Vitis amurensis calcium binding protein gene VamCP1 as well as encoding protein, expression vector and application thereof

Abstract

The invention discloses a vitis amurensis calcium-binding protein gene VamCP1 as well as an encoding protein, an expression vector and application of the vitis amurensis calcium-binding protein gene VamCP1. According to the application, a pCAMBIA2300-VamCP1-GFP overexpression vector is constructed, and is respectively transformed into arabidopsis thaliana and 'Chardonnay' grapes through an agrobacterium tumefaciens-mediated inflorescence infection method and a transient transformation method, and the resistance of a transgenic plant of the calcium binding protein VamCP1 under low-temperature stress is researched. The result shows that the VamCP1 gene can improve the activity of antioxidant enzyme and the content of osmoregulation substances, reduce the damage of plant cell membranes and reduce the permeation of electrolyte. The gene enhances the low-temperature stress resistance of plants by inducing the expression of related cold-resistant genes and eliminating excessive ROS.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and specifically to a gene encoding a calcium-binding protein of Vitis amurensis VamCP1 , its encoded protein, expression vector and application. Background Art

[0002] Grapes and their related products are widely cultivated around the world due to their rich nutritional value and diverse application value. However, extreme temperature conditions, especially cold stress, pose a serious threat to the growth and development of grapes. Cold stress, as one of the main abiotic adversities, is usually divided into two levels: low temperature stress (0℃~15℃) and freezing stress (below 0℃). When encountering low temperature stress, grape plants will experience a series of declines in physiological functions, including reduced membrane fluidity, weakened protein stability, inhibition of enzyme activity, and changes in the expression pattern of cold-resistant genes. These changes often lead to irreversible damage to the plants.

[0003] In order to cope with low temperature stress, plant cells have evolved a complex signal perception and transmission mechanism. When plants receive cold signals, the signal transduction pathways in the cells will be activated. These pathways involve a variety of proteins and non-protein components, which interact with each other to form a complex signal network. Among them, calcium ions (Ca²⁺), as an important second messenger, play a key role in plant growth and development and response to environmental stress. When plants face low temperature stress, Ca²⁺ will rapidly flow from outside the cell into the cytoplasm, resulting in a significant increase in intracellular Ca²⁺ levels. This change is recognized, decoded and transmitted by various Ca²⁺ sensor proteins, which in turn triggers a series of physiological responses to cope with the cold environment.

[0004] In addition to Ca²⁺ signals, low temperature stress is also accompanied by the accumulation of reactive oxygen species (ROS). As a signaling molecule in organisms, ROS plays an important role under normal circumstances, but excessive accumulation can cause serious damage to plant cells. In order to resist this damage, plants have formed an antioxidant defense system, which is mainly composed of antioxidants and antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), which can remove excessive ROS in cells, thereby protecting plants from damage.

[0005] At the molecular level, low temperature stress induces the expression of related cold-resistance genes. Among them, the ICE-CBF-COR pathway is an important cold-resistance signal transduction pathway. The CBF gene in this pathway belongs to the DREB family, and the protein it encodes can activate the expression of the COR gene, thereby improving the frost tolerance of plants. In addition, multiple cold-resistance gene families involved in regulating resistance to low temperature stress have been identified in plants, such as bHLH, MYB, WRKY, etc.

[0006] However, the functional research on cold-resistance genes in grapes is still relatively limited. Vitis amurensis, native to China, is the most cold-resistant variety in the genus Vitis. It is of great significance to explore the cold-resistance-related genes in Vitis amurensis, explore their functions and mechanisms of action, and improve the resistance of non-cold-resistant grapes to low temperature stress through molecular breeding.

[0007] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0008] The inventor cloned a calcium-binding protein gene from the cold-resistant wild grape "Shuangyou" VamCP1 In order to verify the function of this gene in response to low temperature stress, Agrobacterium-mediated genetic transformation technology was used to transform VamCP1 The gene was transformed into the model plant Arabidopsis thaliana and the cultivated grape variety "Chardonnay"; the performance of the transgenic plants under low temperature stress was observed and evaluated and verified. VamCP1 The gene has an excellent effect on improving plant resistance to low temperature stress, thus providing a theoretical basis and practical guidance for conducting molecular breeding of grape stress resistance and increasing grape yield.

