Ctbbx4 protein and gene related to salt tolerance of plants and application thereof

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a CtBBX4 protein, gene, and applications related to plant salt tolerance. This invention provides a CtBBX4 protein related to plant salt tolerance, the amino acid sequence of which is shown in SEQ ID NO.1. The CtBBX4 protein of this invention can improve the salt tolerance of plants. Examples show that overexpression of the CtBBX4 protein in plants... CtBBX4 Genes can increase plant germination rate and biomass, and improve plant salt tolerance by enhancing antioxidant capacity. CtBBX4 Genes are constructed into plant expression vectors and then genetically transformed using Agrobacterium-mediated transformation, which can effectively obtain new germplasm with salt tolerance characteristics, and is of great significance for the breeding of salt-tolerant crops.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a CtBBX4 protein and gene related to plant salt tolerance and its applications. Background Technology

[0002] Soil salinization has become a major threat to global agricultural ecology. Excessive salt disrupts plant metabolic balance through osmotic stress and ion toxicity, leading to stunted crop growth and sharp yield reductions, severely restricting the development and utilization of saline-alkali land. Discovering the functional genes of wild salt-tolerant plants is the core approach to breeding salt-tolerant crops. For example, the salt-tolerant genes of wild plants such as sesquiterpene have been successfully applied to crop improvement.

[0003] As a native plant of arid and saline-alkali regions in northern my country, *Calamus esculenta* exhibits significant salt tolerance. However, current research on the molecular mechanisms of salt tolerance in *Calamus esculenta* is still incomplete, and the isolation, identification, and functional verification of key salt-tolerant genes in its genome remain a gap. Therefore, screening for salt-tolerant genes from *Calamus esculenta* can enrich the plant salt-tolerant gene resource library and provide new gene targets for breeding crops in saline-alkali land, which has important theoretical significance and application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a CtBBX4 protein, gene, and application related to plant salt tolerance. The CtBBX4 protein can improve plant salt tolerance and provide a new pathway for cultivating new salt-tolerant germplasm.

[0005] This invention provides a CtBBX4 protein associated with plant salt tolerance, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] This invention also provides a method related to plant salt tolerance. CtBBX4 Genes, the ones mentioned CtBBX4 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0007] The present invention also provides the CtBBX4 protein described in the above-described scheme or the [other protein described above]. CtBBX4 Application of genes in regulating plant salt tolerance.

[0008] As a preferred embodiment, the regulation includes: increasing the CtBBX4 protein content or increasing... CtBBX4 Gene expression levels increase plant salt tolerance.

[0009] As a preferred embodiment, the improvement of plant salt tolerance includes at least one of the following: (1) increasing germination rate; (2) increasing plant growth; and (3) increasing antioxidant capacity.

[0010] As a preferred embodiment, the plant includes *Gnaphalium affine* and / or *Arabidopsis thaliana*.

[0011] The present invention also provides a recombinant expression vector, wherein the recombinant expression vector comprises the components described above. CtBBX4 Gene.

[0012] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant expression vector described in the above scheme.

[0013] This invention also provides the CtBBX4 protein described in the above-described scheme, and the aforementioned... CtBBX4 The application of the gene, the recombinant expression vector, or the recombinant bacteria in the cultivation of salt-tolerant plant varieties.

[0014] The present invention also provides a method for improving the salt tolerance of plants, comprising the following steps: transferring the recombinant expression vector or the recombinant bacteria described in the above scheme into the plant.

[0015] Beneficial Effects: This invention provides a CtBBX4 protein associated with plant salt tolerance, the amino acid sequence of which is shown in SEQ ID NO.1. The CtBBX4 protein of this invention can improve the salt tolerance of plants. Examples show that overexpression of this protein in plants... CtBBX4 Genes can increase plant germination rate and biomass, and improve plant salt tolerance by enhancing antioxidant capacity. CtBBX4 Genes are constructed into plant expression vectors and then genetically transformed using Agrobacterium-mediated transformation, which can effectively obtain new germplasm with salt tolerance characteristics, and is of great significance for the breeding of salt-tolerant crops. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 ground melon CtBBX4 Subcellular localization map of genes;

[0018] Figure 2 ground melon CtBBX4 A diagram of gene transcriptional activation activity;

[0019] Figure 3 ground melon CtBBX4 The results of functional verification of gene-overexpressing yeast are shown in the figure; where A and B represent the growth and concentration (OD) of overexpressing yeast and empty vector yeast on the culture medium under normal conditions. 600 Figure C and D show the growth and concentration of overexpressed yeast and empty vector yeast on the culture medium under salt stress. 600 In the figure, A and C correspond to results on solid culture medium, and B and D correspond to results on liquid culture medium.

