Sspp2c gene for improving salt tolerance of plant and application thereof

Abstract

The application relates to a SsPP2C gene for improving salt tolerance of plants and application thereof, and belongs to the technical field of genetic engineering. The SsPP2C gene is named SsPP2C21 and codes the amino acid sequence shown in SEQ ID NO. 2. Under the conditions of ABA and NaCl stress, the seed germination rate and seedling rate of transgenic Arabidopsis thaliana overexpressing the gene are significantly higher than those of wild type, and the gene can positively regulate the tolerance of plants to salt stress and ABA stress. The gene provides a new gene resource for plant stress resistance molecular breeding, and can be used for cultivating new crop varieties with salt tolerance, drought resistance or ABA stress resistance, and has important application prospects in improving the non-biological adversity adaptability of crops and stabilizing the yield.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically relating to the SsPP2C gene of Pittosporum tobira that improves the salt tolerance of plants and its application. Background Technology

[0002] Plant growth is severely inhibited in saline soils. High salt concentrations trigger osmotic stress, ion toxicity, and oxidative stress, ultimately leading to decreased crop yield and quality. With the continued expansion of global soil salinization, breeding new salt-tolerant crop varieties has become a crucial direction for sustainable agricultural development. Improving plant salt tolerance through genetic engineering is a core strategy in this field. Currently, the identified resources of plant salt tolerance functional genes are still relatively limited, making it difficult to meet the actual needs of molecular breeding for crop salt tolerance. Therefore, discovering new plant salt tolerance functional genes and clarifying their functions in regulating salt tolerance is of significant theoretical and practical value for enriching plant salt tolerance gene resources and promoting research on salt-tolerant crop breeding.

[0003] *Scaevola sericea* is an erect or spreading shrub, occasionally a small tree, belonging to the genus *Scaevola* in the family Goodeniceae. It typically grows on open sandy beaches or coastal cliffs, exhibiting characteristics of being light-loving, drought-tolerant, salt-tolerant, and tolerant of poor soil. It plays a vital role in coastal vegetation construction and windbreak and sand fixation. Therefore, identifying key salt-tolerant genes in *Scaevola sericea* for molecular design breeding is of great significance for cultivating salt-tolerant plants. Summary of the Invention

[0004] This invention cloned an SsPP2C gene from Pittosporum tobira, naming it SsPP2C21. The SsPP2C21 gene was heterologously transferred into Arabidopsis thaliana, resulting in stable overexpression transgenic plants. Analysis of the transgenic lines' performance under salt stress and exogenous ABA treatment confirmed that enhancing the expression level of the Pittosporum tobira SsPP2C21 gene can improve the plant's resistance to salt and ABA stress. The Pittosporum tobira SsPP2C21 gene and its applications provided by this invention offer key gene resources and technical support for the genetic improvement and breeding of salt-tolerant plants.

[0005] On the one hand, the present invention provides a protein for regulating plant salt tolerance, said protein being any one of the following (a1)-(a3): (a1) A protein with the amino acid sequence shown in SEQ ID NO.2; (a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1); (a3) A protein whose amino acid sequence is at least 99% homologous to the sequence shown in SEQ ID NO.2 and has the same function.

[0006] On the other hand, the present invention provides a gene encoding the protein that regulates plant salt tolerance.

[0007] In a preferred embodiment, the CDS sequence of the gene encoding the protein regulating plant salt tolerance is as shown in SEQ ID NO.1 or a nucleotide sequence having more than 99% homology with SEQ ID NO.1.

[0008] As is known to those skilled in the art, gene sequences can also contain introns, promoters, and various regulatory elements. Therefore, the nucleotide sequences of the aforementioned genes can also contain introns, promoters, and various regulatory elements.

[0009] On the other hand, the present invention provides an expression cassette, recombinant vector, recombinant microorganism, or transgenic plant tissue or transgenic plant containing the gene.

[0010] On the other hand, the present invention provides the application of the protein, gene, expression cassette, recombinant vector, or recombinant microorganism in regulating plant salt tolerance or regulating plant tolerance to ABA.

[0011] In a preferred embodiment, the regulation of plant salt tolerance is manifested by increasing the content or activity of the protein in the plant or increasing the expression level of the gene in the plant to improve plant salt tolerance.

[0012] In a preferred embodiment, the regulation of plant tolerance to ABA is manifested by increasing the content or activity of the protein in the plant or increasing the expression level of the gene in the plant to improve the plant's tolerance to ABA.

