Application of Vitis amurensis calcium-binding protein gene VamCP1 in regulation of plant salt stress resistance

Abstract

The present application relates to ampelopsis japonica calcium binding protein gene VamCP 1In the application in the regulation of plant salt stress resistance, the technical problem of low breeding efficiency caused by the scarcity of grape salt resistance gene resources is solved. Based on the cloning of ampelopsis japonica calcium binding protein gene VamCP 1, the plant salt stress response is regulated by overexpression of the gene; the osmotic regulation ability is enhanced, the water imbalance of the plant is relieved; the antioxidant enzyme activity is improved, the cell membrane damage is reduced; the expression of ion homeostasis related genes is regulated, and the ion toxicity is reduced; and a molecular breeding target is provided for cultivating salt-tolerant grape varieties.

Description

Technical Field

[0001] This invention relates to the field of molecular-assisted breeding technology, and in particular to a calcium-binding protein gene of *Vitis thunbergii*. VamCP 1. Application in regulating plant salt stress resistance. Background Technology

[0002] Grape( Vitis vinifera As a globally important economic fruit tree, its cultivation and the wine industry continue to face the severe challenge of soil salinization. Salt stress inhibits plant growth through multiple mechanisms, mainly including: ① Osmotic stress: leading to cell water imbalance and inhibiting physiological metabolism; ② Ion toxicity: excessive accumulation of Na⁺ ions damages enzyme activity and membrane structure; ③ Oxidative stress: bursts of reactive oxygen species (ROS) trigger membrane lipid peroxidation (such as malondialdehyde accumulation) and cell damage.

[0003] Current research on plant salt tolerance mechanisms focuses on the synthesis of osmotic regulators (such as proline), enhancement of antioxidant enzyme (POD / SOD / CAT) activity, regulation of ion homeostasis (such as the SOS pathway), and induction of salt tolerance gene expression. However, the functional analysis of grape salt tolerance genes still has significant limitations: cultivated grapes ( V. vinifera Salt-sensitive germplasm is dominant, and there is a lack of endogenous strong salt-resistant gene resources; existing salt-resistant breeding targets are scarce and the molecular mechanisms are unclear, which restricts the efficiency of salt-tolerant variety breeding.

[0004] mountain grape ( Vitis amurensis Rupr . As a native salt-tolerant germplasm in my country, it has excellent salt stress adaptability, but the discovery and functional verification of its key salt-resistant genes have not been systematically carried out, which has prevented this genetic resource from being effectively applied to salt-resistant molecular breeding.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The inventor's previous research revealed the calcium-binding protein gene from wild grapes. VamCP 1. It has the function of regulating plant low temperature resistance (see patent document CN119193608A). Further research has found that this gene also has a new function of regulating salt stress resistance.

[0007] According to one aspect of this disclosure, the mountain grape calcium-binding protein gene VamCP 1 or its expression vector is used in at least one of the following (1) to (8): (1) Improve plant salt stress resistance, or prepare preparations to improve plant salt stress resistance; (2) To improve the plant’s ability to scavenge reactive oxygen species and / or its antioxidant capacity, or to prepare a formulation that improves the plant’s ability to scavenge reactive oxygen species and / or its antioxidant capacity; (3) Breeding / identifying varieties / strains of plants with salt stress resistance traits, or preparing preparations for breeding / identifying varieties / strains of plants with salt stress resistance traits; (4) Regulate the expression of plant SOS gene, or prepare reagents to regulate the expression of plant SOS gene; (5) Construct salt-resistant transgenic plants, or prepare reagents for constructing salt-resistant transgenic plants; (6) Reduce the malondialdehyde content and / or conductivity of plants, or prepare reagents to reduce the malondialdehyde content and / or conductivity of plants; (7) To increase the activity of plant antioxidant enzymes and / or the content of proline, or to prepare reagents to increase the activity of plant antioxidant enzymes and / or the content of proline; (8) Reduce the salt sensitivity of plants, or prepare reagents to reduce the salt sensitivity of plants.

[0008] In some embodiments of this disclosure, the grape calcium-binding protein gene is... VamCP1 Overexpression.

[0009] In some embodiments of this disclosure, the plants include grapes and Arabidopsis thaliana.

[0010] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages: 1. The calcium-binding protein gene was discovered and validated. VamCP1 It has a novel function of regulating salt stress resistance. VamCP 1. Overexpression significantly enhances the overall salt tolerance of plants, increases the survival rate of transgenic plants and significantly reduces biomass loss, providing direct phenotypic gains for salt-tolerant breeding.