[0020] Figure 4 ground melon CtBBX4 The results of constructing the Arabidopsis thaliana gene overexpression vector are shown in Figure A; where A is a schematic diagram of vector construction; B is a gel electrophoresis result, where 1287bp is the gene size and 1436bp is the PCR length using the gene forward primer and the vector reverse primer.

[0021] Figure 5 ground melon CtBBX4 Results of gene overexpression analysis in Arabidopsis thaliana plants;

[0022] Figure 6 ground melon CtBBX4 Germination of Arabidopsis thaliana seeds with overexpressed genes under salt stress;

[0023] Figure 7 ground melon CtBBX4 Phenotypic analysis results of Arabidopsis thaliana plants with overexpressing genes under stress;

[0024] Figure 8 ground melon CtBBX4 Analysis results of fresh weight and chlorophyll content of Arabidopsis thaliana plants under stress due to gene overexpression;

[0025] Figure 9 ground melon CtBBX4 Analysis of physiological indicators of Arabidopsis thaliana plants with overexpressing genes under stress;

[0026] Figure 10 ground melon CtBBX4 Results of gene-silenced plant identification; where A represents PCR identification results of the silent fragment; and B represents the expression level analysis results of the silent plant.

[0027] Figure 11 ground melon CtBBX4 Phenotypic analysis results of gene-silenced plants under stress;

[0028] Figure 12 ground melon CtBBX4 Analysis results of fresh weight and chlorophyll content of gene-silenced plants under stress;

[0029] Figure 13 ground melon CtBBX4 Analysis results of relevant physiological indicators of gene-silenced plants under stress;

[0030] In the picture This indicates that the data shows a significant difference. p <0.01. Detailed Implementation

[0031] This invention provides a CtBBX4 protein associated with plant salt tolerance, the amino acid sequence of which is shown in SEQ ID. Shown in NO.1: MGMQEPAAAASGGGANNNSNKSCFSAGWGLVAKLCDYCNCAAALLFCRTDSAFMCMACDSKFHAANIKLGSKHERVWMCEVCEQAPASVTCKADAAALCVTCDRDIHSANPLARRHERSPVVPFYETAESVVKSTAAAASFLVPPMSSGDHTNNNNNNSSDNSVLVMTCSVDHDDIKLGTRLAHDSYVTDSWISSMDPTAPKVPVDAPDIK SMEFLFSDSDNYLDFDYPINSSETRFQQHYGSGTDGVVPVQSFKPPIPAQLPGHSTEKHFEIDFTKSQISSYNNSYNPQSISHSVSSSSLDVGVVPDGSCMSDISYPYG RSMSSSSGLDLNGSVGGINQGSQMVGMDREARVLRYREKRKNRKFEKTIRYASRKAYAETRPRIKGRFAKRTDQIESDIRSIDRMFSPPSSSSSAFYTESRFGVVPSF. The CtBBX4 protein described in this invention is located in the cell nucleus, which can improve the antioxidant capacity of plants and maintain photosynthetic efficiency, effectively reduce the accumulation of ROS (reactive oxygen species) under salt stress, thereby alleviating oxidative damage and improving the salt tolerance of plants. It can be used to cultivate new germplasm of salt-tolerant plants, which is of great significance for carrying out salt-tolerant crop breeding.

[0032] This invention also provides a method related to plant salt tolerance. CtBBX4 Genes, the ones mentioned CtBBX4

[0033] The present invention also provides the CtBBX4 protein described in the above-described scheme or the [other protein described above]. CtBBX4 Application of genes in regulating plant salt tolerance.

[0034] As one implementation method, the regulation includes: increasing the CtBBX4 protein content or increasing... CtBBX4 Gene expression levels increase plant salt tolerance.

