Vitis amurensis transcription factor VaWRKY26 as well as encoding protein, expression vector and application thereof

Abstract

The invention relates to a vitis amurensis transcription factor VaWRKY26 as well as an encoding protein, an expression vector and application thereof, and aims to solve the technical problems of deficiency of negative regulatory factors and unknown antioxidation and CBF pathway synergistic mechanism in cold-resistant breeding of grapes. The vitis amurensis VaWRKY26 gene is separated and cloned, a recombinant expression vector of the vitis amurensis VaWRKY26 gene is constructed, and Arabidopsis thaliana and grapes are transformed through agrobacterium tumefaciens mediation; results show that overexpression of VaWRKY26 significantly inhibits antioxidant enzyme activity of plants and accumulation of osmoregulation substances, and significantly aggravates cell membrane damage and electrolyte permeation; the gene inhibits CBF-COR cold-resistant pathway core gene expression and destroys ROS removal system functions through a dual action mechanism; the invention discloses a new function of enhancing the low-temperature sensitivity of plants as a negative regulatory factor, and provides a reverse molecular design target for cold-resistant breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular-assisted breeding technology, and in particular to a grape transcription factor. VaWRKY 26. Its encoded protein, expression vector and application. Background Technology

[0002] Grape( Vitis As a globally important economic fruit tree, the fruit and its derivatives are widely cultivated due to their nutritional and commercial value. However, low-temperature stress [including chilling injury (<20℃) and freezing injury (<0℃)] severely damages the plant's physiological functions, limiting its geographical distribution and sustainable industrial development. Low temperatures can induce the following cascade of damage: ① Cell membrane damage: reduced membrane fluidity, hindering substance exchange; ② Protein instability: conformational changes in key functional proteins, metabolic disorders; ③ Enzyme activity inhibition: decreased metabolic reaction efficiency; ④ Oxidative stress: excessive accumulation of reactive oxygen species (ROS), exceeding the scavenging capacity of the antioxidant system (SOD / CAT / POD), leading to irreversible cell damage.

[0003] Plants respond to low-temperature stress through the WRKY transcription factor family. By binding target gene promoter W-box elements through the conserved WRKY domain, they regulate downstream cold resistance pathways: ① activate the classical ICE-CBF-COR signaling pathway (ICE→CBF→COR→cold resistance); ② independently regulate the synthesis of osmotic regulators, the expression of antioxidant enzyme genes, and the maintenance of membrane stability.

[0004] The existing technology has the following shortcomings: ① Lack of research on the function of grape cold resistance genes: especially the lack of systematic analysis of WRKY members in highly cold-resistant germplasm (such as wild grape); ② Scarcity of molecular breeding targets: existing cold resistance mechanisms focus on positively enhancing cold resistance, lacking the exploration of "negative regulatory factors", which limits the design of precision breeding strategies; ③ Incomplete understanding of mechanisms: the interaction mode between WRKY and the ICE-CBF-COR pathway and the regulatory mechanism on the ROS scavenging network have not yet been clarified.

[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 "double-excellent" transcription factors of wild grape were screened and identified. VaWRKY26The gene has a complete open reading frame (ORF) sequence of 1686 bp, encoding 562 amino acids, and contains two conserved WRKY domains. Its expression peaked in *Vitis vinifera* in response to 48 h of low-temperature stress, with the highest expression level observed in its leaf tissues. This gene can reduce the content of osmotic regulatory compounds (proline), decrease the activity of antioxidant enzymes (POD, SOD, CAT), increase malondialdehyde (MDA) and relative conductivity levels, and also regulate the expression levels of CBF and COR genes. VaWRKY26 Overexpression of genes can enhance the sensitivity of plants to low-temperature stress by inducing the expression of related cold-resistant genes and inhibiting the plant's ability to scavenge ROS and its antioxidant capacity, thus providing a basis for molecular breeding of grapes for cold resistance.

[0007] According to one aspect of this disclosure, a grapevine transcription factor is provided. VaWRKY26 Its DNA sequence is shown in SEQ ID NO.1.

