Vitis amurensis calcium-binding protein gene VamCP1, its encoded protein, expression vector and application

Abstract

The present invention discloses a Vitis amurensis calcium-binding protein gene VamCP1 , its encoded protein, expression vector and application. The present application constructs an overexpression vector of pCAMBIA2300-VamCP1-GFP, and transforms it into Arabidopsis thaliana and "Chardonnay" grapes respectively through the agrobacterium-mediated floral dip method and transient transformation method to study the resistance of transgenic plants of calcium-binding protein VamCP1 under low temperature stress. The results show that VamCP1 the gene can improve the activities of antioxidant enzymes and the content of osmoregulatory substances, reduce the damage of plant cell membranes and the leakage of electrolytes. This gene enhances the cold stress resistance of plants by inducing the expression of related cold tolerance genes and scavenging excessive ROS.

Description

Technical Field

[0001] This invention application relates to the technical field of genetic engineering, and particularly relates to a Vitis amurensis calcium-binding protein gene VamCP1 , its encoded protein, expression vector and application. Background Art

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

[0003] To cope with low temperature stress, plant cells have evolved a complex signal perception and transduction mechanism. When a plant receives a cold signal, the signal transduction pathways within the cell are activated. These pathways involve a variety of protein and non-protein components that interact with each other to form a complex signal network. Among them, calcium ions (Ca²⁺), as important second messengers, play a key role in the growth and development of plants and their response to environmental stresses. When a plant faces low temperature stress, Ca²⁺ rapidly flows into the cytoplasm from outside the cell, resulting in a significant increase in the intracellular Ca²⁺ level. This change is recognized, decoded, and transmitted by various Ca²⁺ sensor proteins, thereby triggering a series of physiological reactions to cope with the cold environment.

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

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

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

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

[0008] The inventors cloned a calcium-binding protein gene from the cold-resistant Vitis amurensis "Shuangyou". VamCP1 In order to verify the function of this gene in response to low temperature stress, the Agrobacterium-mediated genetic transformation technology was used to transform the VamCP1 gene into the model plant Arabidopsis thaliana and the cultivated grape variety "Chardonnay"; by observing the performance of transgenic plants under low temperature stress, it was evaluated and verified that the VamCP1 gene has excellent effects on improving the cold tolerance of plants, thus providing a theoretical basis and practical guidance for carrying out grape stress-resistant molecular breeding and increasing grape yield.

[0009] Specifically, the present disclosure provides a 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 coding protein of the above-mentioned Vitis amurensis calcium-binding protein gene VamCP1 whose amino acid sequence is shown in SEQ ID NO.2.

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

[0012] According to the fourth aspect of the present disclosure, the Vitis amurensis calcium-binding protein gene VamCP1 the coding protein or the expression vector is applied to improving the antioxidant enzyme activity and / or proline content of plants.

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

[0014] According to the sixth aspect of the present disclosure, the Vitis amurensis calcium-binding protein gene VamCP1The encoded protein or the expression vector is applied to improving the cold stress resistance, reactive oxygen species scavenging ability, and / or antioxidant ability of plants.

[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 applied to breeding / identifying varieties / lines related to the cold stress resistance traits of plants.

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

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

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

[0019] One or more technical solutions provided in the embodiments of the present application have at least any one of the following technical effects or advantages:

[0020] The Vitis amurensis 'Shuangyou' calcium-binding protein gene was screened and identified. VamCP1 The full-length of the complete open reading frame sequence of this gene is 582 bp, encoding 193 amino acids, with three EF hand domains, conforming to the high conservation of calcium-binding proteins. It is localized in the plasma membrane and nucleus, has a high level of transcriptional self-activation activity, reaches the expression peak at 24 h when Vitis amurensis responds to cold stress, and has the highest expression level in the leaf tissue of Vitis amurensis. This gene can increase the content of osmoregulatory compounds (proline), increase the activities of antioxidant enzymes (POD, SOD, CAT), reduce the levels of malondialdehyde and relative electrical conductivity, and can also regulate the expression levels of CBF and COR genes. VamCP1 Overexpression of the gene enhances the cold stress resistance of plants by inducing the expression of related cold-resistant genes and scavenging excessive ROS, providing a basis for grape cold-resistant molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 For an embodiment of the present application VamCP1 Expression level of the gene in Vitis amurensis; wherein, a: VamCP1 Relative expression levels of the gene in Vitis amurensis 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 (roots, stems, leaves, petioles, tendrils, inflorescences) of Vitis amurensis were normalized using the VvACTIN1 (Vitvi04g01613.t01) gene as an internal reference gene, with significant differences ( P < 0.05, one-way ANOVA).

