Application of Pumpkin CmoKARI1 Gene in Promoting Ile Biosynthesis to Enhance Cold Tolerance of Cucumber

Abstract

The present invention relates to the application of the pumpkin CmoKARI1 gene in promoting Ile biosynthesis to enhance the cold tolerance of cucumbers. By overexpressing the CmoKARI1 gene in cucumber plants, the present invention significantly improves the low-temperature tolerance of cucumbers; exogenous spraying of isoleucine significantly further improves the low-temperature tolerance of cucumbers. The new function of the CmoKARI1 gene described in the present invention has good application potential, provides a new method for reducing low-temperature stress and delaying the low-temperature response of cucumbers in production, and has broad application prospects and high application value.

Description

Technical Field

[0001] The invention relates to the field of plant molecular low temperature stress, in particular to an application of a pumpkin CmoKARI1 gene to promote Ile biosynthesis and enhance the cold resistance of cucumber. Background Art

[0002] Cucumber (Cucumis sativus L.), a major vegetable cultivated in greenhouses, is a thermophilic vegetable. However, during winter and spring in northern China, it often experiences low-temperature stress, resulting in significant reductions in yield and quality. To improve cucumber's cold tolerance, grafting onto pumpkin rootstock has become a widely adopted cultivation method during cold weather. However, different rootstocks have varying degrees of cold tolerance for the scion, and different rootstocks produce different cucumber advantages. Therefore, methods are urgently needed to enhance cucumber's cold tolerance while maintaining its original fruit quality.

[0003] Using omics and molecular biology methods, the research team identified the key gene for pumpkin cold tolerance, ketoacid reductoisomerase CmoKARI1. Analysis revealed that its mRNA can be transported long distances through the pumpkin rootstock to the cucumber scion, improving the grafted cucumber's cold tolerance. KARI1 encodes an enzyme involved in the synthesis of branched-chain amino acids (BCAAs). Studies have shown that mRNA movement under cold stress is associated with fatty acid and amino acid metabolism. However, whether overexpressing the CmoKARI1 gene and exogenously spraying fatty acids and amino acids can enhance cucumber cold tolerance remains unknown. Summary of the Invention

[0004] In response to the shortcomings of the prior art, the present invention aims to provide an application of the pumpkin CmoKARI1 gene to promote Ile biosynthesis and enhance the cold tolerance of cucumbers. The present invention constructs a set of methods for the genetic and stable genetic transformation of hairy roots in the vegetable crop cucumber, obtaining transgenic cucumbers that overexpress the CmoKARI1 gene. Experimental results show that overexpressing the CmoKARI1 gene can effectively improve the low-temperature tolerance of cucumbers. The present invention determines the amino acid that plays a major role in low-temperature tolerance by exogenously spraying different amino acids. Finally, the overexpressed cucumbers are combined with the exogenous amino acid spraying to explore the effects of overexpressing the CmoKARI1 gene and exogenous amino acid spraying on the cold resistance of cucumbers.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0006] The application of the pumpkin CmoKARI1 gene to promote Ile biosynthesis and enhance the cold resistance of cucumbers is characterized in that the application improves the low temperature resistance of cucumber plants by overexpressing the CmoKARI1 gene in cucumber plants;

[0007] The nucleotide sequence of the pumpkin CmoKARI1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the pumpkin CmoKARI1 gene is shown in SEQ ID NO.2.

[0008] The primer pair for amplifying the CmoKARI1 gene of claim 1 is characterized in that the nucleotide sequence of the primer pair is shown in SEQ ID NO.9-10.

[0009] A method for breeding low-temperature-resistant cucumbers, characterized by comprising the following steps:

[0010] Step 1, amplifying the CmoKARI1 gene of claim 1 to obtain an amplified product;

[0011] Step 2, confirming that the restriction sites are XbaI and KpnI, ligating the amplified product to the vector pCambia1300-Super:C-GFP to obtain the overexpression vector pCambia1300-Super::CmoKARI1-GFP-NOSter, and extracting the plasmid after sequencing is correct;

[0012] Step 3, the overexpression vector pCambia1300-Super::CmoKARI1-GFP-NOS ter was transferred into the competent cells of Agrobacterium GV3101, and the genetic transformation of cucumber was performed by infecting cucumber cotyledons;

[0013] Step 4: obtain a CmoKARI1 gene overexpression strain by amplifying the GFP sequence on the overexpression vector pCambia1300-Super::CmoKARI1-GFP-NOSter and detecting CmoKARI1 in the plant by qRT-PCR.

