Application of GhSZF3 gene in improving plant salt tolerance

Abstract

The application discloses GhSZF3 Application of a gene in improving plant salt tolerance, and belongs to the technical field of cotton salt tolerance gene research. GhSZF3 The gene can improve plant salt tolerance, and can significantly improve the seed germination ability of transgenic Arabidopsis under salt stress, which has important significance for cultivating new transgenic plant varieties with salt stress resistance. The gene can provide a wider gene source for directional editing, improvement of cotton salt resistance and molecular breeding of various plants and crops.

Description

Technical Field

[0001] This invention relates to the field of cotton salt tolerance gene research technology, and more specifically to... GhSZF3 Application of genes in improving plant salt tolerance. Background Technology

[0002] Soil salinization is a global resource and ecological problem. In-depth research into the salt tolerance mechanisms of plants, enabling them to adapt to saline soil environments, is an important way for humans to utilize saline soil. Cotton is an important economic crop and raw material for the textile industry in my country, playing a vital role in the national economy. Statistics show that there are 14.25 billion mu (approximately 7% of the land area) of saline-alkali land globally, with Asia accounting for 4.8 billion mu (approximately 320 million hectares), more than one-third of the global total. About 22% of irrigated land (providing one-third of the world's food) suffers from salt damage (secondary salinization), and this is continuously expanding with the development of industry and agriculture. Therefore, improving the salt and alkali resistance of crops to increase crop yields is an urgent problem to be solved in agricultural production in my country and the world. Especially at present, under the premise of ensuring national food security, guaranteeing cotton planting area, cultivating salt-tolerant cotton varieties, and introducing cotton planting to saline-alkali wasteland are effective ways to alleviate the conflict between grain and cotton for land.

[0003] Cotton is a relatively salt-tolerant crop, but its salt tolerance is limited, generally not exceeding 0.3%. As salt concentration increases, it significantly affects the physiological functions and metabolism of cotton, thus inhibiting growth and development, leading to decreased yield and altered quality. Traditional breeding methods for developing salt-tolerant cotton varieties have been slow due to a lack of highly resistant upland cotton resources, and cannot yet meet production needs. The development of biotechnology has provided a rapid and effective method for improving cotton salt tolerance through genetic engineering. Given the significant differences in salt tolerance among different cotton species, fully exploring salt-tolerant genes and innovating cotton germplasm resources by utilizing these differences in salt tolerance is an effective way to improve cotton's salt tolerance.

[0004] Therefore, how to provide a salt-tolerant gene and apply it to improve the salt tolerance of cotton is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides GhSZF3 Application of genes in improving plant salt tolerance.

[0006] This invention, through the GhSZF3 Structural analysis, subcellular localization, transgenic analysis, and gene expression silencing analysis revealed that this gene positively regulates the salt tolerance trait in cotton, has the potential to be applied to the breeding of new salt-tolerant varieties, and is of great significance for the targeted genetic improvement of cotton salt tolerance.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] GhSZF3 The application of the gene in improving plant salt tolerance, the nucleotide sequence of which is shown in SEQ ID NO.2;

[0009] Or a nucleotide sequence that is more than 90% homologous to the nucleotide sequence of SEQ ID NO.2 and encodes amino acids as shown in SEQ ID NO.1.

[0010] The application of a protein or a substance that regulates the expression of the protein-encoding gene in improving plant salt tolerance, wherein the amino acid sequence of the protein is shown in SEQ ID NO.1;

[0011] Alternatively, a protein with the same function can be obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1.

[0012] It may be a fusion protein that has the same function as the protein shown in SEQ ID NO.1 and is obtained by attaching a tag to the N-terminus or C-terminus.

[0013] Application of biomaterials related to the aforementioned genes or proteins in improving plant salt tolerance, wherein the biomaterial is any one of the following:

[0014] A: Nucleic acid molecules with nucleotide sequences as described above or nucleic acid molecules encoding the proteins described above;

[0015] B: An expression cassette containing the nucleic acid molecule described in A;

[0016] C: An expression vector containing the nucleic acid molecule described in A, or a recombinant vector containing the expression cassette described in B;

[0017] D: Recombinant microorganisms containing the nucleic acid molecules described in A, or recombinant microorganisms containing the expression cassette described in B, or recombinant microorganisms containing the recombinant vector described in C.

[0018] Furthermore, the plant is cotton or Arabidopsis thaliana.

