Application of cassava MeMYB98 gene in regulation of plant salt stress

Abstract

The invention discloses an application of a cassava MeMYB98 gene in regulation of plant salt stress. The nucleotide sequence of the MeMYB98 gene is as shown in SEQ ID NO. 1. The cassava MeMYB98 gene is cloned and functionally verified, the cassava MeMYB98 gene is proved to play a key role in regulating and controlling the salt tolerance of plants, a new gene resource is provided for molecular breeding of crops, and the cassava MeMYB98 gene can be used for screening and cultivating salt-tolerant varieties, increasing the yield of the crops and effectively relieving the influence of drought and salt stress on the plants and has a good application prospect. The method has important application value and wide agricultural popularization prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to the application of the cassava MeMYB98 gene in regulating plant salt stress. Background Art

[0002] Cassava (Manihot esculenta Crantz) is a perennial shrub of the genus Manihot in the Euphorbiaceae family, with edible, forage and ornamental values. It is also the staple food crop for about 1 billion people worldwide and ranks as the sixth largest food crop in the world. Cassava plays an important role in food security and efficient utilization of resources due to its characteristics of drought tolerance, barren tolerance and non-competition with food crops for land. However, the problem of soil salinization in China is serious, which restricts the growth and yield of crops. Therefore, cultivating salt-tolerant cassava varieties is of great significance for making full use of saline-alkali land resources and improving the level of agricultural sustainable development. Traditional cassava breeding methods have a long cycle, strong randomness and low efficiency, and it is difficult to achieve precise improvement of target traits. In recent years, the development of genetic engineering technology and transgenic technology has provided new technical means for cassava genetic improvement, greatly making up for the deficiencies of traditional breeding. Exploring key genes related to cassava salt tolerance and using molecular breeding technology to cultivate salt-tolerant new varieties have become the key research directions in current cassava genetic breeding.

[0003] Transcription factors (TFs) are the core factors of gene expression regulation and play an indispensable role in the mechanism of plants responding to abiotic stresses. The MYB transcription factor family is a superfamily with rich functions in plants. According to the differences in their conserved domains, it can be divided into four categories: 1R (R1 / 2, R3-MYB), R2R3-MYB, 3R-MYB and 4R-MYB. Among them, MeMYB98 belongs to the R2R3-MYB family.

[0004] Salt stress will have a profound impact on the physiological and biochemical processes of plants, including water balance, seed setting, osmotic regulation, photosynthesis, mineral absorption, metabolic regulation and overall growth and development, etc. To resist salt stress, a series of genes will be activated in plants, encoding ion channels and transporters, chaperone proteins, antioxidant proteins, signal molecules and transcription factors, etc., to maintain ion balance and osmotic homeostasis and enhance their own salt tolerance. Among them, transcription factors play a crucial regulatory role in the salt stress response process. However, the current systematic identification and functional research on cassava MYB transcription factors are still relatively limited. Therefore, in-depth analysis of the regulatory mechanism of MYB transcription factors will provide important theoretical basis and gene resource support for the cultivation of salt-tolerant cassava varieties. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention provides an application of the cassava MeMYB98 gene in regulating plant salt stress.

[0006] The present invention provides an application of the MeMYB98 gene or the protein encoded thereby in regulating plant salt stress, wherein the nucleotide sequence of the MeMYB98 gene is as shown in SEQ ID NO.1.

[0007] Through the analysis of the expression pattern of the MeMYB98 gene in cassava, it is found that salt stress, osmotic stress and cold stress induce the expression of the MeMYB98 gene to a certain extent, among which the induction expression level of MeMYB98 is the highest under salt stress. At the same time, overexpression of the MeMYB98 gene enhances the salt stress resistance of transgenic Arabidopsis thaliana, and the MeMYB98-silenced lines are more sensitive to salt stress, indicating that the MeMYB98 gene may play a positive regulatory role in the salt tolerance signaling pathway of cassava. This study deepens the understanding of the function of the plant MeMYB98 gene and lays an important foundation for further studying the molecular mechanism of cassava's response to salt stress and providing candidate target genes for salt tolerance.

[0008] In some embodiments, the application includes detecting the application of plants in salt-tolerant plant breeding.

