Use of u2af65b in modulating plant salt stress resistance

Abstract

This invention discloses the use of U2AF65b in regulating salt stress resistance in plants, belonging to the field of biotechnology. This invention clones a splicing factor gene, U2AF65b, from grapes. Transgenic experiments confirm that U2AF65b participates in salt tolerance, expanding our understanding of the generation of salt tolerance in plants and providing a theoretical basis for obtaining highly salt-tolerant plants, thus possessing significant application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the use of U2AF65b in regulating plant salt stress resistance. Background Technology

[0002] Soil salinization severely restricts plant growth and development, leading to reduced crop yields. Understanding how plants sense and respond to salt stress is crucial for improving their salt tolerance through appropriate breeding and genetic engineering strategies. Most research on gene expression regulation under plant stress focuses on transcriptional gene expression regulation; in contrast, little is known about salt-induced posttranscriptional gene expression regulation.

[0003] Pre-RNA splicing is a crucial step between transcription and translation in most eukaryotes and is accomplished by the spliceosome. The spliceosome is dynamically composed of small nuclear ribonucleoproteins (snRNPs) and hundreds of non-snRNP proteins, recognizing splicing sites on the RNA precursor and catalyzing the splicing reaction.

[0004] U2AF is an essential cofactor for U2 snRNP to bind to the branching site of precursor mRNA. It consists of two subunits, U2AF65 and U2AF35. U2AF65 interacts with the polypyrimidine region, which is located downstream of the branching site. Studies have shown that U2AF65 not only binds to the polypyrimidine sequence of the intron of pre-mRNA in the first step of spliceosome assembly, but also binds to spliced ​​mRNA. The proteins encoded by these mRNAs are mainly related to cell cycle progression, transcriptional regulation of specific gene expression, and chromatin stability (Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors[J]. Genome Biol, 2006, 7(11): R113). Patent CN107119056A discloses the application of the splicing auxiliary factor AtU2AF65b derived from Arabidopsis thaliana in the regulation of plant flowering time. Increasing the expression level of this gene can delay flowering, while decreasing its expression level can cause annual plants to flower earlier or shorten the juvenile period of perennial plants. However, it remains unclear whether U2AF65b possesses stress-resistance functions in plants.

[0005] Grapes (Vitis vinifera L.) are deciduous vines belonging to the genus Vitis in the family Vitaceae. They are one of the most widely cultivated and historically significant fruits. As a fruit tree with relatively strong salt tolerance, grapes have potential for development in saline-alkali soils. However, there are no reports on the role of grape-derived U2AF65b in regulating plant salt stress resistance. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide the use of U2AF65b in regulating plant salt stress resistance. This invention is the first to discover that overexpression of the U2AF65b gene in grapes can improve the grapes' resistance to salt stress, and can be used in research on soil salinization improvement.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the use of the U2AF65b gene as a positive regulatory gene in the following (1) or (2):

[0009] (1) Improve the salt tolerance of plants;

[0010] (2) Cultivate plant varieties with improved salt tolerance;

[0011] The U2AF65b gene is a nucleic acid molecule as shown in i) or ii) below:

[0012] i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0013] ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.2.

[0014] In a second aspect, the invention provides the use of the protein encoded by the U2AF65b gene as a positive regulator in either (1) or (2) below:

[0015] (1) Improve the salt tolerance of plants;

[0016] (2) Prepare products that improve the salt tolerance of plants.

[0017] Furthermore, the amino acid sequence of the protein encoded by the U2AF65b gene is shown in SEQ ID NO.2.

[0018] In the above applications, the grape U2AF65b gene or the protein U2AF65b encoded by the U2AF65b gene is used as the target. By overexpressing the grape U2AF65b gene or increasing the expression level or activity of the protein U2AF65b, the resistance of grapes to salt stress can be positively regulated, thereby broadening the application of grapes in soil salinization improvement.

