CN116514941B - MsRGP protein, coding gene thereof and application of MsRGP protein in improving drought resistance and salt tolerance of plants - Google Patents
MsRGP protein, coding gene thereof and application of MsRGP protein in improving drought resistance and salt 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
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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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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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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- C12N15/09—Recombinant DNA-technology
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- 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
- C12N15/8291—Hormone-influenced development
- C12N15/8293—Abscisic acid [ABA]
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Abstract
The application discloses MsRGP protein, a coding gene thereof and application thereof in improving drought resistance and salt tolerance of plants, wherein the amino acid sequence of MsRGP protein is shown as SEQ ID NO:1, the nucleotide sequence of MsRGP gene of the protein is shown as SEQ ID NO: 2. MsRGP1 genes are obtained by screening and separating alfalfa, and are found to simultaneously respond to salt, drought and ABA stress, and can regulate and control stress resistance of plants, such as salt tolerance, drought resistance and ABA stress resistance. The application also discloses application of the MsRGP protein, the coding gene, the recombinant vector or the recombinant bacterium carrying the recombinant vector in improving drought resistance and/or salt tolerance of plants. The MsRGP protein and the coding gene thereof can be applied to the cultivation of transgenic plants, wherein the transgenic plants are plants with improved salt stress tolerance and drought resistance or plants with reduced salt stress tolerance and drought resistance, and can be used as new materials for actual production or scientific research application, thereby playing a role.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to MsRGP protein, a coding gene thereof and application thereof in improving drought resistance and salt tolerance of plants.
Background
Land salinization and drought are always important factors limiting agricultural development, about twenty percent of the world's irrigated land is affected by land salinization, and about forty percent of the world's land is affected by drought. On saline-alkali and arid lands, plants face multiple adverse factors such as water shortage, high salt, high pH value and the like, biomass accumulation and growth speed are obviously reduced, the capacity of root systems for absorbing water and growing downwards is limited, osmotic balance and ion balance are imbalance, and finally metabolic disorder, yellowing of leaves, withering and even death of the plants are caused. In China, the saline-alkali soil area and the arid soil area are increased year by year, the crop yield is reduced, and the agricultural development is restricted.
Therefore, how to alleviate land salinization, improve land and restore land productivity is an important problem related to agricultural development in China. Among them, it is particularly important to develop plants having excellent stress resistance, which can be used as one of crops for improving land and restoring land productivity, wherein alfalfa is a typical representative.
Alfalfa (Medicago sativa) is a homeowner, cross-pollinated leguminous alfalfa plant, has the advantages of biological nitrogen fixation capacity, high protein content, good quality, wide planting range and the like, and is known as 'pasture king'. However, with the increasing severity of global climate and environmental conditions, alfalfa growth is subject to extreme environmental effects, including abiotic stresses such as salt and alkali and drought, resulting in reduced yields and reduced quality. The use of molecular biology to increase alfalfa tolerance to salt and alkaline and drought stress is an effective method for enhancing alfalfa yield and improving land.
Therefore, the method has important significance in excavating new genes related to responding to saline-alkali and drought stress and cultivating new plant materials with stronger stress tolerance.
Thus, the prior art is still to be further developed.
Disclosure of Invention
Aiming at the technical problems, the invention provides MsRGP protein separated from alfalfa, a coding gene MsRGP1 gene thereof and application thereof in improving drought resistance and salt tolerance of plants, and expression of the gene in the plants is down-regulated, so that drought resistance and salt tolerance of the plants can be improved, and the novel varieties of stress-resistant plants can be cultivated.
To solve the problems, the applicant analyzes the expression mode of the alfalfa MsRGP gene family members under salt, drought and ABA stress in the alfalfa, combines transcriptome data, finally screens MsRGP1 responding to the salt, drought and ABA stress, and verifies the gene functions of MsRGP1 in the aspects of salt and alkali, drought and ABA stress in the arabidopsis (Arabidopsis thaliana) and the alfalfa through analyzing the over-expression MsRGP1 plant and the gene mutant, and determines the salt and alkali resistance, drought resistance and ABA stress resistance of the plant of the negative regulation of the gene.
