CN117088957B - Application of tomato SlMYB13 protein and encoding gene thereof in regulation and control of salt tolerance and drought tolerance of plants - Google Patents
Application of tomato SlMYB13 protein and encoding gene thereof in regulation and control of salt tolerance and drought tolerance of plants Download PDFInfo
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Abstract
The invention relates to the technical field of biology, in particular to application of tomato SlMYB13 protein and a coding gene thereof in regulating and controlling plant salt tolerance and drought tolerance. The tomato SlMYB13 gene can negatively regulate and control the salt tolerance and drought tolerance of plants, and can effectively improve the salt tolerance and drought tolerance of plants by reducing the expression quantity of the SlMYB13 gene, thereby providing precious gene resources and a new method for cultivating new varieties of plants with high salt tolerance and drought tolerance. The invention utilizes a CRISPR-Cas9 genome fixed-point editing system to mutate the tomato SlMYB13 gene, creates a salt-tolerant and drought-tolerant tomato strain, remarkably improves the salt tolerance and drought tolerance of the tomato strain, and has important application value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of tomato SlMYB13 protein and a coding gene thereof in regulating and controlling salt tolerance and drought tolerance of plants.
Background
Tomato (tomato)Solanum lycopersicum L.) is an annual herb plant of Solanaceae Solanum, the cultivation area is wide, the plant is one of the vegetable crops with the largest cultivation area in China, the production is extremely easy to be influenced by lack of water, and the yield and quality of tomatoes are directly influenced. The soil resource is precious, and the development of saline-alkali resistant crops has important significance for improving the land increment. The breeding of salt-tolerant drought-tolerant tomatoes is urgent.
The traditional breeding method has the disadvantages of long time consumption, large workload and uncertainty of breeding results when improving plant traits, and can not meet the current plant breeding requirements. And molecular breeding can quickly and directionally modify plant genes, so that the breeding efficiency is greatly improved. The molecular breeding method is used for controlling gene expression to improve plant properties, providing theoretical basis and germplasm resources for cultivating good varieties, and being an important direction for plant breeding development.
The current research in this respect shows that: soil salinization is one of important abiotic stresses for limiting plant growth and development, and salt stress can lead to ion stress, unbalanced plant cell permeation and overhigh pH value, and especially the damage of oxidation inside cells to plants is very large. MYB transcription factor is one of the largest transcription factor families in plants, directly participates in the secondary metabolism of phenylpropanes in plants, and regulates the growth and development of plants, the secondary metabolism and the corresponding biotic and abiotic stress. After T2 generation seedlings of SlMYB86-RNAi are subjected to high-concentration salt treatment, the SlMYB86-RNAi seedlings wilt slower than the wild type, which shows that the silencing of the SlMYB86 gene enhances the resistance of tomatoes under salt stress, and the SlMYB86 is a negative regulator of salt resistance. In addition, under the condition of salt stress, the overexpression of the SlMYB102 can improve the activity of ROS scavenging enzyme, the content of antioxidant of an overexpression plant is increased, and the content of proline (Pro) is higher than that of a wild type, so that the expression of the SlMYB102 positively regulates the tomato salt stress reaction. Drought is also osmotic stress, the growth and the development and the yield of plants are seriously affected, the function of the SlMYB14 gene is verified by using a VIGS technology, and compared with a wild type, the drought resistance of a tomato plant with the silenced SlMYB14 gene is reduced, so that the SlMYB14 is a positive regulatory factor of the tomato in the face of drought stress. The drought resistance of the tomato is negatively regulated by the SlMYB86, compared with the wild type seedling, the drought stress tolerance of the SlMYB86-RNAi seedling is stronger, the leaf water loss rate and Malondialdehyde (MDA) content are lower than those of the wild type seedling, and the relative moisture and chlorophyll content are obviously higher than those of the wild type seedling. The tomato MYB transcription factor is encoded by the SlMX1, the drought tolerance of the tomato plants over-expressing the SlMX1 is obviously enhanced, the fruit quality is improved compared with the wild type, and the drought resistance of the tomatoes can be improved by the SlMX 1.