[0035] In one embodiment, the improvement of plant salt tolerance includes at least one of the following: (1) increasing germination rate; (2) increasing plant growth; and (3) improving antioxidant capacity. In another embodiment, the improvement of antioxidant capacity includes at least one of the following: (1) reducing malondialdehyde accumulation; (2) reducing superoxide anion accumulation; (3) reducing hydrogen peroxide accumulation; and (4) increasing antioxidant enzyme activity. In another embodiment, the antioxidant enzyme includes at least one of superoxide dismutase, catalase, and peroxidase. The results of the examples show that overexpression... CtBBX4 Compared to the wild type, the strains showed significantly lower accumulations of malondialdehyde, superoxide anion, and hydrogen peroxide, and higher activities of superoxide dismutase, catalase, and peroxidase. CtBBX4 By enhancing antioxidant capacity and maintaining photosynthetic efficiency, the accumulation of ROS under salt stress conditions was effectively reduced, thereby mitigating oxidative damage and improving the salt tolerance of plants.

[0036] As a preferred embodiment, the plant includes *Gnaphalium affine* and / or *Arabidopsis thaliana*.

[0037] The present invention also provides a recombinant expression vector, wherein the recombinant expression vector comprises the components described above. CtBBX4 Gene. As one implementation, the base vector of the recombinant expression vector includes pYES2.

[0038] The present invention also provides a recombinant bacterium, which comprises the recombinant expression vector described in the above-described scheme. As one embodiment, the base bacterium of the recombinant bacterium includes Agrobacterium. In a specific embodiment of the present invention, the base bacterium of the recombinant bacterium is Agrobacterium GV3103.

[0039] This invention also provides the CtBBX4 protein described in the above-described scheme, and the aforementioned... CtBBX4 The application of the gene, the recombinant expression vector, or the recombinant bacteria in the cultivation of salt-tolerant plant varieties.

[0040] The present invention also provides a method for improving the salt tolerance of plants, comprising the following steps: transferring the recombinant expression vector or the recombinant bacteria described in the above scheme into the plant.

[0041] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1 CtBBX4 Subcellular localization and transcriptional activation

[0043] (1) CtBBX4 Subcellular localization

[0044] Build CtBBX4 The fusion expression vector with green fluorescent protein (GFP) is processed as follows:

[0045] ① Using *Citrus aurantiacus* genomic DNA as a template, PCR amplification was performed using primers CtBBX4-eGFP-F (SEQ ID NO.5) and CtBBX4-eGFP-R (SEQ ID NO.6) listed in Table 1 to obtain samples containing... CtBBX4 The target segment of a gene.

[0046] Table 1 Primer sequence information

[0047]

[0048] ② The PCAMBIA1300-35S-eGFP vector (purchased from Beijing Coollife Technology Co., Ltd., product number: VT119, denoted as 35S:eGFP) was double-digested with restriction endonucleases KpnI and XbaI, and the linearized PCAMBIA1300-eGFP vector was recovered.

[0049] ③The ingredients described in step ① CtBBX4 The target fragment of the gene and step ② linearize the PCAMBIA1300-35S-eGFP vector for homologous recombination to obtain the recombinant vector, denoted as 35S:CtBBX4-eGFP.

[0050] ④ The recombinant vector was transferred into Agrobacterium GV3103 to obtain recombinant Agrobacterium bacterial suspension.

[0051] ⑤ Use a resuspension to measure the OD of the recombinant Agrobacterium tumefaciens culture obtained in step ④. 600 The value was adjusted to 0.8 to obtain the inoculum; the resuspension consisted of the following components at the following concentrations: MgCl2·6H2O 2.033 g / L, 2-(N-morpholine)ethanesulfonic acid (MES) 2.132 g / L and acetylsyl syringone 200 µM.

[0052] ⑥ Mark the leaves with numbers, and use a 1mL syringe to inject the infection solution and the nuclear localization marker mCherry (concentration of OD) from the back of the leaf using pressure. 600=0.8) was injected into tobacco leaves at a volume ratio of 1:1, then the leaves were sprayed with water, covered with a plastic bag, and placed in the dark overnight. On the second day, the plastic bag was opened, and the leaves were observed under a laser confocal microscope; an empty vector (35S:eGFP) was set up as a control group. The results are shown below. Figure 1 From left to right: green fluorescent protein, cell nuclear localization signal, merged image and bright field image, scale bar is 20 μm.

[0053] The results showed that the fluorescence signal of the CtBBX4-GFP fusion protein appeared only in the cell nucleus, while the fluorescence signal of the empty vector control appeared in both the cytoplasm and the cell nucleus, indicating that the CtBBX4 protein is located in the cell nucleus.