[0022] Figure 2 This is for VamCP1 the cloning of genes in an embodiment of this application.

[0023] Figure 3 This is for VamCP1 the sequence analysis of the proteins encoded by genes in an embodiment of this application; wherein, a: VamCP1 chromosomal localization; b: phylogenetic relationships of CP1 proteins in different species; c: amino acid sequence alignment of VamCP1, AtCP1, MdCP1, OsCP1, SlCP1, and CsCP1.

[0024] Figure 4 This is for VamCP1 the subcellular localization and transcriptional self-activation activity assay in an embodiment of this application; wherein, a: subcellular localization of the VamCP1 protein fused with green fluorescent protein (GFP); b: transcriptional self-activation activity assay of the VamCP1 protein in yeast, co-transformation of pGBKT7-53 and pGADT7-T was used as a positive control, and co-transformation of pGBKT7-Lam and pGADT7-T was used as a negative control.

[0025] Figure 5 This is VamCP1 the identification of transgenic Arabidopsis thaliana in an embodiment of this application; wherein, a: PCR detection of transgenic Arabidopsis thaliana; b: RT-qPCR detection of transgenic Arabidopsis thaliana.

[0026] Figure 6 This is VamCP1 the cold tolerance evaluation of transgenic Arabidopsis thaliana in an embodiment of this application; wherein, a: phenotypic changes of wild-type (WT) and transgenic Arabidopsis thaliana lines (OE#1, OE#5, OE#7) before and after treatment at -6°C for 4 h and recovery; b: detection of hydrogen peroxide (H 2 O 2 ) levels in wild-type and transgenic Arabidopsis thaliana lines before and after treatment at 4°C for 3 d by DAB staining; c: H 2 O 2 content in wild-type and transgenic Arabidopsis thaliana lines before and after treatment at 4°C for 3 d; d: determination of physiological and biochemical indexes in wild-type and transgenic Arabidopsis thaliana lines before and after treatment at 4°C for 3 d.

[0027] Figure 7For the detection of the expression levels of cold tolerance-related genes ( AtCBF1 , AtCBF2 , AtCBF3 , AtCOR47 , AtRD29A , AtRD29B ) in transgenic Arabidopsis thaliana lines under non-stress and cold stress conditions in one embodiment of the present application, the AtACTIN2 (AT3G18780.1) gene was used as an internal reference to normalize the expression levels, with significant differences ( P < 0.05, one-way ANOVA).

[0028] Figure 8 Identification of transient transformation of "Chardonnay" grapes in one embodiment of the present application; a: PCR detection of transiently transformed "Chardonnay" grapes; b: RT-qPCR detection of transiently transformed "Chardonnay" grapes.

[0029] Figure 9 Cold tolerance evaluation of transiently transformed "Chardonnay" grapes in one embodiment of the present application; a: Phenotypic changes of wild-type (WT) and transiently transformed "Chardonnay" grape lines (OE#1, OE#3, OE#4) before and after cold treatment at -6°C for 2 h and recovery; b: DAB staining to detect H 2 O 2 levels in wild-type and transiently transformed "Chardonnay" grapes before and after cold treatment at 4°C for 3 d; c: H 2 O 2 content in wild-type and transiently transformed "Chardonnay" grapes before and after treatment at 4°C for 3 d; d: Detection of physiological and biochemical indexes of wild-type and transiently transformed "Chardonnay" grapes before and after cold treatment at -2°C.

[0030] Figure 10 For the detection of the expression levels of cold tolerance-related genes ( VvCBF1 , VvCBF2 , VvCBF3 , VvCBF4 , VvCOR15 , VvKIN2 ) in transgenic grape lines under non-stress and cold stress conditions in one embodiment of the present application, the VvACTIN1 (Vitvi04g01613.t01) gene was used as an internal reference gene to normalize the expression levels, with significant differences ( P < 0.05, one-way ANOVA).

[0031] Figure 11 Mechanism of the calcium-binding protein VamCP1 gene in enhancing the cold stress tolerance of Arabidopsis thaliana and grapes in one embodiment of the present application. Detailed implementation manners

[0032] The following is a description of the specific implementation manners of the present invention application in combination with embodiments. However, the following embodiments are only used to illustrate the present application in detail and do not limit the scope of the present application in any way.