Therefore, improving the salt tolerance level of tomatoes from the gene level, and cultivating new varieties of salt tolerant tomatoes is a fundamental means for solving the problem of saline-alkali cultivation of tomatoes. The research of MYB transcription factors provides a theoretical basis for cultivating new varieties of salt-tolerant tomatoes, and has important guiding and practical significance for tomato production and saline-alkali soil development and utilization.
Disclosure of Invention
In order to further solve the problems in the prior art, on the basis of the existing research, the invention further carries out salt stress treatment on tomato wild type materials, extracts RNA, screens genes capable of obviously responding to salt stress by utilizing transcriptome analysis, obtains the gene SlMYB13 after analysis, and discovers that drought stress also strongly inhibits the expression of the SlMYB13 after other adversity stresses.
The technical scheme of the invention comprises the following steps:
in a first aspect, the invention provides the use of a tomato SlMYB13 protein or a gene encoding the same in modulating salt tolerance in a tomato, in particular by inactivating the tomato SlMYB13 protein or reducing expression or non-expression of the gene encoding the same.
In a second aspect, the invention provides application of a tomato SlMYB13 protein or a coding gene thereof in drought tolerance of tomatoes. Further, the expression or non-expression of the tomato SlMYB13 protein is specifically reduced or prevented by inactivating the coding gene thereof.
In a third aspect, the invention provides the use of a tomato SlMYB13 protein or a gene encoding the same in salt-tolerant or/and drought-tolerant tomato genetic breeding or transgenic plant preparation.
In the invention, the tomato SlMYB13 protein has any one of the following amino acid sequences:
(1) An amino acid sequence as shown in SEQ ID NO. 1;
(2) An amino acid sequence with the same functional protein obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1;
(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 1; preferably, the homology is at least 90%; more preferably 95%.
In the invention, the CDS sequence of the coding gene of the tomato SlMYB13 protein has any one of the following nucleotide sequences:
(1) A nucleotide as shown in SEQ ID NO. 2;
(2) The nucleotide sequence shown as SEQ ID NO.2 is obtained by replacing, inserting or deleting one or more nucleotides.
The nucleotide sequence shown in SEQ ID NO.2 is the CDS sequence of the SlMYB13 protein in tomato. In view of the degeneracy of the codons, all nucleotide sequences encoding the tomato SlMYB13 protein are within the scope of the invention.
In the present invention, it is preferable that the expression of the coding gene of tomato SlMYB13 protein is inhibited by an inhibitor, which is gRNA or interfering RNA,
preferably, the target sequence of the gRNA is the 135 th-154 th exon of the third exon of the coding gene of the tomato SlMYB13 protein.
More preferably, the gRNA comprises the nucleotide sequence shown as SEQ ID NO. 3.
The target site of the gRNA is obtained through a large number of screening, and partial knockout of the coding gene of the tomato SlMYB13 protein can be efficiently realized by using the gRNA, so that the tomato SlMYB13 is inactivated.
In a fourth aspect, the invention provides a gRNA for editing a tomato SlMYB13 gene, wherein the target sequence of the gRNA is 135-154 th position of a third exon of the tomato SlMYB13 gene.
The gRNA can be matched with a CRISPR/Cas9 gene editing system to realize the efficient knockout of the tomato SlMYB13 gene.
Preferably, the gRNA comprises the nucleotide sequence shown as SEQ ID NO. 3.
In a fifth aspect, the invention provides a biological material comprising the gRNA for editing a tomato SlMYB13 gene, the biological material comprising an expression cassette, a vector, a transgenic cell, or an engineered bacterium.
The expression cassette may be an expression cassette comprising a AtU6 promoter and the gRNA;
the vector may be a CRISPR-Cas9 gene editing vector containing a AtU promoter, the gRNA, and a Cas9 expression cassette;
the engineering bacteria can be escherichia coli or agrobacterium containing the CRISPR-Cas9 gene editing vector.