Specifically, the technical scheme provided by the application is as follows:
In a first aspect, the present application provides a MsRGP protein, the amino acid sequence of which is as set forth in SEQ ID NO: 1.
The amino acid sequence comprises the amino acid sequence shown in SEQ ID NO:1, and also homologous sequences having a homology of 95% or more to the protein sequence, which sequences can be found in the sequence set forth in SEQ ID NO:1, and mutating the sequence shown in 1. The protein activity and sequence of these homologous sequences are SEQ ID NO:1 are identical in protein activity.
For the purpose of purification or detection of the above protein, a tag protein may be attached to the amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence shown in SEQ ID No. 1.
Such tag proteins include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his6 tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.
In a second aspect, the present application provides MsRGP gene encoding the above protein, the nucleotide sequence of which is shown in SEQ ID NO: 2.
The gene sequence not only comprises the sequence shown as SEQ ID NO:2, and further comprises homologous sequences having a homology of 95% or more to the gene sequence, which sequences can be found in the sequence set forth in SEQ ID NO:2, and mutating the sequence shown in the figure 2. The activity and sequence of the protein expressed by the homologous sequences are SEQ ID NO:2, and the activity of the expressed protein is the same.
In addition, those nucleotides which have been artificially modified and have 95% or more identity to the nucleotide sequence encoding the protein MsRGP isolated by the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention, as long as the functions and activities of the encoded protein are substantially the same.
According to the application, msRGP genes responding to salt, drought and ABA stress are selected from alfalfa, on one hand, an overexpression vector pFGC-MsRGP1-eYFP is constructed, and an agrobacterium-mediated method is utilized to transform arabidopsis thaliana, and a MsRGP transgenic arabidopsis thaliana strain is obtained after screening and identification; on the other hand, msRGP gene knockout strain of Arabidopsis thaliana was studied simultaneously.
By observing the seed germination period and seedling growth period of arabidopsis under the stress of saline-alkali, drought and ABA, compared with a wild type, the over-expression MsRGP1 reduces the tolerance of the arabidopsis to the saline-alkali and the drought and improves the tolerance to the exogenous ABA stress; whereas the resistance of Arabidopsis mutants to saline-alkali and drought is higher than that of the wild type and the resistance to exogenous ABA stress is lower than that of the wild type. Through observation of arabidopsis seedling stage under drought stress, over-expression MsRGP1 reduces the drought tolerance of arabidopsis, and the drought tolerance of arabidopsis mutant is higher than that of wild type.
In addition, the applicant found through research on alfalfa overexpressing MsRGP that under salt treatment and drought treatment, overexpressing MsRGP reduced the tolerance of alfalfa to salt and drought stress, i.e. MsRGP overexpressing alfalfa was lower in salt and drought stress than wild type, which suggests that overexpressing MsRGP1 reduced the tolerance of alfalfa to salt and drought stress, and therefore, expression of MsRGP1 could be reduced by gene editing to thereby increase the tolerance of alfalfa to salt and drought stress, resulting in new alfalfa materials with better salt and drought stress tolerance, which could be expected.
Therefore, msRGP gene is a new important gene affecting the salt and alkali resistance and drought resistance of alfalfa and Arabidopsis thaliana, can be used for cultivating new plant materials with stronger stress resistance, and has high application value.
In a third aspect, the present application provides a recombinant vector carrying the MsRGP gene described above or the MsRGP protein described above.
Vectors described herein are well known to those of skill in the art and include, but are not limited to: plasmids, phages, cosmids (i.e., cosmids), ti plasmids or viral vectors, which are existing expression vectors such as pFGC or existing cloning vectors such as pMD19-T vectors, are not limited to the specific species of the examples of the present application.
Recombinant expression vectors containing MsRGP genes can be constructed using existing plant expression vectors. When the MsRGP gene is used to construct recombinant plant expression vectors, any one of the enhanced promoters or constitutive promoters may be added before the transcription initiation nucleotide.
In addition, when the gene of the present invention is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic.