[0011] 2. Targeting the three major toxic mechanisms of salt stress, VamCP 1. Achieve synergistic protection through the following targeted regulation: ① Optimization of the osmotic regulation system: Induces the synthesis of the osmotic protectant proline; alleviates cell dehydration and maintains turgor pressure and metabolic stability; ② Efficient scavenging of oxidative stress: Synergistically enhances the activity of POD, SOD, and CAT enzymes; reduces malondialdehyde (MDA) accumulation and relative conductivity, effectively inhibiting membrane lipid peroxidation and cell damage; ③ Precise regulation of ion homeostasis: Activates the expression of key genes in the SOS pathway; promotes Na+... + External discharge and K + Retaining it reduces ion toxicity.

[0012] 3. VamCP1. As a major gene driving the salt tolerance pathway, it provides heritable transgenic salt-tolerant germplasm, provides targets for molecular marker-assisted selection (MAS) or gene editing, and accelerates the breeding process of salt-tolerant grape varieties. Attached Figure Description

[0013] Figure 1 As shown in one embodiment of this application VamCP 1. Expression level of gene in wild grape; among which, in wild grape VamCP The relative expression levels of gene 1 at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h) under salt stress.

[0014] Figure 2 As shown in one embodiment of this application VamCP 1. Identification of salt sensitivity of transgenic Arabidopsis thaliana during germination; where a: phenotypic observation of germination rate of transgenic Arabidopsis thaliana; b: data analysis of germination rate of transgenic Arabidopsis thaliana.

[0015] Figure 3 As shown in one embodiment of this application VamCP 1. Identification of salt sensitivity of transgenic Arabidopsis thaliana during growth period; a: Observation of root length phenotype of transgenic Arabidopsis thaliana; b: Analysis of root length data of transgenic Arabidopsis thaliana.

[0016] Figure 4 As shown in one embodiment of this application VamCP 1. Identification of salt sensitivity of transgenic Arabidopsis thaliana during seedling stage; phenotypic changes of wild-type (WT) and transgenic Arabidopsis thaliana lines (OE#1, OE#5, OE#7) after 7 days of treatment with different concentrations of salt stress (0 mmol / L, 100 mmol / L, 150 mmol / L, 300 mmol / L).

[0017] Figure 5 As shown in one embodiment of this application VamCP1 Salt tolerance evaluation of transgenic Arabidopsis thaliana; including: a) determination of physiological and biochemical parameters of wild-type and transgenic Arabidopsis thaliana lines before and after treatment with 150 mmol / L NaCl; b) H2O2 content of wild-type and transgenic Arabidopsis thaliana lines before and after treatment with 150 mmol / L NaCl; c) detection of hydrogen peroxide (H2O2) levels of wild-type and transgenic Arabidopsis thaliana lines before and after treatment with 150 mmol / L NaCl by DAB staining; d) oxidative damage of cell membranes of wild-type and transgenic Arabidopsis thaliana lines before and after treatment with 150 mmol / L NaCl.

[0018] Figure 6 In one embodiment of this application, salt tolerance-related genes in transgenic Arabidopsis thaliana lines under non-stress and salt stress conditions (…). AtSOS 1 AtSOS 2 AtSOS 3 AtNHX 1 AtP 5 CS 1 AtRD 29 A The expression level of ) was detected, in order to AtACTIN2 (AT3G18780.1) gene was used as an internal reference to normalize expression levels, and significant differences were observed ( P <0.05, two-way ANOVA.

[0019] Figure 7 Calcium-binding protein in one embodiment of this application VamCP 1. Mechanism of gene regulation of salt stress resistance in Arabidopsis thaliana. Detailed Implementation

[0020] To better understand the technical solution of this application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the detection methods involved are all conventional methods unless otherwise specified.

[0022] Example 1 VamCP Functional and characteristic analysis of gene 1 in response to salt stress in wild grape To clarify VamCP The expression pattern of gene 1 in the salt stress response of wild grape was analyzed using RT-qPCR to detect the transcriptional level of wild grape at different time points under salt stress. The results showed that ( Figure 1 ), VamCP1 Gene expression peaked after 12 hours of salt stress, indicating that... VamCP Gene 1 may play an important role in the salt stress response.

[0023] Example 2: Calcium-binding protein VamCP 1. Response of transgenic Arabidopsis to salt stress Example 1 revealed through RT-qPCR analysis VamCP Gene 1 is induced to express under salt stress. Further research is needed. VamCP The function of gene 1 under salt stress; in this example, pCAMBIA2300- was constructed. VamCPTransgenic Arabidopsis thaliana lines were obtained by Agrobacterium-mediated inflorescence impregnation using the 1-GFP overexpression vector. Arabidopsis seeds were then screened on 1 / 2 MS medium containing 75 mg / mL kanamycin (Kan). After vernalization at 4°C for 3 days, the seeds were transferred to a light incubator until T3 generation transgenic Arabidopsis seeds were obtained. Three T3 generation transgenic Arabidopsis thaliana lines (OE#1, OE#5, and OE#7) with good expression were obtained by RT-qPCR screening.