[0009] In the present invention, the application of the MeMYB98 gene in plant salt tolerance is that the MeMYB98 gene is used as a molecular marker for plant salt tolerance, that is, the salt tolerance of a plant is illustrated by detecting whether the plant has the MeMYB98 gene; it can also be breeding according to the presence or absence of the MeMYB98 gene. For example, the salt tolerance of a plant can be increased by introducing the MeMYB98 gene or increasing the expression of the MeMYB98 gene, or a new plant variety can be obtained by inhibiting or knocking out the MeMYB98 gene.

[0010] In some embodiments, the amino acid sequence of the protein encoded by the MeMYB98 gene is as shown in SEQ ID NO.2.

[0011] The present invention also provides a method for regulating plant salt stress, which regulates the expression or activity of the plant MeMYB98 gene or the protein encoded thereby, so as to regulate plant salt stress;

[0012] The nucleotide sequence of the MeMYB98 gene is as shown in SEQ ID NO.1.

[0013] In some embodiments, the method is selected from any one of the following:

[0014] (1) Upregulating the expression or activity of the MeMYB98 gene or the protein encoded thereby, so as to improve plant salt tolerance;

[0015] (2) Down-regulate the expression or activity of the MeMYB98 gene or the protein encoded thereby, thereby reducing the salt tolerance of the plant.

[0016] In some embodiments, the up-regulation of the expression or activity of the MeMYB98 gene or the protein encoded thereby includes:

[0017] (1) Transfer the expression construct or vector of the MeMYB98 gene into the plant;

[0018] (2) Transfer the expression construct or vector containing the MeMYB98 gene into the plant.

[0019] In some embodiments, the down-regulation of the expression or activity of the MeMYB98 gene or the protein encoded thereby includes:

[0020] Knock out or silence the MeMYB98 gene in the plant, or inhibit the activity of the MeMYB98 protein, thereby reducing the expression of the ERF04 gene.

[0021] The present invention also provides a breeding method for a plant, and the method is selected from any one of the following:

[0022] (1) By increasing the activity of the MeMYB98 protein in the target plant, a plant with stronger salt stress tolerance than the target plant is obtained;

[0023] (2) By promoting the expression of the MeMYB98 gene in the target plant, a plant with stronger salt stress tolerance than the target plant is obtained.

[0024] The breeding in the present invention is not limited to different species of cassava, and may also include other plants, such as Arabidopsis thaliana, cucumber, tomato, wheat, oat, rice, corn, poplar, turfgrass, alfalfa, peanut, soybean, sorghum, millet, etc. For example, using the MeMYB98 gene of the present invention for salt-tolerant cassava assisted breeding can early screen target cassava, and has the advantages of saving time and low cost.

[0025] In some embodiments, the MeMYB98 gene can be introduced into the target plant through a recombinant expression vector. In this method, the recombinant expression vector can transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroconductivity, Agrobacterium-mediated, etc., and cultivate the transformed plant tissue into a plant.

[0026] In some embodiments, the implementation manner of "promoting the expression of the MeMYB98 gene in the target plant" can be any one of the following:

[0027] (1) Introduce the MeMYB98 gene into the target plant;

[0028] (2) Introduce strong promoters and / or enhancers;

[0029] (3) Other common methods in the art.

[0030] In some embodiments, the target plant is cassava or Arabidopsis thaliana.

[0031] The present invention also provides a method for screening a regulator for regulating plant salt stress, the method comprising: adding a candidate substance to a system containing the MeMYB98 gene or the protein encoded thereby; detecting the system and observing the expression or activity of the MeMYB98 gene or the protein encoded thereby; the nucleotide sequence of the MeMYB98 gene is as shown in SEQ ID NO.1.

[0032] The present invention also provides a method for producing food, comprising the steps of: planting a crop and harvesting the food of the crop; the expression or activity of the MeMYB98 gene or the protein encoded thereby in the crop is increased.

[0033] As used herein, "plant" includes the whole plant, its parental and progeny plants, and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs, in which the target gene or nucleic acid is present in these different parts. The "plant" mentioned herein also includes plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, and likewise, each of the foregoing objects contains the target gene / nucleic acid.

[0034] The present invention includes any plant cell, or any plant obtained or obtainable by the methods therein, and all plant parts and their propagules. This patent also encompasses transfected cells, tissues, organs or whole plants obtained by any of the foregoing methods. The only requirement is that the progeny exhibit the same genotype or phenotypic characteristics, and the progeny characteristics obtained using the methods in this patent are the same.