[0019] Furthermore, improving plant salt tolerance means increasing seed germination rate and root length under salt stress.

[0020] A method for breeding transgenic plants resistant to salt stress, which will GhSZF3 Genes are transferred into plant tissues or plant cells.

[0021] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention GhSZF3 The gene can enhance plant salt tolerance and significantly improve the seed germination ability of transgenic Arabidopsis thaliana under salt stress, which is of great significance for breeding new transgenic plant varieties resistant to salt stress. It can also provide a broader source of genes for targeted editing, improving cotton salt tolerance, and molecular breeding of various plants and crops. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 This is a predicted structure diagram of the GhSZF3 protein in Example 1 of the present invention, where A: GhSZF3 Gene structure diagram: B: GhSZF3 transmembrane domain, C: GhSZF3 secondary structure, D: GhSZF3 tertiary structure.

[0025] Figure 2 This is a diagram showing the subcellular localization of the GhSZF3 protein in Example 4 of the present invention. In the diagram, 1305k-GFP represents the empty vector localization, GhSZF3-GFP represents the gene localization, GFP represents green fluorescence, mCheey represents the nuclear localization marker, Bright represents the bright field, and Merge represents the overlap field.

[0026] Figure 3 In Embodiment 5 of the present invention GhSZF3 Image of quantitative PCR results for gene tissue fluorescence.

[0027] Figure 4 In Embodiment 6 of the present invention GhSZF3 A diagram showing the gene's response to salt induction.

[0028] Figure 5 Overexpression in Example 7 of the present invention GhSZF3 Positive detection of genes in Arabidopsis thaliana, where M: Marker; 1: Blank; 2: Wild type; 3-11: Arabidopsis thaliana plants of different strains.

[0029] Figure 6 Overexpression in Example 7 of the present invention GhSZF3The diagram shows the effect of genes on the growth status of Arabidopsis thaliana. A: Germination rate of wild-type and overexpression lines under normal growth conditions; B: Germination rate of wild-type and overexpression lines under 200 mM treatment; C: Root length of wild-type and overexpression lines under normal growth and 200 mM treatment; D: Germination rate statistics under normal growth conditions; E: Germination rate statistics under 200 mM treatment; F: Root length statistics under normal growth conditions; G: Root length statistics under 200 mM treatment.

[0030] Figure 7 Silence in Example 8 of the present invention GhSZF3 Identification of salt tolerance function of the gene in cotton, A: TRV2:00 and silence under normal growth and 400mM treatment. GhSZF3 Plant phenotype; B: Silent GhSZF3 C: Determination of expression level; D: Determination of proline content after salt treatment; E: Determination of chlorophyll content after salt treatment. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The cotton variety used in the following examples is cotton variety Ji Mian 172.

[0033] Example 1

[0034] GhSZF3 Gene screening and cloning

[0035] (1) GhSZF3 Gene screening

[0036] Based on the cotton salt stress-related genes previously cloned by our laboratory, we have preliminarily determined... GhSZF3 The gene is a salt tolerance regulatory gene.

[0037] GhSZF3 The amino acid sequence of the gene is shown in SEQ ID NO.1:

[0038] METAGEWLEKALIDLCKKIETGLYLDAEIISGLVSYCELAHPLDAKEYLDNIIGQEAGKVVTEEYLRRRGHLDLCTGNAAIPASKLQAYVKPPSGEISVSGTKKPFKTPKEAAGSSYRAEPKKNVISGNQENRIPNDASDSRNMHKGNQGNSKKKKAGKVVSLAEAAKGSIVFHQGKPCSCQARQHRLVSNCLSCGKIVCEQEGEGPCNFCGALVLREGSTYAGLEGSFTPVSDAEAAAEAYAKRLVEYDRNAAARTTVIDDQSDYYEIEGNSWLSKEEKEFLKKKQEEIEEAERLKRSKVFVTFDLVGRKVLLNEDEVSELESENRILLRPPDEREANRIKPNPNLKLQPVFLSPVPSKKGSKSKQPSKSPVSGLCLEITGRVQHDSNELKYFMTDKKIEAT*, SEQ ID NO.1.

