Application of VvPMA10 gene in improving alkaline salt tolerance of plants or microorganisms

Abstract

The present invention discloses an application of a VvPMA10 gene in improving the alkaline salt tolerance of plants or microorganisms. Experimental verification shows that the VvPMA10 gene can effectively improve the alkaline salt tolerance of grapes, Arabidopsis thaliana and yeast.

Description

Technical Field

[0001] The present invention relates to application of a VvPMA10 gene in improving alkaline salt tolerance of plants or microorganisms, and belongs to the field of biotechnology. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance some understanding of the overall background of the invention and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Soil salinization is a global environmental problem, posing a significant threat to crop yield and quality. Saline-alkali soils contain large amounts of NaHCO₃ and Na₂CO₃, which pose a triple stress to plants: osmotic stress, ion toxicity, and pH stress. Therefore, alkaline salts are far more harmful than neutral salts. However, compared with research on the mechanisms of tolerance to neutral salts, the mechanisms of crop alkaline tolerance urgently require in-depth study. Previous studies have shown that root acid secretion is a fundamental pathway for crops to improve their tolerance to alkaline salts, with plasma membrane H⁺-ATPase playing a central role in this process. However, this pathway has not received sufficient attention in grapevine research.

[0004] Plasma membrane H + -ATPase structure:

[0005] ATPase is widely present in animals, plants, fungi and bacteria. Currently, the ATPases found are mainly divided into four categories, such as P-type ATPase, V-type ATPase, F-type ATPase and ABC transporter. Among them, P-type ATPase is a type of protein that can obtain energy by hydrolyzing ATP and then drive the transport of transmembrane substances, such as plasma membrane H + -ATPase, Na + / K + -ATPase and Ca 2 + -ATPase, etc. V-type ATPase (Vacuolar-type ATPase) is mainly present on the membranes of organelles such as lysosomes, endosomes, and Golgi apparatus, and is involved in regulating the pH and ion balance of these organelles. F-type ATPase is an ATP synthase present in organelles such as bacteria, mitochondria, and chloroplasts. It can use the energy of the proton gradient to synthesize ATP and is one of the important driving forces for energy synthesis in cells. ABC transport proteins are a class of transmembrane proteins that are widely present in various organisms. They are composed of a transmembrane domain on the plasma membrane and an ATP-binding domain in the cytoplasm. They use the ability to hydrolyze ATP to drive the transmembrane transport of specific substances and play an important role in substance transport.

[0006] Plasma membrane H +-ATPase belongs to the P3A subfamily of the P-type ATPase family and is composed of a single polypeptide chain with a size of approximately 100 kDa. + -ATPase contains 10 transmembrane structures (M1-M10) and three intramembrane domains (N-terminal, catalytic and C-terminal autoinhibitory domains). Among them, the catalytic domain contains a conserved Asp residue located within the DKTGTLT sequence motif. In addition, the C-terminal autoinhibitory domain consists of approximately 100 amino acid residues. Previous studies have found that this region contains multiple phosphorylation sites for regulating plasma membrane H + -ATPase activity, the penultimate Thr residue has been of particular interest, as phosphorylation of this site exhibits high affinity binding to 14-3-3 proteins, which eliminates the plasma membrane H + -ATPase autoinhibition. In addition, studies have shown that 10 amino acid residues located in the N-terminal region appear to be involved in the regulation of the autoinhibitory activity of the C-terminal domain.

[0007] PM H + -ATPase participates in the regulation of salt-alkali stress tolerance:

[0008] More and more studies have shown that plasma membrane H+-ATPase plays an important role in regulating plant salt and alkali tolerance. + -ATPase activity contributes to the enhanced tolerance of plants to saline-alkali stress. Overexpression of PMA4 (without the autoinhibitory domain) increases salt tolerance in tobacco seedlings during growth, suggesting that plasma membrane H + -ATPase activity is associated with salt tolerance. SOS2-like protein 5 (PKS5, also named CBL-interacting protein kinase 11, CIPK11) is a plasma membrane H + PKS5 is a negative regulator of ATPase. The pks5 mutant of Arabidopsis thaliana exhibits high tolerance to alkaline stress and has higher proton pump activity. PKS5 negatively regulates plasma membrane H by phosphorylating Ser-931 at the C-terminus of the proton pump AHA2. + -ATPase, thereby inhibiting the interaction between AHA2 and 14-3-3. Lin et al. (2014) found that PKS24 interacts with SCaBP1 and negatively regulates plasma membrane H + In conclusion, the Ser-931 residue at the C-terminus of AHA2 is phosphorylated by PKS5 / 24, which inhibits the interaction between AHA2 and 14-3-3, thereby inhibiting the plasma membrane H + -ATPase activity.

