Application of CsERF113L gene or biological material containing CsERF113L gene in regulation and control of stress resistance and yield of cucumber

Abstract

The invention relates to the field of plant molecular biology, in particular to application of a CsERF113L gene or a biological material containing the CsERF113L gene in regulation and control of stress resistance and yield of cucumbers. The cDNA (complementary deoxyribonucleic acid) sequence of the CsERF113L gene is as shown in SEQ ID NO. 1. The CsERF113L gene is knocked out in cucumber by adopting a CRISPR-Cas9 gene editing technology, the antioxidant capacity and photosynthetic capacity of cucumber plants are remarkably enhanced, the saline-alkaline resistance is remarkably improved, the root growth is accelerated, the cucumber yield is remarkably increased, and knockout strains and over-expression strains have opposite trends. The result shows that the CsERF113L gene affects the regulation and control mechanism of cucumber stress resistance. The CsERF113L gene has important guiding significance and application value for guiding widening of cucumber cultivation conditions in actual production, improvement of cucumber high yield and stress resistance and cucumber seed selection and breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, and specifically relates to a... CsERF113L Genes or containing CsERF113L Application of genetic biomaterials in regulating cucumber stress resistance and yield. Background Technology

[0002] cucumber( Cucumis sativus L. Cucumber is one of the main vegetable crops cultivated in greenhouses in my country. It is widely popular due to its delicious taste and rich nutrition, possessing significant nutritional and economic value. Originating in India, cucumber is a warm-season vegetable. Its sensitivity to abiotic stresses such as low temperatures, salinity, and drought makes the plant susceptible to adverse conditions, leading to the loss of normal physiological functions. This severely impacts fruit quality and the economic benefits for producers. Therefore, to broaden the cultivation conditions for cucumbers in practical production, research has explored specific genes in cucumbers that regulate stress resistance mechanisms, and has sought to improve the stress resistance of cucumbers by modifying or introducing specific genes.

[0003] With the expansion of protected cultivation areas for cucumbers, the increase in soil planting years, and improper fertilizer and water management, soil salinization has become a serious global problem affecting agricultural production and the ecological environment, severely impacting cucumber quality and yield. my country is a major country in terms of saline-alkali soil, making salt-alkali stress one of the main factors limiting cucumber growth and productivity. Furthermore, mixed salt-alkali stress is a widespread phenomenon in nature, and its effects are far greater than those of simple salt or alkali stress alone; that is, the two have a synergistic effect, severely affecting the photosynthetic rate of cucumbers and inhibiting the growth and development of cucumber seedlings. Cucumber salt tolerance is influenced by multiple factors, including responses from the perspectives of photosynthesis, ion toxicity, cell membranes, and antioxidant enzymes, as well as their basic salt tolerance mechanisms. To elucidate the salt tolerance mechanisms of cucumbers and to promote salt-tolerant breeding, thereby improving cucumber salt tolerance and increasing its adaptability and yield in saline-alkali soils, many studies are currently exploring genes involved in regulating cucumber salt tolerance mechanisms. This has significant guiding significance and application value for improving cucumber high-yield and stress-resistant qualities, yield, and cucumber breeding. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the present invention provides a CsERF113L Genes or containing CsERF113L Application of genetically modified biomaterials in regulating cucumber stress resistance and yield. This invention utilizes the construction of... CsERF113L Gene knockout vectors were discovered. CsERF113L Gene knockout can enhance the salt and alkali tolerance of cucumbers and increase cucumber yield.

[0005] The specific technical solution is as follows: A sort of CsERF113LGenes or containing CsERF113L The application of gene-based biomaterials in regulating cucumber stress resistance and yield, the aforementioned CsERF113L The gene sequence number is CsaV3_6G005100, and its cDNA sequence is shown in SEQ ID NO.1.

[0006] As a preferred embodiment of the present invention, regulating the stress resistance of cucumber is to regulate the salt and alkali tolerance of cucumber.