[0033] In the following embodiments, the instrument and equipment involved are all conventional instrument 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 unless otherwise specified.

[0034] Example 1 VamCP1 Analysis of the Function and Characteristics of Genes Responding to Low Temperature Stress in Vitis amurensis

[0035] Based on the previous transcriptome analysis of cold-resistant Vitis amurensis 'Shuangyou' and cold-sensitive Vitis vinifera 'Red Globe', the inventors preliminarily speculated that VamCP1 genes might be involved in the cold stress response. To clarify VamCP1 the expression pattern of genes in Vitis amurensis during the cold stress response, RT-qPCR technology was used to analyze the transcriptional levels of Vitis amurensis leaves at different time points under cold stress and in different tissues of Vitis amurensis. The results showed that ( Figure 1 ), VamCP1 the expression of genes reached a peak at 24 h when Vitis amurensis responded to cold stress, and the expression level of these genes was the highest in the leaf tissues of Vitis amurensis, indicating that VamCP1 genes might play an important role in the cold stress response of leaves.

[0036] To further understand the characteristics of VamCP1 genes, homologous cloning technology was used to design specific primers GC-VamCP1- VamCP1 I-F and GC-VamCP1- Kpn I-R (SEQ ID NO.3 - 4) for the CDS region, and using the leaf cDNA of cold-resistant Vitis amurensis 'Shuangyou' as a template, the BamH genes were successfully obtained by PCR amplification ( VamCP1 ), and its coding region sequence is shown in SEQ ID NO.1. Subsequently, a detailed analysis of the amplified Figure 2 gene sequence was carried out ( VamCP1 ): Chromosome localization analysis showed that Figure 3 , Figure 4 genes were located on chromosome 5; phylogenetic tree analysis indicated that VamCP1 the protein sequence encoded by genes had the highest similarity with MdCP1 of apple, indicating that VamCP1 genes might have certain conservation and functional similarity in the plant kingdom; sequence analysis results showed that VamCP1 VamCP1The protein encoded by the gene has three EF hand domains, which conform to the highly conserved characteristics of calcium-binding proteins, indicating that VamCP1 it plays an important role as a calcium-binding protein in plants; in addition, the results of subcellular localization and transcriptional self-activation activity assays show that VamCP1 the gene is localized to the plasma membrane and nucleus and has a high level of transcriptional self-activation activity.

[0037] Example 2. Calcium-binding protein VamCP1 Response of transgenic Arabidopsis thaliana to low temperature stress

[0038] It was found by RT-qPCR analysis in Example 1 that VamCP1 the gene is induced to express under low temperature stress. To further study the VamCP1 function of the gene under low temperature stress, in this example, an overexpression vector pCAMBIA2300-VamCP1-GFP was constructed, and transgenic Arabidopsis thaliana lines were obtained by Agrobacterium-mediated floral dip method. Arabidopsis thaliana seeds were screened on 1 / 2 MS medium containing 75 mg / mL kanamycin (Kan), vernalized at 4°C for 3 d, and then transferred to a light incubator until T3 generation transgenic Arabidopsis thaliana seeds were obtained. Three T3 generation transgenic Arabidopsis thaliana lines with good expression (OE#1, OE#5, OE#7) were screened by RT-qPCR ( Figure 5 ).

[0039] The screened transgenic Arabidopsis thaliana lines (OE#1, OE#5, OE#7) and wild-type Arabidopsis thaliana (WT) were cold acclimated at 4°C for 3 d and then treated at -6°C for 4 h, and the plant phenotypes were observed ( Figure 6 ). It was found that the wild-type plants were significantly wilted and the wild-type Arabidopsis thaliana died directly after recovery at room temperature, while only a small number of yellow leaves appeared in the transgenic Arabidopsis thaliana. Further, the leaves of wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana before and after low temperature treatment were taken, and DAB staining was carried out respectively, the content of H 2 O 2 was detected, and six physiological and biochemical indexes including antioxidant enzymes (POD, SOD, CAT), proline, malondialdehyde, and conductivity were measured. The results show that ( Figure 7 ) under low temperature stress, VamCP1 the overexpression of the gene will enhance the ROS scavenging ability and antioxidant ability of the plants.

[0040] In addition, RT-qPCR was used to further detect the expression levels of cold-resistant genes in wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana under low temperature stress. The results show that ( Figure 7 ) in the CBF pathway, AtCBF1 , AtCBF2 , AtCBF3The 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.

