Ubiquitin transporter AcRAD23D1, its coding gene and application

Abstract

The application discloses a ubiquitin transporter AcRAD23D1, a coding gene and application thereof, and belongs to the technical field of genetic engineering. The application clones an AcRAD23 family gene AcRAD23D1 from a kiwi fruit, and compares phenotypes and determines physiological parameters under transgenic and drought stress treatment, and identifies biological functions of the gene in positive regulation of drought resistance of the kiwi fruit in detail. The research result has important theoretical significance and practical application value for carrying out molecular directional breeding of the kiwi fruit. The overexpression transgenic kiwi fruit plant obtained by using the agrobacterium-mediated maceration method has significantly increased drought stress resistance, the transgenic kiwi fruit plant with silenced expression has significantly reduced drought stress resistance, and the application has important application value in molecular directional breeding of the kiwi fruit and reduction of cultivation environment limitation.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a ubiquitin transporter AcRAD23D1, its encoding gene, and its applications. Background Technology

[0002] Protein ubiquitination is a ubiquitous post-translational modification. Target proteins are degraded via ubiquitin activator E1, ubiquitin conjugator E2, and ubiquitin ligase E3 through 26S proteasome ubiquitination. In addition to these three enzymes, the ubiquitination degradation pathway requires a class of transport proteins to facilitate substrate delivery to the 26S proteasome. UBL-UBA proteins are important transport proteins in the 26S ubiquitination pathway. They can bind to the proteasome receptor and ubiquitination target through their UBL and UBA domains, respectively. UBL-UBA proteins are classified into five types, with RAD23 (Radiation Sensitive 23) belonging to one of them. RAD23 was initially reported for its involvement in nucleotide excision repair induced by ultraviolet radiation in yeast, and its transport function was subsequently discovered. The RAD23 gene family has been identified in various plants, including carrots, rice, Arabidopsis thaliana, maize, and apples.

[0003] RAD23 is involved in plant growth, development, and responses to various stresses in different species. Two RAD23 isoforms in carrot (Daucus carota) have been shown to rescue the phenotype of UV-sensitive rad23D yeast. RAD23 proteins appear to play important roles in plant cell cycle, morphology, and fertility by transporting UPS substrates to the 26S proteasome. Recombinant RAD23 protein inhibits apoptosis in UV-damaged HeLa cells. RAD23B regulates pollen development by participating in the degradation of KRP1 in Arabidopsis thaliana. Apple RAD23 responds to drought, abscisic acid, chilling injury, high temperature, and oxidative stress. MdRAD23D1 regulates drought response by modulating the degradation of the proline-rich apple protein MdPRP6. MdRAD23D1 also improves water use efficiency in apple plants by enhancing photosynthetic efficiency, stomatal behavior, and free amino acid content under prolonged moderate drought.

[0004] Kiwifruit (Actinidia chinensis) is an important economic crop widely cultivated in more than 30 countries, including China, New Zealand, and Italy. Rich in Vitamin C, dietary fiber, and various minerals, kiwifruit is popular among consumers. However, its unique morphological and structural characteristics, such as large, thin leaves with hairs on the underside, a pithed stem structure, and shallow, fleshy roots, make it highly sensitive to water stress. Drought stress severely impacts the quality and yield of kiwifruit. Therefore, identifying key drought-resistant genes in kiwifruit and exploring its potential drought-resistance mechanisms is of great significance for molecular breeding of kiwifruit and the sustainable development of the entire kiwifruit industry. Summary of the Invention

[0005] The purpose of this invention is to provide a ubiquitin transporter AcRAD23D1, its encoding gene, and its applications to address the problems existing in the prior art. This invention utilizes AcRAD23D1 for VIGS-mediated genetic transformation to obtain transgenic plants. Silencing AcRAD23D1 reduces the drought resistance of kiwifruit plants, while overexpression of AcRAD23D1 improves their drought resistance.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of the present invention is a ubiquitin transporter AcRAD23D1, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The second technical solution of the present invention is the AcRAD23D1 gene encoding the ubiquitin transporter AcRAD23D1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] The third technical solution of the present invention is the application of the ubiquitin transporter AcRAD23D1 or the AcRAD23D1 gene in regulating plant drought resistance.

[0010] The fourth technical solution of the present invention is a method for regulating plant drought resistance by silencing the expression of the AcRAD23D1 gene or reducing the level of the ubiquitin transporter AcRAD23D1 to reduce the drought resistance of plants.