[0035] The present invention also extends to the harvestable parts of the plants as described above, but is not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It further relates to other derivatives after the plants are harvested, such as dry granules or powders, oils, fats and fatty acids, starches or proteins. The present invention also relates to foods or food additives obtained from the related plants.

[0036] The present invention also provides a method for detecting the salt tolerance of a plant, detecting the MeMYB98 gene of a sample to be detected, and if the sample to be detected contains the MeMYB98 target fragment, it is determined to have high salt tolerance.

[0037] Since plants with the MeMYB98 gene, such as cassava, have high salt tolerance, the MeMYB98 gene can be used as an indicator of high salt tolerance. Of course, the sample to be detected can be a naturally occurring plant, and high salt-tolerant plants can be obtained by screening plants containing the MeMYB98 gene; it can also be a sample for transgenic breeding using the MeMYB98 gene. By screening for the presence of the target MeMYB98 gene, plants containing the MeMYB98 gene can be selected, and then high salt-tolerant plants can be obtained.

[0038] Among them, in the present invention, detecting whether the sample to be detected contains the MeMYB98 gene can be carried out in various ways. For example, it can directly detect whether the MeMYB98 gene itself is present, or it can detect the products produced by the MeMYB98 gene. The products include direct products, indirect products, or secondary products, etc. The products can be genes, proteins, or certain compounds, etc.

[0039] For example, to directly detect the MeMYB98 gene itself, it can be detected using specific primer pairs for the MeMYB98 gene, or it can be detected using probes or chips designed for the MeMYB98 gene. Further, the sample to be detected is detected using the primer pairs, probes, or chips of the MeMYB98 gene.

[0040] The primer pairs, probes, or chips for the MeMYB98 gene provided by the present invention can be obtained by designing according to conventional methods. By detecting this gene, it can effectively confirm whether the MeMYB98 gene is present, and to a certain extent, it can illustrate the salt tolerance of the plant to be tested.

[0041] Further, the nucleic acid sequences of the primer pairs are shown in SEQ ID NO.5 and SEQ ID NO.6; the detection primers using cDNA as a template have strong specificity. However, the method of directly detecting the MeMYB98 gene itself is not limited to this, and any method that can be realized in molecular biology is within the protection scope of the present invention.

[0042] Similarly, detecting the products produced by the MeMYB98 gene can also be carried out by various means, such as various ELISA detection kits, etc.

[0043] Further, in different embodiments, the sample to be detected includes materials suitable for sexual reproduction, asexual reproduction, or tissue culture of renewable cells. These samples to be detected can be materials suitable for sexual reproduction, such as selected from pollen, ovary, ovule, embryo sac, etc.; materials suitable for asexual reproduction, such as can be selected from cuttings, roots, stems, protoplasts, etc.; materials suitable for tissue culture of renewable cells, such as can be selected from leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, anthers, flowers, seeds, and stems, etc.

[0044] Specifically, the sample to be detected includes any one of the following materials: seeds, leaves, roots, stems, radicles, and plumules.

[0045] Among them, in the method for detecting the salt tolerance of plants, the plants of the samples to be detected can be various crop plants, flower plants, forestry plants, etc. The plants mentioned above can be (but not limited to): dicotyledonous plants, monocotyledonous plants, or gymnosperms. As a preferred method, the "plants" include but are not limited to: cassava, Arabidopsis thaliana, and all plants with this gene or homologous genes are applicable.

[0046] In summary, compared with the prior art, the present invention has achieved the following technical effects:

[0047] 1. The present invention cloned and functionally verified the cassava MeMYB98 gene, proving that it plays a key role in regulating plant salt tolerance. The MeMYB98 gene can be used as a new gene resource for crop molecular breeding, for screening and cultivating salt-tolerant varieties, increasing crop yields, and effectively reducing the impact of drought and salt stress on plants, and has important application value and broad prospects for agricultural promotion.

