Application of salix psammophila SpsRLCK1 interaction protein SpsBBX3 in improving drought tolerance of plants

Abstract

The invention discloses an application of an interaction protein SpsBBX3 of salix psammophila SpsRLCK1 in improving the drought tolerance of plants. By overexpressing the salix psammophila SpsBBX3 gene in the plant, the drought tolerance of the plant can be remarkably improved. Through contrastive analysis of growth and physiological indexes, including root length, fresh weight, dry weight, plant height, relative water content, relative conductivity, antioxidant enzyme activity and the like, of SpsBBX3 overexpression transgenic poplar and wild 84K poplar under drought stress, it is found that the growth state of the transgenic plant under the drought condition is remarkably superior to that of the wild type poplar; physiological indexes also show that the drought resistance is obviously enhanced. The invention reveals that the SpsBBX3 gene can specifically improve the resistance of plants under drought stress, a new variety of drought-resistant transgenic SpsBBX3 gene can be cultivated, and important theoretical basis and practical value are provided for cultivation of new drought-resistant transgenic varieties in forest molecular breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an interacting protein SpsBBX3 of Salix psammophila SpsRLCK1 and applications thereof. Background Art

[0002] Salix psammophila is a shrub-type plant of the Salicaceae family native to western China. Known for its extensive root system, rapid growth, and excellent resistance to drought, cold, and wind erosion, it is an essential tree species in the "Three Norths Shelterbelt Project" and is widely planted in China's desertified regions. Salix psammophila plays a key role in improving the ecological environment and socioeconomic benefits of western China. Exploring the mechanisms by which Salix psammophila adapts to harsh environments is of great significance for ecological restoration, soil quality improvement, and economic benefits in arid and semi-arid regions.

[0003] RLCKs are a major class of signaling proteins that regulate plant cell activity in response to biotic and abiotic stress signaling molecules and plant growth. Currently, SpsRLCK1 has been shown to enhance plant stress tolerance by regulating antioxidant enzymes and stress-related genes, but its molecular mechanisms remain largely unresolved, particularly with a lack of systematic identification of interacting proteins.

[0004] Members of the B-box (BBX) family participate in responses to abiotic stresses. In atbbx18 mutants, AtBBX18 weakens its repressive effects on genes such as HsfA2 (Heat stress transcription factor A2) and Hsp101 (Heat shock protein 101), positive regulators of heat tolerance, resulting in enhanced heat tolerance. In sweet potato, IbBBX24 binds to the promoter region of the catalase gene IbPRX17 (Peroxidase 17), activating its expression. The dimer formed by IbBBX24's interaction with the IbTOE3 (Target of Early Activation Tagged 3) transcription factor enhances IbBBX24's positive regulation of IbPRX17, increasing antioxidant enzyme activity under drought and salt stresses and improving plant resilience. In chrysanthemum, CmBBX22 negatively regulates drought resistance by regulating abscisic acid response, stomatal conductance, and antioxidant responses. Drought conditions induce the expression of MdBBX7 in apple, which enhances drought tolerance by directly regulating the expression of drought-resistant genes such as MdERF1. Studies have shown that MdBBX7 interacts with MdMIEL1 (MYB30-Interacting E3 Ligase 1), leading to its ubiquitination and degradation. This interaction weakens MdBBX7's ability to activate drought-resistant genes such as MdERF1. Therefore, we hypothesize that BBXs isolated from Salix psammophila may also play an important role in regulating stress and drought tolerance in plants, but their functions remain unknown. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new BBX gene derived from Salix psammophila and its use.

[0006] The technical solution of the present invention is: Salix psammophila SpsBBX3 protein, the amino acid sequence of which is shown in SEQ ID No.2.

[0007] The SpsBBX3 gene encoding the above-mentioned Salix psammophila SpsBBX3 protein.

[0008] Furthermore, the nucleotide sequence of the SpsBBX3 gene is shown in SEQ ID No. 1.

[0009] An expression vector containing the above-mentioned SpsBBX3 gene.

[0010] The use of the above-mentioned SpsBBX3 gene or expression vector in improving plant drought tolerance.