[0024] This example provides a systematic assessment from the germination stage, growth stage, and seedling stage. VamCP The effect of a gene on salt tolerance in Arabidopsis thaliana. First, during the germination period of Arabidopsis thaliana, seeds of transgenic Arabidopsis and WT were sown in different concentrations of NaCl. Germination rates were observed and recorded. The results showed that the germination rate of transgenic Arabidopsis thaliana after NaCl treatment was significantly higher than that of WT, indicating that the gene... VamCP One gene reduces salt sensitivity during Arabidopsis thaliana germination. Figure 2 Secondly, during the growth period of Arabidopsis thaliana, in order to further explore... VamCP The function of gene 1 under salt stress was investigated by observing and statistically analyzing the root length of transgenic Arabidopsis and WT under different concentrations of NaCl stress during their growth stages. The results showed that the root length of transgenic Arabidopsis was significantly higher than that of WT after NaCl treatment, indicating that the function of gene 1 under salt stress was enhanced. VamCP One gene reduces salt sensitivity during Arabidopsis thaliana growth. Figure 3 Finally, during the seedling stage, four-week-old T3 generation transgenic Arabidopsis thaliana (OE#1, OE#5, OE#7) and wild-type (WT) plants were subjected to gradient NaCl stress treatment (0–300 mmol / L); phenotypic observation results showed that as the salt concentration increased, the salt damage symptoms of transgenic plants and WT plants were significantly different. Figure 4 In the absence of NaCl stress treatment, the conversion... VamCP Both the 1-gene Arabidopsis and WT maintained normal growth after 7 days, with leaves showing a tender green color. Under low salt stress (100 mmol / L NaCl) for 7 days, WT leaves showed extensive yellowing, with some wilting and wrinkling, while the transgenic lines only showed yellowing at the leaf edges. Under medium salt stress (150 mmol / L NaCl) for 7 days, all WT leaves turned yellow, some wilted and stunted, and some even died, while the transgenic Arabidopsis showed localized yellowing, with superior plant height and leaf spread. Under high salt stress (300 mmol / L NaCl) for 7 days, WT died completely, while approximately 15% of the transgenic lines survived, demonstrating significant salt tolerance. This indicates that the transgenic... VamCP Gene 1 reduces salt sensitivity in Arabidopsis seedlings.

[0025] Leaves of wild-type Arabidopsis and transgenic Arabidopsis before and after treatment with 150 mmol / L NaCl were further analyzed. DAB staining, H2O2 content detection, and determination of six physiological and biochemical indicators (antioxidant enzymes (POD, SOD, CAT), proline, malondialdehyde, and conductivity) were performed. The results showed that ( Figure 5 Under salt stress, VamCP Overexpression of gene 1 enhances the plant's ability to scavenge ROS and its antioxidant capacity.

[0026] Furthermore, the expression levels of salt-tolerant genes in wild-type Arabidopsis and transgenic Arabidopsis under salt stress were further examined by RT-qPCR. The results showed that ( Figure 6 In the SOS pathway, AtSOS1 , AtSOS 2. AtSOS 3 showed the highest expression level at 3 h; AtNHX 1 (vacuole Na+ compartmentalization gene) expression level significantly increased after 3 h; osmotic regulation gene AtP 5 CS 1 (proline synthesis) peaked at 3 h, consistent with data showing increased proline content in transgenic Arabidopsis; oxidative stress genes AtRD 29 A Significant induction was achieved at 6 h.

[0027] The above results show that VamCP Gene 1 plays an important role in the SOS signaling regulatory network and is overexpressed in Arabidopsis thaliana. VamCP One gene can significantly improve the resistance of transgenic Arabidopsis to salt stress. Figure 7 ).

[0028] Although some preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0029] Obviously, those skilled in the art can make various modifications and variations to this disclosure without departing from the spirit and scope of its inventive concept. Therefore, if such modifications and variations of this application fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A Vitis californica calcium-binding protein gene VamCP 1 or an expression vector thereof in at least one of (1) to (4) below: (1) Improve plant salt stress resistance, or prepare preparations to improve plant salt stress resistance; (2) Activate the expression of the plant SOS gene, or prepare a reagent to activate the expression of the plant SOS gene; (3) Construct salt-resistant transgenic plants, or prepare reagents for constructing salt-resistant transgenic plants; (4) Reduce the salt sensitivity of plants, or prepare reagents to reduce the salt sensitivity of plants; Its features are, The grape calcium-binding protein gene VamCP1 Overexpression; the plant in question is either wild grape or Arabidopsis thaliana.

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