[0011] Overexpression of the AcRAD23D1 gene or increasing the level of the ubiquitin transporter AcRAD23D1 can improve the drought resistance of plants.

[0012] The fifth technical solution of the present invention relates to the application of recombinant expression vectors, overexpression vectors, interference vectors, recombinant viruses, recombinant bacteria, or recombinant gene expression cassettes containing the AcRAD23D1 gene in regulating plant drought resistance.

[0013] The sixth technical solution of this invention is the application of recombinant expression vectors, overexpression vectors, interference vectors, recombinant viruses, recombinant bacteria, or recombinant gene expression cassettes containing the AcRAD23D1 gene in drought-resistant plant breeding.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] 1. This invention identified six AcRAD23 genes in the kiwifruit genome, analyzed their phylogenetic relationships, gene structures, conserved motifs, and cis-acting elements of promoters, and identified the expression patterns of family members in kiwifruit in response to different stresses. The results lay the foundation for the study of the function and mechanism of AcRAD23 in kiwifruit stress response.

[0016] 2. An AcRAD23 family gene, AcRAD23D1, was cloned from kiwifruit. Through phenotypic comparison and physiological parameter determination under transgenic and drought stress treatments, its biological function in positively regulating drought resistance in kiwifruit was identified in detail. The research results obtained have important theoretical significance and practical application value for carrying out molecular-directed breeding of kiwifruit.

[0017] 3. The transgenic kiwifruit plants obtained by the present invention through Agrobacterium-mediated infection show significantly increased sensitivity to drought stress, and have important application value in molecular-directed breeding of drought-resistant kiwifruit and reducing the limitations of cultivation environment. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Chromosomal localization of the identified AcRAD23 gene.

[0020] Figure 2 Phylogenetic analysis, exon / intron structure, and motif distribution of the AcRAD23 gene in kiwifruit were performed. In this analysis, A represents the phylogenetic tree constructed using the neighbor-joining method for the phylogenetic analysis of six kiwifruit AcRAD23 genes; B represents the distribution of introns and exons in the AcRAD23 gene; and C represents the conserved domains of AcRAD23.

[0021] Figure 3 Analysis of cis-acting elements in the AcRAD23 promoter region of kiwifruit.

[0022] Figure 4The expression of the AcRAD23 gene in different tissues of kiwifruit was analyzed. In this study, A represents AcRAD23A, B represents AcRAD23B, C represents AcRAD23C1, D represents AcRAD23C2, E represents AcRAD23D1, and F represents AcRAD23D2.

[0023] Figure 5 The expression patterns of the AcRAD23 gene in response to different stresses are shown. A represents the AcRAD23 gene expression pattern under drought, B under waterlogging, C under salt stress, D under darkness, E under low temperature stress, and F under high temperature stress.

[0024] Figure 6 Subcellular localization analysis of AcRAD23s.

[0025] Figure 7 This invention aims to identify the function of AcRAD23D1 in kiwifruit under drought stress. In this study, A represents PCR verification of VIGS-mediated AcRAD23D1 gene silencing in transgenic kiwifruit plants; B represents qRT-PCR analysis of AcRAD23D1 expression in transgenic plants; CD represents morphological differences between WT and D1-VIGS plants under control and drought conditions; E represents the difference in relative water content between WT and D1-VIGS plants under control and drought conditions; F represents the difference in relative electrical conductivity between WT and D1-VIGS plants under control and drought conditions; and G represents the difference in MDA content between WT and D1-VIGS plants under control and drought conditions.

[0026] Figure 8 This represents the accumulation of ROS in WT and D1-VIGS plants under drought conditions in this invention. AB represents NBT and DAB staining; CD represents hydrogen peroxide and oxygen free radical content; EF represents peroxidase and superoxide dismutase activity.

[0027] Figure 9To enhance the drought resistance of kiwifruit by overexpressing AcRAD23D1, the following data were analyzed: A) qRT-PCR analysis of AcRAD23D1 expression in transgenic lines (L1, L2, L3, and L4); B) phenotypes of WT and AcRAD23D1 overexpressing plants (D1-OE) under normal (CK) and drought conditions; C) relative conductivity of WT and D1-OE under CK and drought conditions; D) hydrogen peroxide content of WT and D1-OE under CK and drought conditions; E) superoxide anion content of WT and D1-OE under normal CK and drought conditions; F) SOD activity of WT and D1-OE under CK and drought conditions; and G) POD activity of WT and D1-OE under CK and drought conditions. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0034] This invention provides a ubiquitin transporter AcRAD23D1, the amino acid sequence of which is shown in SEQ ID NO.2.