In a fourth aspect, the present application provides a recombinant bacterium comprising the recombinant vector described above.
The recombinant bacteria herein may be bacteria, fungi, yeasts or algae. Wherein the bacteria may be derived from Escherichia (Escherichia), agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas (Pseudomonas), bacillus (Bacillus), etc. Specifically, the bacillus coli DH5 alpha and/or the agrobacterium tumefaciens EHA105 can be used.
In a fifth aspect, the application also provides application of MsRGP protein, coding gene thereof, recombinant vector or recombinant bacterium carrying the recombinant vector in improving drought resistance and/or salt tolerance of plants.
Based on the foregoing functions and effects of MsRGP1, a person skilled in the art can apply the foregoing MsRGP protein and its coding gene to the cultivation of MsRGP transgenic plants, which are plants with improved salt stress tolerance and drought resistance or plants with reduced salt stress tolerance and drought resistance, based on conventional experimental methods and prior art in the art, and can act as new materials for practical production or scientific research applications.
Preferably, the plant is alfalfa or arabidopsis thaliana.
Preferably, in the application of improving drought resistance and/or salt tolerance of plants, the drought resistance and salt tolerance of the plants are improved by down-regulating MsRGP gene expression level in target plants or knocking out MsRGP gene, so that dominant new varieties are obtained.
In a sixth aspect, the application also provides an application of the MsRGP protein, the coding gene thereof, the recombinant vector or recombinant bacteria carrying the recombinant vector in improving the tolerance of plants to exogenous abscisic acid stress. Alternatively, the plant is arabidopsis thaliana.
Experiments prove that compared with a wild type, the over-expression MsRGP < 1 > improves the tolerance of the arabidopsis to the stress of the exogenous abscisic acid (ABA).
Preferably, in the use of improving tolerance to exogenous abscisic acid stress in a plant, tolerance to exogenous abscisic acid stress in a plant is improved by up-regulating the amount of expression of MsRGP gene in the plant.
In a seventh aspect, the present application also provides a method for producing a transgenic plant, which is (1) or (2) below:
(1) By reducing the overexpression of MsRGP gene in the target plant, obtaining a plant with salt stress tolerance and drought resistance stronger than the target plant;
(2) By promoting the expression of MsRGP gene in target plant, the plant with lower salt stress tolerance and drought resistance than target plant is obtained.
In the preparation method, the method for reducing the expression quantity of MsRGP gene in the target plant is to utilize the technologies of gene mutation, gene knockout, gene editing or gene knockout and the like to reduce or inactivate the MsRGP gene activity in the genome of the target plant so as to reduce the effect of MsRGP1 expression quantity, thereby obtaining a plant with salt stress tolerance and drought resistance stronger than that of the target plant, and the plant is applied as a dominant plant.
The above promotion of the expression of MsRGP gene in the target plant may be the improvement of the expression level of MsRGP1 in the target plant by using a gene overexpression technique (e.g., introducing an expression vector carrying the coding gene into the target plant).
In the above method, the plant may be a stress-tolerant plant, and further, the stress-tolerant plant may be a transgenic plant with reduced (down-regulated) and/or increased (up-regulated) stress tolerance (e.g., salt tolerance and/or drought tolerance).
The MsRGP protein, the coding gene and the application thereof in improving drought resistance and salt tolerance of plants have the following beneficial effects:
1. The invention firstly screens MsRGP genes from the genome of alfalfa (Medicago sativa), and finds that the genes respond to salt, drought and ABA stress simultaneously, the transgenic arabidopsis thaliana and the transgenic alfalfa which over-express MsRGP genes are obtained by introducing the genes into acceptor plants of arabidopsis thaliana and alfalfa, and the analysis and identification of the saline-alkali resistance, drought resistance and ABA stress resistance of the transgenic plants and MsRGP gene mutant strains of arabidopsis thaliana are carried out, and the invention synthesizes the measurement results of various physiological and biochemical indexes, compared with wild plants, the over-expression MsRGP reduces the saline-alkali and drought resistance of arabidopsis thaliana and alfalfa, and improves the tolerance of arabidopsis thaliana to exogenous ABA stress; whereas the resistance of Arabidopsis mutants to saline-alkali and drought is higher than that of the wild type and the resistance to exogenous ABA stress is lower than that of the wild type.
