Ubiquitin transporter AcRAD23D1 as well as coding gene and application thereof

Abstract

The invention discloses ubiquitin transporter AcRAD23D1 as well as a coding gene and application thereof, and belongs to the technical field of gene engineering. The AcRAD23 family gene AcRAD23D1 is cloned from kiwi fruits, phenotypic comparison and physiological parameter determination under transgenosis and drought stress treatment are utilized, the biological function of the AcRAD23 family gene AcRAD23D1 for positively regulating and controlling drought resistance of the kiwi fruits is identified in detail, and the obtained research result has important theoretical significance and practical application value for developing kiwi fruit molecular directed breeding; the drought stress resistance of the overexpressed transgenic kiwi fruit plant obtained by using an agrobacterium tumefaciens-mediated dip dyeing mode is obviously increased, and the drought stress resistance of the obtained silently expressed transgenic kiwi fruit plant is obviously reduced; the method has an important application value in the aspects of drought-resistant molecular directional breeding of kiwi fruits and reduction of cultivation environment limitation.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a ubiquitin transporter protein AcRAD23D1 and a coding gene and application thereof. Background Art

[0002] Protein ubiquitination is a ubiquitous post-translational modification. The target protein is ubiquitinated and degraded by 26S proteasome through ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. In addition to these three enzymes, the ubiquitin degradation pathway also requires a class of transporters to facilitate the delivery of substrates to the 26S proteasome. UBL-UBA proteins are important transporters in the 26S ubiquitin degradation pathway. They can be connected to proteasome receptors and ubiquitination targets through their UBL domain and UBA domain, respectively. UBL-UBA proteins are divided into five types, and RAD23 (Radiation SENSITIVE23) belongs to one of them. RAD23 was originally reported because they were involved in nucleotide excision repair in yeast caused by ultraviolet light, and their transport function was subsequently discovered. The RAD23 gene family has been identified in many plants, including carrots, rice, Arabidopsis, corn and apple.

[0003] RAD23 is involved in plant growth, development and responses to different 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 an important role in the cell cycle, morphology and fertility of plants by delivering UPS substrates to the 26S proteasome. Recombinant RAD23 proteins inhibit apoptosis in UV-damaged HeLa cells. RAD23B regulates pollen development by participating in the degradation of KRP1 in Arabidopsis. Apple RAD23 responds to drought, abscisic acid, chilling, heat and oxidative stress. MdRAD23D1 regulates drought response by regulating the degradation of apple proline-rich protein MdPRP6. MdRAD23D1 also improves water use efficiency in apple plants by increasing photosynthesis efficiency, stomatal behavior and free amino acid content under long-term moderate drought.

[0004] As an important economic crop, kiwifruit (Actinidia Chinensis) is widely grown in more than 30 countries, including China, New Zealand, and Italy. Due to its rich content of vitamin C, dietary fiber, and various minerals, kiwifruit is very popular among consumers. The unique morphological and structural characteristics of kiwifruit, such as large and thin leaves, hairy leaves on the back, medullary stem structure, and fleshy shallow roots, make kiwifruit highly sensitive to water stress. Drought stress seriously affects the quality and yield of kiwifruit. Therefore, exploring the key genes for drought resistance in kiwifruit and exploring its possible drought resistance mechanism are of great significance to the molecular breeding of kiwifruit and even the sustainable development of the entire kiwifruit industry. Summary of the invention

[0005] The purpose of the present invention is to provide a ubiquitin transporter protein AcRAD23D1 and its encoding gene and application to solve the problems existing in the above-mentioned prior art. The present invention uses AcRAD23D1 to carry out VIGS-mediated genetic transformation to obtain transgenic plants, the silent expression strain of AcRAD23D1 reduces the drought resistance of kiwifruit plants, and overexpression of AcRAD23D1 improves the drought resistance of kiwifruit plants.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a ubiquitin transporter protein AcRAD23D1, whose amino acid sequence 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, and its nucleotide sequence is shown in SEQ ID NO.1.