[0014] A method for improving the low temperature tolerance of cucumbers, characterized in that the method improves the low temperature tolerance of cucumbers by exogenously spraying Ile on the surface of cucumber plants overexpressing the CmoKARI1 gene described in claim 1.

[0015] On the basis of the above scheme, when Ile is exogenously sprayed, the concentration of Ile is 300 μM.

[0016] The application of the pumpkin CmoKARI1 gene of the present invention in promoting Ile biosynthesis and enhancing the cold resistance of cucumber has the following beneficial effects:

[0017] The present invention significantly improves cucumbers' cold tolerance by overexpressing the CmoKARI1 gene in cucumber plants. Exogenous spraying of isoleucine further significantly enhances the cucumbers' cold tolerance. The novel function of the CmoKARI1 gene described in this invention has great potential for application, providing a new method for reducing cold stress and delaying the cucumber's cold response in production, with broad application prospects and high value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention has the following accompanying drawings:

[0019] Figure 1 This is the result of the transport identification experiment of CmoKARI1 mRNA in transgenic hairy root cucumber.

[0020] Figure 2 This is the result of GUS staining experiment of CmoKARI1 transgenic hairy root cucumber.

[0021] Figure 3 This is the result of the GUS transport detection experiment of CmoKARI1 transgenic hairy root cucumber.

[0022] Figure 4 This is the result of GUS staining experiment of CmoKARI1 transgenic hairy root cucumber under low temperature.

[0023] Figure 5 This is the result of the GUS transport detection experiment of CmoKARI1 transgenic hairy root cucumber.

[0024] Figure 6 This is the result of the low temperature treatment phenotypic experiment of CmoKARI1-GUS and GUS transgenic hairy root cucumber plants.

[0025] Figure 7 This is the experimental result of analyzing the REP and MDA contents in CmoKARI1-GUS and GUS transgenic hairy root cucumbers.

[0026] Figure 8 This is the experimental result of gene expression analysis of CmoKARI1-GUS and GUS transgenic hairy root cucumber.

[0027] Figure 9 This is the result of the low temperature treatment phenotypic experiment of CmoKARI1-GFP and GFP transgenic hairy root cucumber plants.

[0028] Figure 10 This is the experimental result of analyzing REP and MDA content in CmoKARI1-GFP and GFP transgenic hairy root cucumber plants under low temperature treatment.

[0029] Figure 11This is the result of the low temperature treatment phenotype experiment on cucumber plants overexpressing the CmoKARI1 gene.

[0030] Figure 12 This is the experimental result of DAB staining analysis of cucumber plants overexpressing the CmoKARI1 gene under low temperature treatment.

[0031] Figure 13 This is the result of NBT staining analysis of cucumber plants overexpressing the CmoKARI1 gene under low temperature treatment.

[0032] Figure 14 This is the experimental result of DAB and NBT staining analysis of cucumber plants overexpressing the CmoKARI1 gene under low temperature treatment.

[0033] Figure 15 This is the experimental result of analyzing the MDA content and chlorophyll content of cucumber plants overexpressing the CmoKARI1 gene under low temperature treatment.

[0034] Figure 16 This is the experimental result of analyzing REP content in cucumber plants overexpressing the CmoKARI1 gene and treated with low temperature.

[0035] Figure 17 This is the experimental result of gene expression analysis in cucumber plants overexpressing the CmoKARI1 gene under low temperature treatment.

[0036] Figure 18 This figure shows the results of the low-temperature phenotypic experiment on cucumber plants sprayed with different concentrations of leucine, isoleucine and valine.

[0037] Figure 19 This is the experimental result of the effect of isoleucine on the low temperature tolerance of cucumber.

[0038] Figure 20 This is the experimental result of REP and MDA content analysis in cucumber plants sprayed with different concentrations of leucine, isoleucine and valine and treated with low temperature.

[0039] Figure 21 This is the experimental result of the effect of isoleucine on the phenotype of cucumber plants overexpressing the CmoKARI1 gene under low temperature treatment.

[0040] Figure 22 This is the experimental result of analyzing the effect of isoleucine on the REP and MDA content of cucumber plants overexpressing the CmoKARI1 gene under low temperature treatment.

[0041] Figure 23 This is the experimental result of analyzing the isoleucine content in cucumber plants overexpressing the CmoKARI1 gene and treated with low temperature.

