Application of chive gene AaWRKY24 in plant salt stress resistance

Abstract

The invention provides an application of a chive gene AaWRKY24 in plant salt stress resistance, belongs to the technical field of plant genetic engineering, and the application of the chive gene AaWRKY24 in any one of the following items (1)-(2): (1) improving the salt stress resistance of plants; (2) cultivating a plant variety with salt stress resistance; the gene can provide gene resources for creation of salt-resistant new germplasm and breeding of special high-resistance new varieties, and has very important scientific and production application significance for promoting resistance breeding, improving the economic benefits of chive and the like.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the application of the scallion gene AaWRKY24 in plant salt stress resistance. Background Technology

[0002] Soil salinization is a significant environmental stressor affecting global agricultural production. With climate change, the development of irrigated agriculture, and the inappropriate use of chemical fertilizers, the area of ​​secondary salinized soils continues to expand, severely impacting crop growth and yield. Improving crop salt tolerance and developing and utilizing salinized land has become an important breeding goal for ensuring food security. Allium fistulosum L. var. caespitosum Makino., a widely cultivated vegetable and condiment crop globally, is also severely affected by salt stress in its growth and quality. Therefore, exploring the salt-tolerant gene resources within Allium species and cultivating salt-tolerant varieties through molecular biology techniques is an effective strategy to address this problem. Currently, research on improving plant salt tolerance mainly focuses on the discovery and utilization of key functional genes. In the complex physiological and molecular network of plant responses to salt stress, a series of functional genes (such as genes for the synthesis of osmotic regulators, ion transporter genes, transcription factors, and signal transduction-related genes) have been shown to play a central role. Traditional genetic improvement methods, such as hybridization breeding, are limited in Allium crops due to their complex genomes and long breeding cycles. Modern genetic engineering offers a more precise and efficient alternative. Existing technologies have involved introducing exogenous salt-tolerant genes (such as genes from microorganisms or other unrelated plants) into plants to improve their salt tolerance. However, these methods primarily rely on the introduction of heterologous genes.

[0003] Defects and shortcomings of existing technology: (1) Reliance on exogenous genes poses potential ecological and food safety risks: Existing technologies mostly use heterologous gene transformation, and the potential long-term impact of their expression products on the local ecosystem and consumer health is still unclear, which to some extent limits the commercial promotion and application of related transgenic crops.

[0004] (2) Insufficient exploration of gene resources and neglect of the advantages of endogenous genes: Existing research has not adequately explored the rich salt-tolerant gene resources contained in Allium species, especially Shallot. Compared with heterologous genes, genetic improvement using the crop's own endogenous genes has obvious advantages because it has better compatibility in the genetic background, more stable expression regulation, and can avoid the controversy caused by exogenous genes.

[0005] Therefore, it is of great significance to explore the salt-resistant gene resources of Allium species to improve the salt stress resistance of crops and to cultivate plant varieties with salt stress resistance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of the scallion gene AaWRKY24 in plant salt stress resistance.

[0007] Specifically, this is achieved through the following technical solutions: One of the objectives of this invention is to provide the application of the scallion gene AaWRKY24 in any of the following (1)-(2): (1) Improve the salt stress resistance of plants; (2) Cultivate plant varieties with salt stress resistance.

[0008] Furthermore, the salt stress resistance includes at least one of the following (1)-(3): (1) Increase plant root length, seedling height, and fresh weight; (2) Increase the content of SS and Pro in plants; (3) Activate CAT activity and reduce the degree of cell damage.

[0009] Furthermore, the scallion gene AaWRKY24 is a nucleic acid molecule as shown in (1) or (2) below: (1) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO. 1; (2) Nucleic acid molecules other than (1) that encode the amino acid sequence shown in SEQ ID NO. 2.

[0010] One of the objectives of this invention is to determine the application of the protein encoded by the scallion gene AaWRKY24 in any of the following (1)-(2): (1) Improve the salt stress resistance of plants; (2) Cultivate plant varieties with salt stress resistance.

[0011] Furthermore, the salt stress resistance includes at least one of the following (1)-(3): (1) Increase plant root length, seedling height, and fresh weight; (2) Increase the content of SS and Pro in plants; (3) Activate CAT activity and reduce the degree of cell damage.

[0012] Furthermore, the protein encoded by the scallion gene AaWRKY24 is the protein shown in (1) below: (1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing.