[0011] Furthermore, the drought tolerance of plants is improved by overexpressing the Salix psammophila SpsBBX3 gene in plants.

[0012] Furthermore, the plant is willow or poplar.

[0013] Furthermore, the plant is Salix psammophila or Populus white poplar.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention successfully identified SpsBBX3 through yeast two-hybrid screening of proteins interacting with SpsRLCK1. By observing the cell damage and physiological indicators of SpsBBX3 transgenic poplars and wild-type poplars under drought stress, it was found that the transgenic poplars suffered significantly less cell damage under drought stress than the wild-type poplars. Physiological indicators also confirmed that the SpsBBX3-overexpressing strains had superior stress resistance to wild-type poplars. This discovery reveals that SpsBBX3 specifically enhances poplar resistance to drought stress and provides important theoretical and practical implications for the development of new drought-resistant transgenic varieties in the field of forest molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the yeast two-hybrid screening result of SpsRLCK1 provided in Example 1 of the present invention.

[0017] Figure 2 This is the result of a dual luciferase assay for detecting the interaction between SpsRLCK1 and SpsBBX3 provided in Example 1 of the present invention.

[0018] Figure 3 The real-time fluorescence quantitative PCR analysis provided in Example 3 analyzed the expression level of the Salix psammophila SpsBBX3 gene under drought stress.

[0019] Figure 4 This is a schematic diagram of the quantitative data of the expression of transgenic SpsBBX3 poplar provided in Example 5 of the present invention.

[0020] Figure 5 This is a phenotypic comparison of the transgenic poplar provided in Example 6 of the present invention and the 84K poplar after drought treatment.

[0021] Figure 6 Comparison of root lengths of the transgenic poplar and 84K poplar after drought treatment provided in Example 6 of the present invention.

[0022] Figure 7 Comparison of fresh weight of transgenic poplars and 84K poplars after drought treatment provided in Example 6 of the present invention.

[0023] Figure 8Comparison of dry weight of the transgenic poplar and 84K poplar after drought treatment provided in Example 6 of the present invention.

[0024] Figure 9 This is a phenotypic comparison of transgenic poplars and 84K poplars under short-term drought and normal growth (control) in the soil provided in Example 6 of the present invention.

[0025] Figure 10 The height comparison of the transgenic poplar and the normal growth (control) of 84K poplar in the soil provided in Example 6 of the present invention is shown.

[0026] Figure 11 This is a comparison of the plant heights of transgenic poplars and 84K poplars in the soil under short-term drought conditions provided in Example 6 of the present invention.

[0027] Figure 12 This is a schematic diagram of the relative electrical conductivity of transgenic poplar and 84K poplar after drought treatment provided in Example 6 of the present invention.

[0028] Figure 13 This is a schematic diagram of the relative water content of transgenic poplar and 84K poplar after drought treatment provided in Example 6 of the present invention.

[0029] Figure 14 Schematic diagram of DAB and NBT staining of leaves of transgenic poplar and 84K poplar after drought treatment provided in Example 6 of the present invention, wherein A is DAB staining and B is NBT staining.

[0030] Figure 15 Schematic diagram of CAT content in leaves of transgenic poplar and 84K poplar after drought treatment provided in Example 6 of the invention.

[0031] Figure 16 Schematic diagram of POD content in leaves of transgenic poplar and 84K poplar after drought treatment provided in Example 6 of the invention.

[0032] Figure 17 Schematic diagram of SOD content in leaves of transgenic poplar and 84K poplar after drought treatment provided in Example 6 of the invention. DETAILED DESCRIPTION

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0034] Example 1 Screening and Verification of the Interacting Protein SpsBBX3 of Salix psammophila SpsRLCK1

[0035] 1.1 Yeast two-hybrid screening of interacting proteins of Salix psammophila SpsRLCK1

[0036] BD-SpsRLCK1 and AD-BBX3 cell cDNA library plasmids were co-transformed into yeast Y2H Gold competent cells; pGBKT7-53 and pGADT7-T were co-transformed into yeast Y2H Gold competent cells as positive controls, and pGBKT7 and pGADT7T were co-transformed into yeast Y2H Gold competent cells as negative controls. The bacterial liquid of each transformant was spread on a selective medium lacking leucine and tryptophan to ensure that the yeast cells could grow. Further screening was performed on a selective medium lacking leucine, tryptophan and histidine to confirm the interaction between proteins. Finally, screening was performed on a selective medium lacking leucine, tryptophan, histidine and adenosine, and X-α-gal dye was added to verify the existence of the interaction. The results are shown in Figure 2. Figure 1 As shown, the experimental group obtained blue positive clones, and the plasmids were extracted and sequenced to obtain the SpsRLCK1 interacting protein SpsBBX3.