[0041] The above results show that VamCP1 Gene 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.

[0042] Example 3: VamCP1 Response of Chardonnay grapes to low temperature stress after transient transformation

[0043] 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 ).

[0044] Similar to the functional verification method of transgenic Arabidopsis in Example 2, the transgenic "Chardonnay" grape lines (OE#1, OE#3, OE#4) obtained by screening were subjected to phenotypic observation, DAB staining, and H 2 O 2 The content of antioxidant enzymes (POD, SOD, CAT), proline, malondialdehyde, and conductivity were measured. The results showed that ( Figure 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.

[0045] 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. VvCBF1 , VvCBF2 , VvCBF3 , VvCBF4 , VvCOR15 , VvKIN2 The expression levels of these six cold-responsive genes ( Figure 10 ).in, VvCBF1 ,VvCBF2 , VvCBF3 and VvKIN2 all showed relatively late responses, with the expression levels reaching their peaks after 6 h of low-temperature treatment. VvCBF4 , VvCOR15 The expression was upregulated at 3 h of low-temperature treatment and then the transcriptional level decreased significantly at 12 h.

[0046] The above results indicate that VamCP1 the gene improves the cold tolerance of transgenic plants under low-temperature stress by enhancing the ROS scavenging ability and antioxidant capacity of the plants and by inducing the expression of related cold-resistant genes ( Figure 11 ).

[0047] Although some preferred embodiments of the present invention application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0048] Obviously, those skilled in the art can make various changes and variations to the present invention application without departing from the spirit and scope of its inventive concept. Thus, if these modifications and variations of this application fall within the scope of the claims of this application and their equivalent technologies, this application also intends to include these modifications and variations.

Claims

1. The DNA sequence of the Vitis amurensis calcium-binding protein gene shown in SEQ ID NO.1 VamCP1 or containing the gene of calcium binding protein of Vitis amurensis VamCP1 Use of an overexpression vector in increasing plant antioxidant enzyme activity and / or proline content, characterized in that: The Vitis amurensis calcium-binding protein gene VamCP1 Overexpression; the plant is Vitis amurensis or Arabidopsis thaliana.

2. The DNA sequence of the Vitis amurensis calcium-binding protein gene shown in SEQ ID NO.1 VamCP1 or containing the gene of calcium binding protein of Vitis amurensis VamCP1 Use of an overexpression vector in reducing the malondialdehyde content and / or electrical conductivity of a plant, characterized in that: The Vitis amurensis calcium-binding protein gene VamCP1 Overexpression; the plant is Vitis amurensis or Arabidopsis thaliana.

3. The DNA sequence of the Vitis amurensis calcium-binding protein gene shown in SEQ ID NO.1 VamCP1 or containing the gene of calcium binding protein of Vitis amurensis VamCP1 Use of an overexpression vector in improving plant low temperature stress resistance, active oxygen scavenging ability and / or antioxidant ability, characterized in that: The Vitis amurensis calcium-binding protein gene VamCP1 Overexpression; the plant is Vitis amurensis or Arabidopsis thaliana.

4. The DNA sequence of the Vitis amurensis calcium-binding protein gene shown in SEQ ID NO.1 VamCP1 or containing the gene of calcium binding protein of Vitis amurensis VamCP1 The use of an overexpression vector in breeding varieties / lines related to plant resistance to low temperature stress is characterized in that: The Vitis amurensis calcium-binding protein gene VamCP1 Overexpression; the plant is Vitis amurensis or Arabidopsis thaliana.

5. The gene of Vitis amurensis calcium-binding protein whose DNA sequence is shown in SEQ ID NO.1 VamCP1 or containing the gene of calcium binding protein of Vitis amurensis VamCP1 The use of an overexpression vector in regulating the expression of plant CBF / COR genes is characterized in that: The Vitis amurensis calcium-binding protein gene VamCP1 Overexpression; the plant is Vitis amurensis or Arabidopsis thaliana.

6. The gene of Vitis amurensis calcium-binding protein whose DNA sequence is shown in SEQ ID NO.1 VamCP1 or containing the gene of calcium binding protein of Vitis amurensis VamCP1 Use of an overexpression vector in the construction or breeding of transgenic plants resistant to low temperature stress, characterized in that: The Vitis amurensis calcium-binding protein gene VamCP1 Overexpression; the plant is Vitis amurensis or Arabidopsis thaliana.

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