Application of RcSAPK2 gene in regulating drought resistance of plants and method for regulating drought resistance of plants

Abstract

The application provides application of RcSAPK2 gene in regulating drought resistance of plants and a method for regulating drought resistance of plants. In a first aspect, the application provides application of RcSAPK2 gene in regulating drought resistance of plants, wherein the RcSAPK2 gene is one of the following: 1) a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 1; 2) a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO. 1 through substitution, deletion or addition of one or more nucleotides; and 3) a nucleotide sequence with at least 80% identity with SEQ ID NO. 1. Experiments show that the RcSAPK2 gene provided by the application plays an important role in resisting drought stress of plants, and provides an important basis for plant breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the RcSAPK2 gene in regulating plant drought resistance and methods for regulating plant drought resistance. Background Technology

[0002] Against the backdrop of global climate change, drought has become a key environmental challenge restricting plant growth, development, and geographical distribution. As a widely used and beloved horticultural plant, the rose (Rosa chinensis Jacq.) benefits greatly from improved drought resistance, which is crucial for its ecological adaptability and landscape value. Therefore, regulating the drought resistance of roses through genetic engineering is not only a significant breakthrough in solving current agricultural and horticultural production problems but also fundamental research for advancing plant drought-resistant breeding practices. Summary of the Invention

[0003] This invention provides the application of the RcSAPK2 gene in regulating plant drought resistance and a method for regulating plant drought resistance.

[0004] The first aspect of this invention provides the application of the RcSAPK2 gene in regulating plant drought resistance, wherein the RcSAPK2 gene is one of 1)-3):

[0005] 1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1;

[0006] 2) Nucleotide sequences derived from the nucleotide sequence shown in SEQ ID NO.1 by substitution, deletion, or addition of one or more nucleotides;

[0007] 3) A nucleotide sequence that is at least 80% identical to SEQ ID NO.1.

[0008] Furthermore, the application of the RcSAPK2 gene in regulating plant drought resistance specifically includes: introducing the RcSAPK2 gene into the starting plant to overexpress the RcSAPK2 gene in the starting plant.

[0009] Furthermore, the application of the RcSAPK2 gene in regulating plant drought resistance includes: silencing the expression of the RcSAPK2 gene in the starting plant and reducing the expression of the RcSAPK2 gene in the starting plant.

[0010] Furthermore, the RcSAPK2 gene is introduced into the starting plant to overexpress the RcSAPK2 gene in the starting plant, specifically including:

[0011] The open reading frame of the RcSAPK2 gene is operatively linked to the pCAMBIA2300 vector to form a pCAMBIA2300-SAPK2 recombinant vector containing the RcSAPK2 gene.

[0012] The pCAMBIA2300-SAPK2 recombinant vector was introduced into Agrobacterium, and transgenic plants overexpressing the RcSAPK2 gene were obtained through Agrobacterium-mediated transformation.

[0013] Furthermore, the plant in question is a rose.

[0014] A second aspect of the present invention provides a method for regulating plant drought resistance, comprising: regulating the expression of the RcSAPK2 gene in the starting plant;

[0015] The RcSAPK2 gene is one of (1)-3):

[0016] 1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1;

[0017] 2) Nucleotide sequences derived from the nucleotide sequence shown in SEQ ID NO.1 by substitution, deletion, or addition of one or more nucleotides;

[0018] 3) A nucleotide sequence that is at least 80% identical to SEQ ID NO.1.

[0019] Furthermore, regulating the expression of the RcSAPK2 gene in the starting plant includes: introducing the RcSAPK2 gene into the starting plant to overexpress the RcSAPK2 gene in the starting plant; or silencing the expression of the RcSAPK2 gene in the starting plant to reduce the expression of the RcSAPK2 gene in the starting plant.

[0020] Furthermore, the RcSAPK2 gene was introduced into the starting plant to overexpress the RcSAPK2 gene in the starting plant, resulting in transgenic plants with better drought resistance than the starting plant.

[0021] Furthermore, the RcSAPK2 gene is introduced into the starting plant to overexpress the RcSAPK2 gene in the starting plant, specifically including:

[0022] The open reading frame of the RcSAPK2 gene is operatively linked to the pCAMBIA2300 vector to form a pCAMBIA2300-SAPK2 recombinant vector containing the RcSAPK2 gene.

[0023] The pCAMBIA2300-SAPK2 recombinant vector was introduced into Agrobacterium, and transgenic plants overexpressing the RcSAPK2 gene were obtained through Agrobacterium-mediated transformation.

[0024] The third aspect of this invention provides the application of the above-mentioned RcSAPK2 gene in plant drought resistance breeding.