In a sixth aspect, the present invention provides a method of modulating salt tolerance, drought tolerance in a plant comprising: regulating and controlling the expression quantity of the SlMYB13 gene.
The coding protein of the tomato SlMYB13 gene has any one of the following amino acid sequences:
(1) An amino acid sequence as shown in SEQ ID NO. 1;
(2) An amino acid sequence with the same functional protein obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1;
(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 1;
preferably, the homology is at least 90%; more preferably 95%.
Preferably, in the above method, the salt tolerance and drought tolerance of the plant are improved by reducing the expression level of the tomato SlMYB13 gene in the plant; alternatively, salt-tolerant, drought-tolerant strains were grown by crossing the inactivated mutant strain of the tomato SlMYB13 gene with other strains.
The reduction of the expression level of the tomato SlMYB13 gene in the plant can be achieved by means of conventional technical means in the field.
Preferably, the reduction of the expression level of the tomato SlMYB13 gene in the plant is achieved by using a CRISPR/Cas9 system, wherein the target sequence of the gRNA used by the CRISPR/Cas9 system comprises a sequence comprising NGG sequence features on the third exon of the tomato SlMYB13 gene, in particular positions 135-154 of the third exon of the SlMYB13 gene.
As a preferred embodiment of the invention, the CRISPR-Cas9 system is constructed by ligating the gRNA to a vector to obtain a recombinant CRISPR/Cas9 vector.
In the present invention, the plant is a monocotyledonous plant or a dicotyledonous plant. Such plants include, but are not limited to, tomato, rice, arabidopsis, grape, soybean, cucumber, wheat, maize, and the like.
The invention provides an application of tomato SlMYB13 protein and a coding gene thereof in regulating and controlling plant salt tolerance and drought tolerance, which has the following beneficial effects:
according to the invention, the screening analysis shows that the tomato SlMYB13 gene can negatively regulate and control the salt tolerance and drought tolerance of plants, and the salt tolerance and drought tolerance of plants can be effectively improved by reducing the expression quantity of the SlMYB13 gene. The discovery of the salt tolerance and drought tolerance regulation function of the SlMYB13 gene provides precious gene resources and a novel method for cultivating new varieties of salt tolerance and drought tolerance plants. On one hand, the tomato strain with stronger salt tolerance and drought tolerance can be obtained by site-directed mutagenesis of the SlMYB13 gene of cultivated tomatoes; on the other hand, the SlMYB13 gene inactivated plant provided by the invention can be hybridized with other tomato varieties to cultivate salt-tolerant and drought-tolerant strains, so that salt-tolerant germplasm resources of tomatoes are enriched. The SlMYB13 gene and the inhibitor thereof have great application value in salt tolerance and drought tolerance breeding of tomatoes.
Drawings
FIG. 1 is a recombinant plasmid map of the SlMYB13 constructed gene editing vector pHSbdcas9i-SlMYB13 in example 1 of the present invention;
FIG. 2 is an identification of E.coli plaques transformed with the gene editing vector pHSbdcas9i-SlMYB13 constructed in example 1 of the present invention; m is DNA Mark, lane number is conversion bacterial plaque number, the band size is 6000bp, 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom in turn;
FIG. 3 is a diagram showing the result of electrophoresis detection of PCR screening of a part of transformed plants containing Cas9 gene in example 1 of the present invention, wherein lane numbers are the transformed plant numbers, CK is a wild type control plant, M is DNA Mark, and the band sizes are 1000bp, 750bp, 500bp, 250bp and 100bp in order from top to bottom;
FIG. 4 shows the gene structure of the SlMYB13 gene and the mutation of the target sequence of the SlMYB13 gene of homozygous mutant plant in example 1 of the present invention, wherein WT is wild type; -representing a base deletion;
FIG. 5 is a graph of seed germination under drought and salt stress treatment for the SlMYB13 knockout strain (myb 13-29) and Wild Type (WT) materials provided in example 2 of the present invention;
FIG. 6 is a graph showing the growth status of the SlMYB13 knockout strain (myb 13-29) and Wild Type (WT) materials provided in example 2 of the present invention before and after drought and salt stress treatments;
FIG. 7 is a graph comparing the activity of related enzymes after drought and salt stress treatment of the SlMYB13 knockout strain (myb 13-29) and Wild Type (WT) materials provided in example 2 of the present invention.