[0042] Figure 24This is the experimental result of gene expression analysis of isoleucine in cucumber plants overexpressing CmoKARI1 gene under low temperature treatment. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below with reference to the accompanying drawings. It should be understood that the following embodiments are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0044] Example 1: Cucumber cultivation and low temperature treatment method

[0045] (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℃ during the day / 18℃ at night.

[0046] (2) 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. The plant materials are measured or sampled at the set treatment time. Each test group is set with at least 3 technical replicates.

[0047] Example 2: Cucumber RNA Extraction

[0048] Take an appropriate amount of cucumber in a 2 mL enzyme-free centrifuge tube, add flame-sterilized steel balls, and grind into powder using a plant grinder;

[0049] RNA extraction by TRizol method:

[0050] (1) Add 1 mL of TRizol extract to the sample tube, vortex to mix, and let stand at room temperature for 5 min;

[0051] (2) Precool the centrifuge at 4°C, centrifuge at 12,000 rpm for 5 min, and add 800 μL of the supernatant to a 1.5 mL centrifuge tube containing RNase-free enzyme.

[0052] (3) Add 160 μL of chloroform to a 1.5 mL centrifuge tube, mix thoroughly by inversion, and let stand at room temperature for 5 min;

[0053] (4) Centrifuge at 4°C and 12,000 rpm for 15 min, and collect 300 μL of the supernatant into a new 1.5 mL centrifuge tube;

[0054] (5) Add 300 μL of isopropanol to the supernatant, mix thoroughly by inversion, and let stand at -20°C for 20 min;

[0055] (6) Centrifuge at 12,000 rpm for 15 min at 4°C and discard the supernatant;

[0056] (7) Wash the RNA pellet with 1 mL of pre-chilled 75% ethanol, centrifuge at 12,000 rpm at 4°C for 5 min, and discard the supernatant.

[0057] (8) Open the centrifuge tube and dry it at room temperature for about 5 minutes to evaporate the remaining ethanol;

[0058] (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 in a -80°C refrigerator. RNA was obtained from the grafted cucumber.

[0059] Example 3: Reverse transcription of cucumber RNA into cDNA

[0060] Novozymes IIQ RT SuperMix for qPCR (+gDNA wiper) (R223-01) kit steps were used.

[0061] Genomic DNA Removal:

[0062] Prepare the reaction solution in an RNase-free centrifuge tube as follows:

[0063]

[0064]

[0065] Mix by gently pipetting at 42°C for 2 min.

[0066] The reverse transcription reaction system was prepared as follows:

[0067] Add 5×HiScriptⅡqRT SuperMixⅡ directly to the reaction tube in the previous step;

[0068]

[0069] The mixture was mixed by pipetting at 50°C for 15 min, and the RNA from the grafted cucumber was reverse transcribed into cDNA at 85°C for 5 s.

[0070] Example 4: Expression detection of CmoKARI1 gene and CBFs-COR cold pathway genes in cucumber overexpressing CmoKARI1 gene at room temperature (26°C) and cold treatment (4°C) The nucleotide sequence of the CmoKARI1 gene in cucumber is shown in SEQ ID NO.1, and its encoded protein sequence is shown in SEQ ID NO.2.

[0071] 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.

[0072] The CmoKARI1 gene in cucumber consists of 3959 bases and 10 exons. The first exon is from base 1 to base 397, the second exon is from base 638 to base 730, the third exon is from base 1264 to base 1329, the fourth exon is from base 1441 to base 1581, and the fifth exon is from base 177 The sixth exon is from base 2073 to base 2213, the seventh exon is from base 2227 to base 2412, the eighth exon is from base 2504 to base 2671, the ninth exon is from base 2838 to base 2993, the tenth exon is from base 3171 to base 3959, and the rest are intron sequences.

[0073] The qRT-PCR primer sequences designed based on the CmoKARI1 gene sequence are as follows:

[0074] CmoKARI1 gene primer sequences:

[0075] qRT-CmoKARI1-F 5'-CGACGATTTTATGAAAAGGAGGGTC-3'(SEQ ID NO.3)

[0076] qRT-CmoKARI1-R 5'-TGCAGGACGTGATTGTCGAA-3'(SEQ ID NO.4)

[0077] CsaCBF1 gene primer sequences:

[0078] qRT-CsaCBF1-F 5'-TACAGAGGAGTCAGGAGGA-3'(SEQ ID NO.5)

[0079] qRT-CsaCBF1-R 5'-AGAATCGGCGAAATTGA-3'(SEQ ID NO.6)

[0080] CsaCOR gene primer sequences:

[0081] qRT-CsaCOR-F 5'-ACTTTGAGAGGACATTTGATG-3'(SEQ ID NO.7)

