Application of catalase and its coding gene in improving salt stress resistance of cucumber

Abstract

This invention provides an application of catalase and its encoding gene in improving the salt stress resistance of cucumber, belonging to the field of plant genetic engineering technology. In this invention, the gene CmoCAT2 from pumpkin is selected. An overexpression vector is constructed, and a pumpkin rootstock overexpressing the CmoCAT2 gene is created using Agrobacterium-mediated transformation. Using cucumber as the scion, it is verified that the mRNA of this gene can be transported long distances from the rootstock to the scion and can be translated locally within the scion, thus improving the salt stress resistance of cucumber. This method can cultivate salt-tolerant cucumber plants, adapting to the needs of cucurbit cultivation in saline-alkali land and saline-alkali soils in protected cultivation facilities.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of a catalase and its encoding gene in improving the salt stress resistance of cucumber. Background Technology

[0002] Salt stress is one of the major abiotic stresses affecting plant growth and development, and it has a significant adverse impact on global food security and sustainable agricultural development. Besides naturally occurring saline-alkali land, the main factors leading to salt stress in crops include secondary soil salinization caused by global climate change and improper irrigation. In my country, cucurbit crops are important crops; however, most cucurbit crops are quite sensitive to salt stress. When the soil salt content exceeds 2‰, the yield and quality of most cucurbit crops are negatively affected.

[0003] Molecular biology studies have shown that plant responses to salt stress are a complex network of multi-pathway synergistic regulation, with key physiological and biochemical processes including ion homeostasis reconstruction, osmotic substance accumulation, and reactive oxygen species (ROS) scavenging at their core. Catalase (CAT) is a core functional enzyme in the plant antioxidant enzyme system, efficiently scavenging excessive H2O2 induced by salt stress and playing an irreplaceable role in maintaining cellular redox homeostasis and mitigating oxidative damage. The catalase encoded by the CmoCAT2 gene is a key antioxidant element in pumpkin's response to salt stress, and its expression level under salt stress is significantly positively correlated with the salt tolerance of pumpkin rootstocks. Previous studies have confirmed that CAT2 (catalase 2) in Arabidopsis thaliana plays a crucial role in maintaining ROS homeostasis and regulating salt tolerance. Proteins such as the dual-specific phosphatase IBR5 (Indole-3-butyric acid response 5) and the cytoplasmic receptor-like kinase CRCK3 (Calmodulin-binding receptor-like cytoplasmic kinase 3) affect plant responses to salt stress by directly or indirectly regulating the activity of CAT2.

[0004] Grafting is an ancient and practical cultivation method that can greatly improve crop resistance to stress and is widely used in cucurbit crops. For example, pumpkin is an excellent rootstock material, characterized by its well-developed root system and strong resistance to stress, playing a significant role in grafting cucumbers, watermelons, and melons. Biomolecules such as mRNA, small RNA, and proteins can be transported bidirectionally and over long distances between the rootstock and scion through the vascular bundle system at the graft union, thereby regulating the growth, development, and stress resistance traits of the scion across organs. However, current methods for improving the salt tolerance of pumpkin rootstocks largely rely on traditional breeding methods, selecting pumpkin germplasm with differences in salt tolerance during germination. This approach is not only time-consuming but also suffers from unsatisfactory resistance to moderate to severe salt stress. Furthermore, the mobility of different mRNAs exhibits significant sequence and tissue specificity, and the improvement in salt tolerance performance of existing improved rootstocks is limited due to the lack of supplementary salt tolerance gene discovery. Currently, there are no clear reports on the mobility and transport regulation mechanisms of CmoCAT2 mRNA in the pumpkin-cucumber grafting system, nor its regulatory function on the salt tolerance of the scion. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides an application of catalase and its encoding gene in improving the salt stress resistance of cucumber. By constructing an overexpression vector, a pumpkin rootstock overexpressing the CmoCAT2 gene was created using Agrobacterium-mediated transformation. Using cucumber as the graft, the study verified that the mRNA of this gene can be transported long distances from the pumpkin rootstock to the cucumber scion and can be translated locally within the cucumber scion, thereby improving the salt stress resistance of cucumber.

[0006] The first objective of this invention is to provide an application of catalase in improving the salt stress resistance of cucumber, wherein the amino acid sequence of the catalase is shown in SEQ ID No. 2.

[0007] A second objective of this invention is to provide an application of a catalase-encoding gene in improving the salt stress resistance of cucumber, wherein the catalase-encoding gene encodes a protein as shown in SEQ ID No. 2.

[0008] In a preferred embodiment, the nucleotide sequence of the gene encoding catalase is shown in SEQ ID No. 1.

[0009] A third objective of this invention is to provide an application of an overexpression vector in enhancing the salt stress resistance of cucumber, wherein the overexpression vector contains a gene encoding the protein shown in SEQ ID No. 2.

[0010] A fourth objective of the present invention is to provide an application of a host cell in enhancing the salt stress resistance of cucumber, wherein the host cell comprises an overexpression vector containing a gene encoding the protein shown in SEQ ID No. 2; or the genome of the host cell contains a gene encoding the protein shown in SEQ ID No. 2.

[0011] The fifth objective of this invention is to provide a method for cultivating salt-tolerant pumpkin rootstock seedlings, comprising the following steps: constructing an overexpression vector containing a gene encoding the protein shown in SEQ ID No. 2; and cultivating stably inherited transgenic pumpkin plants using Agrobacterium-mediated transformation.

[0012] In a preferred embodiment, the Agrobacterium is EHA105 or K599.

[0013] The sixth objective of this invention is to provide a method for cultivating cucumber plants with salt stress resistance, comprising the following steps: constructing an overexpression vector containing a gene encoding the protein shown in SEQ ID No. 2; cultivating transgenic pumpkin plants using Agrobacterium-mediated transformation; and grafting the transgenic pumpkin plants as rootstocks and cucumber plants as scions to obtain grafted plants.