[0019] A third aspect of the present invention provides the use of a recombinant expression vector or engineered bacteria containing the U2AF65b gene in the following (1) or (2):

[0020] (1) Improve the salt tolerance of plants;

[0021] (2) Cultivate plant varieties with improved salt tolerance.

[0022] In the above applications, the recombinant expression vector can be constructed using existing plant expression vectors or prokaryotic expression vectors, such as pHB-gfp, pCXUN, pCAMBIA1300, pTA7001, pTA7002, pBin, PET-30a, PMAL-C2X, pGEX-4T, or other derived vectors.

[0023] A fourth aspect of the present invention provides a method for improving the salt stress tolerance of plants, comprising the step of overexpressing the U2AF65b gene in plants.

[0024] The above methods can induce overexpression of the U2AF65b gene through the following pathways:

[0025] Exogenous transfer of the U2AF65b gene;

[0026] Alternatively, it can upregulate the expression of the U2AF65b gene in the plant genome.

[0027] A fifth aspect of the present invention provides a method for cultivating salt-tolerant grape varieties, comprising the following steps:

[0028] The U2AF65b gene was transferred into wild-type grape plants to overexpress the U2AF65b gene, resulting in transgenic grape plants.

[0029] In the above method, the transgenic grape plants exhibit higher resistance to salt stress than wild-type grape plants.

[0030] The methods described above for transferring the U2AF65b gene into wild-type grape plants include, but are not limited to: polyethylene glycol method, Agrobacterium-mediated method, or gene gun bombardment method.

[0031] The beneficial effects of this invention are:

[0032] In response to the current lack of research on the salt-tolerance function of plant splicing factors, this invention cloned a splicing factor gene, U2AF65b, from grapes. Transgenic experiments confirmed that U2AF65b participates in salt tolerance, expanding our understanding of the generation of salt tolerance in plants and providing a theoretical basis for obtaining highly salt-tolerant plants, thus possessing significant application value. Attached Figure Description

[0033] Figure 1 This is an example of the expression of the grape U2AF65b gene under salt stress according to the present invention.

[0034] Figure 2 The salt tolerance of transgenic grape callus overexpressed with U2AF65b is shown in the figure. In the figure, #1, #2 and #3 represent transgenic grape callus overexpressed with U2AF65b. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] As mentioned earlier, stress response is an important area of ​​plant research. Certain spliceosome protein genes exhibit differential expression under abiotic and biotic stresses, as well as at different growth and development stages. The physiological disorders caused by saline-alkali soils in grapes are becoming increasingly apparent, severely impacting normal growth and development, fruit yield, and quality. However, little is known about how salt stress affects the expression patterns of spliceosome protein genes in plants, and whether these genes play a role in plant salt stress responses remains lacking in transgenic evidence.

[0037] In view of this, this invention conducted a cloning and functional study of the U2AF65b gene derived from grapes. First, the grape U2AF65b gene was cloned from grape tissue culture seedlings; then, the expression of the grape U2AF65b gene at different salt treatment time points was detected, revealing that the grape U2AF65b gene can exhibit differential expression in response to salt stress; further, this invention introduced the grape U2AF65b gene into grape callus tissue, finding that grape callus tissue overexpressing the U2AF65b gene exhibited a significantly higher growth rate under salt stress than the wild type.

[0038] The above results indicate that the grape U2AF65b gene can play a salt-tolerance role in the plant and is a new salt-tolerant gene in grapes that is related to resisting salt stress.