[0013] One of the objectives of this invention is to provide the application of a recombinant expression vector or recombinant bacteria containing the onion gene AaWRKY24 in any of the following (1)-(2): (1) Improve the salt stress resistance of plants; (2) Cultivate plant varieties with salt stress resistance.

[0014] One objective of this invention is to provide a method for improving plant tolerance to salt stress, comprising the following steps: The chive gene AaWRKY24 was transferred into the target plant; Or it could upregulate the expression of the scallion gene AaWRKY24 or its homologs in the plant genome; The scallion gene AaWRKY24 is a nucleic acid molecule as shown in (1) or (2) below: (1) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO. 1; (2) Nucleic acid molecules other than (1) that encode the amino acid sequence shown in SEQ ID NO. 2.

[0015] One objective of this invention is to provide a method for cultivating transgenic plants with enhanced salt tolerance, comprising the following steps: By transferring the chive gene AaWRKY24 into the target plant, plants with better salt tolerance than the wild type were obtained. The scallion gene AaWRKY24 is a nucleic acid molecule as shown in (1) or (2) below: (1) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO. 1; (2) Nucleic acid molecules other than (1) that encode the amino acid sequence shown in SEQ ID NO. 2.

[0016] Furthermore, the plant in question is wheat.

[0017] Furthermore, the spliced ​​sequence of the scallion gene AaWRKY24 is 537 bp in length, and the sequence is shown in SEQ ID NO. 1, as follows: ATGGATCCCTTTGATGCAAAACACCTGCAAACAATCCCTTCAATTAATGCAACTCCTTCAAATCAACAACAGTTTCAACAGCAAGATTCATCACAAGGTTTTGATTGGGCATCATTGTTGCTTAACAATCTCAA TAACGGGGATGAAAGCACGAGCAATATAACTCCCAACAATGTTGGTCATGATGATGGTAGTAACAGAGAAAAGGGATTGATGAATAAGGTTAAAGATGCAAGAAGAAAGAAGAAGATGGAGAAGCCCAGGTTCG AGTTTCATACTCGGAGCGAAAATGATATCCTTGATGATGGTTATCGTTGGCGCAAATATGGTCAGAAATCTGTAAAGAATAGCCAGCATCCAAGGAGCTATTTCAGATGCACGCATCATACATGCAATGTGAAG AAACAAGTGCAAAGGTTAGCCAAGGACACGAGCATTGTGGTTACAACATATGAGGGAGTTCACAATCACCCATGCGAGAAACTCATGGAAGCTCTCACTCCTCTTCTCAAGCAAATTCAGTTTCTTTCTCGCTTT Furthermore, the protein encoded by the scallion gene AaWRKY24 has the sequence shown in SEQ ID NO. 2, as follows: MDPFDAKHLQTIPSINATPSNQQQFQQQDSSQGFDWASLLLNNLNNGDESTSNITPNNVGHDDGSNREKGLMNKVKDARRKKKMEKPRFEFHTRSENDILDDGYRWRKYGQKSVKNSQHPRSYFRCTHHTCNVKKQVQRLAKDTSIVVTTYEGVHNHPCEKLMEALTPLLKQIQFLSRF Beneficial effects The transcriptional level of the scallion gene AaWRKY24 of this invention is induced by salt stress. The AaWRKY24 mutant is significantly intolerant to salt stress, thus proving that the AaWRKY24 gene and its AaWRKY24 protein can increase the root length, seedling height and fresh weight of wheat plants; increase the content of SS and Pro in plants; activate CAT activity and reduce the degree of cell damage.

[0018] Compared to searching for genes from microorganisms or distantly related species, this invention utilizes genes from the equally advanced plant, scallion, whose encoded proteins may exhibit better compatibility and lower unpredictable risks within crop cells. Therefore, it possesses significant theoretical research value and broad agricultural application prospects, providing a new solution for cultivating high-yield crops suitable for saline-alkali soils. This gene can provide genetic resources for the creation of new salt-resistant germplasm and the breeding of distinctive, highly resistant varieties. Simultaneously, it holds crucial scientific and production application significance for advancing resistance breeding and improving the economic benefits of scallions. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Image showing the cloning bands of the AaWRKY24 gene.

[0021] Figure 2 This is a sequence comparison diagram of the AaWRKY24 gene and the isoform_227446 gene.