[0013] On the other hand, the present invention provides a method for cultivating transgenic plants, comprising the step of introducing the gene into a recipient plant to obtain a transgenic plant; the transgenic plant exhibits improved salt tolerance compared to the recipient plant.

[0014] On the other hand, the present invention provides a method for cultivating transgenic plants, comprising the step of introducing the gene into a recipient plant to obtain a transgenic plant; the transgenic plant exhibits increased tolerance to ABA compared to the recipient plant.

[0015] On the other hand, the present invention provides a plant breeding method, comprising the following steps: increasing the content or activity of the protein in the plant or increasing the expression level of the gene in the plant, thereby improving the plant's salt tolerance or tolerance to ABA.

[0016] In a preferred embodiment, in any of the aforementioned applications or methods, the plant is a species of the genus *Arabidopsis* or *Symplocos thaliana*.

[0017] In a further preferred embodiment, in any of the aforementioned applications or methods, the plant is Pittosporum tobira or Arabidopsis thaliana.

[0018] The beneficial effects of this invention are: This invention identifies a novel gene that regulates plant tolerance to abiotic stresses for the first time. Experiments show that overexpression of this gene in Arabidopsis thaliana, followed by ABA and NaCl stress treatment, significantly improved the seed germination and seedling rates of transgenic Arabidopsis compared to the untransgenic wild-type material, effectively enhancing the plant's tolerance to salt and ABA stresses. This gene provides a novel, high-quality gene resource and key technical target for molecular breeding of plant stress resistance. It can be directly applied to the genetic improvement of crop stress resistance, laying the foundation for breeding new salt-tolerant and other stress-resistant crop varieties. It has significant practical application value and promising prospects in the field of agricultural stress resistance breeding. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Differential expression of the SsPP2C21 gene in Pittosporum tobira under salt stress treatment.

[0021] Figure 2 The expression level of SsPP2C21 protein in transgenic Arabidopsis thaliana lines.

[0022] Figure 3 The phenotypes of wild-type and SsPP2C21 transgenic lines on 1 / 2 MS solid medium supplemented with 170 mM NaCl are shown from left to right: phenotype on day 7, germination rate within 5 days, and seedling rate on day 7.

[0023] Figure 4 The phenotypes of wild-type and SsPP2C21 transgenic lines on 1 / 2 MS solid medium supplemented with 1 µM ABA are shown from left to right: phenotype on day 7, germination rate within 5 days, and seedling rate on day 7. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] This invention isolates the SsPP2C21 gene from Pittosporum tobira and obtains transgenic materials overexpressing the SsPP2C21 gene from Arabidopsis thaliana. Phenotypic observation and analysis of transgenic and non-transgenic Arabidopsis thaliana after ABA and NaCl treatment revealed that the seed germination rate and seedling rate of the transgenic material overexpressing the SsPP2C21 gene were significantly higher than those of the non-transgenic material, indicating that overexpression of the SsPP2C21 gene from Pittosporum tobira can improve the plant's tolerance to salt and ABA stress.

[0027] In this invention, when calculating seed germination rate and seedling rate, seed germination is defined as the radicle breaking through the seed coat, and seedling is defined as the cotyledons fully unfolding and turning green.

[0028] In this invention, the nucleotide sequence of the Pittosporum tobira SsPP2C21 gene is shown below (SEQ ID NO.1): > SEQ ID NO.1

[0029] The amino acid sequence encoded by the SsPP2C21 gene of Pittosporum tobira is shown below (SEQ ID NO.2): > SEQ ID NO.2 MWMENPAESDDVDVNAVESSELASNSSVSSIFGSSDVEVPSTGSSGNFSITSSSSGEILAAVVAPRLQPSNHSALGEEEGAIARRNRCVGRHRGVAWGFTSVIGRRREMEDAVAVVPGFMSRTCNRVGGCTAPGSRSSLEISPIHFFGVYDGHGGSQVANFCRERMHRVIAEEWNQEILDRSEWQQRWEAVLCN SFRKVDNEVEAATSEMVGSTAVVVVLSGCQIIVSNCGDSRAVLCKGTETVPLTTDQKPDREDELLRIEQQDGRVINWNGSRVFGVLAMSRAIGDRYFRPWIIPLPEVTFTTRTTDEDECLILASDGLWDVMSNNEVGEVARRLLLRRRRRHVGDGDVPAAQTVADSLTEIAIGRHSSDNISIIVVDLKSRRRRQT.