[0014] In a preferred embodiment, the construction of the overexpression vector includes the following steps: amplifying the gene encoding the protein shown in SEQ ID No. 2, wherein the sequences of the amplification primer pair are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0015] In a preferred embodiment, the Agrobacterium is EHA105 or K599.

[0016] The technical solution of this invention has the following beneficial effects: This invention utilizes the long-distance mobility and salt tolerance regulation function of the pumpkin CmoCAT2 gene. By constructing a gene overexpression vector, a stable transgenic pumpkin plant overexpressing the CmoCAT2 gene was created using an Agrobacterium-mediated transformation method. The salt stress resistance of this transgenic pumpkin plant is significantly improved compared to wild-type pumpkin plants. Using this transgenic pumpkin plant as rootstock and cucumber plants as scions, the resulting grafted plants exhibit significant salt stress resistance. Therefore, this invention provides a new breeding method for preparing cucurbit crops with salt stress resistance, enabling the rapid preparation of pumpkin rootstocks and cucumber plants with high salt stress resistance, suitable for cucurbit cultivation in saline-alkali land and saline-alkali soils in facilities. Attached Figure Description

[0017] Figure 1 This is a vector map of the vector pBSE403R-GFP used in Example 1 of the present invention; Figure 2 This is a vector map of the pANHA-GUS-GFP vector used in Example 1 of the present invention; Figure 3 This is a map of the overexpression vector pBSE403R-GFP-CATCDS constructed in Example 1 of the present invention; Figure 4 This is a map of the fusion expression vector pANHA-CATCDS-GUS constructed in Example 1 of the present invention; Figure 5 This is a spectrum of the vector pANHA-GFP-WZCATCDS used in Example 1 of the present invention; Figure 6 This is an electrophoresis result of RT-PCR verification of the CmoCAT2 gene migration from pumpkin rootstock to cucumber scion in Example 1 of the present invention; Figure 7 The images shown are the fluorescence detection results and electrophoresis results from step 1.5.2 of Embodiment 1 of the present invention. Figure 8 This is a diagram showing the results of the GUS staining verification experiment in Example 1 of the present invention; Figure 9 The image shows the fluorescence detection results of pumpkin plants overexpressing the CmoCAT2 gene in Example 2 of this invention. Figure 10 The images show the phenotypic characteristics of pumpkin plants overexpressing the CmoCAT2 gene and wild-type pumpkin plants under salt stress in Example 2 of this invention, as well as the SPAD value, REC value, MDA content, and O2 content of the leaves. - Bar chart comparing the content of H2O2 and its content; Figure 11 The images show the phenotypic characteristics of cucumber grafted onto pumpkin rootstock overexpressing the CmoCAT2 gene in Example 3 of this invention under salt stress, as well as the SPAD value, REC value, MDA content, and O2 content of the leaves. - Bar chart comparing the content of H2O2 and H2O2. Detailed Implementation

[0018] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.

[0019] In the following embodiments, methods not described in detail are all conventional methods well known to those skilled in the art. The pumpkin materials used were "Fengle Jinjia" (Cucurbita maxima × C. moschata) and "Zhuangshi," both commonly used rootstock varieties with well-developed root systems and strong grafting compatibility. "Fengle Jinjia" was used for gene mobility verification in the rooting grafting system, and its seeds were purchased from Guotou Fengle Seed Industry Co., Ltd. "Zhuangshi" was used for Agrobacterium-mediated genetic transformation, and its seeds were provided by the Institute of Facility Agriculture, Guangdong Academy of Agricultural Sciences. The cucumber material was the salt-stress-sensitive variety "Jinchun 4" (Cucumissativus L.), a commonly used scion material for cucumber grafting, and its seeds were purchased from Tianjin Kerun Cucumber Research Institute. Agrobacterium K599, Agrobacterium EHA105, and Escherichia coli DH5α were purchased from Shanghai Weidi Biotechnology Co., Ltd. The main components of the 1 / 2 modified Hoagland's nutrient solution include: 1 mmol / L MgSO4·7H2O, 4 mmol / L CaCl2, 10 mmol / L KNO3, 0.5 mmol / L Ca(H2PO4)2·H2O, and 74.93 mg / L Coolaber trace element solids (DZPM0059-500G). The pH of the solution is adjusted to 5.85–5.9 with 1 mmol / L KOH.

[0020] Since existing technology has confirmed that catalase encoded by the CmoCAT2 gene is a key antioxidant element in pumpkin's response to salt stress, and its expression level under salt stress is significantly positively correlated with the salt tolerance of pumpkin, but the mobility of different mRNAs exhibits significant sequence specificity and tissue specificity, there are currently no clear reports on the mobility, transport regulation mechanism, and regulatory function of the transcribed RNA (CmoCAT2 mRNA) of the CmoCAT2 gene in the pumpkin-cucumber grafting system. Therefore, the inventors of this application have chosen the CmoCAT2 gene of pumpkin for the following research.

