Vitis vinifera salt stress regulation gene VvPUB23 and application thereof in salt stress regulation

Abstract

The application discloses a grape salt stress regulation gene VvPUB23, and the grape salt stress regulation gene VvPUB23 is a nucleotide sequence as shown in SEQ ID NO. 1. The application adopts the grape salt stress regulation gene VvPUB23 and the application thereof in grape salt stress regulation, and the VvPUB23 gene has the effect of improving the salt stress resistance of plants, and has important significance for breeding of excellent stress-resistant grape varieties and improvement of grape fruit quality.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and biotechnology, and in particular to a grape salt stress regulating gene VvPUB23 and application thereof in salt stress regulation. Background Art

[0002] Grapes (Vitis vinifera L.), a fruit with significant economic, nutritional, and medicinal value, are cultivated extensively across the globe. my country boasts exceptionally high grape production, ranking among the highest in the world. Grapes are cultivated in most regions of the country. The high-quality grapes produced in my country, suitable for both fresh consumption and winemaking, have generated significant economic benefits and earned a high international reputation.

[0003] Soil salinization is one of the main abiotic stresses currently faced by plants worldwide. my country's saline-alkali land is mainly distributed in the northwest, north China, and the middle and lower reaches of the Yangtze River. Its geographical location overlaps with a considerable number of my country's grape-producing areas. Under saline-alkali stress, salt continues to accumulate in the plant body, the ion balance homeostasis is destroyed, the active oxygen content increases, and essential plant physiological activities such as photosynthesis are inhibited, resulting in a decrease in fruit tree yield and quality, or even death.

[0004] U-box proteins are widely present in eukaryotic cells and are directly associated with E3 ubiquitin ligase activity. They possess a β-β-α-β folded structure and over 70 conserved amino acid sequences. Compared to animals, plants are more abundant in U-box proteins, collectively referred to as PUB proteins. These diverse plant U-box proteins play a crucial role in resisting a variety of biotic and abiotic stresses. Currently, numerous PUB proteins that positively or negatively regulate plant stress resistance have been identified and reported in different plant species. Summary of the Invention

[0005] The purpose of the present invention is to provide a grape salt stress regulatory gene VvPUB23 and its application in salt stress regulation. The VvPUB23 gene can improve the ability of plants to resist salt stress and is used for the breeding of excellent stress-resistant grape varieties, thereby improving the ability of crops to resist salt stress, which is of great significance in improving crop quality.

[0006] To achieve the above object, the present invention first provides a grape salt stress regulatory gene VvPUB23, the amino acid sequence of which protein is SEQ ID NO: 2;

[0007] The grape salt stress regulatory gene VvPUB23 has a specific nucleotide sequence as SEQ ID NO: 1.

[0008] In yet another aspect, the present invention provides a recombinant expression vector, which carries the nucleotide sequence shown in SEQ ID NO: 1.

[0009] Preferably, the recombinant expression vector is a vector capable of performing recombinant expression in plant cells.

[0010] As a specific description of the embodiment, the recombinant vector is a pCAMBIA3301-VvPUB23 vector constructed from the pCAMBIA3301 vector.

[0011] The present invention also provides a transformant of a recombinant vector carrying the grape salt stress regulating gene VvPUB23.

[0012] Preferably, the transformant is Agrobacterium tumefaciens and / or a plant cell (or organism); the organism is a transgenic drought-resistant plant, such as apple, tomato, grape, Arabidopsis, etc., with grape being preferred.

[0013] The present invention also provides the use of the grape salt stress regulatory gene VvPUB23 in improving the salt stress resistance of plants.

[0014] The present invention also provides a method for improving plant salt stress resistance, which is to increase the expression level of grape salt stress regulatory genes in plants.

[0015] The present invention constructs the VvPUB23 gene into the expression vector pCAMBIA3301. The VvPUB23 gene carried by pCAMBIA3301 is then transferred into Colombian wild-type Arabidopsis thaliana through Agrobacterium-mediated transformation to obtain transgenic Colombian wild-type Arabidopsis plants. Compared to non-transgenic Colombian wild-type Arabidopsis thaliana (wild type), VvPUB23-transgenic Colombian wild-type Arabidopsis thaliana exhibits a stronger ability to withstand salt stress and grows better under salt stress. Therefore, overexpression of the grape salt stress regulatory gene VvPUB23 can be used to improve plant salt stress resistance, thereby enabling the breeding of high-quality grape varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the gel of the expression vector PCAMBIA3301-VvPUB23 carrying the VvPUB23 gene;

[0017] Figure 2 This is a schematic diagram of the PCR identification results of Columbia wild-type Arabidopsis thaliana successfully transformed with the VvPUB23 gene;

[0018] Figure 3 This is the quantitative expression verification result of Columbia wild-type Arabidopsis thaliana transformed with VvPUB23 gene;

[0019] Figure 4 This is a schematic diagram showing the growth conditions of Columbia wild-type Arabidopsis thaliana transformed with the VvPUB23 gene and the control group under salt stress. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0022] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description. It is intended that all variations within the meaning and range of equivalents of the claims be embraced herein, and any reference signs in the claims should not be construed as limiting the claims to which they relate.