[0030] Example 1: Discovery of Salt Tolerance Genes Using *Scaevola sericea* Vahl seedlings (propagated by cuttings from the same mother plant to ensure genotypic consistency) as experimental material, a method simulating high-salt soil was employed. The seedlings were subjected to salt stress treatment with a 0.5 M NaCl solution, while the control group received deionized water instead of the salt solution. Leaves from both the treatment and control groups were collected at 3h, 6h, 12h, and 24h after stress treatment. Samples were frozen in liquid nitrogen and stored at -80℃. Each treatment was replicated in triplicate, with 5 seedlings per replicate. The collected plant samples were sent to our company for ABA content determination and to Guangzhou Baishu Biotechnology Co., Ltd. for transcriptome analysis.

[0031] The results showed that differentially expressed genes were identified in the leaves of Pittosporum tobira after 24 h of salt stress treatment. The log2FC value of the SsPP2C21 gene was greater than 2, indicating that the gene expression in the leaves of the treated group was significantly higher than that in the untreated group at different time points. Figure 1 ).

[0032] Example 2: Construction of overexpression vector The 35S-SsPP2C21-GFP overexpression vector was constructed using homologous recombination, following these steps: First, the pCAMBIA1302 binary vector carrying the GFP tag was linearized using restriction endonucleases NcoI and SpeI (Takara). The digestion system consisted of 20 μL of vector, 2 μL each of NcoI and SpeI, 5 μL each of 10×K buffer and BSA, and water to a final volume of 50 μL. The digestion conditions were 37°C for 3–4 hours. After the reaction, the digestion products were recovered using a gel extraction kit (Magen). RNA was then extracted from *Pittosporum tobira* leaves using an RNA extraction kit (Vazyme). cDNA was synthesized following the instructions of the Vazyme first-strand reverse transcription kit. Using this cDNA as a template, the cDNA sequence of the SsPP2C21 gene was amplified by PCR using high-fidelity DNA polymerase (Vazyme) with primers F (ACGGGGGACTCTTGACCATGTGGATGGAAAACCCGGCG) and R (AAGTTCTTCTCCTTTACTAGTAGTCTGCTGCCGCCGCCTCCT). The reaction mixture consisted of: 2 × Phanta Max buffer 25 μL, dNTP Mix 1 μL, F and R primers 2 μL each, cDNA template 2 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, and water to a final volume of 50 μL. The reaction program was as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 sec, annealing at 58 ℃ for 15 sec, extension at 72 ℃ for 1 min, 35 cycles; and final extension at 72 ℃ for 5 min. After the reaction, the PCR product, i.e., the target fragment, was recovered using an agarose gel extraction kit (Magen). Finally, the SsPP2C21 gene was inserted into the pCAMBIA1302 binary vector using homologous recombination. The recombination system consisted of: 1 μL of the target fragment, 1.5 μL of the vector fragment, and 2.5 μL of 2× SeamlessCloning Mix (Beijing Bomei Gene Technology Co., Ltd.), for a total volume of 5 μL. The recombination conditions were: 50 ℃ for 1 h. After the reaction, the recombination product was transformed into 50 μL of E. coli competent cells DH5α, and the transformation product was plated onto a substrate containing kanamycin (50 mg·L⁻¹). -1LB solid medium was incubated overnight at 37 ℃. Single colonies growing on the medium were picked and reverse sequenced (using the primer sequence TCACCTTCACCCTCTCCACT). The correctly sequenced colonies were added to 10 ml of LB liquid medium containing kanamycin and incubated overnight. The positive plasmid was then extracted using a plasmid rapid extraction kit (Magen), which is the 35S-SsPP2C21-GFP overexpression vector.

[0033] Example 3: Obtaining transgenic Arabidopsis thaliana The obtained overexpression vectors were transformed into 50 μL of Agrobacterium tumefaciens competent cells GV3101, and then infected into 4-week-old Arabidopsis thaliana 'Col-0' using the flower dipping method. After harvesting T1 seeds, they were seeded into a solution containing 50 mg·L⁻¹ of [a specific solution]. -1 Resistance screening was performed on hygromycin-containing 1 / 2 MS solid medium. The positive seedlings with normal growth were further cultured to obtain T2 generation seeds. After harvesting individual plants, the seeds were again spotted on hygromycin-containing 1 / 2 MS solid medium. If the ratio of resistant seedlings to non-resistant seedlings was 3:1, the line was further cultured to obtain T3 generation seeds. If all seeds could grow normally on hygromycin-containing 1 / 2 MS solid medium, it was proven that the line was a homozygous plant.