[0054] (2) To further verify the transcriptional activation characteristics of CtBBX4, a yeast transcriptional activation experiment was conducted, with the following steps:

[0055] ① Construct the CtBBX4-pGBKT7 recombinant plasmid. The construction method is similar to that of the recombinant vector 35S:CtBBX4-eGFP in step 1. The difference is that the primers used to amplify the target fragment are CtBBX4-BDF (SEQ ID NO.7) and CtBBX4-BDR (SEQ ID NO.8) in Table 1. The PCAMBIA1300-eGFP vector is replaced with the pGBKT7 vector (purchased from Beijing Cooler Master Technology Co., Ltd., product number: VT006). The restriction endonucleases are EcoRI and BamHI.

[0056] ② The recombinant plasmid CtBBX4-pGBKT7 and the empty vector pGBKT7 (negative control) were transformed into yeast Y2Hgold, respectively. After transformation, the yeast was cultured on a medium lacking tryptophan (denoted as SD-Trp) and a medium lacking tryptophan, histidine, and adenine (denoted as SD-Trp-Ade-His). The SD-Trp was purchased from Beijing Coolplay Technology Co., Ltd., product number: PM2252; the SD-Trp-Ade-His was purchased from Beijing Coolplay Technology Co., Ltd., product number: PM2302.

[0057] See results Figure 2 The results showed that both the CtBBX4-pGBKT7 recombinant plasmid and the empty vector could grow normally on SD-Trp medium; however, only yeast cells containing the CtBBX4-pGBKT7 recombinant plasmid could grow on SD-Trp-Ade-His medium, indicating that CtBBX4 has transcriptional activating activity. In conclusion, CtBBX4 is a transcriptional activator located in the cell nucleus.

[0058] Example 2 Overexpression CtBBX4 It enhanced the yeast's resistance to NaCl.

[0059] In containing CtBBX4 Overexpression vector (pYES2- CtBBX4 In yeast, analysis was performed. CtBBX4 The effects of proteins on yeast growth and stress resistance were investigated through the following steps:

[0060] ①Construction CtBBX4 The overexpression vector was constructed using the same method as the recombinant vector 35S in step (1) of Example 1. CtBBX4 Similar to -eGFP, the difference lies in the primers used to amplify the target fragment, which are pYES2- from Table 1. CtBBX4 -F (SEQ ID NO.3) and pYES2- CtBBX4 -R (SEQ ID NO.4), replacing the PCAMBIA1300-eGFP vector with the pYES2 vector (purchased from Beijing Coollife Technology Co., Ltd., product number: VT064), with restriction endonucleases BamHI and EcoRI.

[0061] ② Take 100µL of INVSC1 competent cells thawed on ice (Beijing Cooler Master Technology Co., Ltd., product number: CC303), and add the pre-cooled target plasmid (pYES2-) sequentially. CtBBX4 Add 2~5µg of pYES2), 10µL of carrier DNA, and 500µL of PEG / LiAc. Mix well by pipetting several times, then incubate at 30℃ for 30 min and at 42℃ for 15 min.

[0062] ③ Centrifuge at 10,000 rpm for 30 seconds, discard the supernatant, resuspend in 400 µL of ddH2O, centrifuge for 30 seconds, and discard the supernatant.

[0063] ④ Resuspend the culture in 50 µL of ddH2O, plate it onto an SD / -Ura plate, screen for positive clones, and then inoculate it into 15 mL of SD / -Ura Broth yeast auxotrophic liquid medium. Incubate overnight at 30°C with a shaker. The SD / -Ura plates were purchased from Beijing Cooler Master Technology Co., Ltd., product number: PM2272; the SD / -Ura Broth yeast auxotrophic liquid medium was purchased from Beijing Cooler Master Technology Co., Ltd., product number: PM2271.

[0064] ⑤ Centrifuge at 1,500g for 5 minutes at 4℃ and remove the supernatant.

[0065] ⑥ Resuspend the cell pellet in 50 mL of SG / -Ura liquid medium and incubate in a shaker at 30 °C.

[0066] ⑦OD 600Adjusted to 1, and serially diluted 10-fold, the growth of yeast was evaluated on SD / -Ura Broth yeast auxotrophic solid and liquid media containing different concentrations of NaCl (0 mM, designated as control and 1 M). Results are shown in [Figure number missing]. Figure 3 Table 2 shows the growth and concentration (OD) of overexpressed yeast and empty vector yeast on the culture medium under normal conditions, as well as A and B. 600 Figure C and D show the growth and concentration of overexpressed yeast and empty vector yeast on the culture medium under salt stress. 600 Figure; specifically, A and C correspond to results on solid culture medium, and B and D correspond to results on liquid culture medium.

