Rccbl3 gene and its encoded protein for maintaining sodium ion homeostasis of rosa chinensis cell under salt stress and application
By transiently overexpressing the RcCBL3 gene in rose plants to regulate sodium ion homeostasis, the problem of growth inhibition in roses under salt stress was solved, and their tolerance to salt stress and yield were improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI NORMAL UNIVERSITY
- Filing Date
- 2024-08-07
- Publication Date
- 2026-06-16
AI Technical Summary
Under salt stress, the sodium ion homeostasis of rose plants is disrupted, leading to stunted growth and reduced yield. Existing technologies are insufficient to effectively improve their salt tolerance.
By transiently overexpressing the RcCBL3 gene in rose cells, sodium ion homeostasis was regulated, and the RcCBL3 gene-encoded protein mediated Na+ homeostasis in rose cells under salt stress, thereby improving their stress resistance.
It significantly enhances the tolerance of roses to salt stress, reduces leaf wilting, and improves plant growth and yield under salt stress.
Smart Images
Figure CN118910082B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a gene sequence and its application, specifically to a mechanism for mediating the maintenance of Na+ in rose cells under salt stress. + Steady-state RcCBL3 sequences and their applications. Background Technology
[0002] The rose (Rosa chinensis Jacq.), belonging to the genus Rosa in the family Rosaceae, is a perennial woody plant. Known for its strong fragrance and vibrant colors, it is often called the "Queen of Flowers" and is widely used as a cut flower, potted plant, and in landscaping. Roses are highly adaptable to different environments, have a long flowering period, and come in a variety of shapes and colors, making them widely cultivated throughout my country.
[0003] Salt stress is a significant abiotic factor limiting plant growth and yield. Excessive soil salinity leads to osmotic stress in plants, reducing their absorption of nutrients and water, causing ion imbalances within the plant, resulting in ion toxicity and element deficiencies. The main ion causing salt stress is sodium (Na). + Plants' reliance on Na + and K + The absorption of Na+ is competitive; when intracellular Na+... + Increased concentration leads to K + Exudation disrupts the original ion balance. Salt affects the water potential of roses, reduces leaf water content, and inhibits both plant height and yield; irrigation with a 50mM NaCl solution will hinder rose growth; increased salt concentration leads to a reduction in the number of leaves and flowers. Therefore, tolerance to high concentrations of NaCl is crucial. + The selection and development of rose varieties are indispensable for improving their productivity. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide the RcCBL3 gene, its encoded protein, and its applications for maintaining sodium ion homeostasis in rose cells under salt stress. This gene can enhance the resistance of roses to salt stress, regulate rose signals against abiotic stresses, thereby increasing their resistance and improving their ornamental and practical value.
[0005] To achieve the above objectives, the present invention is implemented through the following solution:
[0006] To achieve the above objectives, a first aspect of the present invention provides an RcCBL3 gene for maintaining sodium homeostasis in rose cells under salt stress, the gene sequence comprising one or more of the following nucleotide sequences:
[0007] 1) The nucleotide sequence shown in SEQ ID NO.1;
[0008] 2) Nucleotide sequences derived from the nucleotide sequence shown in SEQ ID NO.1 by substitution, deletion, or addition of one or more nucleotides;
[0009] 3) A nucleotide sequence that is at least 80% homologous to SEQ ID NO.1.
[0010] A second aspect of the invention provides a protein encoded by the RcCBL3 gene, which mediates the maintenance of sodium homeostasis in rose cells under salt stress. + The gene for homeostasis, the amino acid sequence of the protein, is selected from one or more of the following sequences:
[0011] 1) The amino acid sequence shown in SEQ ID NO.2;
[0012] 2) The amino acid sequence shown in SEQ ID NO.2 is derived from the substitution, deletion, or addition of one or more amino acids;
[0013] 3) An amino acid sequence that has at least 80% homology with SEQ ID NO.2.
[0014] A third aspect of the invention provides the application of the RcCBL3 gene as described above or the protein encoded by the RcCBL3 gene as described above in influencing plant stress resistance.
[0015] Furthermore, the plant in question is a rose.
[0016] Furthermore, the rose in question is the 'Blue Balcony' variety.
[0017] Furthermore, the stress resistance is salt tolerance.
[0018] A fourth aspect of the present invention provides a method for improving plant resistance to salt stress, comprising transiently overexpressing the RcCBL3 gene as described above in a target plant.
[0019] Furthermore, the method described above involves linking the RcCBL3 gene to the pCAMBIA2300 vector via homologous recombination, then transferring the recombinant plant expression vector into the target plant, and obtaining a line with transient overexpression of RcCBL3 through culture.
[0020] Compared with the prior art, the beneficial effects of the present invention are:
[0021] The RcCBL3 described in this invention helps roses resist salt stress and maintain Na+ in rose cells. + It plays an important role in homeostasis. In rose lines that are transiently overexpressed, the roses’ tolerance to salt stress is significantly increased and the wilting of small round leaf discs is significantly reduced, providing an important foundation for rose breeding. Attached Figure Description
[0022] Figure 1 These are pictures of different tissues of the 'Blue Balcony' rose variety.