[0007] As a preferred embodiment of the present invention, by knocking out CsERF113L Genes can be used to improve the stress resistance and yield of cucumbers, or through... CsERF113L Sequence editing of any exon and intron region between the start and stop codons of a gene can induce loss-of-function or de-functional mutations in the gene, thereby improving the stress resistance and yield of cucumbers.

[0008] As a preferred embodiment of the present invention, it contains CsERF113L The biological material of genes is CsERF113L Gene knockout vectors or recombinant engineered bacteria or CsERF113L Gene knockout vector-mediated transgenic plant cell lines.

[0009] In a preferred embodiment of the present invention, the knockout carrier is obtained by... CsERF113L After the dual target sequences of the gene are respectively linked to both ends of the "sgRNA1+gRNA-Sc+U6-26t+U6-29p+sgRNA2" sequence region of the intermediate vector pCBC-DT1T2, the entire gene is inserted into the backbone vector. Bsa It was constructed at the I restriction site; The dual-target sequence is as follows: Target S1 (SEQ ID NO.2): 5'-TGGCGAATCGGCGAGGAAT-3'; Target S2 (SEQ ID NO.3): 5'-GATTGGGGGCTACGGCAGGG-3'.

[0010] In a preferred embodiment of the present invention, the skeleton carrier is a pKSE402 carrier.

[0011] This invention also provides a method for cultivating cucumbers with improved stress resistance or increased yield, characterized by comprising the following steps: Construct the CsERF113L Gene knockout vectors; The knockout vector was transferred into Agrobacterium EHA105 to obtain the inoculum; By using the aforementioned dye solution to inoculate cucumber cotyledon explants, transgenic plants with increased stress resistance or yield can be obtained.

[0012] As a preferred embodiment of the present application, a CRISPR-Cas9 gene editing vector of Cucumis sativus L. is constructed CsERF113L The process of the gene knockout vector is as follows: According to the above-mentioned CsERF113L The single-stranded guide target sequence is designed for the gene, and the single-stranded guide target sequence and the Cas9 coding sequence are inserted into the pKSE402 skeleton vector by PCR reaction, and the CRISPR-Cas9 gene editing vector of Cucumis sativus L. is obtained. The single-stranded guide target sequence is a double-target sequence, and the specific sequence is: Target S1 (SEQ ID NO. 2): 5'-TGGCGAATCGGCGAGGAAT-3'; Target S2 (SEQ ID NO. 3): 5'-GATTGGGGCTACGGCAGGG-3'.

[0013] The constructed CsERF113L The CRISPR-Cas9 gene editing vector of the gene can change CsERF113L The genomic sequence, resulting in weakening or loss of its gene function, thereby significantly improving the salt-tolerant level and yield of Cucumis sativus L.

[0014] As a preferred embodiment of the present application, each 20 μL PCR reaction system includes: 0.4 μL of faded double-stranded primers, 3 μL of 100-500 ng / μL pkSE402, 3 μL of 50-100 ng / μL pCBC-DT1T2, 2 μL of T4 Buffer, 1 μL of 10x BSA, 1.5 μL of BsaI-HF, 1.5 μL of T4 Ligase, and ddH2O. The sequence of the upstream primer of the faded double-stranded primer is shown in SEQ ID NO. 4, and the sequence of the downstream primer is shown in SEQ ID NO. 5.

[0015] As a preferred embodiment of the present application, the PCR reaction conditions are incubated at 37°C for 5 hours, and then heated at 80°C for 10 minutes to inactivate the enzyme.

[0016] As a preferred embodiment of the present application, the maceration is macerated with sterile, 23-27°C, light-avoiding, and 32-48 hours of germination of Cucumis sativus L. cotyledon explants.

[0017] As a preferred embodiment of the present application, the macerated Cucumis sativus L. explants are screened and cultured to grow seedlings, and further cultivation obtains gene editing Cucumis sativus L. germplasm with high resistance to abiotic stress or high yield.