[0025] This invention cloned the RcSAPK2 gene from ancient rose pollen. Transient expression experiments in roses verified that the RcSAPK2 gene plays a crucial role in the rose's response to dehydration stress. Silencing the RcSAPK2 gene makes roses more susceptible to damage under dehydration stress, while overexpression of the RcSAPK2 gene enhances the rose's resistance to dehydration stress. Therefore, the RcSAPK2 gene provided by this invention plays an important role in the rose's resistance to drought stress, providing an important foundation for rose breeding. Attached Figure Description

[0026] Figure 1 The relative expression levels of the RcSAPK2 gene in different tissues of the rose 'Yueyuefen';

[0027] Figure 2 Phenotypic images of leaf discs before and after dehydration in the overexpression and silence groups of 'Early Moon';

[0028] Figure 3 Images showing the staining results of leaf discs of 'Early Moon' before and after dehydration in the overexpression and silence groups;

[0029] Figure 4 Figure 1 shows the results of relative conductivity analysis of leaf discs of 'Zaoyue' 'Early Moon' 'Early Moon' before and after dehydration in the overexpression and silence groups.

[0030] Figure 5 The graphs show the changes in RcSAPK2 gene expression levels in the leaf discs of the 'Zaoyue' variety before and after dehydration in the overexpression and silence groups. In the graphs, A shows the changes in RcSAPK2 gene expression levels in the leaf discs of the overexpression group before and after dehydration, and B shows the changes in RcSAPK2 gene expression levels in the leaf discs of the silence group before and after dehydration. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0032] 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.

[0033] In this invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids.

[0034] Example 1: Cloning of the RcSAPK2 gene

[0035] 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 921 bp band from the rose cDNA. The PCR product was recovered, and the RcSAPK2 gene was obtained. Its nucleotide sequence is shown in SEQ ID NO. 1, and its encoded amino acid sequence is shown in SEQ ID NO. 2, consisting of 306 amino acid pairs with a molecular weight of 75.14 kilodaltons (kDa).

[0036] Example 2: Validation of RcSAPK2 expression profiles in different rose tissues

[0037] Total RNA was extracted from different tissues (roots, stems, leaves, flowers, fruits, and thorns) of the rose 'Yueyuefen' using a commercially available RNAplant extraction kit. The total RNA was then reverse transcribed into cDNA using a commercially available reverse transcription kit. Using the cDNA obtained from the reverse transcription of 'Yueyuefen' from different tissues as templates, amplification was performed using the primer pairs shown in SEQ ID NO. 5 and SEQ ID NO. 6 to verify the expression profiles of RcSAPK2 in different tissues.

[0038] The verification results are as follows Figure 1 As shown, the expression level of RcSAPK2 varies in different tissues, with higher expression in leaves.

[0039] Example 3: Overexpression of the RcSAPK2 gene in the 'Early Moon' rose variety

[0040] 3.1 Obtaining small round discs of rose leaves transiently overexpressing the RcSAPK2 gene

[0041] Step 1: The 921 bp open reading frame of the RcSAPK2 gene was operatively ligated into the pCAMBIA2300 vector. First, RNA was extracted from the petals according to the Steady Pure plant RNA extraction kit procedure. The concentration of the isolated RNA was measured using a concentration detector to ensure the purity ratio (260 / 280 nm) was within the range of 1.8-2.2. Reverse transcription was performed according to the RT kit (Vazyme) procedure to obtain cDNA. PCR amplification was performed using the reverse-transcribed cDNA as a template. The nucleotide sequences of the primers are shown in SEQ ID NO:5 and SEQ ID NO:6. After size detection by 1% agarose gel electrophoresis, appropriately sized specific target bands were excised and recovered from the gel. The recovered gel product was ligated into a blunt-ended vector and transformed into *E. coli*. Single colonies grown on the transformed plates were picked for PCR identification. Positive single colonies were sent to Kexin Technology for sequencing. Sequencing results were compared using DNAMAN software. The amplified DNA and suitable plasmid vector were digested using the same restriction endonuclease to create compatible ends. The required vector volume was determined by the length of the fragment and the vector itself. After a 44-fold dilution, the VIGS system was used to ligate *E. coli*. The required plasmid was extracted using the Nanjing Novizan Bioplasma DNA Extraction Kit. Restriction enzymes were selected according to the experimental objectives to digest the empty plasmid vector. The restriction enzyme sites were Kpn1 and Sal1. The cut linear plasmids were separated and identified by gel electrophoresis. Gel fragments of the corresponding size were cut and recovered. A recombinant vector pCAMBIA2300-SAPK2 containing the gene fragment was formed and transformed into *Agrobacterium* GV3101. *Agrobacterium* GV3101 containing the recombinant vector was added to 5 ml of LB medium containing 100 μM acetylsuccinone and 50 μg / ml kanamycin and cultured at 28℃ and 200 rpm for 16 h. After the culture was completed, the bacterial cells were subcultured in 50 ml LB medium containing 100 μM acetylsyl syringone and 50 μg / ml kanamycin, and cultured at 28℃ and 200 rpm for 13-16 h until the OD600 reached 0.8-1.0.