[0067] Table 2. Functional validation data of yeast overexpression of CtBBX4 gene from *Cucumis melo*.

[0068]

[0069] The results showed that CtBBX4 Overexpression had no significant effect on yeast growth. Figure 3 (A) CtBBX4 Overexpressing yeast cells exhibited significant resistance in media containing NaCl (1M), especially at a 100-fold dilution. Figure 3 (C). Furthermore, under liquid culture conditions, the transformed... CtBBX4 Survival rate tests on the two yeast strains revealed that, under normal, salt-free conditions, their growth trends were essentially the same. Figure 3 (B) Under NaCl stress, CtBBX4 The survival rate of the overexpressing yeast strain was consistently higher than that of the control group. Specifically, under a stress level of 1M NaCl, after 9 hours of stress treatment, CtBBX4 The survival rate of the overexpressing yeast strain was significantly higher than that of the empty vector control strain. Figure 3 (D). These results indicate that CtBBX4 Proteins enhance yeast's ability to resist salt stress.

[0070] Example 3 CtBBX4 Genetic heterologous transformation of Arabidopsis thaliana and screening of positive plants

[0071] Constructed using the method of Example 1 CtBBX4 Plant expression vector 35S:CtBBX4-eGFP, vector map as follows Figure 4 As shown in Figure A, Arabidopsis thaliana (Columbia type) was transformed using an inflorescence immersion method mediated by Agrobacterium GV3103. T0 generation transgenic plants were screened on 1 / 2 MS medium containing 50 mg / L HYG (hygromycin). T1 and T2 generations were screened consecutively using the same method to obtain homozygous transgenic lines.

[0072] To verify the transgenic plants, homozygous samples were extracted in this embodiment. CtBBX4 Genomic DNA from transgenic Arabidopsis and wild-type Arabidopsis was used. CtBBX4 PCR amplification was performed using specific primers (CtBBX4-eGFP-F (SEQ ID NO.5) and p1300-R (SEQ ID NO.11) from Table 1). The PCR reaction system was as follows: 25 μL of 2×TransStart® FastPfu Fly ReactionMix, 1 μL of TransStart® FastPfu Fly DNA Polymerase, 1 μL each of forward and reverse primers, 1 μL of cDNA, and 21 μL of ddH2O. The reaction program was: 98℃ for 1 min; 98℃ for 10 s, 60℃ for 5 s, 72℃ for 10 s, 30 cycles; 72℃ for 1 min; and stored at 4℃. The results are as follows. Figure 4 As shown in B, where +: with CtBBX4 The amplification product of the plant expression vector template; OE1-6: CtBBX4 Transgenic line; WT wild-type Arabidopsis thaliana. All were identified as positive plants.

[0073] Simultaneously, total RNA was extracted from Arabidopsis transgenic homozygous lines that were positive for HYG screening and molecular identification. Using primers CtBBX4-qPCR-F (SEQ ID NO.9) and CtBBX4-qPCR-R (SEQ ID NO.10) listed in Table 1, qRT-PCR was performed to detect RNA. CtBBX4 For gene expression levels, the primers for the internal reference genes were Atactin-F (SEQ ID NO.14) and Atactin-R (SEQ ID NO.15) listed in Table 1. The qRT-PCR reaction system was as follows: 12.5 μL of TB Green Premix Ex Taq II FastqPCR (2×), 1 μL each of forward and reverse primers, 1 μL of cDNA, and 9.5 μL of ddH2O. The reaction program was as follows: 25℃ for 10 min; 95℃ for 30 s; 95℃ for 5 s, 60℃ for 10 s, 40 cycles; 95℃ for 15 s; 60℃ for 1 min; 95℃.

[0074] Test results Figure 5 As shown in Table 3, the results indicate that the overexpressing plants were significantly different from those with WT. p <0.01). This indicates that the overexpressing plants were successfully heterologously transformed. CtBBX4 The positive plants for the gene provided reliable experimental material for subsequent functional analysis.

[0075] Table 3. Expression levels of the CtBBX4 gene overexpressed in Arabidopsis thaliana plants.

[0076]

[0077] Example 4 CtBBX4 Overexpression enhances salt tolerance in Arabidopsis thaliana.