[0008] According to a second aspect of this disclosure, a mountain grape transcription factor is provided. VaWRKY26 The encoded protein has the amino acid sequence shown in SEQ ID NO.2.

[0009] According to three aspects of this disclosure, a method is provided that contains the aforementioned grape transcription factor. VaWRKY26 Or the expressive carrier of its fragments.

[0010] According to the four aspects of this disclosure, the wild grape transcription factor VaWRKY26 The encoded protein or the expression vector is used in at least one of the following (1) to (8): (1) Improve the resistance of plants to low temperature stress, or prepare preparations to improve the resistance of plants to low temperature stress; (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 resistance to low temperature stress, or preparing preparations for breeding / identifying varieties / strains of plants with resistance to low temperature stress; (4) Regulate the expression of plant CBF / COR genes, or prepare reagents to regulate the expression of plant CBF / COR genes; (5) Construct transgenic plants resistant to low temperature stress, or prepare reagents for constructing transgenic plants resistant to low temperature stress; (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) Regulate the low temperature sensitivity of plants, or prepare reagents to regulate the low temperature sensitivity of plants.

[0011] In some embodiments of this disclosure, inhibition or downregulation of the mountain grape transcription factor VaWRKY26 Express.

[0012] This invention has been verified through system function testing. VaWRKY26 As a key negative regulator, the gene significantly improves plant low-temperature sensitivity by inhibiting ROS scavenging ability and interfering with the ICE-CBF-COR pathway. The specific effects are stratified as follows: 1. Gene function VaWRKY26 Overexpression of this substance leads to the following in plants: ① Osmotic imbalance: significantly reduced proline content; ② Inhibition of the antioxidant system: reduced POD, SOD, and CAT enzyme activities; weakened ROS scavenging ability, disrupting redox balance; ③ Exacerbated membrane damage: increased malondialdehyde (MDA) accumulation; increased relative cellular conductivity. This indicates that... VaWRKY26 By inhibiting osmotic protection and antioxidant defense, low-temperature sensitivity is positively regulated.

[0013] 2. Signaling pathway regulation VaWRKY26 The WRKY transcription factor enhances low-temperature sensitivity through the following molecular mechanisms: ① Inhibition of the ICE-CBF-COR pathway: downregulation of CBF and COR cold-resistance gene expression; ② Independent negative regulation: direct targeting of the W-box promoter of antioxidant enzyme genes, inhibiting their transcription. This study is the first to reveal that the WRKY transcription factor enhances low-temperature sensitivity through a dual repression mechanism (classical pathway + antioxidant network).

[0014] 3. Universality of breeding applications ① Cross-species validation: The phenotypic-physiological-molecular three-level negative regulatory effect was reproduced in both Arabidopsis thaliana and grape, proving the conservation of the mechanism; ② Targeted design value: By inhibiting VaWRKY26 Expression (e.g., gene editing / RNAi): can relieve its inhibition of the ROS scavenging system and CBF-COR pathway; and improve the cold resistance of cultivated varieties. Attached Figure Description

[0015] Figure 1 As shown in one embodiment of this application VaWRKY26 Gene expression levels in wild grapes; where a: expression levels in wild grapes VaWRKY26 Relative expression levels of genes at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h) under low temperature conditions (4℃); b: VaWRKY26 The relative expression levels of genes in different tissues of wild grape (roots, stems, leaves, petioles, tendrils, inflorescences) were analyzed. VvACTIN1The expression level of the (Vitvi04g01613.t01) gene was standardized using it as an internal reference gene, and significant differences were observed. P <0.05, one-way ANOVA).

[0016] Figure 2 As shown in one embodiment of this application VaWRKY26 Gene cloning.

[0017] Figure 3 As shown in one embodiment of this application VaWRKY26 Sequence analysis of gene-encoded proteins; where a: VaWRKY26 Chromosomal localization; b: VaWRKY26 , AtWRKY33 , SlWRKY26 , CsWRKY33 , MdWRKY26 and OsWRKY24 Amino acid sequence alignment; c: different species VaWRKY26 Phylogenetic relationships of homologous genes.

