300-35S::CmoCK1-GFP. K599 alone was transformed as a control. 14 days after transformation, RT-PCR was performed using primers for CmoCK1-GFP or GFP to verify the transport of CmoCK1-GFP or GFP.

[0106] Observe GFP under a fluorescent flashlight and select plants with fluorescent roots, such as Figure 1 As shown in A, under a fluorescence microscope, the root tips of wild cucumber hairy roots emit weak fluorescence, while GFP and CmoCK1-GFP transgenic cucumber hairy roots 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 the ImageJ GFP channel. At least 10 replicates independent of transformation were analyzed. Results are shown in Figure 1 As shown in B, fluorescence signals were observed in the roots and leaves of CmoCK1-GFP transgenic hairy root cucumbers. Strong fluorescence signals were observed in the roots of GFP transgenic hairy root cucumbers, but there was basically no fluorescence signal in the aboveground parts.

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

[0109] DNA from cucumber leaves was extracted using the CTAB method, and 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 system, centrifuge and perform PCR. The PCR procedure is as follows:

[0115]

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

[0117] like Figure 1 D shows the results of RT-PCR validation of CmoCK1-GUS or GUS transport using primers fused to CmoCK1-GUS or GUS. No. 1 and No. 2 represent independent transgenic seedlings as biological replicates. Transport rates were analyzed as the number of mobile seedlings / total number of transgenic seedlings. CsaACTIN7 was used as an internal reference for mRNA abundance. HygR was used to exclude Agrobacterium tumefaciens contamination or mobility. The results showed that CmoCK1-GUS mRNA can be transported from roots to the aerial parts.

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

[0119] The hairy root overexpressing plants and wild-type plants of CmoCK1 were simultaneously subjected to 4°C low temperature treatment for 12 h, and their physiological phenotypes were observed and related physiological data were measured. Figure 2 As shown, Figure 2 A is the phenotype of cucumber seedlings (control) with overexpression of 35S::CmoCK1-GFP and 35S::GFP in roots before and after low temperature treatment for 12 hours. After low temperature treatment, the leaves of GFP transgenic hairy root cucumbers showed obvious wilting and curling phenotypes, and some leaves of CmoCK1-GFP transgenic hairy root cucumbers drooped slightly. Figure 2 B is NBT (nitro blue tetrazolium) staining of cucumber leaves. Compared with the leaves of GFP transgenic hairy root cucumbers, the NBT staining area of ​​the leaves of CmoCK1-GFP transgenic hairy root cucumbers was smaller and the staining degree was significantly reduced after low temperature treatment. Figure 2 C and 2D are relative electrolyte permeability (REP) and MDA content; each treatment included 6-12 biological replicates, and each replicate had 3-4 plants (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 is the relative expression of CsaCBF1 and CsaCOR in the leaves of 35S::CmoCK1-GFP and 35S::GFP cucumber seedlings transformed with roots before and after low temperature treatment for 12 hours. Compared with 0 hours of low temperature treatment for 35S::GFP cucumbers transformed with roots, *p<0.05, **p<0.01, ***p<0.001, cucumber ACTIN7 was used as an internal reference. Under low temperature treatment, the relative expression of CsaCBF1 and CsaCOR genes in 35S::CmoCK1-GFP transformed with roots increased significantly.

[0120] The results of low temperature treatment showed that the rooting transformation overexpression plants of the CmoCK1 gene were more cold-resistant, and the degree of NBT staining, relative conductivity and malondialdehyde content (the higher the degree of NBT staining, the higher the relative conductivity and malondialdehyde content, the higher the degree of damage to the cell membrane, the more severe the cold stress, indicating that the plant's cold resistance is weaker) were significantly lower than those of the control plants, and the expression level of the CsaCOR gene was significantly increased. The above indicates that the rooting transformation overexpression of the CmoCK1 gene enhances the cold resistance of cucumber.

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

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

[0123] The plasmid with CmoCK1 gene was transferred into Agrobacterium GV3101 competent cells by chemical transformation, 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, cool them at room temperature and soak them for several hours, peel off the seed coat with tweezers, shake them in 70% alcohol for 30 seconds, shake them in 3% sodium hypochlorite (NaClO) for 8 minutes, sow them in MS medium, about 20 seeds per bottle, and culture them in the dark for 36 hours.