[0039] The sequence of the grape U2AF65b gene is shown in SEQ ID NO.1, as follows:

[0040]

[0041] The amino acid sequence of the protein encoded by the grape U2AF65b gene is shown in SEQ ID NO.2, as follows:

[0042] MPDYEGRYEGNGNGEDLDNYGSSPQPRGSSHGGPDDYSDSKSQHGSREYQRESSKSREREREKGRDKDRERDRDRERDKERDRDRDRDKERDRDRRDRYRDRSDRRERTRDRDDD DFHRSRDYDRRRDFDRDRDDRHKRRSRSKGRSEQRSRSRSASLSKSKRVSGFDMAPPASAMLAGAAAAAGQIPGTTTPTIPGMFPNMFPLASGQFGALPVMPVQAMTQQATRHA RRVYVGGLSPTANEQSVATFFSQVMSAIGGNTAGPGDAVVNVYINHEKKFAFVEMRSVEEASNAMALDGIIFEGAPVKVRRPSDYNPSLAATLGPSQPNPNLNLAAVGLTPGSAG GLEGPDRIFVGGLPYYFTEAQIRELLESFGPLRGFDLVKDRETGNSKGYAFCVYQDLSVTDIACAALNGIKMGDKTLTVRRANQGASQPKPEQENVLLHAQQQIALQRLMFQPGA LATKVVCLTQVVNADELQDDEAYEDIVEDMRIEGGKFGNLVNVAIPRPKPNGEPTPGLGKVFLEYADIDGATKARTGLNGRKFDGNQVVAVFYPENKFSQGEYDG.

[0043] The full-length nucleotide sequence or fragment thereof related to grape U2AF65b of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed in the present invention, and the relevant sequence can be amplified using a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art.

[0044] Once the relevant sequence is obtained, it can be mass-produced using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. Alternatively, mutations can be introduced into the nucleotide sequence of this invention through chemical synthesis.

[0045] In this invention, the expression pattern of grape U2AF65b can be analyzed using real-time quantitative PCR, that is, the presence and quantity of grape U2AF65b RNA transcripts in cell tissues can be analyzed.

[0046] Based on the above-discovered U2AF65b gene, the scope of protection of this invention also includes DNA fragments homologous to the U2AF65b gene.

[0047] These DNA fragments homologous to the U2AF65b gene include alleles, homologous genes, mutant genes, and derived genes corresponding to the nucleotide sequence (SEQ ID NO.1) of this invention, and all fall under the protection of this invention.

[0048] Those skilled in the art can readily mutate the nucleotide sequence of the U2AF65b gene of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 70% or higher identity with the nucleotide sequence of the U2AF65b gene of the present invention, provided their function is equivalent to the nucleotide sequence shown in SEQ ID NO. 1, are derived from and are equivalent to the sequence of the present invention.

[0049] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence shown in SEQ ID NO. 1 of this invention. The equivalence of amino acids or nucleotide sequences can be determined using the BLAST algorithm (Altschul et al. 1990. Journal of Molecular Biology 215:403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90:5873-5877).

[0050] The aforementioned 70% or more identity can be 70%, 75%, 80%, 85%, 90%, or 95% or more identity.

[0051] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0052] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0053] Example 1: Cloning of grape U2AF65b

[0054] 1. Plant materials:

[0055] The material used in this invention is 'Crimson Seedless' grape tissue culture seedlings, and the culture medium is MS medium containing sucrose (30g / L) and plant agar (6g / L).

[0056] 2. RNA extraction and reverse transcription:

[0057] Grape leaves were collected for RNA extraction. Total RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) following the manufacturer's instructions. TaKaRaPrimeScript was used for the extraction. TM The RT reagent kit (Perfect Real Time) reverse transcribes 1 μg of RNA into cDNA.

[0058] 3. Cloning verification of U2AF65b:

[0059] Design primers based on NCBI accession number XM_010650863.2:

[0060] U2AF65b-F: 5′-ATGCCGGACTACGAAGGCA-3′ (SEQ ID NO. 3);

[0061] U2AF65b-F: 5′-CTAGCCATCATATTCCCCTTGG-3′ (SEQ ID NO. 4).

[0062] PCR was performed using PrimeSTAR Max Premix high-fidelity enzyme according to the manufacturer's instructions. The PCR product was then ligated to... The -T1Simple CloningVector (Beijing TransGen Biotech Co., Ltd.) vector was used to transform *E. coli* DH5α. Positive single colonies were screened using colony PCR and then sent to Sangon Biotech Co., Ltd. for sequencing. The nucleotide sequence of the cloned grape U2AF65b is shown in SEQ ID NO.1.