[0035] This invention also provides the AcRAD23D1 gene encoding the ubiquitin transporter AcRAD23D1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0036] This invention also provides the application of the ubiquitin transporter AcRAD23D1 or the AcRAD23D1 gene in regulating plant drought tolerance.

[0037] In some specific implementations, silencing the expression of the AcRAD23D1 gene or reducing the level of the ubiquitin transporter AcRAD23D1 reduces the drought tolerance of plants.

[0038] Overexpression of the AcRAD23D1 gene or increasing the level of the ubiquitin transporter AcRAD23D1 can improve the drought resistance of plants.

[0039] In some specific implementations, the plant includes kiwifruit.

[0040] This invention also provides a method for regulating plant drought resistance by silencing the expression of the AcRAD23D1 gene or reducing the level of the ubiquitin transporter AcRAD23D1 to reduce plant drought resistance.

[0041] Overexpression of the AcRAD23D1 gene or increasing the level of the ubiquitin transporter AcRAD23D1 can improve the drought resistance of plants.

[0042] This invention also provides the application of recombinant expression vectors, overexpression vectors, interference vectors, recombinant viruses, recombinant bacteria, or recombinant gene expression cassettes containing the AcRAD23D1 gene in regulating plant drought resistance.

[0043] This invention also provides the application of recombinant expression vectors, overexpression vectors, interference vectors, recombinant viruses, recombinant bacteria, or recombinant gene expression cassettes containing the AcRAD23D1 gene in drought-resistant plant breeding.

[0044] This invention provides a kiwi ubiquitin transporter AcRAD23D1, its encoding gene, and its applications. Silencing of AcRAD23D1 expression indicates greater sensitivity to drought stress; in addition, it exhibits higher accumulation of reactive oxygen species and lower activity of reactive oxygen species scavenging enzymes.

[0045] Example 1

[0046] I. Genome Identification of Members of the RAD23 Family of Kiwifruit

[0047] After verifying the existence of the UBL-UBA domain on the NCBI-CDD (https: / / www.ncbi.nlm.nih.gov / cdd), Pfam (https: / / pfam.xfam.org / ), and SMART (http: / / smart.embl.de / smart) online search platforms, six members of RAD23 were finally identified in the kiwifruit genome (https: / / kiwifruitgenome.org).

[0048] Based on the naming conventions in the Arabidopsis genome (AtRAD23A, AtRAD23B, AtRAD23C, and AtRAD23D), the RAD23 genes in kiwifruit were named AcRAD23A, AcRAD23B, AcRAD23C1, AcRAD23C2, AcRAD23D1, and AcRAD23D2, respectively. Basic information on these six AcRAD23 genes is shown in Table 1.

[0049] The CDS length, amino acid length, molecular weight (MW), and isoelectric point (pI) of AcRAD23 were predicted using DNAMAN software. The predicted AcRAD23 CDS lengths ranged from 993 bp (AcRAD23A) to 1209 bp (AcRAD23D2), corresponding to protein lengths of 331 aa and 403 aa, respectively. The predicted AcRAD23 molecular weights ranged from 35.44 kDa (AcRAD23A) to 42.85 kDa (AcRAD23D2), and the predicted isoelectric points ranged from 4.19 (AcRAD23A) to 4.76 (AcRAD23D1). Based on the predicted kiwifruit genome nucleotide sequence, the full-length CDS of AcRAD23s was cloned using RT-PCR. Chromosomal location information of the RAD23 gene was extracted from the gff3 file, and MG2C (http: / / mg2c.iask.in / mg2c_v2.0 / ) was used to locate the kiwifruit RAD23 gene on different chromosomes. Six AcRAD23 genes were randomly distributed on the six chromosomes of 29 kiwifruit chromosomes. AcRAD23A to AcRAD23D1 were distributed sequentially on Chr.16, Chr.26, Chr.2, Chr.3, Chr.25, and Chr.16. Figure 1 ).