[0009] Under normal conditions, SCaBP3 / CBL7(Ca 2+ sensor), interacts with AHA2 and blocks the interaction of AHA2 with 14-3-3, leading to plasma membrane H + Furthermore, SCaBP3 enhanced the interaction between PKS5 and AHA2, leading to the decrease of plasma membrane H + -ATPase activity was inhibited to an increased degree. At the same time, SOS2 kinase activity was inhibited by 14-3-3. Under salt-alkali stress, intracellular Ca 2+ Increased Ca concentration leads to the release of SCaBP3 from the C-terminus of AHA2 and relieves the inhibitory interaction between the C-terminus and the central loop. Subsequently, the activity of the SCaBP3-PKS5 complex and the phosphorylation level of Ser-931 are reduced, allowing AHA2 to be activated by 14-3-3. 2+ The interaction between 14-3-3 proteins and PKS5 was also increased, inhibiting the activity of PKS5 and ultimately releasing the inhibition of SOS2 and AHA2 activities. In addition, DnaJ homolog 3 (J3) interacted with PKS5 and inhibited its kinase activity, thereby alleviating the inhibition of proton pump activity. Plasma membrane H was detected in j3 mutants. + -ATPase activity was reduced and the cells were hypersensitive to salt and alkali stress. These studies showed that plasma membrane H + -ATPase plays an important role in improving plant salt tolerance. + It remains to be seen whether ATPase plays an important role in improving grapevine tolerance to alkaline salts. Summary of the Invention

[0010] In response to the deficiencies of the prior art, the present invention provides an application of the VvPMA10 gene in improving the alkaline salt tolerance of plants or microorganisms. Experimental verification shows that the VvPMA10 gene can effectively improve the alkaline salt tolerance of grapes, Arabidopsis thaliana, and yeast.

[0011] The technical solution adopted in the present invention is as follows:

[0012] In a first aspect of the present invention, a use of a VvPMA10 gene in improving alkaline salt tolerance of plants or microorganisms is provided; the plant is grape or Arabidopsis thaliana, and the microorganism is yeast.

[0013] The VvPMA10 gene has the following nucleotide sequence:

[0014] (1) the nucleotide sequence shown by gene ID VIT_209s0002g00130; or

[0015] (2) A derivative sequence obtained by replacing, deleting or adding one or more nucleotides to the nucleotide sequence in (1), and the derivative sequence encodes a polypeptide with the same function as the sequence in (1).

[0016] In a second aspect of the present invention, a use of a polypeptide encoded by the VvPMA10 gene in improving alkaline salt tolerance of plants or microorganisms is provided; the plant is grape or Arabidopsis thaliana, and the microorganism is yeast.

[0017] The VvPMA10 gene has the following nucleotide sequence:

[0018] (1) the nucleotide sequence shown by gene ID VIT_209s0002g00130; or

[0019] (2) A derivative sequence obtained by replacing, deleting or adding one or more nucleotides to the nucleotide sequence in (1), and the derivative sequence encodes a polypeptide with the same function as the sequence in (1).

[0020] In a third aspect of the present invention, there is provided a method for cultivating a highly alkaline salt-tolerant plant, the method comprising the following steps:

[0021] Wild-type grape stem segments, calli or Arabidopsis thaliana were transformed using a vector-mediated method to obtain transgenic lines overexpressing VvPMA10.

[0022] Preferably, the vector-mediated method is Agrobacterium-mediated method.

[0023] The specific steps include:

[0024] First, 5 μL of Agrobacterium containing the VvPMA10 overexpression vector was inoculated into a flask containing 20 mL of LB broth containing kanamycin and rifampicin. The cells were shaken at 220 rpm for 24 hours at 28°C for activation. Subsequently, 100 μL of the activated culture was transferred to 200 mL of LB broth containing kanamycin and rifampicin. The culture was incubated at 28°C and 220 rpm for 24 hours. The OD value was measured. When the OD600 reached between 0.8 and 1.0, the cells were centrifuged at 5000 rpm for 5 minutes to collect the cells. The cells were then resuspended in 200 mL of transformation solution (50 g / L sucrose, 2.2 g / L MS, 0.04 g / L acetosyringone, pH 5.8). The VvPMA10 overexpression vector was then transferred into grape stem segments, calli, and Arabidopsis thaliana (Columbia) using Agrobacterium-mediated infection. Hygroscopious was added to the culture medium for retrograde screening of grape stem segments and calli, and Basta was sprayed on Arabidopsis to screen transgenic lines. The homozygous lines were obtained for phenotypic analysis using the TransDirect Plant Tissue PCR Kit (Beijing Quanshijin) according to the standard operating procedures.