[0022] Figure 3 This is a schematic diagram of the AaWRKY24 gene overexpression vector.

[0023] Figure 4 A schematic diagram for screening AaWRKY24 gene positive strains.

[0024] Figure 5 Figure 1 shows the MDA content analysis of AaWRKY24 gene overexpressing plants and WT plants at different time points.

[0025] Figure 6 Figure 1 shows the analysis of soluble sugar (SS) and proline (Pro) content in AaWRKY24 gene overexpressing plants and WT plants at different growth stages.

[0026] Figure 7 This figure shows the catalase (CAT) activity analysis of AaWRKY24 gene overexpressing plants and WT plants at different growth stages.

[0027] Figure 8 This is a diagram showing the phenotypic analysis of AaWRKY24 gene overexpressing plants and WT plants at different time stages. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be described in detail below with reference to several embodiments and 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.

[0029] 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.

[0030] The test materials used in the embodiments of the present invention, unless otherwise specified, are all conventional test materials in the art and can be purchased through commercial channels.

[0031] The samples in this embodiment of the invention are divided into: Experimental group: AaWRKY24 overexpressing plants; Control group: WT plants Sampling conditions: no stress treatment (0h), salt stress (24h), and recovery after salt stress (R24h); Test method: WT plant: Wild type 1718.

[0032] The AaWRKY24 overexpressing plants in the experimental group were obtained through the following methods: constructing a plant overexpression vector containing the scallion gene AaWRKY24, transforming the constructed plant overexpression vector into wild-type wheat 1718; extracting DNA from wheat using the CTAB method, and verifying the transgene with PCR using specific primers (AaWRKY24-F: Acta ggtctcGCACCATGGATCCCTTTGATGCAAAAC; AaWRKY24-R: Acta ggtctcT cgccAAAGCGAGAAAGAAACTGAATTTGC), thus cultivating salt-tolerant transgenic plants.

[0033] The CTAB method for extracting wheat DNA is as follows: ① Take 2-3 healthy, young wheat leaves, quickly cut them into small pieces, and place them in a 2 mL centrifuge tube pre-filled with steel beads and flash-frozen in liquid nitrogen. Grind thoroughly using a ball mill until the leaf tissue is completely pulverized into a fine powder. Flash freezing in liquid nitrogen effectively inhibits nuclease activity, while grinding with steel beads thoroughly breaks down cell walls, facilitating subsequent DNA release. Add preheated (65℃) mixture (1 mL CTAB and 200 μL β-mercaptoethanol) to each tube, incubate at 65℃ for 30-60 min, inverting and mixing every 5 min, then centrifuge at 12000 rpm for 10 min. ② Collect the supernatant, add an equal volume of chloroform, mix well, let stand for 12 min, then centrifuge at 12000 rpm for 10 min. ③ Repeat step ②, collect the supernatant, add 2 volumes of anhydrous ethanol, mix well, and incubate at -20℃ for 30-60 min. The white solid at the bottom of the centrifuge tube is the genomic DNA precipitate. Centrifuge at 12000 rpm for 10 min and discard the supernatant. ④ Wash the DNA precipitate 2-3 times with 1 mL of 75% anhydrous ethanol, centrifuge at 12000 rpm for 5 min, discard the supernatant, and allow the precipitate to air dry at room temperature until translucent. ⑤ Dissolve the precipitate in 30-50 μL of ddH2O, then add 0.5 μL of RNase and incubate at room temperature for 30 min. ⑥ Perform agarose gel electrophoresis, observe the bands, and detect the genomic DNA. Store the DNA at -20℃ for later use.

[0034] Example 1: Gene Cloning Using scallions as experimental material, RNA was extracted using the Omega EZNA Plant RNA Kit, and then reverse transcribed into cDNA using the Novizan Bio-Reverse Transcription Kit. Using this cDNA as a template, specific primers were designed for PCR amplification. Agarose gel electrophoresis results showed that the amplification bands were consistent with the target bands. Figure 1 As shown, M: D2000 marker, 1-3 are the target bands for amplification; after recovering the target fragment, sequencing was performed, and the sequencing result was 537bp. The gene was named AaWRKY24. Sequence alignment with isoform_227446 showed a consistency of 98.88%, indicating that the AaWRKY24 gene was successfully cloned.