2. The MsRGP protein and the coding gene MsRGP thereof can regulate and control the stress resistance (such as salt tolerance, drought resistance and ABA stress tolerance) of plants, and can cultivate high-quality plants with salt tolerance and/or drought resistance by reducing the content and/or activity of the protein in target plants (such as inhibiting the expression of the gene). The MsRGP and MsRGP coding gene thereof provided by the invention have important theoretical significance and application value in regulating and controlling the salt tolerance and drought resistance of arabidopsis thaliana and medicago sativa and cultivating dominant stress-resistant varieties.
Drawings
FIG. 1 is an analysis of expression patterns of MsRGP gene family members under salt stress; and (3) injection: 0 d: before treatment; 1 d: salt treatment for the first day; 6 d: day 6 of salt treatment;
FIG. 2 is an analysis of the expression pattern of MsRGP gene families in alfalfa roots, stems and leaves;
FIG. 3 is a thermal map of the expression level of MsRGP gene family members under drought and ABA stress;
FIG. 4 is an alfalfa RNA integrity assay;
FIG. 5 shows MsRGP gene fragment amplification results;
FIG. 6 is an identification of pFGC-MsRGP1-eYFP vector; and (3) injection: a: a carrier identification result; m: marker DL2000;1-5: a destination strip; b: pFGC-MsRGP-eYFP vector;
FIG. 7 shows the amino acid sequence alignment of MsRGP and MtRGP;
FIG. 8 is an Arabidopsis resistant plant selection injection: the red circle marks that surviving arabidopsis thaliana is likely to be a positive plant;
FIG. 9 is a MsRGP A transgenic Arabidopsis positive plant identification result; m: marker DL2000; WT: wild type Arabidopsis thaliana; h 2 O: water; the number: different numbers represent different strains.
FIG. 10 shows MsRGP shows the verification of the expression level of transgenic Arabidopsis positive plants; the abscissa is the sequence number of the Arabidopsis positive plant, and the ordinate is the expression level.
FIG. 11 is an identification of homozygous mutants of Arabidopsis; a atrgp. Identification of Arabidopsis homozygous mutant; b: atrgp2 identification of arabidopsis homozygous mutants; m: marker DL2000; WT: wild type Arabidopsis thaliana; 1 or 2: different strains; g: identifying the integrity of the gene sequence; t: T-DNA insertion identification.
FIG. 12 shows germination of wild type Arabidopsis thaliana, msRGP1 transgenic Arabidopsis thaliana and mutants under salt stress; a: germination of 5 lines under 0mM NaCl treatments; b: germination of 5 lines under 125 mM NaCl treatments; c: germination of 5 lines under 150mM NaCl treatments; d: germination rate line plot of 5 lines at 0mM NaCl treatments; e: germination rate line plot of 5 lines at 125 mM NaCl treatments; f: germination rate line plot of 5 lines at 150mM NaCl treatments;
FIG. 13 shows survival of wild type Arabidopsis thaliana, msRGP1 transgenic Arabidopsis thaliana, and mutants under salt stress; and (3) injection: a: survival of 5 strains under 0mM NaCl treatment; b: survival of 5 strains under 125 mM NaCl treatments; c: germination of 5 lines under 150 mM NaCl treatments; d: survival of 5 strains under 0mM NaCl treatment and 125 mM NaCl treatment; e: survival of 5 strains under 0mM NaCl treatment and 150 mM NaCl treatment;
FIG. 14 shows germination of wild type Arabidopsis thaliana, msRGP1 transgenic Arabidopsis thaliana and mutants under drought stress; a: germination of 5 lines under 0mM mannitol treatment; b: germination of 5 lines under 300 mM mannitol treatment; c: germination of 5 lines under 400mM mannitol treatment; d: germination rate line plot of 5 lines under 0mM mannitol treatment; e: germination rate line plot of 5 lines under 300 mM mannitol treatment; f: germination rate line plot of 5 lines under 400mM mannitol treatment;