[0048] 2. Based on the salt resistance of the MeMYB98 gene, the present invention selects salt-tolerant varieties by detecting the MeMYB98 gene, improving the selection efficiency and accuracy. By identifying the MeMYB98 gene to select salt-tolerant plants, such as cassava, the cultivation period of plants can be shortened, providing a new approach for plant salt-tolerant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0050] Figure 1 This is the result of the MeMYB98 protein sequence alignment and phylogenetic tree analysis in Example 2 of the present invention; the black line in the figure represents the R2 conserved domain, and the red line represents the R3 conserved domain;

[0051] Figure 2 This is the analysis of the expression levels of the MeMYB98 gene in different tissue parts of cassava in Example 3 of the present invention;

[0052] Figure 3Analysis of the expression pattern of MeMYB98 under different stress treatments in Example 3 of the present invention; wherein, A is the expression pattern of MeMYB98 under 300 mM NaCl stress; B is the expression pattern of MeMYB98 under 100 μM ABA treatment; C is the expression pattern of MeMYB98 under 20% PEG stress; D is the expression pattern of MeMYB98 under 4°C stress;

[0053] Figure 4 Subcellular localization of MeMYB98 in tobacco epidermal cells in Example 3 of the present invention;

[0054] Figure 5 Growth of Arabidopsis overexpression lines in normal and salt stress media in Example 4 of the present invention; wherein, A is the phenotypes of WT and overexpression lines after treatment with 0, 125, and 150 mM for 7 days, and the scale bar length is 1 cm; B is the measurement of the primary root length of WT, OE#2, OE#3, and OE#; C is the measurement of the number of lateral roots of WT, OE#2, OE#3, and OE#; D is the measurement of the fresh weight of WT, OE#2, OE#3, and OE#;

[0055] Figure 6 Growth of cassava VIGS plants under normal growth and 300 mM NaCl treatment in Example 5 of the present invention; A is the growth of silenced lines and control lines under water treatment; B is the growth of silenced lines and control lines under 300 mM NaCl treatment; C is the change in leaves of silenced lines and control lines under 300 mM NaCl treatment; MeMYB98 is the silenced line, NC is the control line, and the scale bar length is 2 cm;

[0056] Figure 7 Determination of physiological indexes of cassava VIGS plants under normal growth and 300 mM NaCl treatment in Example 6 of the present invention; wherein, A is the APX activity; B is the free proline content; C is the hydrogen peroxide content. Detailed implementation manners

[0057] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0058] Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics techniques that are obvious to those skilled in the art. These techniques have been fully explained in the published literature. In addition, for the methods such as DNA extraction, phylogenetic tree construction, gene editing methods, gene editing vector construction, and obtaining gene-edited plants used in the present invention, except for the methods used in the following examples, the methods disclosed in the existing literature can be used to achieve the same results.

[0059] As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule", or "polynucleotide" mean DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences, and regulatory sequences in non-coding regions, but are not limited thereto. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in the genomic sequence, and / or include the coding sequence in cDNA, and / or include cDNA and its regulatory sequences. In a particular embodiment, for example, regarding an isolated nucleic acid sequence, it is preferably defaulted to be cDNA.

[0060] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources.

[0061] Biological materials:

[0062] The SC8 cassava tissue culture seedlings are preserved in the laboratory; the Arabidopsis Col-0 seeds are preserved in the laboratory; the SC9 cassava is taken from the cassava germplasm resource garden in Haikou City, Hainan Province;

[0063] The overexpression vector pCAMBIA-1300, the subcellular localization vector 35S-GFP, and the VIGS gene silencing vector pCsCMV-NC are preserved in the laboratory;

[0064] Escherichia coli DH5α and Agrobacterium tumefaciens GV3101 are preserved in the laboratory;

[0065] Primer synthesis and sequencing are completed by Tsingke Biological Company.

[0066] Experimental reagents:

[0067] The RNA extraction kit, reverse transcription kit, and fluorescence quantitative kit are purchased from Novoprotein Scientific Inc.

[0068] Common reagents such as Mannitol, NaCl, and PEG were purchased from Solebol;

[0069] Kanamycin was purchased from Solebro Biotech;

[0070] MS culture medium was purchased from Beijing Coolaibo Technology Co., Ltd.;

[0071] Various endonucleases were purchased from Mona Biotechnology Co., Ltd.;

[0072] One-step cloning enzyme was purchased from Novozyme Biotechnology Co., Ltd.;

[0073] Nimble Cloning reagent was purchased from Hainan Nixing Biotechnology Co., Ltd.;

[0074] Plasmid miniprep kit and gel recovery kit were purchased from Beijing Tiangen Biotechnology Co., Ltd.

[0075] Experimental equipment:

[0076] The PCR instrument was purchased from Bio-rad;

[0077] The refrigerated centrifuge was purchased from Eppendorf;

[0078] The quantitative PCR instrument was purchased from Bio-rad;

[0079] The high-temperature and high-pressure sterilizer MLS-3750 was purchased from Sanyo Company, Japan;

[0080] The nucleic acid detector Nanodrop 2000C was purchased from Thermo Scientific;

[0081] The room temperature centrifuge was purchased from Thermo Scientific.