[0009] Specifically, the present invention provides a kind of Vitis amurensis calcium binding protein gene VamCP1 , whose DNA sequence is shown in SEQ ID NO.1.

[0010] According to the second aspect of the present disclosure, there is provided a VamCP1 The encoded protein has an amino acid sequence as shown in SEQ ID NO.2.

[0011] According to the third aspect of the present disclosure, a method is provided comprising the above-mentioned Vitis amurensis calcium-binding protein gene. VamCP1 or an expression vector containing a fragment thereof.

[0012] According to the fourth aspect of the present disclosure, the Vitis amurensis calcium-binding protein gene VamCP1 , the encoded protein or the expression vector is used to increase the activity of plant antioxidant enzymes and / or the proline content.

[0013] According to the fifth aspect of the present disclosure, the Vitis amurensis calcium-binding protein gene VamCP1 , the encoded protein or the expression vector is used to reduce the malondialdehyde content and / or electrical conductivity of plants.

[0014] According to the sixth aspect of the present disclosure, the Vitis amurensis calcium-binding protein gene VamCP1, the encoded protein or the expression vector is used to improve the plant's resistance to low temperature stress, active oxygen scavenging ability and / or antioxidant ability.

[0015] According to the seventh aspect of the present disclosure, the Vitis amurensis calcium-binding protein gene VamCP1 , the encoded protein or the expression vector is used in the breeding / identification of varieties / lines related to plant resistance to low temperature stress.

[0016] According to the eighth aspect of the present disclosure, the Vitis amurensis calcium-binding protein gene VamCP1 Or the expression vector is used in regulating the expression of plant CBF / COR genes.

[0017] According to the ninth aspect of the present disclosure, the Vitis amurensis calcium-binding protein gene VamCP1 Or the expression vector is used in the construction or breeding of transgenic plants resistant to low temperature stress.

[0018] In some embodiments of the present disclosure, the Vitis amurensis calcium-binding protein gene VamCP1 Overexpression in plants.

[0019] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: Screening and identification of the calcium-binding protein gene of Vitis amurensis “double excellence” VamCP1 The complete open reading frame sequence of this gene is 582bp in length, encoding 193 amino acids, with three EF hand domains, which is consistent with the high conservation of calcium-binding proteins. It is located in the plasma membrane and the nucleus, has a high level of transcriptional self-activation activity, reaches the peak expression when Vitis amurensis responds to low temperature stress for 24 hours, and has the highest expression in the leaf tissue of Vitis amurensis. This gene can increase the content of osmotic regulating compounds (proline), increase the activity of antioxidant enzymes (POD, SOD, CAT), reduce the levels of malondialdehyde and relative conductivity, and can also regulate the expression levels of CBF and COR genes. VamCP1 Overexpression of the gene enhances the plant's resistance to cold stress by inducing the expression of related cold-tolerant genes and clearing excess ROS, providing a basis for molecular breeding of grape cold-tolerant plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 In one embodiment of this application VamCP1 The expression level of the fundamental tone in Vitis amurensis; where a: VamCP1 Relative expression levels of genes at different times (0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h) under low temperature conditions (4°C); b: VamCP1The relative expression levels of genes in different tissues of Vitis amurensis (root, stem, leaf, petiole, tendril, inflorescence) were VvACTIN1 (Vitvi04g01613.t01) gene was used as an internal reference gene to standardize the expression level, and the significant difference ( P < 0.05, one-way ANOVA).

[0021] Figure 2 In one embodiment of this application VamCP1 Cloning of genes.

[0022] Figure 3 In one embodiment of this application VamCP1 Sequence analysis of gene-encoded proteins; where a: VamCP1 a: Chromosomal location of CP1 proteins in different species; b: Phylogenetic relationship of CP1 proteins in different species; c: Amino acid sequence alignment of VamCP1, AtCP1, MdCP1, OsCP1, SlCP1 and CsCP1.