[0025] Compared with the related art known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:

[0026] (1) The present invention has shown that overexpression of VvPMA10 improves the alkaline salt tolerance of grape roots and can increase H + -ATPase activity, promoting root acid secretion.

[0027] (2) The present invention has shown that overexpression of VvPMA10 enhances the ability of grape callus to resist NaHCO3 stress. Under saline-alkali stress, the growth of transgenic callus is less inhibited and the MDA content is higher. + -ATPase activity was significantly increased, and oxalate secretion level was significantly increased; overexpression of VvPMA10 also promoted H + secretion.

[0028] (3) Similarly, the present invention has shown that overexpression of VvPMA10 makes Arabidopsis and yeast more tolerant to NaHCO3 stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which constitute a part of the specification of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1Overexpression of VvPMA10 enhances NaHCO3 tolerance in grape roots. qRT-PCR analysis of VvPMA10-OE transgenic grape roots (A). (B) Phenotypes of WT and transgenic roots after 3 days of NaHCO3 treatment. (C) MDA content and H + -ATPase activity (D), oxalate secretion level (E) and H + Determination of efflux rate (F).

[0031] Figure 2 Overexpression of VvPMA10 enhances NaHCO3 tolerance in grape callus. VvPMA10 expression levels in WT and VvPMA10-OE grape callus (A). Growth phenotype (B) and growth increment (C) of WT, VvPMA10-overexpressing (VvPMA10-OE1 and VvPMA10-OE2), and VvPMA10-edited (VvPMA10-Cas9-1 and VvPMA10-Cas9-2) calli under NaHCO3 conditions. MDA content (D), H + -ATPase activity (E) and oxalate secretion level (F) were measured. Acid secretion phenotype of wild-type and VvPMA10 transgenic grape calli (G).

[0032] Figure 3 : Heterologous overexpression of VvPMA10 enhances NaHCO3 tolerance in Arabidopsis thaliana and Saccharomyces cerevisiae. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations and / or combinations thereof.

[0035] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0036] Example 1

[0037] 1. Acquisition of genes:

[0038] The roots of tissue culture seedlings of suitable growth were wrapped in aluminum foil and immediately immersed in liquid nitrogen for RNA extraction. Reverse transcription was performed to convert the cDNA, which was then amplified using specific primers and a high-fidelity enzyme (Novozyme P505). The amplified fragment was then constructed into the plant expression vector PHB using homologous recombination.

[0039] 2. Obtaining VvPMA10 Overexpressing Plant Materials

[0040] Wild-type grape stem segments, calli and Arabidopsis thaliana were transformed by Agrobacterium-mediated method to obtain transgenic lines overexpressing VvPMA10.

[0041] First, 5 μL of Agrobacterium containing the VvPMA10 overexpression vector was inoculated into a flask containing 20 mL of LB broth containing kanamycin and rifampicin. The cells were shaken at 220 rpm for 24 hours at 28°C for primary activation. Subsequently, 100 μL of the activated culture was transferred to 200 mL of LB broth containing kanamycin and rifampicin. The culture was incubated at 28°C and 220 rpm for 24 hours. The OD value was measured. When the OD600 reached between 0.8 and 1.0, the cells were centrifuged at 5000 rpm for 5 minutes to collect the cells. The cells were then resuspended in 200 mL of transformation solution (50 g / L sucrose, 2.2 g / L MS, 0.04 g / L acetosyringone, pH 5.8). The VvPMA10 overexpression vector was then transferred into grape stem segments, calli, and Arabidopsis thaliana (Columbia) using Agrobacterium-mediated infection. Hygroscopious was added to the culture medium for retrograde screening of grape stem segments and calli, and Basta was sprayed on Arabidopsis to screen transgenic lines. The homozygous lines were obtained for phenotypic analysis using the TransDirect Plant Tissue PCR Kit (Beijing Quanshijin) according to the standard instructions for verification.