FIG. 15 is survival of wild type Arabidopsis thaliana, msRGP1 transgenic Arabidopsis thaliana, and mutants under drought stress; a: survival of 5 strains with 0 mM mannitol treatment; b: survival of 5 strains under 300 mM mannitol treatment; c: survival of 5 strains under 400 mM mannitol treatment; d: survival of 5 strains with 0 mM mannitol and 300 mM mannitol; e: survival of 5 strains with 0 mM mannitol and 400 mM mannitol;
FIG. 16 is a phenotypic analysis of wild type Arabidopsis thaliana, msRGP1 transgenic Arabidopsis thaliana, and mutants under ABA stress; a: wild-type arabidopsis thaliana and MsRGP1 overexpressing arabidopsis thaliana germination under 0 μm ABA treatment and under 1 μm ABA treatment; b: germination of wild type arabidopsis and mutants under 0 μm ABA treatment and 1 μm ABA treatment; c: wild type arabidopsis thaliana and MsRGP1 overexpressing the germination rate of arabidopsis thaliana under 0 μm ABA treatment; d: wild type arabidopsis thaliana and MsRGP1 overexpressing the germination rate of arabidopsis thaliana under 1 μm ABA treatment; e: germination rates of wild type arabidopsis and mutants under 0 μm ABA treatment; f: germination rates of wild type arabidopsis and mutants under 1 μm ABA treatment; g: wild-type arabidopsis and MsRGP1 overexpressing arabidopsis green leaf status at 10 d under 0 μm ABA treatment and 1 μm ABA treatment; h: green leaf status of wild type arabidopsis and mutant at 20 d under 0 μm ABA treatment and 1 μm ABA treatment; i: wild-type arabidopsis and MsRGP1 overexpressing arabidopsis green leaf ratio at 10 d under 0 μm ABA treatment and 1 μm ABA treatment; j: wild-type Arabidopsis and mutants have green leaf ratios of 20 d under 0 μM ABA treatment and 1 μM ABA treatment;
FIG. 17 is the effect on Arabidopsis seedlings under abiotic stress; a: growth of seedlings of 5 lines under different treatments; b: root length of 5 lines after growth under different treatments; c: fresh weight of 5 lines after growth under different treatments.
FIG. 18 is an effect of drought stress on Arabidopsis;
FIG. 19 is the survival rate of Arabidopsis after drought treatment; control, before treatment; drought: after drought treatment;
FIG. 20 is a MsRGP1 analysis of expression levels of transgenic alfalfa positive plants;
FIG. 21 is a phenotypic analysis of MsRGP.sup.1 transgenic alfalfa and wild-type alfalfa under salt stress; a: before treatment; b: salt treatment 10 d; c: salt treatment 15 d; d: salt treatment 20 d;
FIG. 22 is a phenotypic analysis of MsRGP.sup.1 transgenic alfalfa and wild-type alfalfa under drought stress; a: before treatment; b: drought treatment 15 d; c: drought treatment 17 d; d: drought treatment 20 d;
Results are expressed as mean ± Standard Deviation (SD), t-test for significance analysis ("x" P < 0.05, "x" P < 0.01).
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1MsRGP screening of Gene
In order to better excavate the saline-alkali tolerance gene of the alfalfa, salt, alkali and saline-alkali mixed treatment of different concentrations is carried out on the alfalfa in the early stage of the experiment, and transcriptome sequencing is carried out on different treatment groups of the alfalfa, so that the transcriptome data analysis shows that: the MsRGP gene responds to the saline-alkali stress, so that a novel gene MsRGP which influences the saline-alkali resistance of alfalfa is obtained, and the nucleotide sequence of the gene is shown as SEQ ID NO:2, the amino acid sequence of the protein expressed by the sequence is shown as SEQ ID NO: 1.
Bioinformatics analysis and expression pattern analysis under different tissues and various stresses were performed for the study MsRGP gene family to predict and further verify the gene function of the gene.