[0037] 1.2 Dual luciferase assay to verify the interaction between SpsRLCK1 and SpsBBX3 proteins

[0038] SpsRLCK1 was fused to a CLuc (C-terminal Luciferase fragment) vector, and SpsBBX3 was fused to an NLuc (N-terminal Luciferase fragment) vector. The constructed vectors and the corresponding empty vectors were transformed into competent Agrobacterium GV3101 cells using the heat shock method. Transformed Agrobacterium were plated onto LB solid medium supplemented with appropriate antibiotics and cultured in a 28°C incubator until colonies formed. After verification, positive bacterial cultures were stored for future use. A single colony from the stored positive colonies was inoculated into LB liquid medium supplemented with antibiotics and cultured in a shaking incubator at 28°C for 6 hours. Next, 20 mL of fresh LB liquid medium supplemented with the same antibiotics was added to a 50 mL centrifuge tube, along with 200 μL of the cultured culture, and cultured for another 6 hours. After the incubation period, the cells were harvested by centrifugation at 6000 rpm for 10 minutes, resuspended twice in 1 / 2 MS liquid medium, and finally adjusted to an OD600 of 1.0 using infiltration buffer. The diluted bacterial solution was mixed in a 1:1 ratio and then allowed to stand in the dark for 2 hours. Four different bacterial solution combinations were prepared: I: SpsRLCK1-CLuc+SpsBBX3-NLuc; II: CLuc+SpsBBX3-NLuc; III: SpsRLCK1-CLuc+NLuc; IV: CLuc+NLuc. Healthy tobacco leaves were selected and a 1mL syringe was used to inject the mixed bacterial solution from the back of the leaves into different areas of the same leaf. Two days after infection, 1mm D-luciferin potassium salt was evenly sprayed on the back of each leaf and treated in a dark environment at 37°C for 7 minutes. The PlantView100 device was then used to scan and record the fluorescence signal intensity on the tobacco leaves. The results are shown as follows. Figure 2 The fluorescence signal intensity of the experimental group was stronger and significantly higher than that of the control group, indicating that there was an interaction between SpsBBX3 and SpsRLCK1.

[0039] Example 2 Cloning of the Salix psammophila SpsBBX3 gene

[0040] 2.1 Obtaining the target gene sequence

[0041] By comparing the sequences of Populus trichocarpa in the Phytozome database, we identified cDNA sequences homologous to Salix psammophila SpsBBX3. Based on the identified homologous sequences, we used Primer5 software to construct suitable primer pairs. These primers were then used with PCR to amplify the complete sequence of the target gene. The primers are shown in Table 1:

[0042] Table 1 Primers for SpsBBX3 gene cloning

[0043]

[0044] Using the cDNA reverse transcribed from total RNA extracted from Salix psammophila leaves as a template, gene cloning was performed using a high-fidelity enzyme PCR reaction system. The specific reaction system is shown in Table 2:

[0045] Table 2 Gene cloning PCR reaction system

[0046]

[0047] The PCR cloning reaction procedure is:

[0048]

[0049] The DNA fragments of the correct size were recovered using the Novezan DNA product purification kit.

[0050] 2.2 Target gene connected to T vector

[0051] The recovered target fragment is connected to the T vector. The reaction system includes:

[0052] Table 3 Reaction system of gene ligation to T vector

[0053]

[0054] The ligation reaction was carried out overnight at 16°C. After completion of the ligation reaction, the product was transformed into E. coli, and individual colonies were selected for PCR verification. Positive colonies were then confirmed by sequencing. Sequencing confirmed the nucleotide sequence shown in SEQ ID No. 1. This gene fragment was named SpsBBX3, consisting of 1113 base pairs and encoding 370 amino acids (SEQ ID No. 2).