[0078] Mature T3 generation seeds and wild-type (WT) Arabidopsis seeds were collected, air-dried, and vernalized at 4℃ for 2 days (in a 4℃ incubator). After vernalization, the seeds were treated with 70% ethanol for 10 min consecutively, followed by anhydrous ethanol for 20 min consecutively, to sterilize the seed surface. The sterilized Arabidopsis seeds were sown on 1 / 2 MS solid medium (10cm × 10cm square plates) with 150mM NaCl added. A stress group (NaCl group) with only 150mM NaCl and a control group without NaCl were also established. Both groups of seeds were placed in a growth chamber at 24℃, 16 h light, 8 h dark, and 70% relative humidity for germination experiments. Each group was tested in triplicate, and the germination rate was recorded every 24 h. The results are shown below. Figure 6 As shown in Table 4.

[0079] Transgenic Arabidopsis thaliana seedlings with consistent growth and wild-type WT seedlings were randomly divided into two groups: a control group and a salt stress group. The Arabidopsis thaliana in the salt stress group was subjected to simulated salt stress by irrigating with 200 mM sodium chloride, and their growth under salt stress was observed. Results are shown below. Figure 7 As shown.

[0080] Table 4 Germination of Arabidopsis seeds overexpressing the CtBBX4 gene.

[0081]

[0082] The results showed that under normal growth conditions, there were no significant differences in growth status and phenotype between wild-type and overexpression lines. However, after salt stress treatment, the growth of both wild-type and overexpression lines was significantly inhibited, manifested as stunted growth and yellowing of leaves due to dehydration, although the overexpression lines experienced less inhibition. Under salt stress, although NaCl treatment inhibited the germination rate of Arabidopsis seeds and prolonged their germination time, the germination rate of transgenic plant seeds was significantly higher than that of wild-type plants. These results indicate that... CtBBX4 Overexpression of this compound significantly enhanced the plant's resistance to salt stress. This indicates that... CtBBX4 It plays an important role in the salt stress response process.

[0083] Example 5 CtBBX4 Enhanced physiological indicators and antioxidant capacity of transgenic Arabidopsis thaliana

[0084] In order to conduct a comprehensive assessment CtBBX4The role of Arabidopsis thaliana in salt tolerance: This example focuses on plants grown in nutrient soil. CtBBX4 The transgenic lines were subjected to salt stress treatment, as follows:

[0085] Mature T3 generation seeds and wild-type (WT) Arabidopsis seeds were collected, air-dried, and vernalized at 4℃. Before sowing onto 1 / 2 MS solid medium, the seeds were surface-sterilized by treatment with 70% ethanol for 10 min followed by anhydrous ethanol for 20 min. The sterilized seeds were sown in 1 / 2 MS medium and transferred to a growth chamber at 24℃, with 16 h of light, 8 h of darkness, and 70% relative humidity. At four true leaves, the seeds were transplanted into a nutrient soil:vermiculite ratio of 1:3 (v / v) for cultivation. Salt stress was applied 14 days after transplanting by irrigating the plants with 150 mM NaCl solution. A control group without salt stress was also included. After 14 days of cultivation, the plants were observed, fresh weight was recorded, and chlorophyll content was measured. The results are shown below. Figure 8 As shown in Table 5.

[0086] Salt stress typically leads to excessive accumulation of reactive oxygen species (ROS) in plants, causing severe oxidative damage. The content of superoxide anion (H2O2) and hydrogen peroxide (O2) in Arabidopsis leaves was detected using a kit from Beijing Solarbio Science & Technology Co., Ltd. - The contents of malondialdehyde (MDA), catalase (CAT) activity, superoxide dismutase (SOD) activity, and peroxidase (POD) activity were measured, and the results are as follows: Figure 9 As shown in Table 6.

[0087] Table 5. Analysis of fresh weight and chlorophyll content of Arabidopsis thaliana plants overexpressing the CtBBX4 gene under stress.

[0088]

[0089] Table 6. Analysis of relevant physiological indicators of Arabidopsis plants overexpressing the CtBBX4 gene under stress.