[0082] qRT-CsaCOR-R 5'-GAAGCTCCAATTTTGACTTG-3'(SEQ ID NO.8)

[0083] The cucumber cDNA product was diluted 5-fold to prepare a diluted cDNA sample. The qRT-PCR reaction system was performed according to the Novozymes Bioluminescence Quantitation Kit (Q711-02) as follows:

[0084]

[0085] After thorough mixing, place the sample in the QuantStudio™ 6Flex real-time PCR instrument for PCR amplification. The qPCR procedure is as follows:

[0086]

[0087] 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 11-17 , Figure 9-10 As shown in the results, CmoKARI1-GFP was overexpressed in cucumber roots or introduced into cucumbers by stable transformation. The results showed that after 12 hours of cold treatment, the levels of CmoKARI1, CsaCBF1 and CsaCOR genes were significantly higher than those of the control, and the levels of REP and DAB were significantly lower than those of the control, indicating that the CmoKARI1 gene responded to 4°C low temperature stress.

[0088] Experimental Example 5: Cucumber Transportability Identification Method

[0089] Using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), set the species to Cucurbitaceae, enter the CDS sequence of CmoKARI1 in the PCR Template field, and select a target product size between 300-500 bp. Using the results from the website, select primers specific for 'Xintai Mici'. Verify primer specificity by RT-PCR to ensure that the CmoKARI1 gene sequence is successfully amplified from both aboveground and underground locations.

[0090] Example 6: Full-length cloning of the CmoKARI1 gene

[0091] Cloning primers were designed based on the CmoKARI1 gene fragment.

[0092] The designed primer sequences are as follows:

[0093] GFP-F 5'-ACACGGGGGACTCTAGAATGGCCGCCGCCACA-3'(SEQ ID NO.9)

[0094] GFP-R 5'-ACCACCCGGGGATCCGTTGCTAGATTGACG-3'(SEQ ID NO.10)

[0095] PCR reaction system: Max DNA Polymerase;

[0096]

[0097] The PCR reaction procedure using cucumber cDNA as template is as follows:

[0098]

[0099] 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 voltage for about 25 minutes, detect the size of PCR product fragments under ultraviolet light, and cut the gel for recovery of the target band.

[0100] Example 7: Construction of 1305-CmoKARI1 overexpression vector

[0101] The restriction sites were XbaI and BamHI. The amplified product in Example 7 was then ligated to the vector pCambia1305-35S::C-GUS-NOSter-35S:C-GFP-NOSter. After correct sequencing, the plasmid was extracted to prepare the overexpression vector pCambia1305-35S::CmoKARI1-GUS-NOSter-35S:C-GFP-NOSter.

[0102] The expression vector was ligated according to the instructions of the NovoRec Plus One-Step PCR Cloning Kit (NR005) of Suzhou Nearshore Protein Technology Co., Ltd. The ligation system (10 μL) was as follows:

[0103]

[0104] Example 8: Hairy Root Genetic Transformation

[0105] Sowing: Select plump cucumber seeds and soak them in 65°C warm water for 30 minutes. Then, cool them to room temperature and soak for several hours. Use tweezers to peel off the seed coat. Shake the seeds in 70% alcohol for 30 seconds, then shake them in 3% sodium hypochlorite (NaClO) for 8 minutes. Sow approximately 20 seeds per bottle in MS medium and incubate in the dark for 4 days.

[0106] Explant acquisition: The seedlings were removed, half of the cotyledons were cut off, and the hypocotyl was cut 0.5 cm below the growing point. The part with the growing point was used as the explant for transformation.

[0107] Cucumber infection: Scrape the Agrobacterium rhizogenes plate, prepared one day in advance, wash with infection solution, and readjust the OD600 to between 0.15 and 0.20. After the adjusted concentration of the culture solution is allowed to stand at 28°C for 1-3 hours, add the explants and incubate at 80-120 rpm for 20 minutes.

[0108] Co-cultivation: After infection, remove excess bacterial suspension from the explants and transfer them to co-cultivation medium covered with sterile filter paper. Co-cultivate at 25°C in the dark for 3 days.

[0109] 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.

[0110] Observe GFP under a fluorescent flashlight and select plants with fluorescent roots.

[0111] Example 9: Identification of positive seedlings of hairy root genetic transformation by overexpression of CmoKARI1 gene

[0112] Hairy roots were transformed with Agrobacterium rhizogenes strain K599 containing either an empty vector (pCambia1300-Super::GFP) or pCambia1300-Super::CmoKARI1-GFP. K599 alone was transformed as a control. 14 days after transformation, RT-PCR was performed using primers specific for CmoKARI1-GFP or GFP to verify the transport of CmoKARI1-GFP or GFP.