[0039] GhSZF3 The nucleotide sequence of the gene is shown in SEQ ID NO.2:

[0040]

[0041] (2) GhSZF3 Cloning of genes

[0042] Specific primers GhSZF3-1F and GhSZF3-1R were designed for amplification using cotton leaf cDNA as a template. The PCR program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 45 s, 58℃ annealing for 45 s, 72℃ extension for 45 s, for a total of 30 cycles; incubation at 72℃ for 10 min. The PCR product was gel-cleaved, purified, and ligated into the T5 vector. The ligation product was transformed into *E. coli* DH5α for blue-white screening. Positive clones identified by PCR were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to verify sequence accuracy.

[0043] GhSZF3-1F: ATGGAGACGGCAGGAGAATGGCT, SEQ ID NO.3;

[0044] GhSZF3-1R: TCAAGTTGCTTCTATCTTTTTATCG, SEQ ID NO.4.

[0045] Example 2

[0046] GhSZF3 Bioinformatics analysis of genes

[0047] Created using GSDS (http: / / gsds.gao-lab.org / ) GhSZF3 Schematic diagrams of exon and intron structures. The molecular weight, theoretical isoelectric point, hydrophobicity, and other physicochemical properties of the cotton GhSZF3 protein sequence were predicted using the ExPASy online tool (http: / / web.expasy.org / protparam / ). The transmembrane structure of the GhSZF3 protein was analyzed using TMHMM and TMpred. The signal peptide of the GhSZF3 protein sequence was predicted using Signal IP4. The secondary and tertiary structures of the protein were predicted using PSIPRED (http: / / bioinf.cs.ucl.ac.uk) and SWISS-MODEL (https: / / swissmodel.expasy.org / interactive / SSmzBZ / models / ), respectively.

[0048] GhSZF3 The gene contains 3 intron sequences ( Figure 1A), in which the CDS sequence contains a C2HC5 type zinc finger domain at positions 526-648. The GhSZF3 protein consists of 403 amino acids, with a predicted molecular weight of 44.50 kDa and an isoelectric point of 7.48. Transmembrane structure analysis of the GhSZF3 protein using TMHMM and TMpred did not reveal any transmembrane structures, indicating that GhSZF3 may not be a transmembrane protein. Figure 1 B). Signal IP4 prediction showed that the protein lacked a signal peptide structure, indicating that GhSZF3 is not a secreted protein. PSIPRED analysis results ( Figure 1 C) indicates that the secondary structure of the GhSZF3 protein consists of 227 randomly coiled amino acid residues, accounting for 56.33%, forming the main structure of the protein; the α-helix contains 149 amino acid residues, accounting for 36.97%; and the extended chain contains 27 amino acid residues, accounting for 6.7%. Further analysis using SWISS-MODEL was conducted to predict the three-dimensional structure of the GhSZF3 protein. Figure 1 (D) The results show that the composition of the tertiary structure of GhSZF3 is basically consistent with the predicted structure of the secondary structure.

[0049] Using the plantCARE website (https: / / www.plantcare.co.uk / ), for GhSZF3 Analysis of cis-elements in the upstream 2000 bp promoter sequence revealed jasmonic acid, abscisic acid, growth hormone, and multiple light-induced cis-regulatory elements. A series of stress-response-related elements, LTR and TC-rich repeats, were also present (Table 1). Therefore, it is speculated that... GhSZF3 Gene expression is likely to be regulated by abiotic stress.

[0050] Table 1 GhSZF3 Stress-related promoter elements in the upstream regulatory region of a gene

[0051]

[0052] Note: " / " in the sequence represents "or".

[0053] Example 3

[0054] Construction of cell fusion expression vector

[0055] Cotton cDNA was amplified using primers F13GhSZF3-1F and F13GhSZF3-R. PCR reaction conditions were the same as in Example 1. The recovered target fragment was ligated into the cell fusion expression vector pBWA(V)HS-linker-eGFP. The ligation product was transformed into TOP10 competent cells for screening and identification, obtaining the cell fusion expression vector pBWA(V)HS-GhSZF3-linker-eGFP.

[0056] Vector construction primers:

[0057] F13GhSZF3-1F:AACACGGGGGACTTTGCAACatggagacggcaggagaatggc, SEQ ID NO.5;

[0058] F13GhSZF3-R: CCTGAAGCGGCCGCTGTACAagttgcttctatctttttatcggtcataaaatatttcaact, SEQ ID NO. 6.