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

[0010] A fourth technical solution of the present invention is a method for regulating plant drought tolerance, which comprises silencing the expression of the AcRAD23D1 gene or reducing the level of the ubiquitin transporter AcRAD23D1 to reduce the drought tolerance of the plant;

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

[0012] A fifth technical solution of the present invention is the use of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette containing the AcRAD23D1 gene in regulating plant drought tolerance.

[0013] A sixth technical solution of the present invention is the use of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette containing the AcRAD23D1 gene in drought-tolerant plant breeding.

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

[0015] 1. The present invention identified 6 AcRAD23 genes from the kiwifruit genome, analyzed their phylogenetic relationships, gene structures, conserved motif compositions and cis-acting elements of promoters, and identified the expression patterns of family members in kiwifruit in response to different adversities. The research results lay the foundation for the study of the function and mechanism of AcRAD23 in kiwifruit adversity response;

[0016] 2. An AcRAD23 family gene, AcRAD23D1, was cloned in kiwifruit. Its biological function in positively regulating kiwifruit drought resistance was identified in detail by comparing phenotypes and measuring physiological parameters under transgenic and drought stress treatments. The research results obtained have important theoretical significance and practical application value for the molecular directed breeding of kiwifruit.

[0017] 3. The transgenic kiwifruit plants obtained by the Agrobacterium-mediated infection method of the present invention have significantly increased sensitivity to drought stress, and have important application value in molecular directed breeding of kiwifruit drought resistance and reducing cultivation environment restrictions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The chromosomal location of the identified AcRAD23 gene.

[0020] Figure 2 Phylogenetic tree analysis, exon / intron structure and motif distribution of kiwifruit AcRAD23 gene. A represents the phylogenetic tree constructed by neighbor-joining method for phylogenetic analysis of 6 kiwifruit AcRAD23 genes; B represents the distribution of introns and exons of AcRAD23 gene; C represents the conserved domain of AcRAD23.

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

[0022] Figure 4The expression analysis of AcRAD23 gene in different tissues of kiwi fruit. 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 AcRAD23 gene in response to different adversities, where A represents the expression pattern of AcRAD23 gene under drought, B represents the expression pattern of AcRAD23 gene under waterlogging, C represents the expression pattern of AcRAD23 gene under salt stress, D represents the expression pattern of AcRAD23 gene under darkness, E represents the expression pattern of AcRAD23 gene under low temperature stress, and F represents the expression pattern of AcRAD23 gene under high temperature stress.

[0024] Figure 6 Analysis of the subcellular localization of AcRAD23s.

[0025] Figure 7 This is the functional identification of AcRAD23D1 in kiwifruit under drought stress in the present invention. A represents the PCR verification of VIGS-mediated AcRAD23D1 gene silencing transgenic kiwifruit plants; B represents the qRT-PCR analysis of AcRAD23D1 expression in transgenic plants; CD represents the morphological differences between WT and D1-VIGS plants under control and drought conditions; E represents the relative water content difference between WT and D1-VIGS plants under control and drought conditions; F represents the relative conductivity difference between WT and D1-VIGS plants under control and drought conditions; G represents the MDA content difference between WT and D1-VIGS plants under control and drought conditions.

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

[0027] Fig. 9To overexpress AcRAD23D1 to improve the drought resistance of kiwifruit, A is the qRT-PCR analysis of the expression of AcRAD23D1 in transgenic lines (L1, L2, L3 and L4); B is the phenotype of WT and AcRAD23D1 overexpressing plants (D1-OE) under normal (CK) and drought conditions; C is the relative conductivity of WT and D1-OE under CK and drought conditions; D is the hydrogen peroxide content of WT and D1-OE under CK and drought conditions; E is the superoxide anion content of WT and D1-OE under normal CK and drought conditions; F is the SOD activity of WT and D1-OE under CK and drought conditions; G is the POD activity of WT and D1-OE under CK and drought conditions. DETAILED DESCRIPTION

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

[0029] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

[0033] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0034] An embodiment of the present invention provides a ubiquitin transporter protein AcRAD23D1, whose amino acid sequence is shown in SEQ ID NO.2.