[0063] Example 2: Expression of grape U2AF65b at different salt treatment time points

[0064] 1. Plant materials:

[0065] 'Crimson Seedless' grape tissue culture seedlings that had been subcultured for one month and showed uniform growth were soaked in NaCl solution. Then, leaves were taken at different time points, with three replicates, and were flash-frozen in liquid nitrogen for preservation.

[0066] 2. RNA extraction and reverse transcription:

[0067] Same as Example 1.

[0068] 3. Changes in U2AF65b expression levels at different salt treatment time points:

[0069] Specific primers were designed to perform real-time quantitative PCR analysis on the expression level changes of U2AF65b at different salt treatment time points. The primers are as follows:

[0070] qU2AF65b-F: 5′-GCTGCTGCAGGTCAGATTCC-3′ (SEQ ID NO. 5);

[0071] qU2AF65b-R: 5′-TACCCGCCGAGCATGTCTAG-3′ (SEQ ID NO. 6).

[0072] The internal reference gene is ACTIN, and the primers are:

[0073] ACTIN-F: 5′-TCCGTTGTCCAGAAGTCCTCTT-3′ (SEQ ID NO. 7);

[0074] ACTIN-R: 5'-GTCAGCAATACCAGGGAACATG-3' (SEQ ID NO. 8).

[0075] 4. Real-time fluorescence quantitative analysis of U2AF65b in the sample:

[0076] Using cDNA as a template, quantitative real-time PCR was performed using specific primers for U2AF65b and ACTIN, respectively. The reaction was performed on a CFX connect Real Time PCR Detection System (Bio-Rad) using a 20 μL system (10 μL SYBR Premix Ex Taq, 1 μL each of forward and reverse primers (10 μM), 1 μL cDNA template, and water to bring the volume to 20 μL). The program was as follows: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 10 s, for 40 cycles.

[0077] 5. Use 2 -△△Ct Relative quantitative analysis of U2AF65b was performed using the following method:

[0078] The results showed that the expression level of U2AF65b gradually increased with the extension of salt treatment time, indicating that U2AF65b can be differentially expressed in response to salt stress. Figure 1 ).

[0079] Example 3: Detection of salt tolerance function in grape U2AF65b callus tissue

[0080] The overexpression vector of U2AF65b was constructed using the pHB-gfp vector (described in the literature "MicroRNA171c-targeted SCL6-II, SCL6-III, and SCL6-IV genes regulate shoot branching in Arabidopsis, doi.org / 10.1093 / mp / ssq042"). The primers are as follows:

[0081] pHBU2AF65b-F: 5′-accagtctctctctcaagcttATGCCGGACTACGAAGGCA-3′ (SEQ IDNO.9);

[0082] pHBU2AF65b-R: 5′-gcccttgctcaccatggatccGCCATCATATTCCCCTTGGG-3′ (SEQ ID NO. 10).

[0083] PCR was performed using PrimeSTAR Max Premix high-fidelity enzyme (in a 20 μL reaction system, 10 μL PrimeSTAR Master Mix, 1 μL each of forward and reverse primers (10 μM), 1 μL template, and water to bring the total to 20 μL). The program was as follows: 98℃ for 10 s, 55℃ for 5 s, 72℃ for 10 s for 34 cycles; extension at 72℃ for 5 mins.

[0084] PCR products were separated by 1.5% agarose gel electrophoresis and purified using a SanPrep DNA gel extraction kit (B518131, Sangon Biotech) according to standard operating procedures.

[0085] The pHB-gfp vector plasmid was subjected to enzyme digestion. The enzyme digestion reaction system is as follows:

[0086]

[0087] The above reaction solution was kept at 37°C for 30 minutes, and then the gel was removed and recovered after agarose gel electrophoresis.