[0042] Step 2: Take the Agrobacterium tumefaciens culture after the culture is completed, centrifuge at 3700 rpm for 9 min at 4℃, and discard the supernatant; resuspend the bacterial cells in 5 mL of MgCl2, centrifuge at 3700 rpm for 9 min at 4℃, discard the supernatant, and collect the bacterial cells.

[0043] The bacterial cells were resuspended in MgCl2 solution to obtain a bacterial suspension with an OD600 of 1.2. 1.5 volumes of AS and 20 volumes of MES were added to resuspend the cell pellet. The suspension was then allowed to stand at room temperature for 2 hours.

[0044] Step 3: Use a hole punch to take tender leaves of the 'Early Moon' rose variety, punch holes in them, and obtain small leaf discs. Use a vacuum instrument to inoculate the small rose leaf discs with Agrobacterium tumefaciens solution and culture for 4-7 days.

[0045] 3.2 Obtaining small round discs of rose leaves that suppress RcSAPK2 gene expression

[0046] The 921 bp open reading frame of the RcSAPK2 gene was operatively ligated into a TRV viral vector to form a TRV-SAPK2 vector containing this gene fragment. The amplified DNA and plasmid vector were digested using the same restriction endonucleases, with compatible ends created at Kpn1 and Sal1 restriction sites. The required vector volume was determined based on the fragment and vector lengths, and after a 44-fold dilution, a VIGS system was obtained for ligation into *E. coli*. The required plasmid was extracted using the Nanjing Novizan bioplasma DNA extraction kit, and the empty plasmid vector was digested with the appropriate restriction enzymes according to the experimental purpose. The cut linear plasmids were separated and identified by gel electrophoresis, and gel fragments of the corresponding size were cut for gel recovery experiments. Using the same method as in 3.1, this vector was transformed into *Agrobacterium* GV3101 to obtain small round discs of rose leaves that inhibited RcSAPK2 gene expression.

[0047] At the same time, TRV-Ev and pCAMBIA2300-Ev control groups were set up.

[0048] Somatic cell embryos were vacuum-permeable to small round leaf discs of roses. The discs were briefly washed with sterile water, then vacuum-treated at 0.7 MPa for 15 minutes, and the water was absorbed with sterile paper towels. The somatic cell embryos were cultured in a dark, low-temperature environment at 18°C ​​in co-culture medium (CM) for 6 days. After 6 days, a portion of the original discs was cryopreserved in liquid nitrogen. The remaining portion was subjected to drought treatment at 26°C for 4 hours. The condition of the rose leaves was observed. The results are as follows: Figure 2 As shown, the rose discs with silenced RcSAPK2 gene did not differ significantly from those of the TRV-Ev control group. However, after dehydration, the TRV-SAPK2 discs exhibited significant wilting and discoloration, while the TRV control group, although showing some wilting, maintained a relatively good green color overall. Furthermore, in plants with transient RcSAPK2 overexpression, the difference between pCAMBIA2300-Ev and pCAMBIA2300-RcSAPK2 was also not significant before dehydration. After dehydration, although the RcSAPK2-overexpressing discs showed slight wilting, the degree of wilting was significantly less than that of the pCAMBIA2300-Ev discs, and the discoloration was less pronounced. This indicates that the RcSAPK2 gene helps improve plant drought resistance.

[0049] Example 4: DNB and NBT staining verification of leaf discs overexpressing and inhibiting the expression of the 'Early Moon' rose.

[0050] Step 1: Prepare PBS solution: In a container, prepare 80 mL of pure water. Add 1.5483 g of disodium hydrogen phosphate heptahydrate and 0.5066 g of sodium dihydrogen phosphate monohydrate to the solution. Adjust the pH to 7, and then add pure water to bring the volume to 800 mL. Prepare two separate containers. Divide the prepared PBS solution into 400 mL portions. Add 0.4 g of DAB and 0.4 g of NBT to each portion, mix well, and store at 4°C protected from light.