[0035] The isoform_227446 gene, with its sequence shown in SEQ ID NO. 3, is as follows: ATGGATCCCTTTGATGCAAAACACCTGCAAACAATCCCTTCAATTAATGCAACTCCTTCAAATCAACAACAGTTTCAACAGCAAGATTCATCACAAGGTTTTGATTGGGCATCATTGTTGCTTAACAATCTCAA TAACGGGGATGAAAGCACGAGCAATATAACTCCCAACAATGTTGGTCATGATGATGGTAGTAACAGAGAAAAGGGATTGATGAATAAGGTTAAAGATGCAAGAAGAAAGAAGAAGATGGAGAAGCCCAGGTTCG AGTTTCATACTCGGAGCGAAAATGATATCCTTGATGATGGTTATCGTTGGCGCAAATATGGTCAGAAATCTGTAAAGAATAGCCAGCATCCAAGGAGCTATTTCAGATGCACGCATCATACATGCAATGTGAAG AAACAAGTGCAAAGGTTAGCCAAGGACACGAGCATTGTGGTTACAACATATGAGGGAGTTCACAATCACCCATGCGAGAAACTCATGGAAGCTCTCACTCCTCTTCTCAAGCAAATTCAGTTTCTTTCTCGCTTT In this invention, the AaWRKY24 gene and the isoform_227446 gene were sequence aligned. Light blue indicates different bases. See details. Figure 2 The fact that only a few bases are different indicates that the cloning was successful.

[0036] Example 2 Construction of AaWRKY24 gene overexpression vector Based on the structural characteristics of the AaWRKY24 gene, an overexpression vector was constructed, and genetic transformation was performed by a commercial institution to obtain the overexpression vector, as shown below. Figure 3 As shown.

[0037] The above conversion method is as follows: Plasmids were extracted according to the instructions of the plasmid miniprep kit (Tiangen, DP103) and transformed into Agrobacterium tumefaciens LBA4404 competent cells using the freeze-thaw method. Wheat coleoptile tips were cut to expose the meristem, and the coleoptiles were immersed in Erlenmeyer flasks containing Agrobacterium, with vacuum permeation for 5 minutes. The AaWRKY24 gene was introduced into wheat “1718” coleoptiles (Yang, et al., 2022) using Agrobacterium-mediated transformation. The infected coleoptiles were covered with plastic bags to maintain high humidity and placed in the dark for 3 days. These coleoptiles were then hydroponically cultured in 1 / 2 Hoagland medium until the two-leaf, one-heart stage. Wheat leaves were then further analyzed using specific primers for PCR identification of transgenic positive plants. Figure 4 As shown, 1-8: screening for AaWRKY24 positive strains; 9: negative control; 10: positive control.

[0038] Example 3 MDA content analysis MDA content determination: The MDA content of the root samples was determined using the Beijing Solarbio MDA kit (catalog number: A007-1-1) according to the kit instructions. MDA is a major product of membrane lipid peroxidation in plants under stress conditions and can serve as an important indicator for evaluating plant stress resistance. Figure 5 The results show that the MDA content in the WT group was significantly higher than that in the overexpressing plants during the three periods of no stress treatment (0h), salt stress (24h), and post-stress recovery (R24h), indicating that the WT plants suffered more severe damage from salt stress. Therefore, the cell membrane damage in WT plants was more severe under salt stress, further demonstrating that AaWRKY24 overexpression can reduce the degree of plant cell membrane damage, thereby improving the plant's tolerance to salt stress.

[0039] Example 4: Analysis of Osmotic Conditioning Substance Content Salt tolerance function analysis of AaWRKY24 gene: Under salt stress, WT plants suffered severe water loss and wilting, with leaves drooping; however, AaWRKY24 overexpressing plants were more upright and less affected by salt stress; therefore, AaWRKY24 gene plants were more salt tolerant than WT plants.

[0040] Soluble sugars (SS) and proline (Pro), osmotic regulators, play important roles in reducing cell osmotic potential, maintaining cell turgor pressure, and resisting salt stress. Physiological analysis of AaWRKY24 overexpressing lines and “1718” WT wheat under no-salt-stress treatment (0h), salt-stress treatment (24h), and post-stress recovery (R24h) conditions revealed that the SS and Pro contents in the overexpressing wheat lines were higher than those in the WT plants at each time point, and their trends were similar. Figure 6As shown, under salt stress and recovery conditions, the SS and Pro contents of the overexpressing lines were higher than those of the WT plants. This indicates that the overexpressing lines can rapidly regulate the content of osmotic regulators in the plant, improve the water absorption and retention capacity of plant tissues, reduce the degree of damage to themselves, and thus improve the plant's tolerance to salt stress.