[0018] Further preferably, after the Cucumis sativus L. explants grow into seedlings, the plant tissues are subjected to CsERF113LThe gene relative expression amount is detected, and the gene with serial number CsaV3_5G031430 as the internal reference gene is screened out CsERF113L The plant with the gene relative expression amount reduced by more than 7 times. CsERF113L The cucumber plant with the gene relative expression amount reduced by more than 7 times has a significantly improved root growth, the root aging process is inhibited, and the salt and alkali tolerance is significantly enhanced.

[0019] More preferably, the plant with the gene relative expression amount reduced by 14-21 times is screened out. CsERF113L The plant with the gene relative expression amount reduced by more than 7 times. CsERF113L When the protein relative expression amount is reduced, the salt and alkali tolerance of the cucumber is further enhanced.

[0020] In the above embodiment, the CsERF113L protein relative expression amount test method comprises: extracting total RNA from the tissue of the cucumber seedling after treatment, obtaining cDNA thereof through reverse transcription, and detecting the expression amount through a detection primer, wherein the upstream sequence of the detection primer is shown in SEQ ID NO. 6, and the downstream sequence is shown in SEQ ID NO. 7.

[0021] The application also provides CsERF113L The CRISPR-Cas9 gene editing vector of the gene is used to improve the salt and alkali tolerance of the cucumber or to make CsERF113L The gene is functionally deleted in the cucumber plant.

[0022] Compared with the prior art, the application uses CsERF113L After the gene is used to construct the CRISPR-Cas9 editing vector and transform the cucumber plant, the gene edited cucumber germplasm is CsERF113L The gene knockout plant, CsERF113L The knockout of the gene significantly promotes the root growth of the cucumber, inhibits the aging process of the cucumber root, enhances the salt and alkali tolerance of the cucumber, and improves the yield of the cucumber. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The gene editing mode (a) of CsERF113L and the expression amount of the three knockout lines (b) are shown, and different letters represent significant differences between lines. P <0.05. DETAILED DESCRIPTION

[0024] The principles and characteristics of the application are described below in combination with the drawings, and the examples are only used to explain the application and not to limit the scope of the application.

[0025] Example 1 The embodiment provides CsERF113L The construction method of the CRISPR-Cas9 gene editing vector of the gene comprises the following steps: The primer FW: 5'-ATATATGGTCTCGATTGTGGCGAATCGGCGAGGAATGTT-3', SEQ ID NO. 4; RV: 5'-ATTATTGGTCTCGAAACGATTGGGGCTACGGCAGGGCAA-3', SEQ ID NO. 5, the sgRNA1+gRNA-Sc+U6-26t+U6-29p+sgRNA2 fragment was cloned from the pCBC-DT1T2 intermediate vector as a template to obtain the target fragment. The PCR amplification system is shown in Table 1, the amplification program is shown in Table 2, and the amplification result is shown in SEQ ID NO. 8.

[0026] Table 1 PCR amplification system Table 2 Amplification program After amplification, the target fragment was recovered by cutting the gel. CsERF113L The cDNA sequence of the gene is shown in SEQ ID NO. 1:

[0027] The sequence of the PCR cloned fragment of interest is shown as SEQ ID NO. 8: GTTGGGACATTCTTCAGTCGGCAAAATCAGAGAACAATTCCAATATGAATTCTCTCAATCTCTCTTAAAAAAAACAACGAATATGACCGACATTTATTAATTTCATATCCAAATATTACTTCCCCTGTTTTTAATCAGTCAAAATAAAATAAAATAAAATAAAAGAACTTTTTTTTTCTCCAGAAAAATCTTTTCCTTTTTGCTTTTGGATTATGGATTTAAGGTTATTACTAATCATCCCAAAAATCTCAAATACAAATTCTTAAAAACAGAGACTTCCACTGTTCATATTTCGGCTCCCCTTCGTGTTTTTAAAAATCCCCAATTTTTCTTCTCTCTTTAAAATCTCCGATTCAAATTCAAATTCAAAAATCCTCTGTGTTTTCCCCTTGTTTTTTTTTTTTTTTCTTTGTTGGGTTTTCGAATTTTTAATAAAGAATTTGTATGTGCTCCTTGAAAGTGGCGAATCGGCGAGGAATTGGAAGTGACTTGGCACAATTTCCAAGTGGCGGCGCCGGAGATGGAGACGGTGACACTGGTGGCGGCGGAGGCGGCGGCAGCAGTAGCAGCGACGACCCTCATAGACTATTATATGGTCAACGTGATGACGGCGATTATTACTCTGCCGTTGGAGAAGTTTCCACGATTGTGTCTACGTTAACTAATGTAATGTCTGGGCAGGCGGCTCCCGATTGGGGCTACGGCAGGGGGCAGGGTTTTCCGAGGGGTTTTGTTTCTTCCTCCTCCTCTTCTTCTTCTTCGACGACATCTGCATCGGGTTCTTCGGGTTCGGAGCTAAGCTATGTTCCGGGGATGAGTAGTTATTGGGTTGGCCAAAAACGAATGCGTGAAGAAGAAATTAGTGTTCAAACACAGCATGATTTTCATTCTGCTTCGAGGGGTTTTAGTTTTATTAGGGGTTTTGATCATCATTTTTCTCAACCACAATCTTCCATTCCTCCA According to CsERF113LThe CDS of the gene was designed to have two specific single-stranded guide target sequences (sgRNA, target S1: 5'-TGGCGAATCGGCGAGGAAT-3', SEQ ID NO. 2; target S2: 5'-GATTGGGGCTACGGCAGGG-3', SEQ ID NO. 3). Cloning primers (FW: 5'-ATATATGGTCTCGATTGTGGCGAATCGGCGAGGAATGTT-3', SEQ ID NO. 4; RV: 5'-ATTATTGGTCTCGAAACGATTGGGGCTACGGCAGGGCAA-3', SEQ ID NO. 5) were designed according to the sequences of SEQ ID NO. 2 and SEQ ID NO. 3 and the homologous sequences of the pCBC-DT1T2 vector. After cloning the Cas9 coding sequence region (gRNA-Sc+U6-26t+U6-29p) in the pCBC-DT1T2 vector using the primers, the following were used Bsa I The pKSE402 vector was digested, and the linearized vector was recovered by gel recovery. The cloning target sequence (SEQ ID NO. 8) and the linearized vector were subjected to homologous recombination ligation. In a 20 μL system, 4 μL of the target fragment, 10 μL of BasicMix (TransGen), and 6 μL of the linearized pKSE402 vector (100-500 ng / μL) were included. After adding the various reaction solutions in order, the reaction solution was mixed gently by pipetting, and then placed in a PCR instrument for 37°C incubation for 5 hours. Subsequently, heating at 80°C for 10 minutes inactivated the enzyme, and the ligation product was obtained, i.e. CsERF113L Gene knockout vector.

[0028] The ligation product was transformed into E. coli, and the E. coli monoclonal with correct sequencing results was expanded. The plasmid was extracted using a DNA small amount extraction kit, and the CsERF113L Gene editing vector cpERF113L-pKSE402 of the gene.

[0029] Example 2 CsERF113L Genetic transformation of the CRISPR-Cas9 gene editing vector of the gene The specific operation steps are as follows: (1) The constructed gene editing vector with correct sequencing was transformed into Agrobacterium competent cells (EHA105) to obtain Agrobacterium containing the cpERF113L-pKSE402 vector.

[0030] (2) The Agrobacterium containing the cpERF113L-pKSE402 vector was used to dip the sterile cucumber cotyledon explants that had germinated for 36 hours at 25°C in the dark.

[0031] (3) Dark culture of the explants after infection.

[0032] (4) Differentiation culture of the screened explants.

[0033] (5) The plants further cultivated are transplanted into nutrient soil for growth, PCR detection is performed using sequencing primers, and the PCR product band is consistent with the CsERF113L gene length, and a positive transgenic line is obtained.