[0113] Observe GFP under a fluorescent flashlight and select plants with fluorescent roots, such as Figure 1 As shown, under a fluorescence microscope, the root tips of wild-type cucumber hairy roots emit weak fluorescence, while GFP and CmoKARI1-GFP transgenic cucumber hairy roots emit strong and stable green fluorescence. 14 days after transformation, RT-PCR was performed using primers recognizing GFP to verify the transport rate of GFP or CmoKARI1-GFP. #1, #2, and #3 represent independent transgenic seedlings as biological replicates. The transport rate was analyzed as the number of moved seedlings / total number of transgenic seedlings. CsaACTIN7 was used as an internal reference for mRNA abundance. Hyg R was used to exclude contamination or transportability of Agrobacterium in the rhizosphere. The results show that CmoKARI1 triggers the movement of GFP mRNA from transgenic roots to non-transgenic new leaves under low temperature conditions.

[0114] DNA was extracted from cucumber leaves using the CTAB method. Primers were designed using the GFP sequence of the overexpression vector pCambia1305-35S::CmoKARI1-GUS-NOSter-35S:C-GFP-NOSter as a template. The primer sequences are as follows:

[0115] CmoKARI1-F 5'-GACTCTAGTCTAGAATGGCCGCCGCCACA-3'(SEQ ID NO.11)

[0116] CmoKARI1-R 5'-GCTCACCATGGTACCGTTGCTAGATTGACG-3'(SEQ ID NO.12)

[0117] Amplification system (20 μL):

[0118]

[0119] After mixing the system, centrifuge and perform PCR. The PCR procedure is as follows:

[0120]

[0121]

[0122] 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).

[0123] like Figure 2 、 Figure 4 Transformed with Agrobacterium tumefaciens K599, containing an empty vector (pCambia1305-35-35S:5-35) or pCamKARI1-Gus. R: Cucumber hairy roots transformed with Agrobacterium, L: New leaves appearing 14 days after transformation. GUS staining showed that the mobile CmoKARI1 in the transformed cucumber hairy roots was translated into new true leaves. Figure 3 、 Figure 5 Shown are RT-PCR results using primers fused to CmoKARI1-GUS or GUS to verify the transportability of CmoKARI1-GUS or GUS. #1, #2, and #3 represent independent transgenic seedlings as biological replicates. Transport rate was analyzed as the ratio of mobile seedlings to the total number of transgenic seedlings. CsaACTIN7 was used as an internal reference for mRNA abundance. HygR was used to exclude contamination or mobility with Agrobacterium tumefaciens. The results demonstrate that CmoKARI1-GUS mRNA can be transported from roots to the aerial part.

[0124] Example 10: Phenotypic and physiological analysis of CmoKARI1 gene overexpression hairy root plants in response to low temperature

[0125] The hairy root overexpressing plants of CmoKARI1-GUS gene and wild-type plants were simultaneously subjected to 4℃ low temperature treatment for 12h, and their physiological phenotypes were observed and related physiological data were measured. Figure 4-10 As shown, Figure 6 Figure 3 shows the phenotypes of cucumber seedlings (control) overexpressing 35S::CmoKARI1-GUS and 35S::GUS in roots before and after 12 hours of low temperature treatment. After low temperature treatment, the degree of leaf wilting of GUS transgenic hairy root cucumbers was higher than that of CmoKARI1-GUS transgenic hairy root cucumbers. Figure 7 Relative electrolyte permeability (REP) and MDA content; each treatment included 3 biological replicates, with 6-9 plants in each replicate (mean ± standard deviation, p < 0.05, Duncan test). Under low temperature treatment, the relative electrolyte permeability (REP) and MDA content of the leaves of CmoKARI1-GUS transgenic hairy root cucumber were significantly lower than those of GUS transgenic hairy root cucumber; Figure 8 Relative expression levels of CsaCBF1 and CsaCOR in leaves of cucumber seedlings transformed with 35S::CmoKARI1-GUS and 35S::GUS before and after 6 and 12 hours of low temperature treatment. Compared with cucumber seedlings transformed with 35S::GUS at 0 hours of low temperature treatment, p < 0.05 (Duncan test), using cucumber ACTIN7 as an internal control, showed that the relative expression levels of CsaCBF1 and CsaCOR genes in root-transformed cucumber seedlings significantly increased under low temperature treatment.