[0059] Example 4

[0060] Transient transformation of tobacco epidermal cells

[0061] The cell fusion expression vector pCAMBIA1305-GhSZF3-EGFP constructed in Example 3 was transformed into Agrobacterium GV3101, and then used to infect tobacco leaves. After 48 hours of infection, the leaves were cut and pressed into slides, and the subcellular localization of the cotton GhSZF3 protein was analyzed under a laser scanning confocal microscope. Figure 2 Fluorescence was observed in both the nucleus and plasma membrane of tobacco cells under a microscope, indicating that the GhSZF3 protein is located in the plant cell nucleus and plasma membrane.

[0062] Example 5

[0063] cotton GhSZF3 Spatiotemporal expression analysis of genes

[0064] In order to find out GhSZF3 Differences in gene expression in different cotton tissues were investigated using quantitative real-time PCR (qPCR) with SYBR Green dye. －ΔΔCt Method calculation GhSZF3 Gene relative expression levels were analyzed to determine expression patterns. Each sample was tested in triplicate, and each PCR reaction was performed in triplicate.

[0065] Quantitative primer sequences:

[0066] Histone-F: AGAAGCTGCTATGGTTGCAAAGC, SEQ ID NO.7;

[0067] Histone-R: TGACCTGTGTGACCGCCATT, SEQ ID NO.8;

[0068] DLGhSZF3-F: GCGAGTTGGCTCATCCTCTT, SEQ ID NO.9;

[0069] DLGhSZF3-R: CCGATAACTAGAGCCCGCAG, SEQ ID NO. 10.

[0070] The roots, stems, and leaves of cotton seedlings (4-leaf stage) were examined. GhSZF3 Gene expression level analysis, quantitative fluorescence results ( Figure 3 )show, GhSZF3 The gene is expressed in cotton roots, leaves, and stems, and the expression differences are quite large, indicating that... GhSZF3 The gene expression level is higher in roots and stems, and relatively lower in leaves. GhSZF3 The gene is predominantly expressed in the cotton rhizome.

[0071] Example 6

[0072] For further analysis GhSZF3 The response of genes to salt induction was detected using real-time quantitative PCR under salt stress. GhSZF3 Gene expression status. The specific steps are as follows:

[0073] Cotton seeds of the Ji Mian 172 variety were sown in sandy soil. After the two cotyledons were fully expanded, they were transplanted into a nutrient solution for hydroponics. The culture conditions were 28 ℃ / light for 16 h, followed by 28 ℃ / dark culture for 8 h, and hydroponics for approximately 30 days. 200 mmol / L NaCl was added to the nutrient solution. Seedling tissues (roots and leaves) were collected at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h, flash-frozen in liquid nitrogen, and stored at -80 ℃ for RNA extraction. The quantitative real-time PCR analysis method was the same as in Example 5.

[0074] The results show ( Figure 4 ), GhSZF3 Gene expression was upregulated in cotton tissues (roots and leaves) under NaCl induction. In roots, NaCl induction showed an increasing trend from 0 to 24 hours, reaching its peak at 3 hours. In leaves, NaCl induction led to a decrease in expression at 3 hours, followed by an increasing trend at other times. This suggests that... GhSZF3 Genes play an important role in cotton's response to abiotic stress, especially in its response to salt stress, and are closely related to cotton salt stress.

[0075] Example 7

[0076] Arabidopsis genetic transformation

[0077] The fusion vector pBWA(V)HS-GhSZF3-linker-eGFP obtained in Example 3 was transformed into Agrobacterium, and Agrobacterium culture was performed. The precipitate was collected by centrifugation and resuspended in the infection solution to OD. 260 Arabidopsis genetic transformation was performed with a value of 0.6–0.8.

[0078] Arabidopsis thaliana was transformed using the flower-dipping method, screened with hygromycin, and detected by PCR using specific primers. The sample size was 354 bp. After obtaining stable plants ( Figure 5 Seeds were sown on 200 mM NaCl 1 / 2 MS medium and the germination rate was observed. At the same time, seedlings that had grown on the medium for 5 days were transplanted to 200 mM NaCl 1 / 2 MS medium and their root length was measured after 5 days.

[0079] Detection primers:

[0080] GhSZF3-1F: ATGGAGACGGCAGGAGAATGGCT, SEQ ID NO.3;

[0081] DLGhSZF3-R: CCGATAACTAGAGCCCGCAG, SEQ ID NO. 10.