[0023] Figure 4 In one embodiment of this application VamCP1 Subcellular localization and transcriptional self-activation activity assay of VamCP1; wherein, a: subcellular localization of VamCP1 protein fused with green fluorescent protein (GFP); b: transcriptional self-activation activity assay of VamCP1 protein in yeast, pGBKT7-53 co-transformed with pGADT7-T was used as a positive control, and pGBKT7-Lam co-transformed with pGADT7-T was used as a negative control.

[0024] Figure 5 In one embodiment of the present application VamCP1 Identification of transgenic Arabidopsis thaliana; wherein, a: PCR detection of transgenic Arabidopsis thaliana; b: RT-qPCR detection of transgenic Arabidopsis thaliana.

[0025] Figure 6 In one embodiment of the present application VamCP1 Evaluation of cold tolerance of transgenic Arabidopsis thaliana; a: phenotypic changes of wild type (WT) and transgenic Arabidopsis lines (OE#1, OE#5, OE#7) treated at -6℃ for 4 h and before and after recovery; b: DAB staining to detect hydrogen peroxide (H2O2) levels of wild type and transgenic Arabidopsis lines before and after treatment at 4℃ for 3 days; c: H2O2 content of wild type and transgenic Arabidopsis lines before and after treatment at 4℃ for 3 days; d: Determination of physiological and biochemical indicators of wild type and transgenic Arabidopsis lines before and after treatment at 4℃ for 3 days.

[0026] Figure 7 In one embodiment of the present application, the cold tolerance-related gene ( AtCBF1 , AtCBF2, AtCBF3 , AtCOR47 , AtRD29A , AtRD29B ) expression level detection, AtACTIN2 (AT3G18780.1) gene was used as an internal reference to normalize the expression level, and the significant difference ( P < 0.05, one-way ANOVA).

[0027] Figure 8 This is the identification of transiently transformed "Chardonnay" grapes in an embodiment of the present application; a: PCR detection of transiently transformed "Chardonnay" grapes; b: RT-qPCR detection of transiently transformed "Chardonnay" grapes.

[0028] Fig. 9 Evaluation of cold tolerance of transiently transformed "Chardonnay" grapes in an embodiment of the present application; a: Phenotypic changes of wild type (WT) and transiently transformed "Chardonnay" grape lines (OE#1, OE#3, OE#4) after cold treatment at -6°C for 2 h and before and after recovery; b: DAB staining to detect H2O2 levels of wild type and transiently transformed "Chardonnay" grapes before and after cold treatment at 4°C for 3 days; c: H2O2 content of wild type and transiently transformed "Chardonnay" grapes before and after treatment at 4°C for 3 days; d: Detection of physiological and biochemical indicators of wild type and transiently transformed "Chardonnay" grapes before and after cold treatment at -2°C.

[0029] Fig.10 In one embodiment of the present application, the cold-resistance-related gene ( VxF , VxF , VxDV , VxDV , VvCOR15 , VvKIN2 ) expression level detection, VvACTIN1 (Vitvi04g01613.t01) gene was used as an internal reference gene to standardize the expression level, and the significant difference ( P < 0.05, one-way ANOVA).

[0030] Fig.11 In one embodiment of the present application, calcium binding protein VamCP1 The mechanism of action of genes improving resistance to cold stress in Arabidopsis and grape. DETAILED DESCRIPTION

[0031] The specific implementation methods of the present invention are described below in conjunction with examples, but the following examples are only used to illustrate the present application in detail and do not limit the scope of the present application in any way.

[0032] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents involved are all commercially available conventional reagents; unless otherwise specified, the detection methods involved are all conventional methods.

[0033] Embodiment 1 VamCP1 Function and characteristics analysis of genes responding to low temperature stress in Vitis amurensis Based on the previous transcriptome analysis of the cold-resistant wild grape "Shuangyou" and the non-cold-resistant European grape "Red Globe", the inventors preliminarily speculated that VamCP1 Genes may be involved in low temperature stress response. VamCP1 The expression pattern of genes in response to low temperature stress in Vitis amurensis was analyzed by RT-qPCR technology at different time points in Vitis amurensis leaves and in different tissues of Vitis amurensis under low temperature stress. The results showed that ( Figure 1 ), VamCP1 The expression of the gene reached its peak at 24 h after Vitis amurensis was exposed to low temperature stress, and the gene was expressed at the highest level in the leaf tissue of Vitis amurensis, indicating that VamCP1 The gene may play an important role in the response of leaves to low temperature stress.