[0090]

[0091] The results showed that under normal conditions, there were no significant changes in leaf fresh weight and chlorophyll content between wild-type and overexpression lines, while the fresh weight and chlorophyll content of the overexpression lines were significantly higher than those of the wild-type lines after stress. Figure 8Salt stress significantly increased the levels of malondialdehyde (MDA), superoxide anion (SAO), and hydrogen peroxide (HPO) in Arabidopsis leaves. Compared with wild-type plants, the overexpression lines showed significantly lower accumulations of MDA, SAO, and HPO. Furthermore, salt stress significantly increased the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase in both wild-type and overexpression lines. Compared with wild-type, the overexpression lines showed significantly higher activities of these antioxidant enzymes. Figure 9 These results indicate that CtBBX4 By enhancing antioxidant capacity and maintaining photosynthetic efficiency, the accumulation of ROS under salt stress conditions was effectively reduced, thereby mitigating oxidative damage and improving the salt tolerance of plants.

[0092] Example 6 CtBBX4 Silent carrier construction

[0093] In containing CtBBX4 Silencing vector (pTRV2-) CtBBX4 In the ground-top melons, analysis was performed. CtBBX4 The effects of protein on the growth and stress resistance of ground-shoot cucumbers were investigated through the following steps:

[0094] Build CtBBX4 The method for constructing the silencing vector is the same as that for the recombinant vector 35S in Example 1: CtBBX4 Similar to -eGFP, the difference lies in the primers used to amplify the target fragment, which are pTRV2- from Table 1. CtBBX4 -F (SEQ ID NO.12) and pTRV2- CtBBX4 -R (SEQ ID NO.13) replaces the PCAMBIA1300-eGFP vector with the pTRV2 vector, and the restriction endonucleases are XbaⅠ and KpnⅠ.

[0095] TRV vector systems typically contain two plasmids: pTRV1 (helper vector, providing replication and movement proteins) and pTRV2 (vector backbone, used for inserting the target gene fragment). The target gene fragment needs to be cloned into pTRV2, working together with pTRV1 to achieve silencing. Fragment selection: A specific 300bp fragment of the target gene is selected. The ligation product is transformed into E. coli, positive clones are selected by antibiotic resistance plate screening, and after sequencing verification, the recombinant pTRV2 plasmid is extracted (pTRV1 plasmid is prepared simultaneously).

[0096] The TRV2 plasmid was double-digested with restriction endonucleases KpnI and Xba1. The vector was ligated using a homologous recombination kit and transformed into competent *E. coli* cells. After antibiotic selection, positive single colonies were picked and detected by PCR using vector primers. The TRV2-Ct vector successfully ligated the insert fragment... BBX4 The product obtained by PCR amplification of the recombinant plasmid is 300 bp in length (the insert fragment is 300 bp in length). Figure 10In A, where +: TRV2-Ct BBX4 Recombinant plasmid; pTRV:CtBBX4: silent plant, WT: wild-type ground squash), the inserted fragment is SEQ ID NO.16: 5'-TCAGATAACAGCGTGCTAGTCATGACTTGTAGCGTCGACCACGATGATATCAAGCTCGGCACGCGCCTTGCACATGATAGTTACGTCACCGATTCGTGGATCTCGTCGATGGATCCAACGGCTCCCAAAGTTCCGGTGGATGCTCCGGACATTAAATCCATGGAGTTTCTGTTTTCTGATTCGGACAATTATCTCGATTTCGATTACCCAATTAATTCGTCCGAAACTCGTTTTCAACAACATTACGGTTCGGGAACAGACGGCGTCGTTCCGGTACAGTCATTCAAACCCCCAATTCCT-3'.

[0097] Will carry TRV1 and TRV2-Ct BBX4 Agrobacterium GV3101 was cultured overnight at 28°C with shaking at 180 rpm in LB medium containing kanamycin (50 μg / mL) and rifampin (25 μg / mL). The bacterial cells were collected by centrifugation, resuspended in an invasion solution (containing 10 mmol / L MgCl2, 10 mmol / L MES, and 200 μmol / L acetylsyleugenone), and the OD600 was adjusted to approximately 1.0. The cells were then incubated in the dark for 4–6 h in preparation for injection infection.