[0050] Table 1

[0051]

[0052] II. Evolutionary and Structural Analysis of AcRAD23

[0053] To investigate the characteristics of the AcRAD23 protein, a neighbor-joining phylogenetic tree of the AcRAD23 protein sequence was constructed using MEGA 6.06, and its gene structure and conserved motifs were analyzed using the online Gene Structure Display Server (http: / / gsds.gaolab.org / ) and MEME (http: / / meme-suite.org / ). The phylogenetic tree showed that all AcRAD23 proteins are highly conserved. Figure 2 (A). All AcRAD23 genes have similar gene structures, with AcRAD23B having the longest gene length. Figure 2 (B) Fourteen conserved motifs of AcRAD23 were identified using online MEME. All 14 predicted conserved motifs appeared only once in each AcRAD23 protein. Except for eight motifs that were absent in AcRAD23A, all other AcRAD23 proteins were present. Figure 2 (C)

[0054] III. Analysis of Cis-Components in AcRAD23

[0055] To elucidate the possible mechanisms of AcRAD23's response to different stresses, stress-related cis-acting elements were predicted in the 2kb promoter region upstream of each AcRAD23 transcription start site (ATG) using the online PlantCare database (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). These included ABRE (ABA response element), LTR (cis-acting element involved in the low-temperature response), MYB (MYB binding site), and MYC (MYC binding site). Figure 3 Hormone-related cis-elements were also identified, such as the CGTCA-Motif (a cis-acting element involved in the MeJA response), the P-box (a gibberellin response element), and the TGACG-Motif (a cis-acting element involved in the MeJA response). Among these, MYC and MYB showed the highest frequency in each promoter. These results suggest that the AcRAD23 gene may be involved in stress response and plant hormone signal transduction.

[0056] IV. Expression patterns of AcRAD23 gene in different tissues

[0057] Total RNA was extracted from the roots, stems, leaves, petioles, flowers, and fruits of kiwifruit using a plant genomic total RNA extraction kit (Qingke, Beijing, China). The quality and concentration of the extracted total RNA were assessed using a BioPhotometer (D30, Eppendorf, Germany), and the RNA was reverse transcribed into cDNA using a reverse transcription kit (Thermo Sciences, Waltham, CA, USA). Gene expression levels were detected using a CFX 96 instrument (Bio-Rad, CA, USA). Actine (EF 063572) was used as an internal reference gene to calculate the relative expression of target genes.

[0058] The results showed that all AcRAD23 genes were expressed in most tissues, but the expression levels of each AcRAD23 gene were lower in flower tissues. Figure 4 Of the AcRAD23 compounds, AcRAD23A showed the highest expression level in roots, while AcRAD23B and AcRAD23D1 showed the highest expression levels in stems. Furthermore, AcRAD23C1 and AcRAD23C2 showed the highest expression levels in shoot tips. Among all AcRAD23 compounds, AcRAD23B had the highest expression level, primarily in roots, stems, and shoot tips.

[0059] V. Expression patterns of the AcRAD23 gene under different stresses

[0060] To further understand the potential role of RAD23 under different stress conditions, the transcriptional level of AcRAD23 under different types of abiotic stress was detected using qRT-PCR. Kiwi seedlings were planted in plastic pots (10×10×10cm) filled with a substrate / vermiculite / perlite (3:1:1, v:v:v) mixture and placed indoors with a 14h light / 10h dark photoperiod and 40%–60% relative humidity. Seedlings of similar growth stage, approximately 4 months old, were selected for stress treatment. For heat stress and low temperature stress, seedlings were transferred to incubators at 48℃ and 4℃ for 6h and 24h, respectively; for drought treatment, watering was stopped for 8 days; for darkness treatment, seedlings were exposed to continuous darkness for 24h; salt stress was applied to the seedlings by adding 120mM sodium chloride to the nutrient pots; for waterlogging stress, seedlings were immersed in water for 24h. Kiwifruit leaves were collected at 0, 1, 2, 3, and 5 h of heat treatment. For the other five treatments, leaves were collected at 0, 4, 8, 12, and 24 h. All samples were stored at -80 °C for subsequent experiments.

[0061] Under drought treatment, all AcRAD23 genes except AcRAD23C2 were induced to express 4 hours after drought treatment, but the expression of AcRAD23 genes did not change significantly during the drought treatment process. Figure 5 (A). The expression trends of AcRAD23A and AcRAD23C1 were similar. At 4 h after drought induction, the expression level of AcRAD23D1 was the highest, about 2.5-7 times that of other genes at the same time point.