Detailed Description
The invention will be further described with reference to specific examples, which are intended to enable those skilled in the art to further understand the invention, but are not intended to be limiting, and all techniques based on the principles of the invention fall within the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 tomato SlMYB13 Gene site-directed mutagenesis based on CRISPR-Cas9 System
(1) Selection of gRNA targets
The NCBI website (https:// www.ncbi.nlm.nih.gov /) is logged in, and the sequence of the tomato SlMYB13 gene is inquired (CDS sequence is shown as SEQ ID NO.2, and encoding protein sequence is shown as SEQ ID NO. 1). According to the sequence of the SlMYB13 gene, the gene contains three exons, and based on the characteristics of the gRNA sequence of CRISPR-Cas9, the sequence containing the NGG sequence characteristics on the sense strand of the third exon of the SlMYB13 gene is selected by screening and analyzing, and specifically the 135 th to 154 th positions: 5'-TGTAGATGTACATTTGGAGG-3' (SEQ ID NO. 3), the RNA strand formed after transcription can specifically bind to the SlMYB13 gene and guide the CRISPR-Cas9 system to carry out efficient site-directed mutagenesis on the tomato SlMYB13 gene.
(2) Gene editing vector construction
Synthesizing primer gRNA-F according to the selected gRNA target sequence target (SEQ ID NO. 3) cagtggtctcatgcaTGTAGATGTACATTTGGAGG (SEQ ID NO. 4); gRNA-R cagtggtctcaaaacCCTCCAAATGTACATCTACA (SEQ ID NO. 5) was amplified by PCR, the reaction procedure was: 94 ℃ for 5min;94℃30s,50℃45s,72℃46s,30 cycles; 72℃for 10min and 16℃for 30min.100V,200mA electrophoresis was performed on the PCR amplification product for 30min, and recovery was performed using a DNA gel recovery kit (Axygen), and the recovered product was labeled rDNA1.
The pHSbdcas9I plasmid was digested with BsaI/Eco31I and ligated to rDNA1, and the double-stranded target sequence was ligated to the plasmid vector using T4 ligase to obtain MYB13 editing vector pHSbdcas9I-SlMYB13, see FIG. 1 recombination map.
After transformation of E.coli DH 5. Alpha. The detection primers JC-F GTAAAACGACGGCCAGT (SEQ ID NO. 6) and JC-R CCAGAAATTGAACGCCGAAG (SEQ ID NO. 7) were designed, the reaction procedure was 94℃for 5min;94℃30s,50℃45s,72℃54s,30 cycles; the amplification sequence 850bp is 10min at 72 ℃ and 30min at 16 ℃, and the detection result of PCR amplification electrophoresis is shown in FIG. 2.
(3) Agrobacterium tumefaciens transformed with pHSbdcas9i-SlMYB13 plasmid
And (3) transforming the recombinant pHSbdcas9i-SlMYB13 vector in the step (2) into an agrobacterium GV3101 strain by a heat shock method to obtain recombinant agrobacterium. The specific method comprises the following steps: 50 mu L of Agrobacterium GV3101 competent cells are taken in a centrifuge tube, 2 mu L of pHSbdcas9i-SlMYB13 plasmid is added, the mixture is subjected to ice bath for 45min, liquid nitrogen is frozen for 1min, water bath is carried out at 37 ℃ for 3min, 1mLYEB liquid culture medium is added, the culture is carried out at 28 ℃ for 3h at 125rpm, concentrated bacterial liquid is centrifuged at 12000rpm for 1min, 400 mu L of LYEB liquid culture medium back-melted bacterial liquid is added, and the liquid is coated on YEB solid culture medium (containing Kan50 mg.L -1 And Rif70mg.L -1 ) Culturing in an incubator at 28 ℃ in a dark inversion mode for 2-3 days; and (3) carrying out PCR detection on the selected bacterial plaque by using JC-F, JC-R primer, and carrying out shaking preservation on the correct bacterial plaque.