[0034] In Example 1 and Example 2, plant positive detection, E. coli positive detection, and Agrobacterium positive detection are all detected by taq-PCR, sequencing primers shown as SEQ ID NO. 4 and SEQ ID NO. 5 are used for sequencing of the positive clones, and primers shown as SEQ ID NO. 6 and SEQ ID NO. 7 are used for detection of the positive plants and clones.

[0035] Upstream primer (SEQ ID NO. 6): 5'-GTTGGGACATTCTTCAGTCGG-3'; Downstream primer (SEQ ID NO. 7): 5'-TGGAGGAATGGAAGATTGTGG-3'.

[0036] The taq-PCR detection system is shown in Table 3.

[0037] Table 3 taq-PCR detection system The taq-PCR reaction program is shown in Table 4.

[0038] Table 4 taq-PCR reaction program The sequencing result is shown as SEQ ID NO. 8.

[0039] Plant tissue DNA extraction method: (1) 0.1 g of young leaves of the plant seedlings is placed in a 2 mL centrifuge tube, ground in about 750 µL of CTAB extraction solution (β-mercaptoethanol is added), and then placed in a 65 ℃ water bath for more than half an hour; (2) After water bath, about 750 µL of chloroform and isopropyl alcohol mixture (volume ratio of 24:1) is added to the centrifuge tube, inverted 100 times, and centrifuged at 10,000 r / min for 10 min; (3) 450~500 µL of supernatant is taken into a new 1.5 mL centrifuge tube, an equal volume of cold isopropyl alcohol is added, mixed, and then placed in a -20 ℃ refrigerator for 10 min, and then centrifuged at 12,000 r / min for 10 min. (4) Discard the supernatant, add 800 μL of 75% ethanol to wash the precipitate, centrifuge instantaneously, discard the ethanol, and dry until colorless and transparent; (5) Add 200 μL of ddH2O (different amounts for different purposes), dissolve the DNA, and store at -20 °C for standby.

[0040] Example 3 CsERF113L Gene expression test The following methods are used to test the expression of transgenic positive plants: Take the young leaves of transgenic plants to extract RNA.

[0041] The expression of the gene in the positive gene editing line (cpERF113L-1, cpERF113L-2, cpERF113L-3) is detected. CsERF113L Gene expression detection.

[0042] The expression detection steps include RNA extraction, RNA reverse transcription, and expression determination.

[0043] RNA extraction: (1) Grind the plant tissue in liquid nitrogen, take about 100 mg of sample into a 2.0 mL centrifuge tube cooled in liquid nitrogen, add 1 mL of Trizol, invert and extract for a few minutes, and then stand for 5 min; (2) Add 200 μL of chloroform, vigorously invert and extract for 15 s, and stand at room temperature for 2-3 min; (3) Centrifuge at 4 °C and 13000 r / min for 10 min; (4) Transfer 300-400 μL of supernatant to a new 1.5 ml centrifuge tube (try not to suck the lower layer, and reduce the amount of supernatant to ensure purity), add an equal volume of ice-cold isopropanol (-20 °C), invert and mix, then stand at -20 °C for 10 min; (5) Centrifuge at 4 °C and 13000 r / min for 10 min; (6) Discard the supernatant, add 1 mL of 75% ethanol (DEPC water diluted), and suspend the precipitate; (7) Centrifuge at 12000 r / min for 1 min; (8) Discard the supernatant, repeat steps (6) and (7) once to improve RNA purity, and dry the centrifuge tube in a fume hood for 5-10 min; (9) Add about 20 μL of DEPC water (adjust according to the amount of RNA extracted); (10) RNA quality was detected by agarose gel electrophoresis, and the absorbance method was used to determine the RNA concentration, and then stored at -80 ℃ for standby.