[0082] Under normal growth conditions, the germination rate and root length of WT and transgenic lines were basically the same. On a 200mM medium, the germination rate of WT was approximately 17.8%, and the root length was approximately 1.25cm; GhSZF3 The germination rates of the transgenic Arabidopsis thaliana OE-1 and OE-2 lines were 96.7% and 75.6%, respectively, with root lengths of approximately 1.73 cm and 1.77 cm. This indicates that the germination rate of the transgenic Arabidopsis thaliana lines was significantly higher than that of the wild type, and their root lengths were also significantly longer. It can be inferred that... GhSZF3 Genes can enhance a plant's salt tolerance and have a positive regulatory effect on a plant's salt tolerance. Figure 6 ).

[0083] Example 8

[0084] cotton GhSZF3 Salt treatment experiment and determination of physiological indicators in gene-silenced plants

[0085] Building TRV2- GhSZF3 The carrier was silenced using VIGS technology. GhSZF3 Genes, the process is as follows:

[0086] The silenced fragment of the gene designed using SGN VIGS TooL is 544~843bp of SEQ ID NO.2.

[0087] The silenced fragments were constructed into the TRV2 vector to obtain TRV2- GhSZF3 The cells were then transferred to competent Agrobacterium cells for Agrobacterium culture, and the OD... 260 The value is 0.8~1.0, and TRV2 is collected. GhSZF3 TRV2:00, TRV2:CLA cells, mixed with the infection solution, for subsequent injection.

[0088] After germinating cotton seeds, select those showing signs of whitening and sow two seeds per plastic pot, covering them with a layer of vermiculite and covering with plastic wrap. Cultivate until the cotton seedlings grow to the point where the seed coat falls off. Provide 12 hours of light, daytime temperature of 27-28℃, nighttime temperature of 20-22℃, and humidity of 30%-40%. Inject when the two cotyledons have unfolded but before the first true leaf has emerged. Mix the injection solution with the 192 bacterial culture medium at a 1:1 ratio. TRV2:00 serves as the control, and TRV2:CLA serves as the albino control.

[0089] Quantitative results showed that VIGS- GhSZF3 plant GhSZF3 Expression levels were significantly reduced. This was observed in TRV2 and VIGS- GhSZF3 The cotton seedlings were treated with 400 mg / L NaCl irrigation, and the chlorophyll and proline content of the leaves were measured. Figure 7 It can be seen that the control group was more silent after 5 days of salt treatment. GhSZF3 Transgenic strain VIGS- GhSZF3 The leaves showed obvious yellowing, and the chlorophyll value of the silent plants was significantly lower than that of the control, with significantly lower proline content, indicating that the plant tissues in the control group were relatively aged. These results all indicate that VIGS- GhSZF3 The salt tolerance of the plants was significantly reduced compared to the control.

[0090] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0091] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Overexpression GhSZF3 The application of genes in improving plant salt tolerance is characterized by, The nucleotide sequence of the gene is shown in SEQ ID NO.2; The plant in question is cotton or Arabidopsis thaliana.

2. The application of increasing GhSZF3 protein expression in improving plant salt tolerance, characterized in that, The amino acid sequence of the GhSZF3 protein is shown in SEQ ID NO.1; The plant in question is cotton or Arabidopsis thaliana.

3. The claim 1 GhSZF3 The application of the gene or the GhSZF3 protein-related biomaterials described in claim 2 in the positive regulation of plant salt tolerance, characterized in that... The biomaterial is any one of the following: A: The nucleotide sequence is the nucleic acid molecule as described in claim 1 or the nucleic acid molecule encoding the protein as described in claim 2; B: An expression cassette containing the nucleic acid molecule described in A; C: An expression vector containing the nucleic acid molecule described in A, or a recombinant vector containing the expression cassette described in B; D: Recombinant microorganisms containing the nucleic acid molecules described in A, or recombinant microorganisms containing the expression cassette described in B, or recombinant microorganisms containing the recombinant vector described in C; The plant in question is cotton or Arabidopsis thaliana.

4. The application according to any one of claims 1 to 3, characterized in that, Improving plant salt tolerance refers to increasing seed germination rate and root length under salt stress.

5. A method for cultivating transgenic plants resistant to salt stress, characterized in that, Will GhSZF3 Genes are transferred into plant tissues or plant cells; The GhSZF3 The nucleotide sequence of the gene is shown in SEQ ID NO.2; The plant in question is cotton or Arabidopsis thaliana.

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