[0021]

[0022] The amino acid sequence of the protein encoded by the nucleotide sequence shown in SEQ ID No. 1 (SEQ ID No. 2) is as follows: MDPYKYRPSSAYNTPFCTTNSGAPIWNNTAVMSVGERGPILLEDYQLIEKIATFTRERIPERVVHARGASAKGFFEVTHDVSDLSCADFLRAPGVQTPVIVRFSTVIHERGSPETLRDPRGFAVKFYTREGNFDLVGNNFPVFFVRDAMQFPDVIRAFKPNPKSHLQESWRFLDFCSYHPESLLSFAWFYDDVGIPINYRHMEGFGVQAYSLINKAGKARLVKFHWKPTCGVKSMLEEEAIR VGGSNHSHATQDLYESIAAGNFPEWRLYIQTIDYEDQNNYDFEPLDTTIAWPEDVVPLRPVGRLVLNKNIDNFFAENEMLAFSMSLVPGIHYSDDKMLQARSFAYADTQRHRLGPNYLQLPVNAP KCPHHNNHEGFMNFMHRDEEVNYFPSRYDACRHAEKYPMPPNVLSGKRERCVIPKENHNFKQAGDRYRSWAPDRQERFVNRFVEALSDPKVTHEVRNIWISYWTQADRSLGQKIASRMNARPNM.

[0023] Example 1: Vector Construction and Validation of CmoCAT2 Gene Mobility 1.1 CmoCAT2 gene fragment amplification 1.1.1 RNA Reverse Transcription to Obtain cDNA: Total RNA was extracted from the tissue of "Fengle Jinjia" pumpkin using the TranZol RNA Extraction Kit (purchased from Beijing TransGen Biotechnology Co., Ltd.). RNA reverse transcription was then performed using the Novizan HiScript III kit to obtain pumpkin cDNA. All procedures were performed according to the kit instructions.

[0024] 1.1.2 CmoCAT2 Gene Amplification: Using pumpkin cDNA as a template, the CDS fragment of the CmoCAT2 gene was amplified by PCR using specific primers CATCDS-CZ-F and CATCDS-CZ-R. Using pumpkin cDNA as a template, the same PCR amplification reaction system and program as for the CmoCAT2 gene CDS fragment amplification were employed, and the CATCDS-GUS fusion fragment was amplified using primer pairs CmCATCDS-GUS-CZ-F and CmCATCDS-GUS-CZ-R. The PCR amplification reaction system was as follows: 1 μL pumpkin cDNA, 25 μL 2×Phanta Flash Master Mix, 2 μL 10 μmol / L upstream primer, 2 μL 10 μmol / L downstream primer, and 20 μL ddH2O. The PCR amplification program was as follows: 95℃ pre-denaturation for 30 s, 34 cycles (95℃, 15 s; 57℃, 30 s; 72℃, 30 s), and extension at 72℃ for 5 min. PCR amplification products were detected by 1% agarose gel electrophoresis. The amplified product of the CmoCAT2 gene CDS fragment showed a specific band of 1479 bp, consistent with the size of the target fragment. After electrophoresis, the fragments were purified using a liquid purification kit (purchased from Aisjin Biotechnology (Hangzhou) Co., Ltd., catalog number AP-PCR-50) to obtain the CmoCAT2-CDS fragment and the CATCDS-GUS fusion fragment. The nucleotide sequence (SEQ ID No. 3) of the upstream primer CATCDS-CZ-F for amplifying the CmoCAT2 gene CDS fragment is as follows: gctgtacagatctgagctcatggatccctacaagtaccg; the nucleotide sequence (SEQ ID No. 4) of the downstream primer CATCDS-CZ-R is as follows: cgaacgaaagctctgagctcttacatgtttggtctc. The nucleotide sequence (SEQ ID No. 5) of the upstream primer CmCATCDS-GUS-CZ-F for amplifying the CATCDS-GUS fusion fragment is as follows: cgaacgatagtcgactctagaatggatccctacaagtaccgg; the nucleotide sequence (SEQ ID No. 6) of the downstream primer CmCATCDS-GUS-CZ-R is as follows: ggtttctacaggacgtaacatcatgtttggtctcgcattcatacg.

[0025] 1.2 Construction of recombinant vector: The pBSE403R-GFP vector was digested with restriction endonuclease Sac I (vector map shown). Figure 1 The pANHA-GUS-GFP vector was digested with the restriction endonuclease Xba I (vector diagram shown). Figure 2The enzyme digestion reaction system and conditions for both reactions were identical, as follows: 5 μL vector, 1 μL restriction endonuclease, 5 μL 10×Cutsmart buffer, 39 μL ddH2O; incubated overnight at 37℃. The purified CmoCAT2-CDS fragment from step 1.1.2 was homologously recombinated with the enzyme-digested pBSE403R-GFP linear vector to obtain the root overexpression vector pBSE403R-GFP-CATCDS. The vector map is shown below. Figure 3 This vector, based on the pBSE403R-GFP backbone, inserts the CDS coding sequence of CmoCAT2 and carries a dual-fluorescent reporter gene (GFP, DsRed) and a kanamycin (Kan) resistance gene (NPTII). It is suitable for overexpression and fluorescence tracking of the CmoCAT2 gene in root grafting systems. The vector's restriction enzyme site is Sac I. The purified CATCDS-GUS fusion fragment was homologously recombinated with the restriction-digested pANHA-GUS-GFP linear vector to obtain the fusion expression vector pANHA-CATCDS-GUS. The vector map is shown below. Figure 4 This vector, based on the pANHA-GUS-GFP backbone, inserts a CATCDS-GUS fusion fragment and carries a GFP fluorescent reporter gene and a Kan resistance gene NPTII. It is suitable for GUS histochemical staining verification of CmoCAT2 gene mobility. The vector's restriction enzyme site is Xba I. Homologous recombination was performed using the ClonExpress II One Step Cloning Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The recombination reaction system and conditions were as follows: 3 μL linear vector, 2 μL target fragment, 2 μL 5×CE II Buffer, 1 μL Exnase II, 2 μL ddH2O; reaction at 37℃ for 30 min.