[0055] Example 3 Analysis of expression characteristics of Salix psammophila SpsBBX3 gene (qRT-PCR)

[0056] 3.1 Primer design

[0057] Fluorescence quantitative PCR (qRT-PCR) was performed based on the obtained complete gene sequence, and quantitative primers and internal reference primers were designed using Primer3 software. The primers are shown in Table 4:

[0058] Table 4 Primer design for expression pattern analysis

[0059]

[0060] 3.2 Drought stress treatment of Salix psammophila tissue culture seedlings

[0061] Normally grown Salix psammophila tissue culture seedlings were transferred to a medium containing 100 mM Mannitol. Total RNA was extracted from the roots and reverse-transcribed into cDNA. qRT-PCR reactions were then performed using the following reaction system (20 μl): SYBR qPCR Master Mix, 10 μl; Quantitation F, 0.4 μl; Quantitation R, 0.4 μl; Salix psammophila cDNA, 2 μl; and ddH2O, 7.2 μl.

[0062] The PCR reaction program was set as: 95°C for 30 seconds, 95°C for 10 seconds, and 60°C for 30 seconds, for 39 cycles, and the instrument's default melting curve acquisition program was used.

[0063] The obtained data were analyzed, such as Figure 3 The results showed that the expression level of SpsBBX3 gene in Salix psammophila increased under drought stress treatment, so it was speculated that this gene might be induced by drought stress.

[0064] Example 4 Construction of Salix psammophila SpsBBX3 gene expression vector

[0065] 4.1 Primer design

[0066] To construct a plant overexpression vector, primers with homology arms to pCAMBIA1302 were designed. The primers are shown in Table 5:

[0067] Table 5 Primer sequences

[0068]

[0069] 4.2 Construction of pCAMBIA1302-SpsBBX3 overexpression vector

[0070] The SpsBBX3 gene was amplified using primers Overexpression F and Overexpression R. The product was recovered and the pCAMBIA1302 vector was double-digested and linearized. The target gene was then constructed into the vector using Novion's native recombinase. The reaction system is shown in Table 6:

[0071] Table 6 Reaction system

[0072]

[0073] The reaction procedure was: 37°C for 30 min and then cooled to 4°C or immediately placed on ice for cooling.

[0074] The ligated vector was transformed into E. coli, and single clones were selected for PCR verification and sequencing of positive colonies. The pCAMBIA1302-SpsBBX3 overexpression vector was successfully constructed.

[0075] Example 5 Genetic transformation of poplar SpsBBX3 gene

[0076] BBX3 was transformed into 84K poplar leaf discs via Agrobacterium-mediated transformation. BBX3-infected leaf discs were cultured on MS minimal medium (adventitious bud induction medium) supplemented with 0.5 mg / L 6-BA and 0.05 mg / L NAA in the dark (22 ± 2°C) for three days.

[0077] 2. After co-cultivation, the leaf discs were transferred to a co-culture medium containing 3 mg / L hygromycin and 200 mg / L timentin, and resistant adventitious buds were induced and screened under the conditions of a culture temperature of 23-25°C and a light intensity of 16 hours of light and 8 hours of darkness.

[0078] 3. After about 30 days of induction culture, the resistant adventitious buds were transferred to rooting medium (1 / 2 MS basic medium supplemented with 0.05 mg / L IBA and 0.02 mg / L NAA, pH = 5.8) containing 3 mg / L hygromycin and 200 mg / L timentin until adventitious roots were induced.

[0079] 4. cDNA was extracted from the leaves of rooted plants using the CTAB method and verified by PCR.

[0080] 5. Extract RNA from the 20 positive seedlings screened, and verify the transgenic expression level by qRT-PCR.

[0081] The results are as follows Figure 4 As shown, OE-1, OE-23, and OE-28 plants with appropriate expression levels were selected for subsequent phenotypic identification and analysis of drought resistance.