[0023] Figure 2 This represents the content of RcCBL3 in different tissues of the 'Blue Balcony'. (ab represents the significance of differences between groups, t-test, p<0.05)
[0024] Figure 3 This is an electrophoresis diagram of the RcCBL3 gene identification provided in Embodiment 3 of the present invention.
[0025] Figure 4 The phenotypes of 'Blue Balcony' leaf discs were obtained by vacuum infection with pCAMBIA2300-RcCBL3 at 25℃ and under 16h light / 8h dark conditions, followed by water treatment and treatment with 100mM and 200mM NaCl for 0 and 7 days, respectively, as well as control and overexpression leaf disc phenotypes.
[0026] Figure 5 The control and overexpression leaf discs were obtained by vacuum infection of 'Blue Balcony' leaf discs in pCAMBIA2300-RcCBL3, and then subjected to water treatment and 100mM and 200mM NaCl treatment for 7 days under 25℃, 16h light / 8h dark conditions. DAB and NBT were measured.
[0027] Figure 6 The RcCBL3 expression levels in control and overexpression leaf discs of 'Blue Balcony' were compared after vacuum infection with pCAMBIA2300-RcCBL3 at 25℃ under 16h light / 8h dark conditions, followed by water treatment and treatment with 100mM and 200mM NaCl for 7 days. (ab represents significant difference between groups, t-test, p<0.05). Detailed Implementation
[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in this technical field. Of course, the instruments and materials used in the embodiments are not limited to the examples listed herein, but are based on their ability to solve the technical problems of the present invention and achieve the corresponding technical effects.
[0029] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, all reagents used were commercially available or publicly available.
[0030] In this invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids.
[0031] Studies have revealed that the RcCBL3 gene in roses affects the salt stress tolerance of roses and positively regulates the Na+ ionization of rose cells. + In steady state, plants with transient overexpression of the 'Blue Balcony' RcCBL3 gene under the same salt concentration showed less wilting of leaflets compared to the control group, significantly lighter DAB and NBT staining, and a larger green area on leaflets.
[0032] Example 1: Cloning of the RcCBL3 gene in roses
[0033] Total RNA was extracted from the petals of *Rosa rugosa* (commercially available) using an RNAplant extraction kit. The total RNA was then reverse transcribed into cDNA using a commercially available reverse transcription kit. Primers were designed based on the transcriptome sequencing results, and their sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. An RT-PCR method was used to amplify a 693 bp band from the rose cDNA. The PCR product was recovered to obtain the RcCBL3 gene, whose nucleotide sequence is shown in SEQ ID NO.1. The amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO.2, consisting of 230 amino acid pairs with a molecular weight of 25.30 kilodaltons (kDa).
[0034] Example 2: Validation of RcCBL3 expression profiles in different rose tissues
[0035] (1) Extract as shown Figure 1 RNA was extracted from different tissues of the rose 'Blue Balcony' using an RNAplant (commercially available) extraction kit. The total RNA was then reverse transcribed into cDNA using a reverse transcription kit (commercially available).
[0036] (2) Primers were designed based on transcriptome sequencing data. The primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6.
[0037] (3) Using cDNA obtained by reverse transcription from 'Blue Balcony' at different developmental stages as templates, the expression profiles of RcCBL3 in different tissues were verified, and the results are as follows: Figure 2As shown, RcCBL3 expression varies in different tissues. It is expressed at a higher level in leaves and is widely expressed in rose tissues.
[0038] Example 3: Overexpression of the RcCBL3 gene in the 'Blue Balcony' rose.
[0039] The 690bp open reading frame of the RcCBL3 gene was operatively ligated into the pCAMBIA2300 vector to form the pCAMBIA2300-RcCBL3 vector containing this gene fragment. This vector was transformed into Agrobacterium GV3101, and rose leaf discs were vacuum-infected using Agrobacterium-mediated transformation. After 7 days of culture, rose leaf discs with transient overexpression were obtained. These discs were then treated with water and 100mM and 200mM NaCl for 7 days under conditions of 25℃, 16h light / 8h dark. The results are as follows: Figure 4 As shown, the wilting of the transiently overexpressed material was significantly reduced, indicating that this gene can maintain the Na+ level in rose cells by mediating salt stress. + Steady state. The specific steps are as follows:
[0040] (1) The open reading frame 690 bp of the RcCBL3 gene was operatively linked into the pCAMBIA2300 vector to form the pCAMBIA2300-RcCBL3 vector containing the gene fragment.
[0041] (2) Transform the vector from step (1) into Agrobacterium (GV3101).
[0042] (3) Take Agrobacterium (GV3101) from step (2) and culture it in 5 ml of LB medium containing 100 μM acetylsuccinone and 50 μg / ml kanamycin at 28°C and 200 rpm for 16 h.