[0026] The CmoCAT2 gene overexpression vector pANHA-GFP-WZCATCDS used in pumpkin genetic transformation is based on the pANHA-GUS-GFP vector, with the GUS gene replaced by the CmoCAT2 gene and 3×HA (hemagglutinin tag). It carries a GFP fluorescent reporter gene and the Kan resistance gene NPTII, and is used for Agrobacterium-mediated pumpkin genetic transformation to achieve stable overexpression of the CmoCAT2 gene. The pANHA-GFP-WZCATCDS vector was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and its vector map is shown below. Figure 5 As shown.

[0027] 1.3 Positive Clone Identification and Recombinant Plasmid Extraction: The three recombinant vectors constructed in step 1.2 were each transformed into *E. coli*, specifically as follows: The recombinant vectors were transformed into *E. coli* DH5α using the heat shock method, plated on LB solid medium containing 50 mg / L kanamycin (Kan), and incubated overnight at 37°C. Single colonies were picked for colony PCR identification (PCR reaction system and conditions were the same as in step 1.1.2). Positive colonies were sent for sequencing, and sequence alignment was performed using Geneious software. The colony PCR identification and sequencing results showed that the CmoCAT2 gene sequence had no mutations. Positive clones with correct sequence alignment were used to extract positive plasmids using the Tiangen Plasmid Mini-Prep Kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP103), and stored at -20°C for later use.

[0028] 1.4 Agrobacterium Transformation: The extracted positive plasmids pBSE403R-GFP-CATCDS and pANHA-CATCDS-GUS were transformed into Agrobacterium K599 using the freeze-thaw method, respectively. The transformed plasmids were plated on LB solid medium containing 50 mg / L Kan and 25 mg / L rifampin (Rif) and incubated at 28°C for 2 days. Single colonies were picked for PCR identification (PCR reaction system and conditions were the same as in step 1.1.2). Positive Agrobacterium cultures were stored at 4°C for short-term storage; for long-term storage, an equal volume of 50% sterile glycerol was added, and the culture was stored at -80°C. The extracted positive plasmid pANHA-GFP-WZCATCDS was transformed into Agrobacterium EHA105 using the freeze-thaw method, following the same procedure as the transformation of the two positive plasmids described above.

[0029] 1.5 Verification of long-distance mobility of CmoCAT2 gene: The cross-species long-distance mobility of CmoCAT2 mRNA from pumpkin rootstock to cucumber scion was verified using a triple technique of RT-PCR, root grafting, and GUS histochemical staining.

[0030] Seed germination: Seeds of "Fengle Jinjia" pumpkin and "Jinchun No. 4" cucumber were treated as follows: Plump seeds were selected, soaked in 55℃ hot water for 8 hours, wrapped in moist gauze, and placed in a petri dish. Germination was carried out in a 28℃ constant temperature incubator for 48 hours. After germination, the seeds were sown in seedling trays filled with Shangdao substrate soil and grown in an artificial climate chamber. The artificial climate chamber conditions were: 14h light / 10h darkness, temperature (25±2)℃, and relative humidity (65±5)%. The cucumber seeds were germinated and sown in the same manner after the pumpkin seeds were sown.

[0031] Agrobacterium infection: 10 μL of Agrobacterium K599 positive bacterial suspension was added to 15 mL of LB liquid medium containing 50 mg / L Kan and 25 mg / L rifampin (Rif), and incubated at 28℃ and 200 rpm for 12-16 h. Then, the suspension was centrifuged at 5000 rpm for 10 min, the supernatant was removed, and the suspension was resuspended in 15 mL of MS liquid medium containing 80 mg / L AS (acetylsyleugenol) to obtain a resuspension containing Agrobacterium K599. Pumpkin seedlings with newly unfolded cotyledons were selected as rootstocks, and the hypocotyl of the pumpkin seedlings was obliquely cut 2-3 cm below the cotyledons. The cut root tips were used for grafting, and cucumber scions were grafted onto the pumpkin rootstock using the insertion-hole grafting method. Fresh grafted plants were used as explants for Agrobacterium K599-mediated transformation. The hypocotyl incision was immersed in 1 mL of the above-mentioned Agrobacterium K599 resuspension for 30 minutes for infection. The infected explants were then cultured in a box containing sterilized vermiculite moistened with MS medium at 23°C in the dark for 4 days, and fluorescence was detected. Afterward, the infected grafted plants were transferred to seedling trays and cultured in the dark for 3 days while maintaining a certain level of humidity, then gradually exposed to light and ventilation. Once the grafted seedlings had established themselves, non-fluorescent roots were removed every 7 days, while fluorescent roots were retained.

[0032] 1.5.1 Preliminary RT-PCR validation: Once the pumpkin seeds germinated and grew to the point of having one leaf and one bud, and the cucumber cotyledons had fully expanded, a heterologous grafting system (Csa / Cmo) was constructed using 'Fengle Jinjia' pumpkin (Cmo) as rootstock and 'Jinchun No. 4' cucumber (Csa) as scion. Cucumber self-grafting (Csa / Csa) served as a negative control, and pumpkin self-grafting (Cmo / Cmo) as a positive control. Grafted plants were cultured as follows: top grafting was performed, and post-grafting care was taken to maintain moisture and avoid light. The healing of the graft union was observed daily. Successfully grafted plants were transplanted into hydroponic pots and managed with 1 / 2 modified Hoagland nutrient solution, with the nutrient solution changed every 3 days. After the plants stabilized, root tissues from the pumpkin rootstock and leaf tissues from the cucumber scion were collected. Total RNA was extracted from each using the TranZol kit (purchased from Beijing TransGen Biotech Co., Ltd.), and then reverse transcribed into cDNA using the Novizan HiScript III kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). Using cDNA obtained from each grafting group as a template, RT-PCR amplification was performed using CmoCAT2 gene-specific primers (PCR reaction system and conditions were the same as in step 1.1.2) to detect the presence of pumpkin-derived CmoCAT2 transcripts in cucumber scions. The RT-PCR identification results are as follows: Figure 6 As shown. Figure 6From left to right, lanes 1 and 2 show the leaves and roots of cucumber self-grafted (Csa / Csa), lanes 3 and 4 show the leaves and roots of cucumber / pumpkin grafted (Csa / Cmo) scion, and lanes 5 and 6 show the leaves and roots of pumpkin self-grafted (Cmo / Cmo). Specific target bands were observed in lane 3, while no target bands were observed in lanes 1 and 2, confirming that endogenous CmoCAT2 mRNA can migrate from pumpkin rootstock to cucumber scion.