[0035] The embodiment of the present invention also provides the AcRAD23D1 gene encoding the ubiquitin transporter AcRAD23D1, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0036] The embodiment of the present invention also provides the use of the ubiquitin transporter protein AcRAD23D1 or the AcRAD23D1 gene in regulating plant drought tolerance.

[0037] In some specific embodiments, silencing the expression of the AcRAD23D1 gene or reducing the level of the ubiquitin transporter AcRAD23D1 reduces the drought tolerance of the plant;

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

[0039] In some specific embodiments, the plant comprises kiwi fruit.

[0040] The embodiment of the present invention also provides a method for regulating plant drought tolerance, silencing the expression of the AcRAD23D1 gene or reducing the level of the ubiquitin transporter AcRAD23D1, thereby reducing the drought tolerance of the plant;

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

[0042] The embodiments of the present invention also provide the use of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette comprising the AcRAD23D1 gene in regulating plant drought tolerance.

[0043] The embodiments of the present invention also provide the use of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette containing the AcRAD23D1 gene in drought-tolerant plant breeding.

[0044] The invention provides a kiwifruit ubiquitin transporter protein AcRAD23D1 and a coding gene and application thereof. The silent expression of the AcRAD23D1 shows a phenomenon of being more sensitive to drought stress; in addition, it shows higher accumulation of active oxygen and lower activity of active oxygen scavenging enzymes.

[0045] Example 1

[0046] 1. Genome-wide identification of kiwifruit RAD23 family members

[0047] After verifying the presence 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 confirmed in the kiwifruit genome (https: / / kiwifruitgenome.org).

[0048] According to the nomenclature in the Arabidopsis genome (AtRAD23A, AtRAD23B, AtRAD23C and AtRAD23D), the RAD23 in kiwifruit was named AcRAD23A, AcRAD23B, AcRAD23C1, AcRAD23C2, AcRAD23D1 and AcRAD23D2. The basic information of these six AcRAD23 is shown in Table 1.

[0049] The CDS length, amino acid length, protein molecular weight (MW) and isoelectric point (pI) of AcRAD23 were predicted using DNAMAN software. The predicted length of AcRAD23 CDS ranged from 993 bp (AcRAD23A) to 1209 bp (AcRAD23D2), corresponding to protein lengths of 331 aa and 403 aa, respectively. The predicted molecular weight of AcRAD23 ranged from 35.44 kDa (AcRAD23A) to 42.85 kDa (AcRAD23D2), and the predicted isoelectric point ranged from 4.19 (AcRAD23A) to 4.76 (AcRAD23D1). Based on the predicted nucleotide sequence of the kiwifruit genome, the full-length CDS of AcRAD23s was cloned using RT-PCR technology. The 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. The six AcRAD23 genes were randomly distributed on the six chromosomes of 29 kiwifruits. AcRAD23A to AcRAD23D1 were distributed on Chr.16, Chr.26, Chr.2, Chr.3, Chr.25 and Chr.16 in turn ( Figure 1 ).

[0050] Table 1

[0051]

[0052] 2. AcRAD23 evolution and structural analysis

[0053] To study the characteristics of AcRAD23 protein, a neighbor-joining phylogenetic tree of AcRAD23 protein sequence was constructed using MEGA 6.06, and its gene structure and conserved motifs were analyzed using the online websites Gene Structure Display Server (http: / / gsds.gaolab.org / ) and MEME (http: / / meme-suite.org / ). The phylogenetic tree showed that all AcRAD23 proteins were highly conserved ( Figure 2 All AcRAD23 genes have similar structures, among which AcRAD23B has the longest gene length ( Figure 2 (B). 14 conserved motifs of AcRAD23 were identified using online MEME. All 14 predicted conserved motifs were present only once in each AcRAD23 protein. Except for 8 motifs that were absent in AcRAD23A, they were present in all AcRAD23 proteins ( Figure 2 Middle C).