[0041] Example 5: Catalase (CAT) Activity Analysis CAT is widely present in plant and animal tissues, and its activity is related to the metabolic intensity and cold resistance and disease resistance of organisms. For example Figure 7 As shown, during the R24h period, the CAT activity of the AaWRKY24 overexpressing lines was significantly higher than that of the WT lines, indicating that AaWRKY24 can activate CAT activity under salt stress, reduce the degree of cell damage, and thus give the transgenic materials stronger salt tolerance.

[0042] Example 6: Analysis of Relevant Phenotypic Traits Phenotypic analysis of seedlings revealed that, for example Figure 8 As shown, under normal conditions, the green leaves of WT plants and overexpression lines were upright and vigorous. However, the root development and plant height growth of the overexpression lines were significantly better than those of WT plants. At each stage, the root length, seedling height, and fresh weight of the AaWRKY24 overexpression lines were significantly greater than those of WT plants; and at the 0h and Rh stages, the increase in seedling height of the overexpression lines was significantly greater than that of the WT plants. It can be seen that the dry and fresh weights of the AaWRKY24 overexpression lines were higher than those of WT plants at 0h, 24h, and Rh stages, especially at the Rh stage, where the increase in dry and fresh weights of the AaWRKY24 overexpression lines was the greatest. After 24h of salt stress, the overexpression lines were in better growth condition than WT plants. Therefore, AaWRKY24 overexpression significantly promoted the growth of wheat seedlings, and AaWRKY24 overexpression indeed enhanced the salt tolerance of wheat.

Claims

1. Application of the scallion gene AaWRKY24 in any of the following (1)-(2): (1) Improve the salt stress resistance of plants; (2) Cultivate plant varieties with salt stress resistance.

2. The application as described in claim 1, characterized in that, The salt stress resistance includes at least one of the following (1)-(3): (1) Increase plant root length, seedling height, and fresh weight; (2) Increase the content of SS and Pro in plants; (3) Activate CAT activity and reduce the degree of cell damage.

3. The application as described in claim 1 or 2, characterized in that, The scallion gene AaWRKY24 is a nucleic acid molecule as shown in (1) or (2) below: (1) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO. 1; (2) Nucleic acid molecules other than (1) that encode the amino acid sequence shown in SEQ ID NO.

2.

4. The application of the protein encoded by the scallion gene AaWRKY24 in any of the following (1)-(2): (1) Improve the salt stress resistance of plants; (2) Cultivate plant varieties with salt stress resistance.

5. The application as described in claim 4, characterized in that, The salt stress resistance includes at least one of the following (1)-(3): (1) Increase plant root length, seedling height, and fresh weight; (2) Increase the content of SS and Pro in plants; (3) Activate CAT activity and reduce the degree of cell damage.

6. The application as described in claim 4 or 5, characterized in that, The protein encoded by the scallion gene AaWRKY24 is as shown in (1) below: (1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing.

7. The application of recombinant expression vectors or recombinant bacteria containing the scallion gene AaWRKY24 in any of the following (1)-(2): (1) Improve the salt stress resistance of plants; (2) Cultivate plant varieties with salt stress resistance.

8. A method for improving plant tolerance to salt stress, characterized in that, Includes the following steps: The chive gene AaWRKY24 was transferred into the target plant; Or it could upregulate the expression of the scallion gene AaWRKY24 or its homologs in the plant genome; The scallion gene AaWRKY24 is a nucleic acid molecule as shown in (1) or (2) below: (1) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO. 1; (2) Nucleic acid molecules other than (1) that encode the amino acid sequence shown in SEQ ID NO.

2.

9. A method for cultivating transgenic plants with improved salt tolerance, characterized in that, Includes the following steps: By transferring the chive gene AaWRKY24 into the target plant, plants with better salt tolerance than the wild type (WT) were obtained. The scallion gene AaWRKY24 is a nucleic acid molecule as shown in (1) or (2) below: (1) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO. 1; (2) Nucleic acid molecules other than (1) that encode the amino acid sequence shown in SEQ ID NO.

2.

10. The method as described in claim 8 or 9, characterized in that, The plant in question is wheat.

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