[0033] 1.5.2 Root Grafting Verification: When the pumpkin seeds germinate and grow to one leaf and one bud, the hypocotyl of the pumpkin seedling is obliquely cut 2-3 cm below the cotyledon to obtain the pumpkin rootstock. Agrobacterium K599 carrying pBSE403R-GFP-CATCDS is used to infect the pumpkin rootstock, and then cucumber is grafted onto the pumpkin rootstock using a top grafting method to obtain the grafted cucumber plant CmoCAT OE. The pumpkin rootstock is then infected with Agrobacterium K599 carrying the empty vector pBSE403R-GFP (without the CmoCAT2 gene), and cucumber scions are grafted onto it to construct the control plant EV. The culture conditions for the cucumber plant CmoCAT OE and the control plant EV are as follows: The plants are cultured in boxes containing sterilized vermiculite moistened with MS medium at 23℃ in the dark for 4 days, and fluorescence is detected. Afterwards, the infected grafted plants are transferred to seed trays and cultured in the dark for 3 days while maintaining a certain level of humidity, and then gradually exposed to light and ventilation. Every 7 days, non-fluorescent roots were removed, and fluorescent roots were retained. When the fluorescent roots exceeded 3 cm in length and the plants were growing well, the seedlings were carefully transplanted into hydroponic pots, with 5 L of 1 / 2 concentration modified Hoagland nutrient solution added to each pot, and cultured in a growth chamber. When the grafted seedlings developed to the three-leaf-one-heart stage and their physiological state stabilized, the root fluorescence intensity and distribution uniformity were reconfirmed using a handheld fluorescent protein observation lamp, and plants with uniform growth and clear fluorescence signals were selected for salt stress treatment. Subsequently, NaCl was added to the hydroponic nutrient solution to a final concentration of 100 mM to apply acute salt stress. 24 hours after treatment, rootstock root and scion leaf tissues were collected, and total RNA was extracted using the TranZol kit (purchased from Beijing TransGen Biotech Co., Ltd.), and reverse transcribed into cDNA using the Novizan HiScriptIII kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). RT-PCR amplification was performed using GFP-specific primers. The amplified products were detected by 1% agarose gel electrophoresis to verify the mobility of GFP and CmoCAT2 transcripts. The NPTII gene was used to distinguish between true mRNA movement and potential false positive signals caused by Agrobacterium rhizogenes. The pumpkin Actin gene (Cucurbitaceae Crop Gene Database gene ID: CmoCh02G006200) or cucumber Actin gene (Cucurbitaceae Crop Gene Database gene ID: CsaV3_6G041900.1) were used as internal controls to correct for technical deviations and eliminate interference from sample differences and experimental procedures, thus providing reliable evidence for true mRNA movement. The RT-PCR amplification reaction system was as follows: 1 μL cDNA, 5 μL 2×Taq Master Mix, 0.5 μL 10 μmol / L upstream primer, 0.5 μL 10 μmol / L downstream primer, and 3 μL ddH2O.The RT-PCR amplification reaction program was as follows: pre-denaturation at 95℃ for 30 s, 37 cycles (95℃, 15 s; 57℃, 30 s; 72℃, 30 s), extension at 72℃ for 5 min. The nucleotide sequence of the GFP upstream primer GFP-F (SEQ ID No. 7) is as follows: ggagaggacctcgacct; the nucleotide sequence of the GFP downstream primer GFP-R (SEQ ID No. 8) is as follows: agaggccacgatttgacac. The RT-PCR identification results are shown below. Figure 7 As shown. Figure 7 The image above (a) shows the fluorescence and electrophoresis results of the control plant EV after treatment with 100 mmol / L NaCl for 24 h, with #1 and #2 representing two plants; the image above (b) shows the fluorescence and electrophoresis results of the plant CmoCAT OE after treatment with 100 mmol / L NaCl for 24 h, with #1 and #2 representing two plants; L represents the scion leaf tissue, R represents the rootstock root tissue; Kan R represents the NPTII band. Figure 7 As can be seen above, the mRNA of the CmoCAT2 gene or its encoded protein can be transported long distances from the pumpkin rootstock to the cucumber scion via the vascular system, carrying the fused GFP along with it. This result directly confirms that the CmoCAT2 gene has the ability to move long distances across the grafting interface, and that this characteristic depends on the CmoCAT2 gene itself, rather than on the vector or the GFP tag.

[0034] 1.5.3 GUS Staining Verification: Using a grafting method similar to that in 1.5.2, Agrobacterium K599 carrying pANHA-CATCDS-GUS was used to infect pumpkin rootstock, resulting in grafted cucumber plants CATCDS-GUS. Pumpkin rootstock was then infected with Agrobacterium K599 carrying the empty vector pANHA-GUS-GFP (without the CmoCAT2 gene), and cucumber scions were grafted onto it to construct control plants. GUS histochemical staining was performed on these CATCDS-GUS plants. After incubation at 37℃ in the dark overnight, the stain was decolorized with 70% ethanol. The presence of a blue GUS signal in the cucumber scions was observed under a stereomicroscope (results are shown in Figure 1). Figure 8 As shown in the figure, the mobility and local translation ability of CmoCAT2 mRNA were verified. Figure 8 The top left image shows a pumpkin / cucumber grafted plant with the empty vector pANHA-GUS-GFP, and the right image shows a pumpkin / cucumber grafted plant with root overexpression of pANHA-CATCDS-GUS. From Figure 8 As can be seen above, the right image shows obvious blue GUS signals in the pumpkin rootstock roots, hypocotyls, and cucumber scion leaves, while the left image shows no blue signal in the leaves, confirming that CmoCAT2 mRNA can move from the pumpkin rootstock to the cucumber scion and complete local translation.