[0042] Real-time PCR was used to detect the expression of VvPMA10 in the obtained lines. The specific primers used for quantitative analysis of the grape VvPMA10 gene in real-time PCR were dF (5′-TGATCCAGGCAGCTCATACG-3′) and dR (5′-GCAAAGTCTCTGTTCCCTGG-3′). The reference gene for actin in grape stem segments and calli was as follows:

[0043] Actin-F: 5′-GAGATTCCGTTGTCCAGAAGTC-3′;

[0044] Actin-R: 5′-CAATGTTGCCATAGAGGTCCTT-3′.

[0045] 3. Obtaining VvPMA10 Overexpressing Yeast Materials

[0046] Specific primers were used to amplify the fragment using a high-fidelity enzyme (Novozyme P505). The amplified fragment was then inserted into the yeast expression vector PYES2 by homologous recombination. The PYES2 vector carrying the VvPMA10 gene was transformed into BY4741 yeast cells using the PEG / LiAc method. Transformed yeast cells were plated onto SD / -U medium and cultured at 30°C for 3–5 days. Positive clones were identified by PCR. Subsequently, a single positive clone was inoculated into a flask containing 20 ml of SD / -U medium and shaken at 30°C at 220 rpm for 24 hours for primary activation. Subsequently, 100 μL of the activated culture was transferred to 200 ml of SD / -U medium and cultured at 28°C at 220 rpm. The OD value was measured and, when the OD600 reached between 0.8 and 1.0, the cells were collected by centrifugation at 5000 rpm for 5 minutes. Treat with 100 mM NaHCO₃ for 3 hours, then dilute and spot-plate the bacterial suspension onto SD / -U medium. Incubate at 30°C for 3–5 days and observe the growth of the yeast strain. Use the empty PYES2 vector as a control.

[0047] Example 2

[0048] 1 Materials and Methods

[0049] 1.1 Test materials

[0050] 1.1.1 Plant test materials

[0051] In this experiment, the experimental materials used include: grape tissue culture seedlings, 'red Gamay' grape callus, BY4741 cerevisiae yeast and Arabidopsis thaliana;

[0052] Grape tissue culture seedlings: SA15 tissue culture seedlings preserved in the laboratory, subcultured every six weeks, cultured at 24°C, 16h / 8h (light / dark) environment, one-month-old healthy and uniform tissue culture seedlings were used for the experiment;

[0053] The culture method of 'red Gamay' grape callus was similar to that of previous studies ( Gollop et al., 2002 ): culture was carried out under sterile conditions at 24°C in the dark, and subcultured every three weeks;

[0054] 1.2 Treatment Method

[0055] Transgenic roots: 50 mM NaHCO3 treatment

[0056] Arabidopsis: 4 mM NaHCO3 treatment

[0057] Saccharomyces cerevisiae: 100 mM NaHCO3 treatment

[0058] 2 Results

[0059] Table 1 Analysis of physicochemical properties of grape VvPMA10

[0060]

[0061] 2.1 VvPMA10 overexpression in roots promotes acid secretion and improves NaHCO3 tolerance

[0062] Next, this study used Agrobacterium tumefaciens to generate grape root lines overexpressing VvPMA10 to evaluate its function. qRT-PCR analysis revealed that VvPMA10 expression was significantly upregulated in seven roots, indicating that seven VvPMA10-overexpressing root lines were successfully obtained. Two root lines (OE2 and OE5) with the highest expression levels were selected for further study. Figure 1 Middle A).

[0063] The overexpression roots and wild-type roots were treated in a NaHCO3 environment for 3 days. Figure 1 As shown in Figure B, the WT roots showed browning and were more severely damaged, while the transgenic roots showed a less damaged phenotype. Under alkaline salt stress, the MDA content in the VvPMA10-OE roots was significantly lower than that in the WT roots, indicating that VvPMA10 overexpression improved the alkaline salt tolerance of grape roots ( Figure 1 At the same time, we evaluated the organic acid secretion level of the transgenic roots. Compared with the control, the VvPMA10 transgenic roots under NaHCO3 stress significantly increased the secretion of H + -ATPase activity and oxalate secretion level ( Figure 1 D, E). Effect of NMT technology on the H of VvPMA10 transgenic roots. + The efflux rate test found that VvPMA10 overexpression promoted the proton efflux rate compared with the wild-type roots. When NaHCO3 was treated for less than 6 h, the H + There was no significant difference in the outflow rate. As the treatment time was extended to 12 and 24 hours, NaHCO3 significantly promoted the secretion of hydrogen ions ( Figure 1 In conclusion, overexpression of VvPMA10 increased H + -ATPase activity, promoting root acid secretion.