[0054] 3. Analysis of cis-elements in AcRAD23

[0055] To elucidate the possible mechanism of AcRAD23 response to different stresses, the online PlantCare (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) database was used to predict the cis-acting elements related to stress for each 2 kb promoter region upstream of the transcription start point (ATG) of AcRAD23, such as ABRE (ABA response element), LTR (cis-acting element involved in low temperature response), MYB (MYB binding site) and MYC (MYC binding site). Figure 3 ). Hormone-related cis-elements were also found, such as CGTCA-Motif (cis-acting element involved in MeJA response), P-box (gibberellin response element), and TGACG-Motif (cis-acting element involved in MeJA response). Among them, MYC and MYB appeared most frequently in each promoter. These results suggest that the function of the AcRAD23 gene may be related to stress response and plant hormone signal transduction.

[0056] 4. Expression patterns of AcRAD23 gene in different tissues

[0057] Total RNA from roots, stems, leaves, petioles, flowers and fruits of kiwifruit was extracted using a plant genome total RNA extraction kit (Qingke, Beijing, China). The quality and concentration of the extracted total RNA were detected 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 was 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 the target gene.

[0058] The results showed that all AcRAD23 genes were expressed in most tissues, but the expression levels of each AcRAD23 gene in flower tissue were low ( Figure 4 ). Among them, AcRAD23A had the highest expression level in roots, and AcRAD23B and AcRAD23D1 had the highest expression levels in stems. In addition, AcRAD23C1 and AcRAD23C2 had the highest expression levels in the shoot apex. Among all AcRAD23, AcRAD23B had the highest expression level, mainly in roots, stems, and shoot apex.

[0059] 5. Expression patterns of AcRAD23 gene under different stresses

[0060] To further understand the potential role of RAD23 under different stress conditions, the transcription level of AcRAD23 under different types of abiotic stress was detected by qRT-PCR. Kiwifruit seedlings were planted in plastic pots (10×10×10 cm) filled with a mixture of substrate / vermiculite / perlite (3:1:1, v:v:v) and placed in a room with a 14 h light / 10 h dark photoperiod and 40%-60% relative humidity. Seedlings of approximately 4 months old with the same growth status were selected for stress treatments. For heat stress and low temperature stress, the seedlings were transferred to a constant temperature box at 48°C and 4°C for 6 h and 24 h, respectively; for drought treatment, watering was stopped for 8 d for drought-stressed kiwifruit seedlings; for darkness treatment, kiwifruit seedlings were exposed to continuous darkness for 24 h; salt stress was applied to the seedlings by applying 120 mM sodium chloride to the nutrient pots; for waterlogging stress, kiwifruit seedlings were immersed in water for 24 h. 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, except for AcRAD23C2, all AcRAD23 genes were induced to express 4 h after drought treatment, but the expression of AcRAD23 genes did not change significantly during the drought treatment ( 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 inhibited under waterlogging stress. The expression level of AcRAD23A decreased slightly at 4 h, then gradually increased until reaching a peak at 24 h ( Figure 5 (B) The expression of AcRAD23B, AcRAD23C1, AcRAD23C2, AcRAD23D1 and AcRAD23D2 decreased continuously from 4h to 24h.

[0063] Under salt stress, the expression profile of AcRAD23 was significantly different ( Figure 5 (C). The expression of AcRAD23A was significantly downregulated, and its expression level dropped to 60% of that at 0 h after 4 h. The remaining five genes showed significant upregulation. Compared with 0 h 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] The dark-induced expression of AcRAD23 genes, although to different extents, showed similar expression patterns under dark stress conditions ( Figure 5 (D) The expression levels of these genes showed an initial increase, peaked at different time points, and then decreased. Notably, AcRAD23D1 showed the highest expression level during the entire stress period.

[0065] Cold stress induced the expression of AcRAD23D1, but inhibited the expression of the other five AcRAD23 genes ( Figure 5 Middle E). AcRAD23D1 responded rapidly to cold stress, with the highest expression level at 4 h and a peak at 8 h, which was about 8.5 times the expression level at 0 h. The expression level decreased after 12 h. The other five genes showed similar expression patterns under cold treatment, with down-regulation at 4 h and up-regulation at 8 h.