[0035] Example 2: Agrobacterium-mediated genetic transformation of pumpkin and identification of positive plants Using the cotyledons of the "Zhuangshi" pumpkin as explants, stable transformation of the CmoCAT2 gene was achieved via Agrobacterium EHA105-mediated transformation. The specific procedures are as follows: 2.1 Seed Disinfection and Germination: Select plump pumpkin seeds, soak them in 55℃ warm water for at least 30 minutes, remove the shells, and then disinfect them by surface sterilization with 75% ethanol for 30 seconds, soaking them in 0.5% NaClO aqueous solution for 15 minutes, and rinsing them 6 times with sterile water. Sow the disinfected seeds on seed germination medium and germinate them in the dark at 28℃ for 36-48 hours. The seed germination medium is MS liquid medium containing 1 mg / L 6-BA (6-benzylaminopurine) and 1 mg / L ABA (abscisic acid).

[0036] 2.2 Explant preparation: When the seed coat is removed and the vascular bundle ridges of the cotyledons are clearly visible, about 1 / 3 of the distal end of the cotyledons is cut off, the hypocotyl is removed, and the two cotyledons are separated to obtain cotyledon explants with U-shaped wounds.

[0037] 2.3 Agrobacterium infection: Inoculate 1 μL of positive Agrobacterium EHA105 into 1 mL of liquid LB medium containing 50 mg / L Kan and 25 mg / L LRift, and shake at 28°C (200 rpm) for 24 hours. Transfer the mixed bacteria to 15 mL of liquid LB medium and incubate until OD500. 600nm =0.6~0.8, centrifuge, collect bacterial cells, and dilute to OD using IM liquid medium (MS liquid medium containing 1 mg / L 6-BA, 1 mg / L LABA, 1.25 mmol / L 2-(N-morpholino)ethanesulfonic acid and 200 μmol / L acetylsylphenol). 600nm =0.2, add 0.1% acetylsyl syringone and culture in the dark for 1h. Immerse the explants in the bacterial solution, treat with 100W ultrasound for 10s, and then enhance the infection effect by vacuum filtration (0.8MPa, 90s).

[0038] 2.4 Co-culture and Differentiation: After infection, the explants were dried by aspirating the surface bacterial solution and placed in co-culture medium (MS liquid medium containing 1 mg / L 6-BA, 1 mg / L ABA, 1.25 mmol / L 2-(N-morpholino)ethanesulfonic acid, 200 μmol / L acetylsylphenol ketone and 250 μmol / L L-glutamine) and cultured in the dark at 23°C for 4 days. After rinsing 8 times with sterile water, they were transferred to differentiation medium (MS liquid medium containing 1 mg / L 6-BA, 1 mg / L ABA and 200 mg / L trimethyltin chloride) and cultured under light at 28°C. Subculture was performed every 10 days, and transgenic regenerated shoots were screened.

[0039] 2.5 Rooting and Hardening-off: When the transgenic regenerated shoots grow to 2-4 cm, they are transferred to rooting medium (MS liquid medium of 200 mg / L trimethyltin chloride) to induce rooting. After hardening-off, well-rooted transgenic plants are transplanted into sterile substrate and cultured in an artificial climate chamber (under the same conditions as in step 1.5) until T0 generation seeds are harvested. T0 generation seeds are soaked in 55℃ warm water for 8 hours, then wrapped in moist gauze and placed in petri dishes for germination in a 28℃ constant temperature incubator for 48 hours. After germination, they are sown in seedling trays containing Agrobacterium tumefaciens substrate and grown in an artificial climate chamber (under the same conditions as in step 1.5) to obtain T1 generation plants. Wild-type (WT) explants not infected with Agrobacterium EHA105 are used as controls and cultured according to steps 2.4-2.5.

[0040] 2.6 Molecular identification of pumpkin plants overexpressing the CmoCAT2 gene: Using a handheld fluorescent protein light (LUYOR-3415RG), fluorescence detection was performed on transgenic pumpkin plants, T0 generation seeds, and T1 generation seedlings in a dark environment. Bright-field and fluorescence photographs were taken to assist in the identification of positive transgenic plants. The results are as follows: Figure 9 As shown. Figure 9 The above figures (a) show the fluorescence detection results of transgenic T0 generation (CmoCAT-OE) explants overexpressing the CmoCAT2 gene and wild-type (WT) explants; (b) show the fluorescence detection results of transgenic T0 generation (CmoCAT-OE) plants overexpressing the CmoCAT2 gene and wild-type (WT) plants; (c) show the fluorescence detection results of harvested pumpkins from transgenic T0 generation (CmoCAT-OE) plants overexpressing the CmoCAT2 gene and wild-type (WT) plants; and (d) show the fluorescence detection results of harvested seeds from transgenic T0 generation (CmoCAT-OE) plants overexpressing the CmoCAT2 gene and wild-type (WT) plants. "Bright" indicates a bright-field image, and "GFP" indicates a fluorescent image. As can be seen from the figures, clear green GFP fluorescence signals were detected in the leaves, stems, and T0 generation seeds of the transgenic pumpkin plants, while no fluorescence signal was detected in the wild-type pumpkin plants, confirming that the CmoCAT2 gene was successfully overexpressed in pumpkins.