[0082] Example 6 Analysis of Drought Resistance of Poplar SpsBBX3 Transgenic Plants

[0083] 6.1 Root length, fresh weight, and dry weight of transgenic poplars

[0084] Poplars 84K and poplars transfected with the SpsBBX3 gene were cultured in culture bottles containing 0 mM and 200 mM mannitol (mannitol simulated drought), and their root lengths were observed and recorded. Figure 5 、 6 As shown in Figures 7 and 8, under normal conditions, there was a significant difference in root length between 84K poplars and transgenic SpsBBX3 poplars. Under drought conditions, the root length of transgenic SpsBBX3 poplars was significantly longer than that of 84K poplars. Measurements of the fresh and dry weights of the poplars revealed that both the fresh and dry weights of drought-exposed poplars were lower than those of poplars grown under normal conditions. Furthermore, after drought stress, the fresh and dry weights of the transgenic poplars were significantly higher than those of 84K poplars. This suggests that transgenic SpsBBX3 poplars are more likely to survive drought and are more drought-tolerant.

[0085] 6.2 Phenotypic analysis of transgenic poplars

[0086] 84K poplars and transgenic poplars OE-1, OE-23, and OE-28 were transplanted into peat soil: vermiculite = 3:1 and grown under drought treatment (the soil water content was maintained at 30-40% of field capacity (FC) for 20 days to induce drought. The soil RWC was maintained at 30-40% of field capacity, while the control group maintained the soil RWC at ≥70% of field capacity). After 20 days, the phenotypes of the poplars were observed and recorded, as shown in Figure 2. Figure 9 、 10 As shown in Figures 11 and 2, the roots of the transgenic poplars under drought stress were found to be more developed than those of the 84K poplars. Furthermore, the plant height of the poplars increased over time, with the transgenic poplars being taller than the 84K poplars. Furthermore, the plant height of the drought-treated poplars was higher than that of the poplars grown under normal conditions. This suggests that the transgenic poplars are more drought-tolerant than the 84K poplars.

[0087] 6.3 Determination of relative electrical conductivity of transgenic poplars

[0088] 1. Collect the normal growth and drought-treated poplar leaf samples in 6.2. Wash the dust on the leaf surface with tap water, then rinse with double-distilled water several times, and absorb excess water with filter paper.

[0089] 2. Place the leaf in a flask containing 50 ml of ultrapure water and vacuum immerse for 20 minutes. Measure the initial conductivity value (denoted as R1) using a conductivity meter.

[0090] 3. Place the flask in a water bath and boil it. After cooling, measure the final conductivity value (recorded as R2).

[0091] The relative conductivity percentage is calculated by the formula: relative conductivity (%) = R1 / R2×100%. The result is as follows Figure 12 As shown in the results, under normal treatment, the relative electrical conductivity of 84K poplars and transgenic SpsBBX3 poplars was similar. However, under drought stress, the relative electrical conductivity of the poplars overexpressing SpsBBX3 was significantly lower than that of 84K poplars, with significant differences between the two strains. This indicates that the stress tolerance of these three strains far exceeds that of 84K poplars. These results suggest that the SpsBBX3 gene plays a key role in enhancing the drought resistance of poplars.

[0092] 6.4 Determination of relative water content of transgenic poplars

[0093] 1. The leaves of 84K poplar and transgenic poplar collected in 6.2 were rinsed with clean water, and the surface moisture was absorbed with filter paper. The fresh weight (FW) was recorded.

[0094] 2. Soak the leaves in a ziplock bag filled with tap water for 6 hours to ensure that they are fully absorbed by water and reach saturation. Then weigh them again to obtain the saturated weight (TW).

[0095] 3. Place these leaves in an electric heated forced air drying oven at 80°C and dry them to constant weight, then weigh them to obtain the dry weight (DW).

[0096] The relative water content is calculated as follows: relative water content of leaves (%) = (FW-DW) / (TW-DW) × 100%. The results show that Figure 13 Under normal conditions, the relative water content of 84K poplar and transgenic poplar is not much different and remains at a high level. However, under drought conditions, the relative water content of poplar leaves decreases, and the relative water content of 84K poplar is even lower, indicating that the SpsBBX3 gene can enable poplar to maintain a higher water content, making the transgenic poplar more drought-resistant.