[0126] The hairy root overexpressing plants of CmoKARI1-GFP gene and wild-type plants were simultaneously subjected to 4℃ low temperature treatment for 12h, and their physiological phenotypes were observed and related physiological data were measured. Figure 9-10 As shown, Figure 9 The phenotype of the leaves of Super::GFP and Super::CmoKARI1-GFP cucumber seedlings transformed with root hairs was analyzed by low temperature treatment at 28℃ and 4℃ for 12h. After low temperature treatment, the wilting degree of the leaves of CmoKARI1-GFP transgenic hairy root cucumbers was lower than that of GFP transgenic hairy root cucumbers. Figure 10 Relative electrolyte permeability (REP) and MDA content; each treatment included 3 biological replicates, with 6-9 plants in each replicate (mean ± standard deviation, p < 0.05, Duncan test). Under low temperature treatment, the relative electrolyte permeability (REP) and MDA content of the leaves of CmoKARI1-GFP transgenic hairy root cucumber were significantly lower than those of GFP transgenic hairy root cucumber.

[0127] The results of low-temperature treatment showed that the root-transformed overexpression plants of the CmoKARI1 gene were more cold-resistant, and their relative conductivity and malondialdehyde content (the higher the relative conductivity and malondialdehyde content, the higher the degree of damage to the cell membrane, the more severe the cold stress, and the weaker the plant's cold resistance) were significantly lower than those of the control plants. The expression levels of the CsaCBF1 and CsaCOR genes were significantly increased. The above indicates that the root-transformed overexpression of the CmoKARI1 gene enhances the cold resistance of cucumber.

[0128] Example 11: Stable genetic transformation by overexpression of CmoKARI1 gene

[0129] The full-length sequence of 3969 bp of the CmoKARI1 gene was amplified by RT-PCR to obtain an amplified product (Example 7). The restriction sites were XbaI and KpnI. The amplified product was then ligated to the vector pCambia1300-Super:CmoKARI1-GFP. After correct sequencing, the plasmid was extracted to prepare the overexpression vector pCambia1300-Super:C-GFP (same as Example 8).

[0130] The plasmid containing the CmoKARI1 gene was chemically transformed into competent Agrobacterium GV3101, and genetic transformation of cucumber was performed by infecting cucumber cotyledons:

[0131] Sowing: Select plump cucumber seeds and soak them in 65°C warm water for 30 minutes. Then, cool them to room temperature and soak for several hours. Use tweezers to peel the seed coat. Shake the seeds in 70% alcohol for 30 seconds, then shake them in 3% sodium hypochlorite (NaClO) for 8 minutes. Sow the seeds in MS medium, approximately 20 seeds per bottle, and incubate in the dark for 36 hours.

[0132] Explant acquisition: Use tweezers to remove the radicle of the previously prepared 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.

[0133] Cucumber infection: Scrape the Agrobacterium tumefaciens plate prepared 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 culture solution is allowed to stand at 28°C for 1-3 hours, inoculate twice using a syringe to vacuum for 1.5 minutes each time.

[0134] Co-cultivation: After infection, remove excess bacterial suspension from the explants and transfer them to co-cultivation medium covered with sterile filter paper. Co-cultivate at 25°C in the dark for 4 days.

[0135] Shoot induction: Transfer the explants to budding medium and culture for three weeks.

[0136] 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 to induce root formation.

[0137] Three CmoKARI1 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 CmoKARI1 gene in the plant by qRT-PCR (qRT-CmoKARI1-F / R).

[0138] Example 12: Phenotypic and physiological analysis of CmoKARI1 gene overexpression plants in response to low temperature