[0082] Example 1 Construction of MeMYB98 overexpression plants

[0083] 1. Planting and processing of plant materials

[0084] SC8 cassava tissue samples from different tissue sites during the tuber expansion phase were collected from the Hainan Cassava Resource Garden. Samples were collected from mature leaves, young leaves, phloem, xylem, tubers, and fibrous roots. Samples were quickly frozen in liquid nitrogen and stored at -80°C. After 30 days of growth, SC8 cassava tissue culture seedlings were acclimated and hydroponically treated with 150 mM NaCl, 20% PEG6000, and 50 μM ABA at 4°C for 0, 1, 3, 6, 12, 24, and 48 hours. Samples were then collected, quickly frozen in liquid nitrogen, and stored at -80°C until further use.

[0085] 2. RNA Extraction and Real-Time Quantitative RT-PCR (qRT-PCR)

[0086] The cassava RNA extraction kit was purchased from Nanjing Novoprotein Scientific Inc., with the product number RC401-01, and RNA was extracted. The RNA content was detected by a Nanodrop 2000 nucleic acid analyzer, and the quality was checked by gel electrophoresis. The reverse transcription kit DRR047A (Takara, Dalian, China) was used to synthesize cDNA. The qRT-PCR primers were designed using Primer3 software (http: / / frodo.wi.mit.edu / ) and the primer specificity was verified by NCBI-primer blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi). MeACTIN was used as the internal reference gene for qRT-PCR analysis. The qRT-PCR experiment was carried out using a CFX96 real-time fluorescence quantitative PCR detection system (Bio-Rad, Hercules, CA, USA) and SYBR Premix Ex Taq reagent TM Kit (Takara). Each sample was subjected to three biological replicates and three technical replicates. The amplification parameters were as follows: 95°C for 30 seconds, 95°C for 5 seconds, 60°C for 30 seconds, for 40 cycles.

[0087] 3. Construction of the MeMYB98 Overexpression Vector

[0088] To further analyze the function of MeMYB98, the overexpression vector pCAMBIA-1300-MeMYB98 of MeMYB98 was constructed, and overexpressing Arabidopsis plants were obtained. The gene sequence number (Sequence ID) of the MeMYB98 gene in NCBI is XM_021739446.1. The messenger RNA (mRNA) sequence length of the MeMYB98 gene is 3183 bp, and the coding sequence length of the MeMYB98 gene is 1362 bp, including 453 amino acids. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ IDNO.2.

[0089] The specific construction process is as follows:

[0090] (1) Primers with restriction enzyme KpnI and SpeI cleavage sites were designed, and the sequences are as follows:

[0091] P1300-MeMYB98-F: SEQ ID NO.1;

[0092] P1300-MeMYB98-R: SEQ ID NO.2.

[0093] (2) Using the cDNA sample as a template, perform PCR amplification and purify and recover the amplified product;

[0094] (3) Double-digest the pCAMBIA-1300 vector with KpnI and SpeI, and purify the digested product;

[0095] (4) Carry out homologous recombination ligation of the PCR amplified product and the digested vector to construct the pCAMBIA-1300-MeMYB98 overexpression vector;

[0096] (5) Using the heat shock transformation method, transform the ligation product into Escherichia coli DH5α, perform Kan (100 mg / L) resistance screening, select positive colonies for PCR detection, amplify the colonies correctly identified by PCR detection, send for sequencing, and extract the plasmid from the correctly sequenced bacterial solution for standby;

[0097] (6) Transform the extracted plasmid into the Agrobacterium competent cell GV3101 and store it at -80 °C for standby.

[0098] (7) Use the Agrobacterium floral dip method to transform wild-type Arabidopsis thaliana (Col-0, WT), screen the harvested seeds on 1 / 2 MS medium containing Kan, harvest the seeds of potential transgenic plants individually, and screen again on the medium containing Kan until the T3 generation to screen for potential homozygous transgenic plants.