(4) Agrobacterium-mediated tomato transformation
Selecting full, uniform and fresh tomato seeds, repeatedly washing the tomato seeds with sterile water for several times, sterilizing the tomato seeds with 70% alcohol for 30s and 10% sodium hypochlorite for 20min, washing the sterilized seeds with sterile water for 4-5 times, sucking water to dryness by using sterile filter paper, and inoculating the seeds in an MS culture medium, wherein the illumination intensity is 1600-1800 lx, the illumination is 25 ℃/16h and the darkness is 18 ℃/8h.
50. Mu.L of recombinant Agrobacterium solution was taken in 50mLYEB liquid medium (Kan50mg.L -1 And Rif70mg.L -1 ) In the triangular flask, the shaking table is set at 28 ℃, and the shaking table is cultured at 200rpm until the OD600 is 0.6-0.8. Centrifuging at 4000rpm at 4 ℃ for 15min, and collecting thalli; the thalli are blown uniformly by MS liquid culture medium, resuspended until the OD600 is about 0.5, and poured into a culture dish for standby.
Selecting tomato cotyledon as explant for transformation, cutting cotyledon into 0.5cm×0.5cm leaf pieces, horizontally placing in preculture medium (MS+ZT2.0mg.L) -1 +IAA0.2mg·L -1 ) 15-20 pieces per dish. The culture conditions are the same as above, and the culture is performed for 1-2 days. And taking the explant out of the pre-culture medium, putting the explant into a culture dish containing the resuspended bacterial liquid, infecting for 30min, taking the explant out, sucking the explant on sterile paper, putting the explant back into the pre-culture medium, and co-culturing for 1-2 days. After the co-culture explant is transferred into selectionCulture medium selection (MS+ZT2.0 mg.L) -1 +IAA0.2mg·L -1 +Kan50mg·L -1 +Carb500mg·L -1 ) After a few days of differentiation culture, the cotyledons begin to thicken and the explants will form callus and adventitious buds on the selection medium. And subculturing every 7-10 days. When the adventitious bud length is about 1 cm-2 cm, selecting healthy regenerated buds, cutting off bud callus, and transferring to rooting medium (MS+IAA0.1mg.L) -1 +Carb300mg·L -1 +Kan50mg·L -1 ) And (3) allowing them to form whole plants. After a large number of lateral roots of the seedlings grow, the bottle cap of the culture bottle is opened, the seedlings are placed in a shade for hardening, the culture medium on the root system is cleaned after 3 days, and the seedlings are transferred into a plastic basin containing nutrient media.
(5) Identification of transgenic tomato and mutation site detection
The detection primers cas9-F GCACCCGGTGGAGAACACGC (SEQ ID NO. 8) and cas9-R GTTCAGGTACGCGTCATGGGC (SEQ ID NO. 9) are used for carrying out carrier sequence detection, positive plants are identified, and the identification PCR amplification electrophoresis detection results of the positive plants are shown in figure 3.
And then the positive plant genome is used as a template, primers SlMYB13-F (SEQ ID NO.10 CGCGGATCCATGGGGAGATCTCCTTGCTGTGA) and SlMYB13-R (SEQ ID NO.11 TCCCCCGGGTCATAATTCAGGCAATTCTAACATCAACTC) are used for amplifying the whole sequence of the SlMYB13 gene, the PCR product is used for sequencing, and whether mutation occurs in a target site is judged through the sequencing sequence.