[0125] Explant acquisition: Use tweezers to remove the previously prepared radicle of the germinated cucumber seeds in a clean bench, taking care not to damage the surrounding tissues. Then cut off one-third of the cotyledon away from the radicle, remove the endocarp, and completely separate the two cotyledons and place them in MS liquid culture medium for later use.

[0126] Cucumber infection: scrape the Agrobacterium tumefaciens plate that was coated one day in advance, wash it with infection solution and readjust the OD600 to between 0.2-0.3. After the adjusted concentration of the bacterial solution is left to stand at 28℃ for 1-3h, use a syringe to vacuum and infect twice, each time for 1.5 minutes.

[0127] Co-cultivation: After infection, remove excess bacterial liquid from the explants and transfer them to co-cultivation medium covered with sterile filter paper. Co-cultivate at 25℃ in the dark for 4 days.

[0128] Shoot induction: The explants were transferred to budding medium and cultured for three weeks.

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

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

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

[0132] The CmoCK1 gene overexpression plants and wild-type plants were simultaneously subjected to 4°C low temperature treatment for 12 h, and their physiological phenotypes were observed and related physiological data were measured. Figure 3 As shown, Figure 3 A is the phenotype of two-leaf-age cucumber seedlings overexpressing 35S::CmoCK1-GFP transgenic lines (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 the wild-type, the degree of wilting of cucumber seedlings overexpressing the CmoCK1 gene was lower; Figure 3 B is relative electrolyte permeability (REP) and MDA content; each treatment included 4 biological replicates, 3-4 plants in each 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 overexpressing 35S::CmoCK1-GFP transgenic lines (OE#1, #2 and #4) were significantly lower than those of the wild-type cucumber seedlings; Figure 3 C is the relative expression levels of CmoCK1, CsaCBF1 and CsaCOR in the leaves of transgenic cucumber seedlings overexpressing 35S::CmoCK1-GFP (OE#2 and #4) before and after 12 hours of low temperature treatment. Compared with the wild type at 0 hours of low temperature treatment, *p<0.05, **p<0.01, ***p<0.001. Cucumber ACTIN7 was used as an internal reference. Under low temperature treatment, the expression level of CmoCK1 gene in transgenic lines overexpressing 35S::CmoCK1-GFP (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, and the relative conductivity and malondialdehyde content of the three overexpression strains were significantly lower than those of the wild-type plants. The above indicated that overexpression of the CmoCK1 gene enhanced the cold resistance of cucumber.

[0134] Example 14: Phenotypic and physiological analysis of response to low temperature in wild-type plants and plants overexpressing CmoCK1 as rootstocks and wild-type rootstock-scion grafts

[0135] The CmoCK1 gene overexpression plants and wild-type plants were used as rootstocks and wild-type rootstocks and scions were grafted, respectively, and subjected to 4°C low temperature treatment to observe their physiological phenotypes and measure relevant physiological data. Figure 4 As shown, Figure 4 A shows the phenotype of two-leaf wild-type cucumber seedlings grafted onto 35S::CmoCK1-GFP overexpressing transgenic lines (OE#2 and #4) and wild-type cucumber rootstocks before and after 12 hours of cold treatment. The arrows indicate the connected parts after grafting. After low temperature treatment, the degree of leaf wilting of WT / OE#2 and WT / OE#4 cucumbers was lower than that of grafted WT / WT. Figure 4 B is 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: cucumber wild-type scion, H2O: negative control; asterisks indicate the transport mRNA of CmoCK1-GFP; each replicate included 9 grafted plants, and the results showed that CmoCK1-GFP mRNA could be transported from the rootstock to the scion. Figure 4 C is the result of Western blot analysis of protein levels in rootstocks and scions grafted onto OE#2 / #4 rootstocks by wild type before and after 12 hours of cold treatment; CmoCK1 monoclonal antibody was used to detect the fused CmoCK1-GFP protein (about 70KDa); Coomassie brilliant blue staining was used as a control; each replicate included 9 independent grafted plants. R: rootstock, S: scion, the results showed that a specific band could be detected near 70kDa in WT / OE#2 and WT / OE#4 cucumber scions. Figure 4 D is the relative electrolyte permeability (REP) and MDA content of wild type grafted onto OE#2 / #4 and wild type cucumber rootstocks under 12 hours of cold treatment. Each treatment included 6 biological replicates, with 3-4 plants in each 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 WT / OE#2 and WT / OE#4 were significantly reduced compared with WT / WT; Figure 4E is the relative expression of CsaCBF1 and CsaCOR in leaves of wild-type grafted cucumber rootstocks OE#2 / #4 and wild-type before and after 12 hours of cold treatment. Each condition included 4 replicates, and each replicate contained 3 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 reference. Under low temperature treatment, the relative expression of CsaCBF1 and CsaCOR genes in leaves of wild-type grafted cucumbers OE#2 / #4 was significantly enhanced compared with wild-type and wild-type grafted cucumbers; PC / PE ratios of OE#2 and wild-type cucumbers overexpressing CmoCK1 at two leaf ages before and after 12 hours of cold treatment. Each condition included three replicates, and each replicate contained 9 plants (mean ± SD, one-way ANOVA, t-test, *p<0.05, **p<0.01, ***p<0.001). Under low temperature treatment, the PC / PE ratio of leaves of OE#2 and wild-type cucumber grafted plants was significantly higher than that of wild-type and wild-type grafted plants.