[0043] (4) The Agrobacterium from step (3) was subcultured in 50 ml LB medium containing 100 μM acetylsuccinone and 50 μg / ml kanamycin and incubated at 28℃ and 200 rpm for 13-16 h until the OD600 reached 0.8-1.0.
[0044] (5) Take the Agrobacterium tumefaciens bacterial suspension from step (4), centrifuge at 3700 rpm for 9 min at 4℃, and discard the supernatant; in a clean bench, resuspend the bacterial cells with 5 mL MgCl2, centrifuge at 3700 rpm for 9 min at 4℃, and discard the supernatant (repeat once).
[0045] (6) Add an appropriate amount of MgCl2 to resuspend the bacterial cells. The OD600 of the dual-carrier injection is 1.2. Add 1.5 times the volume of AS and 20 times the volume of MES to the bacterial solution to resuspend the cell pellet. Let stand at room temperature for 2 hours.
[0046] (7) Use a hole punch to take rose leaves, punch holes, and obtain small round leaf pieces.
[0047] (8) Use a vacuum instrument to transfer the Agrobacterium transformation solution successfully transformed in step (6), such as... Figure 3 The results showed that the RcCBL3 gene was verified by PCR in the bacterial culture. The transformed bacterial culture contained the RcCBL3 target fragment, while the PCR of the bacterial culture transformed with the EV vector did not contain the corresponding fragment.
[0048] (9) Under conditions of 25℃, 16h light / 8h darkness, the rose leaves were treated with water and 100mM and 200mM NaCl for 7 days, and the leaves were observed. Figure 4 The image shows the phenotypes of the control and overexpressing leaf discs after 0 and 7 days of culture treatment with infected leaf discs; Figure 4 As shown, the transient overexpression material exhibited significantly reduced wilting and significantly enhanced salt stress tolerance.
[0049] Example 4: DNB and NBT staining verification of the 'Blue Balcony' rose overexpression leaf disc control group and the RcCBL3 gene overexpression experimental group.
[0050] (1) Select as follows Figure 5 The control and overexpressing leaf discs shown were subjected to DNB and NBT staining experiments.
[0051] (2) Preparation of PBS solution: Prepare 80 mL of pure water in a container, add 1.5483 g of disodium hydrogen phosphate heptahydrate to the solution, then add 0.5066 g of sodium dihydrogen phosphate monohydrate to the solution, adjust the pH to 7, and add pure water to make up to 800 mL. Then prepare two separate containers, divide the prepared PBS solution into 400 mL portions, add 0.4 g of DAB and NBT to each, mix well, and store at 4°C protected from light.
[0052] (3) Randomly select 10 leaf discs and place them into 50ml centrifuge tubes. Add NBT / DAB to each tube, vacuum for 15 minutes, and incubate overnight in the dark. The next day, pour the solution into a waste container, add anhydrous ethanol, and boil until the leaves are no longer green. Replace the anhydrous ethanol continuously during this process. After the color fades, prepare 50% glycerol, pour it into the tube, and incubate until the leaves unfold. Let it stand for 1-2 hours, remove it, wipe off the glycerol on the surface of the leaves with a paper towel, and take a picture.
[0053] The results are as follows Figure 5 As shown, the color of the RcCBL3 overexpression experimental group was significantly lighter than that of the control group, proving that RcCBL3 can improve tolerance to rosé salt stress and has a high overexpression efficiency.
[0054] Example 5: Verification of RcCBL3 expression levels in the 'Blue Balcony' rose overexpression leaf disc control group and the RcCBL3 gene overexpression experimental group under salt stress.
[0055] Select as Figure 6 The control and overexpression petal discs shown were used to extract total RNA using RNAplant (commercially available) extraction kit. The total RNA was then reverse transcribed into cDNA using a commercially available reverse transcription kit.
[0056] (1) Primers were designed based on transcriptome sequencing data. The primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6.
[0057] (2) Using cDNA obtained from reverse transcription of control and overexpression leaf discs as templates, the overexpression efficiency of the RcCBL3 gene was verified. The results are as follows: Figure 6 As shown, the expression of RcCBL3 in the overexpression leaves of 'Blue Balcony' increased with the degree of salt stress, while the expression in the control group remained lower than that in the overexpression group and did not increase with the degree of salt stress. This proves that the high salt stress tolerance of the overexpression group is due to the increased expression of RcCBL3, and that the expression of RcCBL3 in the overexpression group responds positively to salt stress.
[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. Maintaining sodium homeostasis in rose cells under salt stress RcCBL3 The application of genes in improving the salt tolerance of roses is characterized by, The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. Maintaining sodium homeostasis in rose cells under salt stress RcCBL3 The application of gene-encoded proteins in improving the salt tolerance of roses is characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.
2.
3. A method for improving the resistance of roses to salt stress, characterized in that, Transient overexpression in target plants RcCBL3 The gene, the target plant is the rose, the RcCBL3 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.