[0062] Except for AcRAD23A, the expression of almost all AcRAD23 genes was suppressed under waterlogging stress. The expression level of AcRAD23A decreased slightly at 4 hours, then gradually increased, reaching a peak at 24 hours. Figure 5 (Middle B). The expression of AcRAD23B, AcRAD23C1, AcRAD23C2, AcRAD23D1 and AcRAD23D2 decreased continuously from 4h to 24h.

[0063] The expression profile of AcRAD23 varies considerably under salt stress. Figure 5 (C) AcRAD23A expression was significantly downregulated, decreasing to 60% of the level at 0h after 4h. The remaining five genes showed significant upregulation. Compared to 0h after salt treatment, the expression levels of AcRAD23D1, AcRAD23C2, and AcRAD23D2 were approximately 1.8, 4, 2.8, and 3.8 times higher, respectively.

[0064] AcRAD23 gene expression, although varying in degree, exhibited similar expression patterns under dark stress conditions. Figure 5 (Middle D). The expression levels of these genes showed an initial increase, peaked at different time points, and then declined. Notably, AcRAD23D1 showed the highest expression level throughout the stress period.

[0065] Cold stress induced the expression of AcRAD23D1, while inhibiting the expression of the other five AcRAD23 derivatives. Figure 5 AcRAD23D1 responded rapidly to cold stress, with the highest expression level at 4 h, peaking at 8 h (approximately 8.5 times the expression level at 0 h), and expression decreasing after 12 h. The other five genes showed similar expression patterns under cold treatment, with downregulation at 4 h and upregulation at 8 h.

[0066] The expression levels of AcRAD23A, AcRAD23C2, and AcRAD23D2 were all induced by heat stress, with AcRAD23A showing the highest expression level at 1 hour during the stress period. Figure 5 (F). Conversely, AcRAD23B, AcRAD23C1, and AcRAD23D1 showed a tendency to be inhibited by heat treatment. The expression level of AcRAD23B continued to decrease from 0 h.

[0067] VI. Subcellular localization of AcRAD23s

[0068] The CDS of AcRAD23 was cloned into the pCambina2300 vector to construct the 35S::GFP-AcRAD23s fusion expression vector. The empty GFP and 35S::GFP-AcRAD23s fusion expression vector was transformed into Agrobacterium strain EHA105 and cultured overnight in an incubator (28°C, 180 rpm) until OD... 600 =0.6. Bacteria were then collected and resuspended in the same volume of MES solution (10 mM MES, 10 mM MgCl2, 150 mM acetylsuccine, pH 5.8). Empty GFP and recombinant 35S::GFP-AcRAD23s were transiently expressed in tobacco epidermis using a 1 ml syringe. 4,6-Diamino-2-phenol dihydrochloride (DAPI) was used as a nuclear marker. All fluorescence images were obtained using confocal microscopy (FK 1000; Olympus, Tokyo, Japan). The results showed that the AcRAD23 gene was strongly expressed in both the nucleus and cell membrane of tobacco cells, indicating that AcRAD23 is localized in the nucleus and cell membrane of tobacco cells. Figure 6 ).

[0069] VII. VIGS-mediated AcRAD23D1 silencing enhances the drought sensitivity of kiwifruit.

[0070] The nucleotide sequence of AcRAD23D1 is shown in SEQ ID NO.1:

[0071]

[0072] The amino acid sequence of AcRAD23D1 is shown in SEQ ID NO.2:

[0073] MKIFVKTLKGSHFEIEVKPEDTVVDVKKNIESVQGADVYPASQQMLIYQGKVLKDGTTLEENKVTENNFVVIMLMKNKGSSSGGSITSTAPAAKVPQTSAPPPTAAPQAPSATLGSPRSVPAPAPAPAPAPAAAPTTTVTAASESNVYGQAASNLVAGSTLEGTIQQILDMGGGTWDRDTVVRALRAAFNNPERAVEYLY SGIPEQVEVPPVARAPASGLAANSPAQPPQSAQPAPAPAPAPAPVPLSGPNANPLDLFPQGLPNMGSNAAGAGTLDFLRNSQQFQALRTMVQANPQILQPMLQELGKQNPHLMRLIQEHQADFLRLINEPAEGVEGNILEQLTAAMPQAVTVTNEEREAIERLEAMGFDRALVLEVFFACNKNEELAANYLLDHMHEFED.