[0023] Furthermore, it should be understood that although this specification describes the embodiments, not every embodiment includes only one independent technical solution. This description is for clarity only. Those skilled in the art should consider the specification as a whole. The technical solutions in the various embodiments may also be appropriately combined to form other embodiments that are understandable to those skilled in the art. These other embodiments are also encompassed within the scope of protection of the present invention.

[0024] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, which should be covered by the scope of protection of the present invention / invention.

[0025] Example 1: Obtaining the grape salt stress regulatory gene VvPUB23

[0026] Leaf RNA was extracted according to the instructions of Tiangen Company's plant RNA extraction kit, and cDNA synthesis was carried out according to the instructions of Takara Company's reverse transcription synthesis kit.

[0027] First, based on the CDS sequence of the VvPUB23 gene, primers were designed using Primer Premier 5 to amplify its complete CDS sequence. The primer sequences (including modified bases) and names are as follows:

[0028] VvPUB23-F: 5'-AGAACACGGGGGACTCTTGACCATGGATGGATGAAATCGATGTTCCTTC-3' (SEQ ID NO. 3),

[0029] VvPUB23-R: 5'-ATGGATGAAATCGATGTTCCTTCTCAAGAAGATGGATAAGATGAGAACA-3' (SEQ ID NO. 4);

[0030] Secondly, taking the leaves of Cabernet Sauvignon grape, total RNA of the leaves is extracted, cDNA is generated by reverse transcription, and the reverse-transcribed cDNA is used as a template to perform RT-PCR amplification by using the primer pair VvPUB23-F / R, the amplified fragment is sequenced, and the VvPUB23 gene sequence is obtained, and the nucleotide sequence is as follows:

[0031] ATGGAGCCTAGTCAACATGGCCTCTTAGAAGAGTTTCTAGCTCTAAGAAGAGACACTTGCAGCACTTTCACTTCTGCAGTGAATGAGTTCTTCCCCAATGGGTGGAACTTTGATGCTTTTGATGAGAACCAAGTTTTGTCCACATCAAATCCTTCATTTGAAGGATTCTCCACCCCAACAGAACCTATCTTCGAATGTCCTTTCAGTGAAGTCTATCCTTCTGTTGATGGGTTCACCGTAGCAGAGATTGATTCATCTTACCACAAGAATGACGGCACACCCCCATTTCCAATTCAAGAAGAGTACCCTTCACTGGTTGAGGATGAAGATATCGGTCTCCTCAACAGTGATCTTCATGGGTTGGAAGAGAGGAATACCAGCTGTAAAGTTGAGATGGAACAAGCCATGGACGCTCCAGTTTTCAACCTAGGTTTGTGTGGAGAGAGAAAGGCTCGAGTTAAGAAGCTTGAGGGTCAGCCCTCCAAGAATCTAATGGCAGAAAGAAGGCGAAGGAAGCGACTCAATGATCGCCTTTCCATGCTCAGATCAATTGTTCCTAAGATCAGCAAGATGGACAGAACATCTATACTTGGAGATACCATAGATTATATGAAAGAGCTCCTAGAGAAAATCAACAAGTTGCAAGAAGAAGAAATCGAAGTGGGCTCGGATCAAACCAACCTAATGGGCATCTTCAAGGAGCTAAAGCCAAATGAAGTACTAGTGAGAAATTCTCCCAAGTTTGATGTGGAAAGGAGAAACATGGATACCCGGATCGAGATTTGCTGTGCAGCAAAGCCAGGACTGTTGCTGTCGACAGTGAACACACTGGAACTTCTAGGCCTTGAAATTCAACAGTGCGTTATAAGTTGCTTCAATGATTTTTCAATGCAAGCATCT(SEQID NO.1);

[0032] 其编码的蛋白的氨基酸序列如下:

[0033] MDEIDVPSHFLCPISLQLMRDPVTVATGITFDRENIERWLFSCKNNTCPFTKQVLVDTDLTPNHTLR

[0034] RLIQAWCIVNACHGVERIPTPKPPIDKAQIIKLFNDAIKFPQMQLKCLQRLRSIAFESDRNKKCLEAAGA

[0035] VEFLASIIKKDESAVIEVVLEDGSREFTRASDEALSILYQLETSEAALKSLVSSNYGFIESLVHVLKCGNY

[0036] QSRAYAAMLLKSIFQVADPIQLINASPELFTEIVHVLRDGISQQASKAALKLLVELCPWGRNRIKAVVAG

[0037] VSHVLIEHLLDTSEKRTCELILVVLDQLCSCAEGRAELLKHGAGLAIVSKKILRVSQVGTDRAVKILAS

[0038] VSKFSATSRVLQEMLQVGVVSKLCLVLQVDSSKKTKEKTREILNLHSRVWKNPSCIPARLLSSYPSS

[0039] (SEQ ID NO.2)

[0040] Example 2: Construction and genetic transformation of VvPUB23 gene overexpression vector

[0041] 1. Construction of overexpression vector

[0042] The salt stress regulation-related grape gene VvPUB23 cloned in Example 1 was connected to the linearized vector PCAMBIA3301 by homologous recombination to construct a plant expression vector named PCAMBIA3301-VvPUB23 ( Figure 1 ), the specific operations are as follows:

[0043] 1) Linearized vector was first obtained using double digestion with Nco I and Bgl II (Takara). The linearized vector was then recovered by agarose gel electrophoresis and gel recovery kit (Tiangen Biochemical Technology Co., Ltd.). The concentration and purity of the recovered product were checked using a nucleic acid protein analyzer before proceeding to the next step.

[0044] 2) Recombination reaction of the target fragment DNA and linearized vector by one-step directional cloning kit (Coastal Protein Technology Co., Ltd.), add the recombination product to 50 μL of DH5α competent cells, mix gently with pipette, incubate on ice for 30 min, heat shock at 42℃ metal bath for 60 s, then quickly cool on ice for 2 min.

[0045] 3) Add 700 μL of LB liquid medium, incubate at 37℃ for 60 min. Centrifuge at 5000 rpm for 5 min, collect the bacteria, discard part of the supernatant, resuspend the bacteria with the remaining medium, use a sterile swab to evenly spread on the LB solid medium containing Kan resistance, and invert in a 37℃ incubator for 12-16 h.

[0046] 4) Pick several clones on the recombination reaction transformation plate for colony PCR identification, pick the corresponding single colony in the liquid LB medium containing Kan antibiotic, incubate at 37℃, 200 rpm in an incubator overnight, extract the plasmid or directly sequence the bacterial solution, and identify the correctness of the vector by enzyme digestion electrophoresis.

[0047] II. Genetic transformation

[0048] The above prepared pCAMBIA3301-VvPUB23 vector is introduced into Agrobacterium tumefaciens EHA105 (Shanghai Sungene Bioengineering Co., Ltd.), and then introduced into Columbia wild-type Arabidopsis thaliana, and the specific operation is as follows:

[0049] 1) Recombinant vector into Agrobacterium EHA105

[0050] a. Add 1 μg of plasmid DNA to each 50 μL of EHA105 Agrobacterium competent cells, mix gently with the bottom of the tube, and sequentially stand on ice for 5 min, liquid nitrogen for 5 min, 37℃ water bath for 5 min, and ice bath for 5 min.

[0051] b. Add 700 μL of YEB liquid medium without antibiotic, and incubate at 28℃ for 2-3 h. Centrifuge at 6000 rpm for 1 min, collect the bacteria, take about 100 μL of supernatant, resuspend the bacteria by blowing gently, and evenly spread on YEB solid medium containing Kan and Rif, and invert in a 28℃ incubator for 2 d, and pick several positive clones for simple verification by colony PCR.

[0052] 2) Genetic transformation:

[0053] a. Agrobacterium activation: inoculate Agrobacterium in 10 mL of YEB liquid medium containing Rif and Kan antibiotics, 28℃, 180 rpm shaking for 2 d.

[0054] b. Agrobacterium expansion: Inoculate 1 mL of the culture into 50 mL of fresh antibiotic-containing liquid medium. Shake the culture at 180 rpm at 28°C for 16-24 hours. Measure the OD value to maintain it between 1.5 and 2.0. Centrifuge at 5000 rpm for 10 minutes at room temperature and discard the supernatant.