[0034] To further understand VamCP1 The characteristics of the gene were designed by using homologous cloning technology. VamCP1 The CDS region specific primers GC-VamCP1- Kpn IF and GC-VamCP1- Bm IR (SEQ ID NO.3~4), and the leaf cDNA of cold-resistant Vitis amurensis “Shuangyou” was used as template, and the cDNA was successfully obtained by PCR amplification. VamCP1 Gene( Figure 2 ), whose coding region sequence is shown in SEQ ID NO.1. VamCP1 The gene sequences were analyzed in detail ( Figure 3 , Figure 4 ): Chromosome localization analysis showed that VamCP1 The gene is located on chromosome 5; phylogenetic tree analysis shows that VamCP1 The protein sequence encoded by the gene has the highest similarity with MdCP1 of apple, indicating VamCP1 The gene may have certain conservation and functional similarity in the plant kingdom; sequence analysis results show that VamCP1 The protein encoded by the gene has three EF hand domains, which are consistent with the highly conserved characteristics of calcium-binding proteins, indicating that VamCP1 As a calcium-binding protein, it plays an important role in plants; in addition, the results of subcellular localization and transcriptional autoactivation activity assays showed that VamCP1The gene is localized to the plasma membrane and the nucleus and has a high level of transcriptional autoactivation activity.

[0035] Example 2: Calcium Binding Protein VamCP1 Response of transgenic Arabidopsis to low temperature stress Example 1 RT-qPCR analysis revealed VamCP1 Gene expression is induced by low temperature stress. VamCP1 The function of the gene under low temperature stress, in this case, the pCAMBIA2300-VamCP1-GFP overexpression vector was constructed, and the transgenic Arabidopsis strains were obtained by Agrobacterium-mediated inflorescence infection. Arabidopsis seeds were screened on 1 / 2MS medium containing 75 mg / mL kanamycin (Kan), and after vernalization at 4°C for 3 days, they were transferred to a light incubator until T3 transgenic Arabidopsis seeds were obtained. Three T3 transgenic Arabidopsis strains with good expression (OE#1, OE#5, OE#7) were screened by RT-qPCR ( Figure 5 ).

[0036] The transgenic Arabidopsis lines (OE#1, OE#5, OE#7) and wild-type Arabidopsis (WT) were cold-acclimated at 4°C for 3 days and then treated at -6°C for 4 h to observe the plant phenotypes ( Figure 6 ), and found that the wild-type plants wilted significantly. After returning to normal temperature, the wild-type Arabidopsis died directly, while the transgenic Arabidopsis only had a small number of yellow leaves. The leaves of wild-type Arabidopsis and transgenic Arabidopsis before and after low temperature treatment were further taken for DAB staining, detection of H2O2 content, and determination of six physiological and biochemical indicators such as antioxidant enzymes (POD, SOD, CAT), proline, malondialdehyde, and conductivity. The results showed that ( Figure 7 ), under low temperature stress, VamCP1 Overexpression of the gene will enhance the plant's ROS scavenging and antioxidant capabilities.

[0037] In addition, RT-qPCR was used to further detect the expression levels of cold-resistance genes in wild-type Arabidopsis and transgenic Arabidopsis under low temperature stress. The results showed that ( Figure 7 ), in the CBF pathway, AtCBF1 , AtCBF2 , AtCBF3 The expression level increased at 3 h and 6 h; in the cold response group COR gene family, AtCOR47 , AtRD29A , AtRD29B After 3 h, the expression level gradually increased. AtRD29A The gene expression level decreased again at 12 h.

[0038] The above results show that VamCP1Gene plays an important role in the CBF-COR signaling regulatory network and is overexpressed in Arabidopsis VamCP1 The gene can significantly improve the resistance of transgenic Arabidopsis to low temperature environments.