[0018] Figure 4 As shown in one embodiment of this application VaWRKY26 Identification of transgenic Arabidopsis thaliana; where, a: PCR detection of transgenic Arabidopsis thaliana; b: RT-qPCR detection of transgenic Arabidopsis thaliana.

[0019] Figure 5 As shown in one embodiment of this application VaWRKY26 Evaluation of cold tolerance in transgenic Arabidopsis thaliana: a) Phenotypic changes in wild-type (WT) and transgenic Arabidopsis thaliana lines (OE#6, OE#7, OE#12) after treatment at -6℃ for 4 h and before and after recovery; b) Peroxidase activity levels in wild-type and transgenic Arabidopsis thaliana lines before and after treatment at 4℃ for 3 d by DAB staining; c) Hydrogen peroxide (H2O2) content in wild-type and transgenic Arabidopsis thaliana lines before and after treatment at 4℃ for 3 d; d) Determination of physiological and biochemical indicators in wild-type and transgenic Arabidopsis thaliana lines before and after cold treatment at -6℃ for 4 h.

[0020] Figure 6 In one embodiment of this application, cold-resistance-related genes in transgenic Arabidopsis thaliana lines under non-stress and low-temperature stress conditions (…). AtCBF1 , AtCBF2 , AtCBF3 , AtCOR47 , AtRD29A , AtRD29B 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, one-way ANOVA).

[0021] Figure 7This application describes the identification of transiently transformed "Chardonnay" grapes in one embodiment; wherein, a: PCR detection of the transiently transformed "Chardonnay" grapes; b: RT-qPCR detection of the transiently transformed "Chardonnay" grapes.

[0022] Figure 8 This application provides an embodiment for evaluating the cold tolerance of transiently converted "Chardonnay" grapes; wherein: a: phenotypic changes of wild-type (WT) and transiently converted "Chardonnay" grape lines (OE#1, OE#2, OE#3) after treatment at -2℃ for 3 days and before and after recovery; b: peroxidase activity levels of wild-type and transiently converted "Chardonnay" grapes before and after cold treatment at 4℃ for 3 days, detected by DAB staining; c: hydrogen peroxide (H2O2) content of wild-type and transiently converted "Chardonnay" grapes before and after treatment at 4℃ for 3 days; d: physiological and biochemical indicators of wild-type and transiently converted "Chardonnay" grape lines before and after cold treatment at -2℃ for 3 days.

[0023] Figure 9 In one embodiment of this application, cold-resistance-related genes in transgenic grape lines under non-stress and low-temperature stress conditions ( VvCBF1 , VvCBF2 , VvCBF3 , VvCBF4 , VvKIN2、VvCOR15 The expression level of ) was detected, in order to VvACTIN1 The expression level of the (Vitvi04g01613.t01) gene was standardized using it as an internal reference gene, and significant differences were observed. P <0.05, one-way ANOVA).

[0024] Figure 10 In one embodiment of this application, the mountain grape transcription factor VaWRKY26 Mechanism of action in enhancing the sensitivity of Arabidopsis thaliana and grape to low-temperature stress. Detailed Implementation

[0025] The specific implementation of this application will be described below with reference to the embodiments. However, the following embodiments are only used to illustrate this application in detail and do not limit the scope of this application in any way.

[0026] 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.

[0027] Example 1 VaWRKY26 Analysis of the function and characteristics of genes in response to low temperature stress in wild grapes Based on previous transcriptome analysis studies of the cold-resistant wild grape variety "Shuangyou" and the non-cold-resistant European grape variety "Red Globe," the inventors made preliminary inferences. VaWRKY26The gene may be involved in the low-temperature stress response. RT-qPCR technology was used to clarify... VaWRKY26 The expression pattern of genes in *Vitis vinifera* during low-temperature stress was investigated, and the transcriptional levels of *Vitis vinifera* leaves at different time points and in different tissues under low-temperature stress were analyzed; the results showed that ( Figure 1 ), VaWRKY26 Gene expression peaked in *Vitis thunbergii* after 48 h of response to low-temperature stress, and the highest expression level was observed in the leaf tissues of *Vitis thunbergii*, indicating that... VaWRKY26 Genes may play an important role in the low-temperature stress response of leaves.