[0055] c. Resuspend the bacterial suspension in 5% sucrose solution and add 0.05% Silwet L-77. Using 4-week-old Arabidopsis plants, cut open flowers with scissors and immerse the remaining inflorescence in the infection solution. Gently rotate for 15-30 seconds. Once fully infected, wrap the inflorescence in plastic wrap and incubate in the dark for 24 hours. Remove the plastic wrap and incubate in an incubator for one week. Re-infect one or two times. After infection, incubate as usual, watering every two days. Collect seeds after one month.

[0056] 3. Screening and identification of overexpression plants

[0057] 1) Screening of homozygous transgenic Arabidopsis plants

[0058] Evenly sow T0 generation seeds on a substrate. When two cotyledons have grown (approximately 7 days), spray with a 0.1‰ glyphosate solution for three consecutive days. Transplant surviving plants to the substrate for further growth, and harvest seeds from individual plants, which are the T1 generation. Repeat the above steps to screen for the T3 generation.

[0059] Among the four positive lines obtained, DNA was extracted from the leaves of the selected positive plants, and the VvPUB23 gene was identified by PCR to perform molecular verification of the target gene of the transgenic plants ( Figure 2 ), and finally confirmed that the gene had been transferred into the Columbia wild-type Arabidopsis. Figure 2 In the figure, M is DL 2000 Marker, and lines 1-4 are transgenic T3 plants.

[0060] 2) Quantitative expression verification of transgenic Columbia wild-type Arabidopsis

[0061] RNA was extracted from callus tissue using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Beijing). 1 μg of each RNA was used as a template and reverse transcribed into first-strand cDNA using the iScript II Reverse Transcriptase Kit. The DNA was then stored at -20°C. Primers were designed using actin2 as an internal reference gene. The cDNA was diluted 10-fold and the reaction system was prepared using the SYBR qPCR Master Mix (Novagen, Nanjing) kit. Gene expression was determined using a BIO-RAD Cycler IQ6 fluorescence quantitative PCR instrument, with three replicates.

[0062] The qRT-PCR primer pair for the Arabidopsis actin 2 internal reference is:

[0063] aF: 5′-TATGAATTACCCGATGGGCAAG-3′ (SEQ ID NO.5);

[0064] aR: 5′-TGGAACAAGACTTCTGGGCAT-3′ (SEQ ID NO. 6);

[0065] The target gene quantitative qRT-PCR primer pairs are:

[0066] VvPUB23-F: 5′-CTGTCATGGGGTTGAACGGA-3′ (SEQ ID NO.7);

[0067] VvPUB23-R: 5′-GAGCTGCATCTGTGGGAACT-3′ (SEQ ID NO. 8).

[0068] The results showed that the expression of the VvPUB23 gene in the four transgenic lines tested was significantly increased, compared with the non-transgenic wild-type Colombian Arabidopsis thaliana as the control. This indicates that the VvPUB23 gene was transformed and inserted into the corresponding Colombian wild-type Arabidopsis genome and expressed. The three lines with the highest expression of the VvPUB23 gene were selected, namely OE#1, OE#3, and OE#4 ( Figure 3 ).

[0069] Example 3: Determination of Salt Stress Regulation Performance of VvPUB23 Overexpressing Transgenic Colombian Wild-Type Arabidopsis

[0070] Three transgenic wild-type Arabidopsis thaliana strains overexpressing the VvPUB23 gene in Example 2 were selected to observe their growth conditions under salt stress.

[0071] The results showed that the growth potential of the three transgenic lines overexpressing the cloned VvPUB23 gene of the present invention was better than that of the control. That is, the ability of VvPUB23 transgenic Colombia wild-type Arabidopsis to resist salt stress was significantly higher than that of the wild-type Colombia wild-type Arabidopsis control ( Figure 4 ), overexpression of the gene VvPUB23 can improve the salt stress resistance of Arabidopsis thaliana.

[0072] Therefore, the present invention adopts the above-mentioned grape salt stress regulatory gene VvPUB23 and its application in salt stress regulation. The VvPUB23 gene can improve the salt stress resistance of plants, which is of great significance for the breeding of excellent grape varieties and improving the stress resistance quality of crops.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for improving the salt stress resistance of Arabidopsis thaliana, characterized in that: The method is to transfer a recombinant expression vector carrying a nucleic acid fragment of a grape salt stress regulatory gene with a nucleotide sequence of SEQ ID NO: 1 into Arabidopsis thaliana to improve the salt stress resistance of Arabidopsis thaliana.

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