[0041] 2.7 Salt tolerance determination of pumpkin plants overexpressing the CmoCAT2 gene Salt tolerance was evaluated in T1 generation pumpkin plants overexpressing the CmoCAT2 gene, and morphological and physiological indicators were measured. The specific steps are as follows: 2.7.1 Material cultivation: Transgenic pumpkin seeds and wild-type pumpkin seeds were soaked in 55℃ warm water for 8 hours and germinated in a 28℃ constant temperature incubator for 48 hours. After germination, they were sown in substrate soil and cultured in an artificial climate chamber (under the same conditions as in step 2.5) until they had two leaves and one heart. They were then transplanted into hydroponic pots and cultured with 1 / 2 modified Hoagland nutrient solution. The nutrient solution was changed every 3 days.

[0042] 2.7.2 Salt stress treatment: After the plants have grown steadily, NaCl was added to 1 / 2 of the modified Hoagland nutrient solution to a final concentration of 100 mM as the salt stress treatment group, and the experiment without sodium chloride was used as the control group. The index was measured after 7 days of treatment.

[0043] 2.7.3 Indicator Testing: Morphological indicators: Observe the plant's growth vigor, the degree of leaf wilting, leaf color and other phenotypic characteristics and take photos to record them; Physiological indicators: SPAD values ​​(chlorophyll content) were measured using a SPAD-502 chlorophyll meter; malondialdehyde (MDA) content was determined using the thiobarbituric acid method; relative conductivity (REC), reflecting cell membrane integrity, was measured using a conductivity meter; and O2 was measured using the hydroxylamine oxidation method. - The content of H2O2 was determined using a hydrogen peroxide reagent kit (purchased from Nanjing Jiancheng Bioengineering Institute, catalog number A064-1), which reflects the plant's antioxidant capacity.

[0044] The measurement results are as follows Figure 10 As shown. Figure 10 The figure above (a) shows the phenotypic diagrams of wild-type (WT) plants and plants overexpressing the CmoCAT2 gene (CmoCAT2-OE) after 7 days of treatment with the control group (CK) and the salt stress treatment group (NaCl). Figures (b) to (f) show the SPAD value, REC value, MDA content, and O2 content of the leaves of wild-type (WT) plants and plants overexpressing the CmoCAT2 gene (CmoCAT2-OE) after 7 days of treatment with the control group (CK) and the salt stress treatment group (NaCl). - Bar chart comparing the content of H2O2 and its content; ns in the graph indicates no significant difference (P≥0.05). This indicates a highly significant difference (P<0.01). The difference is considered highly significant (P<0.0001); the bar chart represents the mean ± standard error of the data obtained from the three parallel experiments. As can be seen from the graph, under salt stress, wild-type plants exhibited severe wilting and yellowing, while plants overexpressing the CmoCAT2 gene showed good growth with only slight wilting. The SPAD value of wild-type (WT) plants decreased from 43.2 to 28.7, a decrease of 33.6%; the SPAD value of plants overexpressing the CmoCAT2 gene (CmoCAT2-OE) decreased from 42.9 to 40.6, a decrease of 5.4%, significantly less than that of wild-type (WT) plants. The relative electrical conductivity of wild-type (WT) plants increased from 9.5% to 30.1%, an increase of 3.2 times; the relative electrical conductivity of plants overexpressing the CmoCAT2 gene (CmoCAT2-OE) increased from 8.1% to 10.6%, a much smaller increase than that of wild-type (WT) plants. The MDA content of wild-type (WT) plants increased from 2.18 mmol / (g fresh weight) to 5.98 mmol / (g fresh weight), an increase of 174%; the MDA content of plants overexpressing the CmoCAT2 gene (CmoCAT2-OE) increased from 2.06 mmol / (g fresh weight) to 3.46 mmol / (g fresh weight), an increase of 68%, which was 60.9% lower than that of wild-type (WT) plants. Under 100 mol / L NaCl treatment, the O2 content of plants overexpressing the CmoCAT2 gene (CmoCAT2-OE) decreased. - The salt content was reduced by 30.8% compared to wild-type (WT) plants; the H2O2 content was reduced by 17.7% compared to wild-type (WT) plants. These results indicate that overexpression of the CmoCAT2 gene can significantly improve the salt tolerance of pumpkin without affecting normal growth.

[0045] Example 3: Verification of the effect of pumpkin rootstock overexpressing the CmoCAT2 gene on improving cucumber salt tolerance during grafting. Using pumpkin plants overexpressing the CmoCAT2 gene as rootstock and cucumber 'Jinchun 4' as scion, the top grafting method was employed to verify the effect of pumpkin rootstock overexpressing the CmoCAT2 gene on improving the salt tolerance of cucumber scions. The specific steps are as follows: 3.1 Grafting Procedure: T0 generation pumpkin seeds were sown in advance. When the pumpkin seedlings reached two leaves and one bud, and the cucumber seedlings had fully developed cotyledons, top grafting was performed to obtain CmoCAT2-OE / Csa plants. After grafting, the plants were kept moist (90%-95% humidity) and cultured in the dark. The healing of the graft union was observed daily. After successful grafting, the plants were transplanted to hydroponic pots and cultured using 1 / 2 modified Hoagland nutrient solution. Using wild-type "Zhuangshi" pumpkin as rootstock and "Jinchun No. 4" cucumber as scion, WT / Cas plants were obtained and grafted and cultured using the same method described above.