Sequencing results show that the myb13-29 in the transformed plant is a homozygous strain with two chromosomes simultaneously mutated, and the sequencing results are shown in FIG. 4, and the third exon 144-149 is deleted in ACATTTT.
The DNA sequence after deletion is shown as SEQ ID NO.12, and the amino acid sequence is shown as SEQ ID NO.13 (MGRSPCCEKLGLKRGPWSKEEDYLLINYIKKNGHPNWRALPKLAGLLRCGKSCRLRWTNYLRPDIKRGNFTHQEEDTIIKLHQVLGNSWSAIAARLPGRTDNEIKNIWHTRLKKKRNESQLKETQSEPENTNVDGRRPTILMINIPKYRILK-; myb13-29 causes sequence translation dislocation after PAM site due to base deletion, premature termination of protein translation and functional change. Selection of myb13-29，Selfing was performed for subsequent experiments.
Example 2 transgenic lines tomato salt tolerance assay
(1) And (3) selecting the homozygous seeds of the knockout strain slmyb13-29 verified to be homozygous by sequencing and wild type for germination experiments, wherein 10 seeds of each variety are respectively placed in culture dishes containing filter papers for germination, wherein the dishes contain NaCl solutions (0, 50mM and 60 mM) and PEG6000 solutions (5% and 10%) with different concentrations, the photoperiod is 16 hours under illumination, 8 hours under darkness and the temperature is 28+/-1 ℃. Seed germination was measured for seven days, and three biological replicates were performed for each group. As shown in FIG. 5, the germination rate of slmyb13-29 seeds was higher under salt stress and PEG simulated drought conditions compared to WT.
(2) Selecting a knockout strain SlMYB13-29 which is verified to be homozygous by sequencing, and performing 300mM high-salt and 10% PEG6000 simulated drought treatment on the strain, so as to analyze the functions of the slMYB13 in regulating the salt tolerance and drought resistance of tomato plants.
Seedlings of the wild tomato line, the gene knockout line slmyb13-29, were selected to be consistent in growth vigor. Photographs were taken as controls prior to salt stress and drought stress treatment.
Salt stress treatment, 200mL of 300mM NaCl solution was poured into each pot.
Drought stress treatment was simulated, each pot was watered with 200mL of 10% peg6000 solution.
The growth status of each strain is observed until obvious difference appears in growth status among each strain in the fifth day of treatment, and the result is shown in figure 6, and compared with WT, the growth status of slmyb13-29 after salt stress and drought stress treatment is better.
And (3) measuring the activities of catalase, peroxidase and superoxide dismutase and the contents of malondialdehyde and proline by selecting the slmyb13-29 subjected to salt stress and drought treatment and the Wild Type (WT) in the step (2). As shown in FIG. 7, the activity of slmyb13-29 Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) was significantly higher, and the proline content was higher and the malondialdehyde content was lower than that of WT.
POD, SOD and CAT are main enzymes in a plant antioxidant system, and the activity of the POD, SOD and CAT can reflect the influence degree of the plant from external stress. SOD can catalyze the conversion of superoxide anions into H 2 O 2 And O 2 Is the primary substance for eliminating free radicals in organisms, CAT and POD are the substances for eliminating H 2 O 2 The three maintain the stable level of free radicals in plants through the synergistic effect, and the physiological and biochemical changes of the plants caused by the free radicals generated by stress of adversity are prevented. MDA is a product of lipid peroxidation, and can destroy the cell membrane structure of plants under adverse stress to cause toxic action on the plants, thus being used as an important physiological index of membrane damage. Proline is a compatible permeate, free proline in plants is one of main osmotic adjusting substances, the content of proline is low under normal conditions, the content of proline is obviously increased only under adverse stress, free radicals in cells can be effectively reduced, the pH value is adjusted, when the plants are subjected to salt stress and drought stress, the osmotic potential of the cells can be reduced by the proline, the homeostasis of the plant cells is maintained, and the influence of the stress on the plants is reduced. Under salt and drought stress, the knockout of the SlMYB13 gene enhances the capability of the mutant in scavenging active oxygen, the integrity of cell membranes is good, the content of proline is increased, and the maintenance of cell homeostasis and osmotic potential is facilitated, so that the expression of the SlMYB13 negatively regulates the salt tolerance and drought tolerance of tomatoes is shown.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, all changes and modifications that may be made without departing from the spirit of the invention are intended to be within the scope of the invention as claimed.