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

[0137] Example 15: NBT staining

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

[0139] (2) Take the first true leaf of the cucumber plant, immerse the plant material in the prepared NBT staining solution in a centrifuge tube away from light, evacuate for half an hour, and place it in a dark place away from light for 4 hours after evacuation;

[0140] (3) After 4 hours, pour off the NBT dye solution, soak in anhydrous ethanol and place in a boiling 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 cork punch to take the first true leaf of the cucumber plant. Ten leaves are used as a biological replicate (avoid the main vein when taking leaf discs). Each experimental group has three or more biological replicates and is placed in a centrifuge tube containing 20 ml of deionized water.

[0144] (2) Measure the conductivity of deionized water without blades, recorded as S0, and shake the centrifuge tube with blades on a horizontal shaker for 2 h, and measure the conductivity of the solution, recorded as S1;

[0145] (3) Place the centrifuge tube containing the blade in a boiling water bath for 15 min. After cooling to room temperature, measure the conductivity of the solution, recorded as S2, and calculate REL according to the following formula:

[0146]

[0147] Example 17: Malondialdehyde (MDA) content detection

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

[0149] (2) Transfer 2 mL of the supernatant to a new 10 mL centrifuge tube, add 2 mL of distilled water to the control group, then add 2 mL of 0.6% TBA to each tube, boil in a boiling 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 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 measurement, 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 belong to the prior art known to professional and technical personnel in this field.

[0154] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of CmoCK1 gene in low temperature tolerance of cucumber, characterized in that: The nucleotide sequence of the CmoCK1 gene is shown in SEQ ID NO.

1.

2. Application of CmoCK1 gene in creating cucumber cold-resistant rootstock, characterized in that: The nucleotide sequence of the CmoCK1 gene is shown in SEQ ID NO.

1.

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

2.

4. The use according to claim 1 or 2, characterized in that By increasing the expression level of the CmoCK1 gene in cucumber plants, the low temperature resistance of cucumber plants is improved.

5. A primer pair for amplifying the CmoCK1 gene as claimed in claim 1 or 2, characterized in that: The nucleotide sequences of the primer pairs are shown in SEQ ID NOs. 9-10.

6. A method for breeding low-temperature resistant cucumbers, characterized in that: By increasing the expression level of the CmoCK1 gene in the cucumber plant, the low temperature resistance of the cucumber plant is improved; the nucleotide sequence of the CmoCK1 gene is shown in SEQ ID NO.

1.

7. The breeding method of low temperature resistant cucumber according to claim 6, characterized in that: The steps include: 1) Amplifying the CmoCK1 gene to obtain an amplified product; 2) Determine the restriction sites as XbaI and KpnI, connect the amplified product to 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, and extract the plasmid after sequencing is correct; 3) The overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter was transferred into the competent cells of Agrobacterium GV3101, and the genetic transformation of cucumber was performed by infecting cucumber cotyledons; 4) The CmoCK1 gene overexpression strain was obtained by amplifying the GFP sequence on the overexpression vector pCambia1305-35S::CmoCK1-GUS-NOSter-35S:C-GFP-NOSter and detecting the CmoCK1 in the plant by qRT-PCR.

8. A method for creating a low-temperature resistant cucumber rootstock, characterized in that: Cucumbers that overexpress the CmoCK1 gene are obtained through genetic transformation, and the rootstocks that overexpress the CmoCK1 gene are grafted to obtain low-temperature-resistant rootstocks; The nucleotide sequence of the CmoCK1 gene is shown in SEQ ID NO.1.

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