[0099] Example 2 Cassava MeMYB98 sequence alignment and phylogenetic tree analysis

[0100] To study the functional characteristics of MeMYB98, in this example, through literature search, homologous sequences of MeMYB98 and reported R2R3-MYB protein sequences involved in plant salt stress response were collected, including Arabidopsis thaliana (AtMYB30, AtMYB111, AtMYB64, AtMYB118), foxtail millet (SiMYB16), wheat (TaMYB33), trifoliate orange (PtrMYB3), and poplar (PdMYB118). Use DNAMAN to perform sequence alignment of these protein sequences with the MeMYB98 protein sequence, and analyze the homologous evolutionary relationship of MeMYB98 with proteins of selected species such as Arabidopsis thaliana, wheat, and foxtail millet by MEGA7.

[0101] The results showed that the MeMYB98 protein had a high degree of similarity with other MYB proteins in the conserved sequence, both containing two incomplete repeat sequences, R2 and R3, and these two sequences were separated by tryptophan. However, in the non-conserved region, the sequences differed greatly. These characteristics indicate that MeMYB98 belongs to the R2R3-MYB family. Figure 1Phylogenetic tree analysis showed that MeMYB98 was closely related to PdMYB118 of Populus trichocarpa.

[0102] Example 3 Expression pattern of cassava MeMYB98 gene under abiotic stress

[0103] To explore the expression pattern of MeMYB98 in different tissues of cassava, quantitative analysis was performed in this example.

[0104] Figure 2 The results showed that the MeMYB98 gene had a relatively high expression level in mature leaves, but relatively low expression in xylem and tuberous roots.

[0105] To further explore the expression pattern of cassava MeMYB98 under different abiotic stresses, hydroponic cassava seedlings with consistent growth were subjected to stress treatments in this example: 20% PEG6000, 300 mM NaCl, 100 μM ABA, and 4°C. Samples were taken at 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h after treatment, quickly frozen in liquid nitrogen, and then stored in an -80°C refrigerator. After sampling, RNA was extracted and reverse transcribed into cDNA for quantitative analysis.

[0106] The results showed that MeMYB98 could respond to abiotic stresses. Under NaCl and ABA stresses, the expression level of MeMYB98 was generally up-regulated; under 4°C and PEG stresses, the expression level of MeMYB98 was generally down-regulated, among which, MeMYB98 had a higher response to NaCl stress ( Figure 3 ). In addition, subcellular localization analysis of MeMYB98 in tobacco epidermal cells found that the MeMYB98 transcription factor was mainly localized in the nucleus ( Figure 4 ).

[0107] Example 4 Overexpression of MeMYB98 can significantly improve the salt tolerance of Arabidopsis thaliana seedlings

[0108] To further study the biological function of MeMYB98, the recombinant pCAMBIA-​1300-​MeMYB98 vector was introduced into wild-type Arabidopsis thaliana in this example to generate 7 transgenic lines. The expression levels of MeMYB98 in these transgenic lines were detected by qRT-PCR, and three homozygous MeMYB98 lines (OE#2, OE#3, and OE#7) representing low, medium, and high transgenic expression levels were screened for subsequent studies.

[0109] To deeply explore the effects of overexpression of the MeMYB98 gene on the growth and development of Arabidopsis thaliana under normal and salt stress conditions, in this example, wild-type plants grown on 1 / 2 MS medium for 3 days and 3 overexpression lines were respectively transferred to 1 / 2 MS medium containing 125 mM NaCl, 150 mM NaCl, and 1 / 2 MS medium for salt stress treatment. After 7 days of treatment, the plant phenotypes were observed, and the changes in growth indexes such as root length, plant fresh weight, and number of lateral roots were statistically analyzed.

[0110] The results showed that under salt stress conditions, the root lengths and fresh weights of the three overexpression lines were higher than those of the wild type ( Figure 5 A), and the reduction rates of root length and biomass reduction were relatively low ( Figure 5 B, D). These results indicate that the MeMYB98 gene can enhance the resistance of Arabidopsis thaliana to salt stress environment.

[0111] Example 5 Gene-silenced line MeMYB98 can significantly reduce the salt stress resistance of cassava

[0112] To further study whether silencing of the MeMYB98 gene would reduce the salt stress tolerance of transgenic cassava, in this example, the recombinant pCsCMV-NC-MeMYB98 vector was injected into the leaves of SC9 cassava, and 8 gene-silenced lines were generated. Subsequently, the expression level of MeMYB98 in these transgenic lines was detected by qRT-PCR, and its expression level was lower than that of the control group. To determine the role of MeMYB98 under salt stress, transgenic plants and control lines were treated with 300 mM NaCl and water for 0 h, 24 h, 36 h, 48 h, and 60 h and sampled.