[0139] The CmoKARI1 gene overexpressing 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 11-17 As shown, Figure 11 The phenotypes of two-leaf-stage cucumber seedlings overexpressing Super::CmoKARI1-GFP (OE#1, #2, and #4) and wild-type cucumber seedlings before and after 12 hours of low temperature treatment. Under low temperature treatment, the degree of wilting in cucumber seedlings overexpressing the CmoKARI1 gene was lower than that in wild-type. Figure 12 、 Figure 13 and Figure 14 Figure 3 shows NBT and DAB staining, as well as the DAB and NBT intensities of cucumber leaves relative to the control (WT at day 0). The blue color on the leaves represents the extent of damage, with darker staining indicating more severe damage. The results show that under low temperature treatment, the staining level of overexpressing plants was significantly lower than that of the control. Figure 15 、 Figure 16 MDA, chlorophyll, and relative electrolyte osmotic pressure (REP) contents were measured; each treatment included three biological replicates, with 5-6 plants per replicate (mean ± SD, p < 0.05, Duncan test). Under low temperature treatment, the MDA content of the transgenic lines overexpressing Super::CmoKARI1-GFP (OE#1, #2, and #4) was significantly lower than that of the wild-type cucumber seedlings, while the chlorophyll content was significantly higher than that of the wild-type cucumber seedlings. Figure 17Figure 3. Relative expression levels of CsaCBF1, CsaCOR, and CsaMYC2 in leaves of transgenic cucumber seedlings overexpressing Super::CmoKARI1-GFP (OE#1, #2, and #4) before and after 12 hours of low temperature treatment, compared with wild-type seedlings treated with low temperature at 0 hours. (p < 0.05, Duncan's test). Cucumber ACTIN7 was used as an internal control. Under low temperature treatment, the expression levels of CsaCBF1, CsaCOR, and CsaMYC2 genes in transgenic lines overexpressing Super::CmoKARI1-GFP (OE#1, #2, and #4) were significantly higher than those in wild-type cucumber seedlings.

[0140] The results of low temperature treatment showed that plants overexpressing the CmoKARI1 gene were more cold-resistant. The NBT, DAB staining degree, relative conductivity and malondialdehyde content of the three overexpression strains were significantly lower than those of the wild-type plants, and the chlorophyll content was significantly higher than that of the wild-type plants. The above indicated that overexpression of the CmoKARI1 gene enhanced the cold resistance of cucumber.

[0141] Example 13: Application of leucine, isoleucine and valine on cucumber seedling leaves under normal and low temperature conditions

[0142] Exogenous spraying of 0, 100, 200, 300, 400 μM isoleucine, leucine and valine was performed, and the cells were treated at 4°C. The physiological phenotypes at 0 and 12 h were observed and the relevant physiological data were measured. Figure 18-24 As shown, Figure 18 The phenotypic changes of leaves sprayed with different concentrations of branched-chain amino acids under normal and 12-hour low temperature conditions were shown. When sprayed with 300 μM isoleucine, the degree of leaf wilting was the lowest. Figure 19 This is the phenotype after spraying 300 μM isoleucine for 12 hours. Spraying isoleucine significantly improves the low temperature tolerance of cucumber. Figure 20 The REP and MDA contents were measured after 12 hours of cold treatment. The results showed that only 300 μM isoleucine significantly improved the cold tolerance of cucumber seedlings.

[0143] Example 14: Phenotypic and physiological analysis of CmoKARI1 gene overexpressing plants in response to low temperature after exogenous spraying of isoleucine.

[0144] The CmoKARI1 gene overexpressing plants and wild type plants were sprayed with isoleucine, and the control was treated with water. At the same time, they were subjected to a low temperature treatment of 4°C. Their physiological phenotypes were observed and related physiological data were measured. Figure 19-24 As shown, Figure 21 The figures show the phenotypes of wild-type and transgenic cucumbers before and after 12 hours of cold treatment after exogenous spraying of isoleucine. After low temperature treatment, the degree of wilting of transgenic plants sprayed with isoleucine was significantly lower than that of wild-type plants and transgenic plants not sprayed with isoleucine. Figure 22 Figure 2 shows the relative electrolyte permeability (REP) and MDA content of wild-type and transgenic cucumbers sprayed with isoleucine under a 12-hour cold treatment. Each treatment included six biological replicates, with 3-4 plants per replicate (mean ± SD, two-way ANOVA followed by t-test, *p < 0.05, **p < 0.01, ***p < 0.001). Under low temperature treatment, the electrolyte permeability (REP) and MDA content of transgenic cucumbers sprayed with isoleucine were significantly reduced. Figure 23 Isoleucine content in leaves of two-leaf-stage CmoKARI1-overexpressing transgenic cucumbers OE#2 and OE#4 and wild-type cucumbers sprayed with isoleucine before and after 12 hours of cold treatment. Figure 24 Relative expression levels of CsaCBF1 and CsaCOR in wild-type and transgenic cucumber plants sprayed with isoleucine before and after 12 hours of cold treatment. Water was used as the control. Each treatment included four replicates, each containing 12-16 plants (mean ± SD, 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, isoleucine content in the leaves of transgenic cucumber plants sprayed with isoleucine was significantly increased, as was the relative expression of CsaCBF1 and CsaCOR genes.