[0051] Step 2: Randomly select 10 pieces each Figure 2 The experimental and control group leaf discs were placed in 50ml centrifuge tubes, and the NBT and DAB solutions prepared in step 1 were added respectively. The tubes were then vacuum-sealed for 15 minutes and incubated overnight in the dark. The next day, the solutions were poured into a waste container, anhydrous ethanol was added, and the mixture was boiled until the leaves lost their green color, with the anhydrous ethanol being replaced continuously. After decolorization, 50% glycerol was prepared and poured into the tubes until the leaves fully expanded. The tubes were then left to stand for 1-2 hours. Afterward, the glycerol on the leaf surface was wiped off with a paper towel, and the leaves were photographed.

[0052] like Figure 3 As shown, after dehydration treatment, the leaves of the TRV-Ev group exhibited slight to moderate deposition, while the TRV-RcSAPK2 group showed significant brown and blue deposition, and the pCAMBIA2300-RcSAPK2 group showed relatively light deposition. This confirms that the leaves of the overexpression groups had less dehydration.

[0053] Example 5: Determination of relative conductivity of leaf discs overexpressing and inhibiting the expression of the 'Early Moon' rose.

[0054] Select as Figure 2 The small discs of the leaf shown were added to sterile water, vacuum-sealed, and incubated overnight in the dark. The following day, conductivity was measured using a conductivity meter. After boiling for 20 minutes, the sample was cooled for 5-10 minutes and then measured again. The relative values ​​were then taken. The experiment was repeated three times.

[0055] Test results are as follows Figure 4 As shown in the analysis of relative conductivity changes, compared with the control group TRV-Ev, plants treated with TRV-RcSAPK2 exhibited significantly increased relative conductivity, indicating that RcSAPK2 gene silencing may lead to decreased cell membrane stability, making plants more susceptible to damage under abiotic stress. While compared with the control group pCAMBIA2300-Ev, the relative conductivity of plants treated with pCAMBIA2300-RcSAPK2 decreased, but the difference was not statistically significant.

[0056] Example 6: Analysis of RcSAPK2 expression levels in overexpressing and suppressed leaf discs of the 'Early Moon' rose variety.

[0057] Select as Figure 2 The leaf discs shown were used to extract total RNA using RNAplant (commercially available). The total RNA was then reverse transcribed into cDNA using a commercially available reverse transcription kit.

[0058] Using cDNA obtained by reverse transcription from leaf discs as a template, and with GAPDH and β-actin as internal reference genes, the relative expression levels of the RcSAPK2 gene after 0 h and 4 h of dehydration were verified.

[0059] like Figure 5 As shown, under drought conditions, compared to the control group, the expression level of the RcSAPK2 gene in the overexpressing materials was significantly upregulated to twice that of the control, revealing the active state of this gene in response to drought stress. The expression level of RcSAPK2 in the silenced lines was significantly lower than that in the control. This confirms that the RcSAPK2 gene plays a crucial role in plant response to drought stress, and changes in its expression level directly affect the drought resistance of plants.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. RcSAPK2 The application of genes in improving the drought resistance of roses is characterized by, The RcSAPK2 Genes are nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, RcSAPK2 The application of genes in improving the drought resistance of roses specifically includes: introducing genes into the starting plant. RcSAPK2 Genes, making RcSAPK2 The gene is overexpressed in the starting plant.

3. The application according to claim 2, characterized in that, Introduce into the starting plant RcSAPK2 Genes, making RcSAPK2 Genes are overexpressed in the starting plant, specifically including: The RcSAPK2 The open reading frame of the gene is operatively linked to the pCAMBIA2300 vector to form a vector containing the gene described above. RcSAPK2 The pCAMBIA2300-SAPK2 recombinant vector of the gene; The pCAMBIA2300-SAPK2 recombinant vector was introduced into Agrobacterium, and overexpression was obtained through Agrobacterium-mediated transformation. RcSAPK2 Genetically modified plants.

4. A method for improving the drought resistance of roses, characterized in that, include: Improve the starting plant RcSAPK2 Gene expression; The RcSAPK2 Genes are nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that, Improve the starting plant RcSAPK2 Gene expression includes: introducing gene into the starting plant. RcSAPK2 Genes, making RcSAPK2 The gene is overexpressed in the starting plant.

6. The method according to claim 5, characterized in that, Introduce into the starting plant RcSAPK2 Genes, making RcSAPK2 Genes are overexpressed in the starting plant, specifically including: The RcSAPK2 The open reading frame of the gene is operatively linked to the pCAMBIA2300 vector to form a vector containing the gene described above. RcSAPK2 The pCAMBIA2300-SAPK2 recombinant vector of the gene; The pCAMBIA2300-SAPK2 recombinant vector was introduced into Agrobacterium, and overexpression was obtained through Agrobacterium-mediated transformation. RcSAPK2 Genetically modified plants.

7. The claim 1 RcSAPK2 Application of genes in drought-resistant breeding of roses.

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