[0113] Figure 6 The results showed that compared with non-transgenic plants, under 300 mM NaCl stress, the leaves of MeMYB98 gene-silenced plants showed phenotypes of curling and withering.

[0114] Example 6 Silencing of MeMYB98 expression significantly reduces oxidative stress tolerance

[0115] To further understand the effects of MeMYB98 on plant salt tolerance, in this example, the activities of ascorbate peroxidase (APX), proline, and hydrogen peroxide contents between SC9 and MeMYB98 gene-silenced lines were studied.

[0116] The results of physiological index determination showed that after 60 h of treatment, the APX activity of the silenced lines decreased significantly, while the APX activity of the control lines was higher than that of the silenced lines at 48 h and 60 h of treatment ( Figure 7 A); compared with the control lines, the proline content in the silenced lines showed a decreasing trend (Figure 7 B); Meanwhile, the hydrogen peroxide content of the silenced lines was higher than that of the control lines, showing obvious differences ( Figure 7 C).

[0117] Innovatively, this invention uses cassava SC8 as a template to clone the MeMYB98 gene and constructs its heterologous expression vector. The vector is transformed into wild-type Arabidopsis thaliana (Col-0, WT) by the Agrobacterium-mediated floral dip method to obtain heterologous expression plants. The analysis results show that, compared with the wild type, the MeMYB98 overexpression lines exhibit stronger salt tolerance. In addition, after silencing the MeMYB98 gene by VIGS technology, the gene-silenced lines are more sensitive to salt stress, manifested as leaf curling and water loss. Thus, it can be seen that the MeMYB98 gene plays an important role in regulating plant salt tolerance and provides an important gene resource for crop molecular breeding.

[0118] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0119]

[0120]

Claims

1. Use of the MeMYB98 gene or the protein encoded thereby in regulating plant salt stress, wherein the nucleotide sequence of the MeMYB98 gene is as shown in SEQ ID NO.

1.

2. The application according to claim 1, wherein The amino acid sequence of the protein encoded by the MeMYB98 gene is as shown in SEQ ID NO.

2.

3. A method for regulating plant salt stress, characterized in that, Regulating the expression or activity of the plant MeMYB98 gene or the protein encoded thereby, thereby regulating plant salt stress; The nucleotide sequence of the MeMYB98 gene is as shown in SEQ ID NO.

1.

4. The method according to claim 3, wherein The method is selected from any one of the following: (1) Up-regulating the expression or activity of the MeMYB98 gene or the protein encoded thereby, thereby improving the salt tolerance of plants; (2) Down-regulating the expression or activity of the MeMYB98 gene or the protein encoded thereby, thereby reducing the salt tolerance of plants.

5. The method according to claim 3, characterized in that, The up-regulation of the expression or activity of the MeMYB98 gene or the protein encoded thereby includes: (1) Transferring an expression construct or vector of the MeMYB98 gene into a plant; (2) Transferring an expression construct or vector containing the MeMYB98 gene into a plant.

6. The method according to claim 3, wherein The down-regulation of the expression or activity of the MeMYB98 gene or the protein encoded thereby includes: Knocking out or silencing the MeMYB98 gene in a plant, or inhibiting the activity of the MeMYB98 protein, thereby reducing the expression of the ERF04 gene.

7. A breeding method of a plant, characterized in that, The method is selected from any one of the following: (1) By increasing the activity of the MeMYB98 protein in the target plant, obtaining a plant with stronger salt stress tolerance than the target plant; (2) By promoting the expression of the MeMYB98 gene in the target plant, obtaining a plant with stronger salt stress tolerance than the target plant.

8. The breeding method according to claim 7, characterized in that, The target plant is cassava or Arabidopsis thaliana.

9. A method for screening a regulator for regulating plant salt stress, characterized in that, The method includes: adding a candidate substance to a system containing the MeMYB98 gene or the protein encoded thereby; detecting the system and observing the expression or activity of the MeMYB98 gene or the protein encoded thereby; the nucleotide sequence of the MeMYB98 gene is as shown in SEQ ID NO.

1.

10. A method for producing grain, characterized in that, Including the following steps: planting crops and harvesting the grains of the crops; the expression or activity of the MeMYB98 gene or the protein encoded thereby in the crops is increased.

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