[0044] RNA reverse transcription: According to the concentration of the RNA sample, the RNA sample was uniformly diluted to a specific concentration with DEPC water. Reverse transcription was performed using a reverse transcription kit, and the reverse transcription process was as follows:

[0045] 1) Genomic DNA removal Prepare the mixture as shown in Table 5 in an RNase-free centrifuge tube: Table 5 Mixture After mixing, place it in a PCR instrument at 42 ℃ for 2 min.

[0046] 2) First strand cDNA synthesis Add 2 μl of 10×RT Mix and 2 μl of HiScript II Enzyme Mix to the mixture in the previous step.

[0047] After mixing, place it in a PCR instrument. Set the reaction program of the PCR instrument: 50 ℃, 15 min; 85℃, 2 min.

[0048] After the reaction program is completed, the product can be immediately used for qPCR reaction, or stored at -20 ℃.

[0049] CsERF113L Relative expression amount of gene determination: Determine the relative expression amount of the gene according to the following procedure and SYBR mix system CsERF113L The relative expression amount of the gene, the selected internal reference gene sequence number is CsaV3_5G031430, and the relative expression amount of the gene is tested according to the conventional detection method in the art.

[0050] The qPCR reaction system is shown in Table 6, and the SYBR mix system is shown in Table 7.

[0051] Table 6 qPCR reaction system Note: Q_FW and Q_RV in Table 6 are the upstream primer and downstream primer of the expression detection primer, respectively.

[0052] The expression detection primer of the internal reference gene CsaV3_5G031430 is: Q_FW: 5'-CACCAAGCCCAAGAAGATC-3', SEQ ID NO. 9; Q_RV: 5'-TAAACCTAATCACCACCAGC-3', SEQ ID NO. 10; CsERF113L The expression amount of the gene was detected by the primer: Q_FW: 5'-TGCTTCGAGGGGTTTTAGTT-3', SEQ ID NO. 11; Q_RV: 5'-CGTACTCCTCTGTATCGTCG-3', SEQ ID NO. 12.

[0053] Table 7 SYBR mix system The test results are shown in Table 8. Figure 1 The relative expression amount of the gene was greatly reduced, and the specific CsERF113L The relative expression amount of the gene was greatly reduced, and the specific CsERF113L The relative expression amount of the gene was greatly reduced, and the specific CsERF113L The relative expression amount of the gene was greatly reduced, and the specific

[0054] Table 8 CsERF113L The relative expression amount of the gene was greatly reduced, and the specific The wild type control strain (CK) and the cpERF113L gene edited strain were cultured to one leaf and one heart according to the conventional seedling raising method, transplanted into a flower pot, and continued to be cultured to three leaves and one heart, and then subjected to salt stress treatment. The salt stress solution used was a 300 mM composite salt solution, and the molar ratio was NaCl:Na2SO4:NaHCO3:Na2CO3=1:9:9:1, and the pH was 9.0. In the control group, 150 mL of sterile water was poured on each strain, and in the treatment group, 150 mL of composite salt solution was poured on each strain. During the treatment, the management mode of each strain was uniformly carried out by conventional cultivation management. The physiological indicators of the plants were measured on the 3rd day after salt treatment, and the survival rate of the plants was counted on the 7th day. The results are shown in Tables 9-13.

[0055] Table 9 Effect of salt stress on photosynthetic parameters of cucumber leaves Table 10 Effect of salt stress on H2O2 and MDA content in cucumber leaves Table 11 Effect of salt stress on antioxidant enzyme activity in cucumber leaves Table 12 Influence of saline-alkali stress on survival rate of cucumber Table 13 Influence of knocking out CsERF113L on root morphological development of cucumber As shown in Tables 9-13, knocking out CsERF113L gene significantly improves photosynthetic parameters and antioxidant capacity of the plant under saline-alkali stress, and finally improves the survival rate and saline-alkali tolerance of the plant. Meanwhile, as shown in Table 13, knocking out CsERF113L gene significantly promotes the root development of the plant, which helps to promote the transportation of water and nutrients, and thus promotes the growth of the plant and improves the stress resistance of the plant.