[0098] Take equal volumes of pTRV1 Agrobacterium resuspension and recombinant viral vector pTRV2-Ct BBX4Agrobacterium resuspension was thoroughly mixed and used for injection infection. Ground elm seedlings cultured to the cotyledon stage in a light incubator were used as the infection material. A disposable 1mL needleless syringe was used to inject the bacterial solution into the apical meristem of the seedlings. Before injection infection, slight wounds could be created before injecting the bacterial solution into the apical meristem. Infected plants were then cultured in a light incubator at 22℃, 50% humidity, and a photoperiod of L:D (light:dark) -16h:8h. The expression level of CtBBX4 was detected by RT-qPCR. The primers used are shown in Table 1: CtBBX4-qPCR-F (SEQ ID NO. 9) and CtBBX4-qPCR-R (SEQ ID NO. 10). The qRT-PCR reaction system was as follows: 12.5 μL of TBGreen Premix Ex Taq II Fast qPCR (2X), 1 μL of each of the forward and reverse primers, 1 μL of cDNA, and 9.5 μL of ddH2O. The reaction program was as follows: 25℃ for 10 min, 95℃ for 30 s, [95℃ for 5 s, 60℃ for 10 s] for 40 cycles, 95℃ for 15 s, 60℃ for 1 min, and 95℃.

[0099] Test results as follows Figure 10 As shown in Table B and Table 7, the results indicate that silencing CtBBX4 The gene strain was successfully constructed.

[0100] Table 7 Ground-top melon CtBBX4 Gene silencing plant expression level analysis

[0101]

[0102] Example 7: Silence CtBBX4 It reduced the resistance of ground cucumber to NaCl.

[0103] Operational investigation based on Example 5 CtBBX4 The phenotype, physiological indicators, and antioxidant capacity of *Melon simonii* after gene silencing were analyzed, and the results are as follows: Figures 11-13 As shown in Tables 8 and 9.

[0104] Table 8 Ground-top melon CtBBX4 Analysis of fresh weight and chlorophyll content of gene-silenced plants under stress

[0105]

[0106] Table 9 Ground-top melon CtBBX4 Analysis of relevant physiological indicators of gene-silenced plants under stress

[0107]

[0108] The results indicate that TRV-mediated basalt melon... CtBBX4In gene silencing experiments, under normal control conditions, there were no significant differences in growth phenotype between wild-type (WT), empty vector TRV-infected plants, and pTRV2-CtBBX4 plants; however, after salt stress treatment, the growth of pTRV2-CtBBX4 plants was more significantly inhibited, exhibiting more severe wilting symptoms. Figure 11 Physiological analysis further showed that the fresh weight and chlorophyll content of pTRV2-CtBBX4 plants under salt stress were significantly lower than those of WT and TRV plants. Figure 12 ), malondialdehyde (MDA), superoxide anion (O2) 2- Oxidative damage indicators such as hydrogen peroxide (H2O2) were significantly elevated, while the antioxidant enzyme activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were significantly reduced. Figure 13 ).

[0109] In conclusion, CtBBX4 Genes play a crucial role in regulating salt stress tolerance in *Melia azedarach*. Silencing these genes reduces salt stress tolerance by influencing the activity of antioxidant enzyme systems and the degree of oxidative damage. Overexpression of these genes in plants can also contribute to salt stress tolerance. CtBBX4 Genes can improve a plant's salt tolerance.

[0110] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A CtBBX4 protein associated with plant salt tolerance, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. A CtBBX4 gene associated with plant salt tolerance, the nucleotide sequence of which is shown in SEQ ID NO.

2.

3. The application of the CtBBX4 protein of claim 1 or the CtBBX4 gene of claim 2 in regulating plant salt tolerance; wherein the plant is Cucurbita moschata and / or Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, The regulation includes increasing the CtBBX4 protein content or increasing the CtBBX4 gene expression level to improve plant salt tolerance.

5. The application according to claim 4, characterized in that, The improvement of plant salt tolerance includes at least one of the following: (1) increasing germination rate; (2) increasing plant growth; and (3) improving antioxidant capacity.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains the CtBBX4 gene as described in claim 2.

7. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the recombinant expression vector of claim 6.

8. The application of the CtBBX4 protein of claim 1, the CtBBX4 gene of claim 2, the recombinant expression vector of claim 6, or the recombinant bacteria of claim 7 in the cultivation of salt-tolerant plant varieties; wherein the plant is Cucurbita spp. and / or Arabidopsis thaliana.

9. A method for improving the salt tolerance of plants, characterized in that, The procedure includes the following steps: transferring the recombinant expression vector of claim 6 or the recombinant bacteria of claim 7 into a plant; wherein the plant is Cucurbita moschata and / or Arabidopsis thaliana.

10. A transgenic plant, characterized in that, The transgenic plant overexpresses the CtBBX4 gene; the nucleotide sequence of the CtBBX4 gene is shown in SEQ ID NO.2; the plant is Cucurbita moschata and / or Arabidopsis thaliana.

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