[0028] To further understand VaWRKY26 Based on the characteristics of the gene, homologous cloning technology was used to design a gene targeting... VaWRKY26 CDS region-specific primers GC- VaWRKY26 - Kpn IF and GC- VaWRKY26 - BamH IR (SEQ ID NO. 3-4), and using leaf cDNA of the "Shuangyou" wild grape variety as a template, was successfully obtained by PCR amplification. VaWRKY26 Gene( Figure 2 Its coding region sequence is shown in SEQ ID NO.1. Subsequently, the amplified... VaWRKY26 The gene sequence was analyzed in detail. Figure 3 Chromosomal localization analysis showed that VaWRKY26 The gene is located on chromosome 8; phylogenetic analysis shows that... VaWRKY26 The gene-encoded protein sequence is similar to that of soybeans. GmWRKY49 And Raymond's cotton GrWRKY24 Close kinship indicates VaWRKY26 Genes may exhibit a certain degree of conservation and functional similarity within the plant kingdom; sequence analysis results indicate that... VaWRKY26 The gene-encoded protein possesses two WRKY domains, which conform to the highly conserved characteristics of WRKY transcription factors, indicating that... VaWRKY26 It plays an important role in plants as a WRKY transcription factor.

[0029] Example 2 VaWRKY26 The response of transgenic Arabidopsis thaliana to low temperature stress Example 1 revealed through RT-qPCR analysis VaWRKY26 Gene expression was induced by low-temperature stress. Further research is needed. VaWRKY26To investigate gene function under low-temperature stress, this study constructed the pCAMBIA2300-VaWRKY26-GFP overexpression vector. Transgenic Arabidopsis lines were obtained via Agrobacterium-mediated inflorescence impregnation. Arabidopsis seeds were screened on 1 / 2 MS medium containing 75 mg / mL kanamycin (Kan). After vernalization at 4℃ for 3 days, the seeds were transferred to a light incubator until T3 generation transgenic Arabidopsis seeds were obtained. Three T3 generation transgenic Arabidopsis lines (OE#6, OE#7, and OE#12) with good expression were obtained by RT-qPCR screening. Figure 4 ).

[0030] Transgenic Arabidopsis thaliana lines (OE#6, OE#7, OE#12) and wild-type Arabidopsis thaliana (WT) were subjected to cold acclimatization at 10℃ for 1 day and treatment at -6℃ for 4 hours. Plant phenotypes were observed, and it was found that wild-type plants only showed slight wilting of leaves, while transgenic lines showed severe wilting. After recovery and growth for 3 days in a 22℃ light incubator, the growth vigor of transgenic lines was significantly worse than that of wild-type plants. Further analysis of leaves from wild-type and transgenic Arabidopsis thaliana before and after low-temperature treatment, including DAB staining, H2O2 content detection, and determination of six physiological and biochemical indicators (antioxidant enzymes (POD, SOD, CAT), proline, malondialdehyde, and conductivity), showed that... Figure 5 Under low temperature stress, VaWRKY26 Overexpression of genes can inhibit the plant's ability to scavenge ROS and its antioxidant capacity.

[0031] Furthermore, the expression levels of cold-resistance genes in wild-type and transgenic Arabidopsis thaliana under low-temperature stress were further detected by RT-qPCR. The results showed that ( Figure 6 In the CBF pathway, AtCBF1 , AtCBF2 , AtCBF3 Expression levels increased at 3 h and 6 h; in the COR gene family of cold-responsive groups. AtCOR47 , AtRD29A , AtRD29B Expression levels gradually increased after 3 hours, but AtCOR47 and AtRD29A Gene expression levels decreased again at 12 h.