[0064] 2.2 Overexpression of VvPMA10 improves alkaline salt tolerance in grape callus

[0065] To further confirm the function of VvPMA10, we obtained genetic transformation materials of 'red Gamay' callus tissue. Through RNA level identification, we successfully obtained two independent VvPMA10 overexpressing transgenic callus lines (VvPMA10-OE1 and VvPMA10-OE2) ( Figure 2 Subsequently, alkaline salt tolerance experiments and acid secretion level analysis were carried out using transgenic calli. Figure 2 As shown, overexpression of VvPMA10 enhances the tolerance of grape callus to NaHCO3 stress. Under NaHCO3 stress, the growth of WT callus was significantly inhibited, the growth of overexpressing transgenic callus was less inhibited, and the growth of gene-edited callus (VvPMA10-Cas9-1 and VvPMA10-Cas9-2) was more severely inhibited ( Figure 2 Middle B), the growth increment of VvPMA10-OE callus was significantly higher than that of WT callus ( Figure 2 In addition, NaHCO3 treatment increased the MDA content of callus tissue, while the MDA content in overexpression transgenic callus was significantly lower than that in wild-type callus, while the loss-of-function callus showed the opposite phenotype ( Figure 2 Middle D).

[0066] At the same time, it was found that under normal growth conditions, there was no significant difference in proton pump activity and oxalic acid secretion between transgenic callus and WT callus; however, after alkaline salt treatment, H + -ATPase activity was significantly increased, and oxalate secretion level was significantly increased ( Figure 2 In addition, in the medium containing bromocresol purple, it was observed that NaHCO3 treatment caused a clear yellow area to appear around the VvPMA10-OE callus, indicating that overexpression of VvPMA10 also promoted H + Secretion ( Figure 2 Middle G).

[0067] Therefore, we concluded that overexpression of VvPMA10 increases H + -ATPase activity, promoting oxalic acid secretion, and thus enhancing the alkaline salt tolerance of callus tissue.

[0068] 2.3 Overexpression of VvPMA10 improves alkaline salt tolerance in Arabidopsis and yeast

[0069] This study also heterologously overexpressed the VvPMA10 gene in the model plants Arabidopsis thaliana and yeast to further analyze its function in resisting alkaline salt stress. Alkaline salt tolerance experiments were conducted on homozygous T3 transgenic Arabidopsis thaliana. Figure 3As shown, when cultured on 1 / 2MS medium, the growth phenotypes of wild-type and transgenic Arabidopsis were consistent, while when grown in a NaHCO3 environment, it was found that the roots of VvPMA10 transgenic Arabidopsis were longer. In addition, the growth trends of control yeast (carrying an empty pYES2 plasmid) and transgenic yeast (carrying a VvPMA10-pYES2 plasmid) were consistent under normal growth conditions, while under NaHCO3 stress, the growth of VvPMA10 transgenic yeast was less inhibited than that of control yeast ( Figure 3 Thus, overexpression of VvPMA10 renders Arabidopsis and yeast more tolerant to NaHCO3 stress.

[0070] In summary, VvPMA10 overexpression improves alkaline salt tolerance in grape roots, calli, Arabidopsis, and yeast, indicating that VvPMA10 is a positive regulator of alkaline salt tolerance.

[0071] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Overexpression VvPMA10 Application of a gene in improving alkaline salt tolerance of plants or microorganisms, wherein the plant is grape or Arabidopsis thaliana, and the microorganism is yeast; VvPMA10 The specific information of the gene is: Gene ID: VIT_209s0002g00130, Location: chr9:122206-139084.

2. An overexpression according to claim 1 Vv Application of a polypeptide encoded by the PMA10 gene in improving the alkaline salt tolerance of plants or microorganisms; the plant is grape or Arabidopsis thaliana, and the microorganism is yeast.

3. A method for cultivating highly alkaline salt-tolerant plants, characterized in that: The method comprises the following steps: The overexpression gene of claim 1 is obtained by transforming wild-type grape stem segments, calli or Arabidopsis thaliana using a vector-mediated method. VvPMA10 Genetic transgenic lines.

4. The method for cultivating highly alkaline salt-tolerant plants according to claim 3, wherein: The vector-mediated method is an Agrobacterium-mediated method.

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