[0056] The wild type control strain (CK) and the cpERF113L gene edited strain were cultured to three-leaf-one-heart according to the conventional seedling raising method, and then planted into the soil on March 22, 2025. The water and fertilizer management of each treatment was uniformly managed using water and fertilizer integrated automatic irrigation equipment. The harvest time of cucumber was from April 20 to June 26. Four biological replicates were set for each strain, and six randomly selected plants were used for each biological replicate to determine the yield. The results are shown in Table 14.

[0057] Table 14 Influence of knocking out CsERF113L gene on yield of cucumber CsERF113L As shown in Table 14, knocking out CsERF113L gene significantly improves the fruit number and fruit yield of the plant.

[0058] The above description is only the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A kind CsERF113L Genes or containing CsERF113L The application of gene-based biomaterials in regulating cucumber stress resistance and yield is characterized by, The CsERF113L The gene sequence number is CsaV3_6G005100, and its cDNA sequence is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, Regulating the stress resistance of cucumbers involves regulating their salt and alkali tolerance.

3. The application according to claim 1, characterized in that, By knocking CsERF113L Genes can be used to improve the stress resistance and yield of cucumbers, or through... CsERF113L Sequence editing of any exon and intron region between the start and stop codons of a gene can induce loss-of-function or de-functional mutations in the gene, thereby improving the stress resistance and yield of cucumbers.

4. The application according to claim 1, characterized in that, contain CsERF113L The biological material of genes is CsERF113L Gene knockout vectors or recombinant engineered bacteria or introduction CsERF113L Transgenic plant cell lines with gene knockout vectors.

5. The application according to claim 4, characterized in that, The knockout vector is obtained by... CsERF113L After the dual target sequences of the gene are respectively linked to both ends of the "sgRNA1+gRNA-Sc+U6-26t+U6-29p+sgRNA2" sequence region of the intermediate vector pCBC-DT1T2, the entire gene is inserted into the backbone vector. Bsa It was constructed at the I restriction site; The dual-target sequence is as follows: Target S1: 5'-TGGCGAATCGGCGAGGAAT-3'; Target S2: 5'-GATTGGGGGCTACGGCAGGG-3'.

6. The application according to claim 5, characterized in that, The skeletal carrier is a pKSE402 carrier.

7. A method for cultivating cucumbers with improved stress resistance or increased yield, characterized in that, Includes the following steps: Construct the structure described in claim 1 CsERF113L Gene knockout vectors; The knockout vector was transferred into Agrobacterium EHA105 to obtain the inoculum; By using the aforementioned dye solution to inoculate cucumber cotyledon explants, transgenic plants with increased stress resistance or yield can be obtained.

8. The method according to claim 7, characterized in that, Build CsERF113L The process of using a gene knockout vector is as follows: According to the above CsERF113L A single-stranded guide target sequence was designed, and the single-stranded guide target sequence and the Cas9 coding sequence were inserted into the pKSE402 backbone vector by PCR reaction to obtain the gene. The single-chain guide target sequence is a dual-target sequence, specifically as follows: Target S1: 5'-TGGCGAATCGGCGAGGAAT-3'; Target S2: 5'-GATTGGGGGCTACGGCAGGG-3'.

9. The method according to claim 8, characterized in that, Each 20 μL PCR reaction system includes: 0.4 μL of annealed double-stranded primers, 3 μL of 100~500 ng / μL pkSE402, 2 μL of T4 Buffer, 1 μL of 10 x BSA, 1.5 μL of BsaI-HF, 1.5 μL of T4 Ligase, and ddH2O to make up the difference; The upstream primer sequence of the annealed double-stranded primer is shown in SEQ ID NO.4, and the downstream primer sequence is shown in SEQ ID NO.

5.

10. The method according to claim 8 or 9, characterized in that, The PCR reaction conditions were: incubation at 37°C for 5 hours, followed by heating at 80°C for 10 minutes to inactivate the enzyme.

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