[0074] AcRAD23D1 was expressed at the highest level under drought stress. Therefore, this invention uses VIGS-mediated silencing of AcRAD23D1 in kiwifruit plants (named D1-VIGS) to study its potential role in drought response.

[0075] To construct the VIGS vector, a 300-base fragment from the 5' end of AcRAD23D1 was introduced into the pTRV 2 vector (tobacco mosaic virus). All successfully constructed vectors were transformed into Agrobacterium tumefaciens strain EHA 105. The AcRAD 23D1-VIGS line was obtained from one-month-old tissue-cultured 'Hongyang' kiwifruit. Kiwifruit plants were immersed in Agrobacterium suspensions containing 1) pTRV 2 and pTRV 1 (1:1) and 2) pTRV 2-AcRAD 23D1 and pTRV 1 (1:1), and the mixture was vacuumed at 0.08 MPa for 10 minutes. After infection, all plants were transferred to MS medium for further growth. Positive lines were identified at both the DNA and mRNA levels. Figure 7 (AB).

[0076] Subsequently, the plants were subjected to drought stress by adding 45% polyethylene glycol to MS medium. Under normal conditions, there was no significant difference in growth phenotype between WT and D1-VIGS plants. However, after drought stress, D1-VIGS plants exhibited more severe necrosis symptoms than WT plants. Figure 7 (CD). Furthermore, the RWC of D1-VIGS plants was lower than that of WT plants ( Figure 7 (E). Meanwhile, VIGS plants exhibited higher REL and MDA contents than WT plants under drought stress. Figure 7 (FG).

[0077] 8. VIGS-mediated AcRAD23D1 silencing in kiwifruit leads to increased ROS accumulation under drought stress.

[0078] Drought stress typically leads to the accumulation of reactive oxygen species (ROS), resulting in oxidative damage. ROS accumulation was observed in both the WT and D1-VIGS lines under both control and drought stress conditions. First, ROS accumulation was qualitatively detected by immersing leaves from the 3rd-4th nodes (top to bottom) of the WT and D1-VIGS lines in DAB and NBT staining solutions. Under control conditions, there was no significant difference in DAB and NBT staining between the WT and D1-VIGS lines; however, under drought conditions, the brown and blue spots on D1-VIGS leaves were deeper than those on WT leaves. Figure 8 (AB). Meanwhile, ROS accumulation was quantitatively detected using the corresponding kit (Nanjing Jiancheng Biotechnology Institute, Nanjing, China), and it was found that the H2O2 and OFR contents of the D1-VIGS strain were higher than those of WT (AB). Figure 8 (CD). Conversely, lower ROS scavenging enzyme activity was found in the D1-VIGS line (CD). Figure 8 Medium EF).

[0079] The inventors also conducted overexpression experiments, which confirmed that overexpression of the AcRAD23D1 gene reduced ROS accumulation in plants under drought stress and enhanced drought resistance. Figure 9 (China AG).

[0080] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A ubiquitin shuttle protein AcRAD23D1, characterized in that, The amino acid sequence thereof is shown as SEQ ID NO.

2.

2. A gene encoding the ubiquitin transporter AcRAD23D1 of claim 1. AcRAD23D1 characterized in that, The nucleotide sequence thereof is shown as SEQ ID NO.

1.

3. The ubiquitin shuttle AcRAD23D1 of claim 1 or the nucleic acid molecule of claim 2 AcRAD23D1 Use of the gene for increasing drought tolerance in plants, characterized in that, The plant is kiwifruit.

4. Use according to claim 3, characterized in that, Overexpression of said AcRAD23D1 genes or increasing the level of said ubiquitin transporter AcRAD23D1 increases the drought tolerance of plants.

5. A method for increasing drought tolerance in plants, characterized by, overexpressing the gene of claim 2 AcRAD23D1 overexpressing the gene of claim 1 or increasing the level of the ubiquitin transporter AcRAD23D1 in a plant, said plant being a kiwifruit.

6. A composition comprising the recombinant virus of claim 2 AcRAD23D1 Use of a recombinant expression vector, recombinant virus, recombinant bacteria or recombinant gene expression cassette of a gene for increasing drought tolerance in plants, characterized in that, The plant is kiwifruit.

7. A composition comprising the recombinant virus of claim 2 AcRAD23D1 The use of a recombinant expression vector, recombinant virus, recombinant bacteria or recombinant gene expression cassette of the gene of claim 2 in breeding drought-tolerant plants, characterized in that, The plant is kiwifruit.

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