[0088] The digested pHB-gfp and U2AF65b were ligated according to the standard operating procedure of the ClonExpress II One Step Cloning Kit, and the recombinant plasmid was transformed into *E. coli* DH5α. Positive clones were screened by colony PCR and sent to Sangon Biotech for sequencing. For single colonies with correct sequencing, the colonies were cultured overnight by shaking, and the plasmid was extracted according to the standard operating procedure of the SanPrep Column Plasmid DNA Mini-Extraction Kit (Sangon Biotech). This yielded the overexpression vector plasmid containing U2AF65b.

[0089] The U2AF65b overexpression vector plasmid was transformed into grape callus using the Agrobacterium infection method. The method is as follows: Agrobacterium containing the U2AF65b overexpression vector plasmid was shaken and the bacterial cells were collected. Resuspension liquid (MS + 3% sucrose + 200ul / LAS (100mM); pH 5.8-6.0) was added, and the OD 600nm was adjusted to 0.6. Grape callus was added (the preparation of grape callus is based on the method in the reference "Induction of callus in grape tissue culture [J]. Hubei Agricultural Sciences, 2012, 51(04): 827-830. DOI: 10.14088 / j.cnki.issn0439-8114.2012.04.052."). The mixture was shaken in a shaker for 30 minutes, filtered through gauze, and transferred to B5 medium with filter paper. Two days later, it was transferred to B5 medium containing 10mM hygromycin (the newly grown callus is transgenic; if it does not grow, the transformation has failed). Positive transgenic callus was obtained and used for salt tolerance identification.

[0090] Grape callus was subcultured on B5 medium and used as experimental material after 20-25 days of growth. Grape callus of the same size was transferred to B5 medium containing 150 mM NaCl and cultured for 15 days before being photographed. Wild grape callus (WT) without the U2AF65b overexpression vector plasmid was used as a control.

[0091] The results are as follows Figure 2 As shown, the results indicate that under salt stress, the growth of grape callus overexpressing U2AF65b was significantly higher than that of wild type, meaning that U2AF65b can positively regulate salt tolerance.

[0092] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. U2AF65b Use of the gene as a positive regulatory gene in (1) or (2) below: (1) Improve the salt tolerance of plants; (2) Cultivate plant varieties with improved salt tolerance; The U2AF65b Gene is a nucleic acid molecule as indicated in i) or ii): i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) Nucleic acid molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 2; The plant in question is a grape.

2. U2AF65b Use of a gene-encoded protein as a positive regulatory factor in (1) or (2) below: (1) Improve the salt tolerance of plants; (2) To prepare products that improve the salt tolerance of plants; The U2AF65b The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 2; The plant in question is a grape.

3. A composition comprising U2AF65b Use of a recombinant expression vector or engineered bacteria of a gene in (1) or (2) below: (1) Improve the salt tolerance of plants; (2) Cultivate plant varieties with improved salt tolerance; The U2AF65b The gene is a nucleic acid molecule as indicated in i) or ii): i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) Nucleic acid molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 2; The plant in question is a grape.

4. A method for increasing the tolerance of a plant to salt stress, characterized in that, include: steps for overexpressing a gene in a plant U2AF65b steps for overexpressing a gene in a plant The U2AF65b Gene is a nucleic acid molecule as indicated in i) or ii): i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) Nucleic acid molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 2; The plant in question is a grape.

5. The method of claim 4, wherein, By the following route U2AF65b Gene overexpression: exogenous introduction U2AF65b gene; Alternatively, up-regulating expression of a gene in the plant genome. U2AF65b of a gene.

6. A method of breeding a salt tolerant grape variety, characterized in that, Includes the following steps: Will U2AF65b Genes were transferred into wild-type grapevines, enabling... U2AF65b Gene overexpression was used to obtain transgenic grape plants. The U2AF65b Gene is a nucleic acid molecule as indicated in i) or ii): i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) Nucleic acid molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 2; The transgenic grape plants exhibited higher resistance to salt stress than wild-type grape plants.

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