Claims (10)
1. The application of the tomato SlMYB13 protein or the coding gene thereof in regulating salt tolerance and/or drought tolerance of tomatoes is characterized in that the expression or non-expression of the coding gene of the tomato SlMYB13 protein is reduced by inactivating the tomato SlMYB13 protein or inactivating the coding gene thereof;
the amino acid sequence of the tomato SlMYB13 protein is shown as SEQ ID NO. 1;
the CDS sequence of the coding gene of the tomato SlMYB13 protein is shown as SEQ ID NO. 2.
2. Use of the tomato SlMYB13 protein or a gene encoding it according to claim 1 for the genetic breeding of salt tolerant or/and drought tolerant tomatoes or the preparation of transgenic plants, wherein the expression of the gene encoding the tomato SlMYB13 protein is reduced.
3. Use of the tomato SlMYB13 protein or a gene encoding it in salt-tolerant or/and drought-tolerant tomato genetic breeding or transgenic plant production according to claim 2, characterized in that the expression of the gene encoding the tomato SlMYB13 protein is inhibited by an inhibitor, which is a gRNA or an interfering RNA, the target sequence of which is position 135-154 of the third exon of the gene encoding the tomato SlMYB13 protein.
4. Use of the tomato SlMYB13 protein or a gene encoding the same according to claim 3 in salt-tolerant or/and drought-tolerant tomato genetic breeding or transgenic plant production, wherein the nucleic acid sequence of the gRNA is shown in SEQ ID No. 3.
5. Editing a gRNA of a tomato SlMYB13 gene, wherein the target sequence of the gRNA is 135-154 th position of a third exon of the tomato SlMYB13 gene; the nucleic acid sequence of the gRNA is shown as SEQ ID NO. 3.
6. A biological material comprising the gRNA of the editing tomato SlMYB13 gene of claim 5, wherein the biological material comprises an expression cassette, a vector, a transgenic cell, or an engineered bacterium;
the nucleic acid sequence of the gRNA is shown as SEQ ID NO. 3;
the expression cassette is an expression cassette containing a AtU promoter and the gRNA;
the vector is a CRISPR-Cas9 gene editing vector containing a AtU6 promoter, the gRNA and a Cas9 expression cassette;
the engineering bacteria are escherichia coli or agrobacterium containing the CRISPR-Cas9 gene editing vector.
7. A method for regulating and controlling salt tolerance and/or drought tolerance of plants, which is characterized in that the expression quantity of a tomato SlMYB13 gene is reduced;
the amino acid sequence of the coding protein of the tomato SlMYB13 gene is shown as SEQ ID NO. 1.
8. The method for regulating salt tolerance and/or drought tolerance of a plant according to claim 7, wherein salt tolerance and drought tolerance of the plant are improved by reducing expression level of tomato SlMYB13 gene in the plant; alternatively, salt-tolerant, drought-tolerant strains were grown by crossing the inactivated mutant strain of the tomato SlMYB13 gene with other strains.
9. The method of modulating salt tolerance and/or drought tolerance in a plant according to claim 7, wherein reducing the expression level of a tomato SlMYB13 gene in said plant is achieved using a CRISPR/Cas9 system, wherein the target sequence of a gRNA used by said CRISPR/Cas9 system comprises a sequence comprising NGG sequence features on the third exon of said tomato SlMYB13 gene.
10. The method of claim 9, wherein the CRISPR/Cas9 system uses a gRNA with a target sequence of position 135-154 of the third exon of the SlMYB13 gene.
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