[0039] Example 3: VamCP1 Response of Chardonnay grapes to low temperature stress after transient transformation To further investigate overexpression VamCP1 The effect of genes on plant cold tolerance, in this case transformation VamCP1 Agrobacterium was overexpressed in Chardonnay grapes, and three transgenic Chardonnay grape lines with good expression (OE#1, OE#3, OE#4) were obtained by RT-qPCR screening ( Figure 8 ).

[0040] The same method as the functional verification of transgenic Arabidopsis in Example 2 was used to observe the phenotype of the transgenic "Chardonnay" grape lines (OE#1, OE#3, OE#4) obtained by screening, DAB staining, detection of H2O2 content, and determination of six physiological and biochemical indicators, including antioxidant enzymes (POD, SOD, CAT), proline, malondialdehyde, and conductivity. The results showed that ( Fig. 9 ), under low temperature stress, the wild-type grape strains wilted significantly. After returning to normal temperature, all the leaves of the wild-type grape strains turned yellow and the plants died, while the leaves of the transgenic "Chardonnay" grape strains were normal or slightly yellowed; under low temperature stress, the overexpression VamCP1 The genes can increase the activity of antioxidant enzymes in the "Chardonnay" grape strain, regulate the content of osmotic regulating substances, reduce damage to plant cell membranes and reduce the penetration of electrolytes.

[0041] At the same time, in transiently transformed Chardonnay grapes, RT-qPCR was used to detect the regulatory mechanism of ICE-CBF-COR to resist low temperature stress. VxF , VxF , VxDV , VxDV , VvCOR15 , VvKIN2 The expression levels of these six cold-responsive genes ( Fig.10 ).in, VxF , VxF , VxDV and VvKIN2 All of them showed a relatively late response, with the expression level reaching a peak after 6 h of low temperature treatment. VxDV , VvCOR15 The expression was upregulated at 3 h after low temperature treatment, and then the transcription level was significantly decreased at 12 h.

[0042] The above results show that VamCP1The gene improves the resistance of transgenic plants to low temperature stress by improving the ROS scavenging and antioxidant capacity of the plants and inducing the expression of related cold-resistant genes ( Fig.11 ).

[0043] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

[0044] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A calcium-binding protein gene from Vitis amurensis VamCP1 , whose DNA sequence is shown in SEQ ID NO.

1.

2. The Vitis amurensis calcium-binding protein gene of claim 1 VamCP1 The encoded protein has an amino acid sequence as shown in SEQ ID NO.

2.

3. A protein containing the Vitis amurensis calcium-binding protein gene according to claim 1 VamCP1 or an expression vector containing a fragment thereof.

4. The Vitis amurensis calcium-binding protein gene of claim 1 VamCP1 , Use of the encoded protein according to claim 2 or the expression vector according to claim 3 in improving plant antioxidant enzyme activity and / or proline content.

5. The Vitis amurensis calcium-binding protein gene according to claim 1 VamCP1 , Use of the encoded protein according to claim 2 or the expression vector according to claim 3 in reducing the malondialdehyde content and / or electrical conductivity of plants.

6. The Vitis amurensis calcium-binding protein gene of claim 1 VamCP1 , the use of the encoded protein according to claim 2 or the expression vector according to claim 3 in improving the plant's resistance to low temperature stress, active oxygen scavenging ability and / or antioxidant ability.

7. The Vitis amurensis calcium-binding protein gene of claim 1 VamCP1 , use of the encoding protein described in claim 2 or the expression vector described in claim 3 in the breeding / identification of varieties / lines related to plant resistance to cold stress.

8. The Vitis amurensis calcium-binding protein gene of claim 1 VamCP1 Or use of the expression vector according to claim 3 in regulating the expression of plant CBF / COR genes.

9. The Vitis amurensis calcium-binding protein gene of claim 1 VamCP1 Or use of the expression vector according to claim 3 in the construction or breeding of transgenic plants resistant to low temperature stress.

10. The use according to any one of claims 4 to 9, characterized in that: The Vitis amurensis calcium-binding protein gene VamCP1 Overexpression.

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