[0032] The above results show that VaWRKY26 This gene plays an important role in the CBF-COR signaling regulatory network and is overexpressed in Arabidopsis thaliana. VaWRKY26 The gene can significantly increase the sensitivity of transgenic Arabidopsis to low-temperature environments.

[0033] Example 3 VaWRKY26 Response of Chardonnay grapes to low-temperature stress after transient conversion To further study overexpression VaWRKY26 The influence of genes on the cold resistance of plants, in this example, transformation. VaWRKY26 Agrobacterium GV3101 (pSoup-p19) bacterial culture was overexpressed into Chardonnay grapes, and three transgenic Chardonnay grape lines (OE#1, OE#2, and OE#3) with good expression were obtained by RT-qPCR screening. Figure 7 ).

[0034] Similar to the functional verification method of transgenic Arabidopsis thaliana in Example 2, the selected transgenic "Chardonnay" grape lines (OE#1, OE#2, OE#3) and wild-type Arabidopsis thaliana (WT) were subjected to cold acclimatization at 10℃ for 1 day and treatment at -2℃ for 3 days. Phenotypic observation, DAB staining, detection of H2O2 content, and determination of six physiological and biochemical indicators, including antioxidant enzymes (POD, SOD, CAT), proline, malondialdehyde, and conductivity, were then performed. The results showed that ( Figure 8 Under low-temperature stress, the transgenic Chardonnay grape lines showed significant wilting. After recovery to normal temperature, all leaves of the transgenic Chardonnay grape lines turned yellow and the plants died, while the leaves of wild-type grape lines remained normal or showed slight yellowing. Overexpression under low-temperature stress VaWRKY26 The gene reduces the activity of antioxidant enzymes in Chardonnay grape vines, regulates the content of osmotic regulators, and increases electrolyte permeability.

[0035] Meanwhile, in transiently transformed Chardonnay grapes, the regulatory mechanism of ICE-CBF-COR resistance to low-temperature stress was detected by RT-qPCR. VvCBF1 , VvCBF2 , VvCBF3 , VvCBF4 , VvKIN2、VvCOR15 The expression levels of these six cold-response groups ( Figure 9 ).in, VvCBF1 , VvCBF2 and VvKIN2 All showed a relatively late response, with expression levels reaching their peak after 6 h of low-temperature treatment; VvCBF3 , VvCBF4 The expression reached its peak after 3 hours of low-temperature treatment; VvCOR15 The level of expression gradually increases over time.

[0036] The above results indicate that VaWRKY26 The gene enhances the low-temperature sensitivity of transgenic plants by inhibiting the plant's ROS scavenging and antioxidant capacity, and by inducing the expression of related cold-resistance genes. Figure 10 ).

[0037] 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 application.

[0038] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of its inventive concept. Therefore, if such modifications and variations 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 grape transcription factor VaWRKY26 Its DNA sequence is shown in SEQ ID NO.

1.

2. The grapevine transcription factor of claim 1 VaWRKY26 The encoded protein has the amino acid sequence shown in SEQ ID NO.

2.

3. Containing the grapevine transcription factor as described in claim 1 VaWRKY26 Or the expressive carrier of its fragments.

4. The grapevine transcription factor of claim 1 VaWRKY26 The use of the protein encoded by claim 2 or the expression vector by claim 3 in at least one of the following (1) to (8): (1) Improve the resistance of plants to low temperature stress, or prepare preparations to improve the resistance of plants to low temperature stress; (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 resistance to low temperature stress, or preparing preparations for breeding / identifying varieties / strains of plants with resistance to low temperature stress; (4) Regulate the expression of plant CBF / COR genes, or prepare reagents to regulate the expression of plant CBF / COR genes; (5) Construct transgenic plants resistant to low temperature stress, or prepare reagents for constructing transgenic plants resistant to low temperature stress; (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) Regulate the low temperature sensitivity of plants, or prepare reagents to regulate the low temperature sensitivity of plants.

5. The application according to claim 4, characterized in that, Inhibit or downregulate grapevine transcription factors VaWRKY 26 expressions.

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