[0066] The expression levels of AcRAD23A, AcRAD23C2, and AcRAD23D2 were all induced by heat stress, among which AcRAD23A had the highest expression level at 1 h during the stress period ( Figure 5 (F). In contrast, AcRAD23B, AcRAD23C1, and AcRAD23D1 tended 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 vectors were transformed into the Agrobacterium strain EHA105 and cultured overnight in an incubator (28°C, 180 rpm) until the OD 600 =0.6. Then, the bacteria were collected and the same volume of MES solution (10 mM MES, 10 mM MgCl 2 , 150 mM acetosyringone, pH 5.8). Empty GFP and recombinant 35S::GFP-AcRAD23s were transiently expressed in tobacco epidermis using a 1 ml syringe. 4,6-diamidino-2-phenol dihydrochloride (DAPI) was used as a nuclear maker. All fluorescent images were obtained using a confocal microscope (FK 1000; Olympus, Tokyo, Japan). The results showed that the AcRAD23 gene was strongly expressed in the nucleus and cell membrane of tobacco, indicating that AcRAD23 is localized in the nucleus and cell membrane of tobacco ( Figure 6 ).

[0069] VIGS-mediated silencing of AcRAD23D1 enhances kiwifruit's sensitivity to drought

[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, so the present invention used VIGS-mediated silencing of AcRAD23D1 kiwifruit plants (named D1-VIGS) to study its potential role in drought response.

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

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

[0077] VIGS-mediated AcRAD23D1 silencing in kiwifruit accumulates more ROS under drought stress

[0078] Drought stress usually leads to the accumulation of ROS, which causes oxidative damage. ROS accumulation was observed between WT and D1-VIGS lines under both control and drought stress conditions. First, the leaves of WT and D1-VIGS lines at nodes 3-4 from top to bottom were immersed in DAB and NBT dyes to qualitatively detect the accumulation of ROS. Under control conditions, there was no significant difference in DAB and NBT staining between WT and D1-VIGS lines, but under drought, the brown and blue spots on the leaves of D1-VIGS were darker than those of WT ( Figure 8 At the same time, the accumulation of ROS was quantitatively detected using the corresponding kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and it was found that the H 2 O 2 and OFR content was higher than WT ( Figure 8 In contrast, lower ROS scavenging enzyme activity was found in the D1-VIGS line ( Figure 8 Medium EF).

[0079] The inventors also conducted an overexpression experiment, which confirmed that overexpression of the gene AcRAD23D1 reduced ROS accumulation in plants under drought stress and enhanced drought resistance ( Fig. 9 (in 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 methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A ubiquitin transporter AcRAD23D1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

2. The AcRAD23D1 gene encoding the ubiquitin transporter AcRAD23D1 according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

3. Use of the ubiquitin transporter AcRAD23D1 according to claim 1 or the AcRAD23D1 gene according to claim 2 in regulating plant drought tolerance.

4. The use according to claim 3, characterized in that: Silencing the expression of the AcRAD23D1 gene or reducing the level of the ubiquitin transporter AcRAD23D1 to reduce the drought tolerance of the plant; Overexpressing the AcRAD23D1 gene or increasing the level of the ubiquitin transporter AcRAD23D1 can improve the drought resistance of plants.

5. The use according to claim 4, characterized in that: The plants include kiwi fruit.

6. A method for regulating plant drought tolerance, characterized in that: Silencing the expression of the AcRAD23D1 gene or reducing the level of the ubiquitin transporter AcRAD23D1 to reduce the drought tolerance of the plant; Overexpressing the AcRAD23D1 gene or increasing the level of the ubiquitin transporter AcRAD23D1 can improve the drought resistance of plants.

7. Use of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette comprising the AcRAD23D1 gene in regulating plant drought tolerance.

8. Use of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette comprising the AcRAD23D1 gene in drought-tolerant plant breeding.

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