[0034] Protein expression levels in transgenic Arabidopsis lines were analyzed using Western blot with Actin and GFP antibody (Abmart). Results showed that, using Actin as a plant internal control, transgenic lines #3 and #7 stably expressed the SsPP2C21 protein fused with the GFP tag. Figure 2 This indicates that the transgenic process was successful and that the genetic material can be stably inherited and expressed.

[0035] Example 4: Phenotypic analysis of the sensitivity of Arabidopsis thaliana overexpressing SsPP2C21 to high salt stress Seeds of wild-type (WT) and SsPP2C21-overexpressing transgenic Arabidopsis thaliana were spotted on 1 / 2 MS solid medium plates containing different concentrations of NaCl. The performance of each material under different salt stress concentrations was observed and statistically analyzed. The results showed that in medium containing 170 mM NaCl, the germination rate and seedling establishment rate of the SsPP2C21 overexpressing line were 91% and 98% on day 3 and day 7, respectively, both higher than the germination rate (0%) and seedling establishment rate (31%) of the control group at the same time point. Figure 3 The above phenotypic differences demonstrate that transgenic Arabidopsis thaliana with overexpression of the SsPP2C21 gene can significantly enhance its tolerance to salt stress.

[0036] Example 5: Phenotypic analysis of the sensitivity of Arabidopsis thaliana overexpressing SsPP2C21 to exogenous ABA stress Wild-type (WT) and SsPP2C21-overexpressing transgenic Arabidopsis seeds were spotted on 1 / 2 MS solid medium plates containing 1 µM ABA. The performance of each material under ABA stress was observed and statistically analyzed. Figure 4 The results showed that under ABA treatment, WT seeds barely germinated and formed seedlings, exhibiting significant stress sensitivity. In contrast, the SsPP2C21 overexpressing material achieved a germination rate of 88% on day 3, significantly higher than WT. Furthermore, after 7 days of 1 μM ABA stress treatment, the seedling formation rate of the SsPP2C21 overexpressing material reached 88%, far exceeding that of the control group. These results indicate that the SsPP2C21 gene in *Pittosporum tobira* has a significant advantage in improving the stress tolerance of *Arabidopsis thaliana* and plays a crucial role in regulating seed germination and stress resistance.

[0037] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A protein that regulates plant salt tolerance, characterized in that, The protein is any one of the following (a1)-(a3): (a1) A protein with the amino acid sequence shown in SEQ ID NO.2; (a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1); (a3) A protein whose amino acid sequence is at least 99% homologous to the sequence shown in SEQ ID NO.2 and has the same function.

2. A gene encoding the protein that regulates plant salt tolerance as described in claim 1.

3. An expression cassette, recombinant vector, recombinant microorganism, or transgenic plant tissue or transgenic plant containing the gene of claim 2.

4. The use of the protein of claim 1, the gene of claim 2, or the expression cassette, recombinant vector, or recombinant microorganism of claim 3 in regulating plant salt tolerance or regulating plant tolerance to ABA.

5. The application according to claim 4, characterized in that, The regulation of plant salt tolerance is manifested in increasing the content or activity of the protein in the plant or increasing the expression level of the gene in the plant to improve plant salt tolerance.

6. The application according to claim 4, characterized in that, The regulation of plant tolerance to ABA is manifested by increasing the content or activity of the protein in the plant or increasing the expression level of the gene in the plant to improve the plant's tolerance to ABA.

7. A method for cultivating transgenic plants, characterized in that, The method includes the step of introducing the gene of claim 2 into a recipient plant to obtain a transgenic plant; compared with the recipient plant, the transgenic plant has improved salt tolerance.

8. A method for cultivating transgenic plants, characterized in that, The method includes the step of introducing the gene of claim 2 into a recipient plant to obtain a transgenic plant; compared with the recipient plant, the transgenic plant has increased tolerance to ABA.

9. A plant breeding method, characterized in that, The method includes the following steps: increasing the content or activity of the protein of claim 1 in the plant or increasing the expression level of the gene of claim 2 in the plant, thereby improving the plant's salt tolerance or tolerance to ABA.

10. The application according to any one of claims 4-6 or the method according to any one of claims 7 to 9, characterized in that, The plants in question are either *Scabiosa* or *Arabidopsis*.

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