[0145] The above low temperature treatment results showed that the relative conductivity and malondialdehyde content of the two overexpression lines sprayed with isoleucine were significantly lower than those of the wild-type plants and the overexpression plants not sprayed with isoleucine. The CmoKARI1 gene overexpression plants sprayed with isoleucine were more resistant to cold.

[0146] Example 15: NBT staining

[0147] (1) Dissolve 0.1 g of NBT in 50 ml of 50 mM phosphate solution to prepare a 0.2% NBT staining solution. Wrap the centrifuge tube with tin foil.

[0148] (2) Take the first true leaf of the cucumber plant, soak the plant material in the prepared NBT staining solution in a centrifuge tube in the dark, vacuum for half an hour, and place it in the dark for 4 hours after vacuuming;

[0149] (3) After 4 hours, discard the NBT dye solution, soak in anhydrous ethanol and place in a boiling water bath for 10 minutes, shaking several times during the process;

[0150] (2) Carefully remove the plant material and take photos for observation.

[0151] Example 16: DAB staining

[0152] (1) Prepare a 50 ml centrifuge tube and add 50 mg of DAB to 45 ml of sterile water until the DAB is completely dissolved.

[0153] (2) Adjust the pH to 3.0 with NaOH;

[0154] (3) Wrap the centrifuge tube with tin foil and store it away from light;

[0155] (4) Add 25 μl of Tween-20 and 2.5 ml of 200 mM Na2HPO4 to a centrifuge tube to prepare a 10 mM Na2HPO4-DAB solution;

[0156] (5) Prepare plant materials;

[0157] (6) Soak the prepared plant material in DAB solution, vacuum it for half an hour, and then place it in a dark place for 4 hours;

[0158] (7) Replace the DAB dye solution with bleach solution (ethanol: acetic acid: glycerol = 3:1:1), and then incubate in a 95°C water bath for 15 min;

[0159] (8) After the water bath, bleach with fresh bleach solution at room temperature for 30 minutes;

[0160] (9) Carefully remove the plant material and take photos for observation.

[0161] Example 17: Detection of relative conductivity

[0162] (1) Use a hole punch to remove the first true leaf of the cucumber plant. Ten discs 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.

[0163] (2) Measure the conductivity of deionized water without blades, which is recorded as S0. Shake the centrifuge tube with blades on a horizontal shaker for 2 h and measure the conductivity of the solution, which is recorded as S1.

[0164] (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, which is recorded as S2. Calculate REL according to the following formula:

[0165]

[0166] Example 18: Malondialdehyde (MDA) content detection

[0167] (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 collect the supernatant;

[0168] (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.

[0169] (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:

[0170]

[0171] 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.

[0172] Finally, it should be noted that the above description is merely a preferred embodiment 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 will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

[0173] The contents not described in detail in this specification belong to the prior art known to those skilled in the art.

Claims

1. Use of the pumpkin CmoKARI1 gene in enhancing the cold tolerance of cucumbers, characterized in that, The application improves the low-temperature tolerance of cucumber plants by overexpressing the CmoKARI1 gene in cucumber plants; The nucleotide sequence of the pumpkin CmoKARI1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the pumpkin CmoKARI1 gene is shown in SEQ ID NO.

2.

2. A breeding method for low-temperature resistant cucumbers, characterized in that, It includes the following steps: Step 1, amplify the CmoKARI1 gene described in claim 1 to obtain an amplification product; Step 2, determine that the restriction enzyme sites are XbaI and KpnI, ligate the amplification product to the vector pCambia1300-Super:C-GFP to obtain the overexpression vector pCambia1300-Super::CmoKARI1-GFP-NOSter, and extract the plasmid after correct sequencing; Step 3, transfer the overexpression vector pCambia1300-Super::CmoKARI1-GFP-NOS ter into the competent cells of Agrobacterium tumefaciens GV3101, and perform genetic transformation of cucumber by the method of infecting cucumber cotyledons; Step 4, obtain the overexpression lines of the CmoKARI1 gene by amplifying the GFP sequence on the overexpression vector pCambia1300-Super::CmoKARI1-GFP-NOSter and qRT-PCR detection of CmoKARI1 in plants.

3. A method for improving the low-temperature tolerance of cucumbers, characterized in that, The method improves the low-temperature tolerance of cucumber by externally spraying Ile on the surface of cucumber plants overexpressing the CmoKARI1 gene described in claim 1.

4. The method for improving the low-temperature tolerance of cucumbers according to claim 3, characterized in that, When externally spraying Ile, the concentration of Ile is 300 μM.

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