[0097] 6.5 NBT and DAB analysis of transgenic poplars

[0098] 1. Accurately weigh 0.788 g of Tris-HCl using a balance and dissolve it in ultrapure water. Then, dilute the volume to 100 ml, ensuring the pH of the solution is 5.5.

[0099] 2. Add 0.1 g of 3,3'-diaminobenzidine (DAB) powder to the solution and shake thoroughly to form a DAB working solution with a concentration of 1 mg / ml.

[0100] 3. Accurately weigh 0.5 g of Nitroblue Tetrazolium (NBT) and dissolve it in a mixture of 22.87 ml of phosphate buffer A and 21.25 ml of buffer B. The final volume is adjusted to 100 ml to obtain a 1 mg / ml NBT working solution.

[0101] 4. In a 10 ml centrifuge tube, add the staining solution and the leaf sample to be analyzed, ensuring that the staining solution completely covers the leaf. Vacuum the tube containing the leaf and staining solution for approximately 30 minutes to ensure that the leaf is fully immersed in the staining solution.

[0102] 5. Wrap the test tube with tin foil and place it in a 37°C incubator in the dark for 8 hours of staining reaction.

[0103] 6. Remove the staining solution and replace it with fresh decolorizing solution. Then place the test tube in a 95°C water bath to decolorize until the decolorization is complete. After decolorization, the leaves should be cooled back to room temperature and washed with anhydrous ethanol. At the same time, use filter paper to gently absorb excess water from the leaf surface to more clearly observe the color development of reactive oxygen species on the leaves.

[0104] The results are as follows Figure 14 As shown in the figure, under normal treatment, there was no significant difference between 84K poplar and transgenic poplar. However, under drought stress, DAB and NBT showed a darker blue coloring effect on the leaves of 84K poplar, while the coloring effect of SpsBBX3 transgenic poplar was lighter. The results showed that overexpression of SpsBBX3 can reduce intracellular hydrogen peroxide H2O2 and superoxide anion O2 - content, thereby improving the drought resistance of poplar.

[0105] 6.7 Antioxidant Enzyme Activity Analysis

[0106] The activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were detected by ultraviolet spectrophotometer using the Solebro kit. Figure 15 、 16 As shown in Figure 17, under normal conditions, there were no significant differences in POD, SOD, and CAT activity between SpsBBX3 transgenic poplars and 84K poplars. However, after drought stress, the POD, SOD, and CAT activities of transgenic poplars were significantly higher than those of 84K poplars, indicating that the drought resistance of SpsBBX3 transgenic poplars was significantly stronger than that of 84K poplars.

[0107] These results suggest that overexpressing the SpsBBX3 gene can enhance poplar trees' antioxidant defense mechanisms under drought conditions, effectively improving their drought tolerance. Specifically, the higher POD, SOD, and CAT activities in the transgenic poplar trees help scavenge reactive oxygen species (ROS), reduce oxidative damage, and better cope with the stress of drought.

[0108] In summary, poplars transformed with the SpsBBX3 gene can participate in drought stress by maintaining a stable state of reactive oxygen species (ROS), thereby improving the drought tolerance of poplars.

Claims

1. Salix psammophila SpsBBX3 protein, the amino acid sequence of which is shown in SEQ ID No.

2.

2. The SpsBBX3 gene encoding the Salix psammophila SpsBBX3 protein according to claim 1.

3. The SpsBBX3 gene according to claim 2, characterized in that The nucleotide sequence of the SpsBBX3 gene is shown in SEQ ID No.

1.

4. An expression vector containing the SpsBBX3 gene according to claim 2 or 3.

5. Use of the SpsBBX3 gene according to claim 2 or 3 or the expression vector according to claim 4 in improving drought tolerance of plants.

6. The use according to claim 5, characterized in that By overexpressing the Salix psammophila SpsBBX3 gene in plants, the drought tolerance of plants is improved.

7. The use according to claim 5 or 6, characterized in that The plant is a willow plant or a poplar plant.

8. The use according to claim 5 or 6, characterized in that The plant is Salix psammophila or Populus white poplar.

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