[0046] 3.2 Salt Stress Treatment: When the grafted plants reached the three-leaf stage, NaCl was added to half of the modified Hoagland nutrient solution to a final concentration of 100 mM as the salt stress treatment group. The experiment without sodium chloride was used as the control group. After 7 days of continued cultivation, various physiological indicators of the cucumber scions were measured using the same methods as in step 2.7.3. The results are as follows: Figure 11 As shown. Figure 11 The figure above (a) shows the phenotypic diagrams of WT / Cas and CmoCAT2-OE / Csa plants after 7 days of treatment with the control group (CK) and the salt stress treatment group (NaCl). Figures (b) to (f) show the SPAD value, REC value, MDA content, and O2 content of the leaves of WT / Cas and CmoCAT2-OE / Csa plants after 7 days of treatment with the control group (CK) and the salt stress treatment group (NaCl), respectively. - Bar chart comparing the content of H2O2 and its content; ns in the graph indicates no significant difference (P≥0.05). The difference is significant (P<0.05). This indicates a highly significant difference (P<0.01). This indicates a highly significant difference (P<0.001). The difference is considered highly significant (P<0.0001). The data in the bar graph represent the mean ± standard error of the data obtained from the three parallel experiments. The graph shows that in the control group without sodium chloride treatment, the growth vigor of the grafted plants obtained by the two grafting methods was consistent. In the salt stress treatment group treated with 100mM NaCl, the cucumber grafts of WT / Cas plants were severely wilted, and the leaf edges turned yellow, while the cucumber grafts of Csa / CmoCAT2-OE plants grew normally, with only slight wilting and green leaves. The SPAD value of WT / Cas plants decreased from 41.5 to 24.4, a decrease of 41.2%; the SPAD value of Csa / CmoCAT2-OE plants decreased from 42.1 to 39.2, a decrease of 6.9%, significantly less than that of WT / Cas plants. The relative electrical conductivity of WT / Cas plants increased from 8.13% to 36.20%, an increase of 345.3%; the relative electrical conductivity of Csa / CmoCAT2-OE plants increased from 5.77% to 9.37%, an increase of 62.4%, with the increase being 81.9% lower than that of WT / Cas plants. The MDA content of WT / Cas plants increased from 6.45 mmol / (g fresh weight) to 13.96 mmol / (g fresh weight), an increase of 116.4%; the MDA content of Csa / CmoCAT2-OE plants increased from 7.29 mmol / (g fresh weight) to 9.40 mmol / (g fresh weight), an increase of 28.9%, with the increase being 75.1% lower than that of WT / Cas plants. The O2 content of Csa / CmoCAT2-OE plants... - The content of CmoCAT2 was reduced by 28.0% compared to WT / Cas plants; the H2O2 content was reduced by 13.9% compared to WT / Cas plants. These results indicate that pumpkin rootstocks overexpressing the CmoCAT2 gene can significantly improve the salt tolerance of cucumber scions and effectively alleviate the oxidative damage and ion toxicity of cucumber scions under salt stress.

[0047] In summary, this invention experimentally verifies that the mRNA of the CmoCAT2 gene can be transported long distances from the pumpkin rootstock to the cucumber scion via the vascular bundle system of the pumpkin-cucumber grafting system, and then translated locally in the scion tissue to synthesize a biologically active catalase. This mobility is determined by the CmoCAT2 gene sequence itself and is independent of the vector backbone and tag protein, overcoming the limitations of tissue-specific gene expression and achieving systemic salt tolerance regulation between rootstock and scion. The catalase encoded by the CmoCAT2 gene is a core enzyme in the plant reactive oxygen species scavenging system, capable of efficiently removing O2 accumulated in plants under high salt stress. - It can reduce membrane lipid peroxidation damage caused by reactive oxygen species, maintain cell membrane integrity, reduce MDA content and relative conductivity, and alleviate the damage of oxidative stress to plants.

[0048] The embodiments described above are merely preferred embodiments of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by anyone skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection claimed in this application and the content of the specification should be included within the scope of protection of this application.

Claims

1. The application of a catalase in improving the salt stress resistance of cucumber, characterized in that, The amino acid sequence of the catalase is shown in SEQ ID No.

2.

2. The application of a catalase-encoding gene in improving the salt stress resistance of cucumber, characterized in that, The gene encoding the catalase encodes the protein shown in SEQ ID No.

2.

3. The application according to claim 2, characterized in that, The nucleotide sequence of the gene encoding the catalase is shown in SEQ ID No.

1.

4. The application of an overexpression vector in improving the salt stress resistance of cucumber, characterized in that, The overexpression vector contains a gene encoding the protein shown in SEQ ID No.

2.

5. The application of a host cell in enhancing the salt stress resistance of cucumber, characterized in that, The host cell contains the overexpression vector as described in claim 4, or the genome of the host cell contains the gene encoding catalase as described in claim 2.

6. A method for cultivating salt-stress-tolerant pumpkin rootstock seedlings, characterized in that, The process includes the following steps: constructing the overexpression vector as described in claim 4, and cultivating transgenic pumpkin plants using Agrobacterium-mediated transformation.

7. The method according to claim 6, characterized in that, The Agrobacterium is EHA105 or K599.

8. A method for cultivating cucumber plants with salt stress resistance, characterized in that, The process includes the following steps: constructing the overexpression vector as described in claim 4, cultivating transgenic pumpkin plants using Agrobacterium-mediated transformation, using the transgenic pumpkin plants as rootstocks and cucumber plants as scions for grafting, and obtaining grafted plants.

9. The cultivation method according to claim 8, characterized in that, The construction of the overexpression vector includes the following steps: amplifying the gene encoding the protein shown in SEQ ID No. 2, with the sequences of the amplification primer pair shown in SEQ ID No. 3 and SEQ ID No.

4.

10. The cultivation method according to claim 8, characterized in that, The Agrobacterium is EHA105 or K599.

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