1.1 Experimental methods
1.1.1 Stress treatment of alfalfa
Alfalfa seeds were placed in petri dishes with filter paper laid and kept moist, and after dark cultivation 5 d, the germinated seedlings were transferred to Hoagland nutrient solution for hydroponic culture with nutrient solution changes every 3: 3 d. The culture condition is 16 h light/8 h dark, the day and night temperature is 25 ℃/22 ℃, and the relative humidity is 60-70%. Alfalfa of the same growth potential at 30 days of age was selected for treatment, and a control group (Hoagland nutrient solution) and a treatment group (salt, mannitol and ABA) were set, and three replicates were set, and the treatment method and sampling time were as follows:
salt stress: treatment with Hoagland's nutrient solution containing 100mM NaCl was performed and samples were taken on days 0 h, 3 h, 6h, 12 h, 24 h and six of the treatments.
(2) Osmotic stress: samples were taken at treatments 0 h, 3h, 6h, 12 h, 24h and 2d using a Hoagland nutrient solution treatment containing 300mM Manntiol.
(3) ABA stress: treatment with Hoagland nutrient solution containing 10. Mu.M ABA was performed with samples taken at treatments 0h, 3h, 6 h, 12 h and 24 h. The sampling parts are roots, stems and leaves. All samples were flash frozen using liquid nitrogen and stored in a-80 ℃ refrigerator until use.
1.1.2 QRT-PCR analysis of MsRGP Gene family
A more comprehensive analysis of expression patterns was performed on MsRGP gene families by qRT-PCR (SCHMITTGEN ET al., 2008). Specific primers for qRT-PCR were designed using PRIMER PREMIER software (Table 1). Alfalfa RNA was extracted according to the instructions and reverse transcribed. qRT-PCR assays were performed according to the procedure instructions. Three replicates were set up for each sample and the data was analyzed using the 2 fatter CT method. Results are expressed as mean ± Standard Deviation (SD). Significant differences were calculated using t-test ("×" represents P <0.05, "×" represents P < 0.01). Expression levels under drought and ABA stress are presented in the form of heat maps.
TABLE 1 qRT-PCR primers
1.2 Experimental results and analysis
1.2.1 Analysis of expression patterns of MsRGP Gene families under salt stress
(1) Expression pattern analysis was performed on MsRGP gene family members before and after 100 mM NaCl stress by transcriptome data (fig. 1). The expression levels of MsRGP1, msRGP2, and MsRGP5 exhibited a tendency to be up-regulated on the first and sixth days under salt stress, and had similar expression patterns. MsRGP3 showed a down-regulation trend on the first day under salt stress and an up-regulation trend on the sixth day. MsRGP4 and MsRGP a showed a trend of down-regulating the expression level on both the first and sixth days under salt stress. It was demonstrated that MsRGP gene family responded to salt stress.
(2) QRT-PCR analysis of MsRGP Gene family
As shown in FIG. 2, msRGP.sup.1 and MsRGP.sup.2 are expressed in roots and stems more than other family members. The highest expression level in the leaves was MsRGP, msRGP times. Overall, msRGP1 was expressed at higher levels in roots, stems or leaves than other MsRGP gene family members.
1.2.2 Analysis of expression patterns of MsRGP Gene family members under drought and ABA stress
Under drought stress, the expression patterns of MsRGP and MsRGP2 under drought stress are substantially identical, and the expression levels in roots and stems are down-regulated as a whole, but the expression levels in leaves are up-regulated. MsRGP4 showed various levels of up-regulation of the expression levels in roots, stems and leaves as a whole. Furthermore, the expression levels of MsRGP and MsRGP5 in the leaves were both up-regulated (fig. 3A).
Under ABA stress, 5 MsRGP gene family members showed a tendency to rise and fall after each other when the amount of expression of root, stem and leaf was 3 h to 12h, except MsRGP expression in root. Among them, msRGP1 was expressed in the highest amount in roots, stems and leaves at ABA stress treatment of 3 h (fig. 3).
From the above results, msRGP and MsRGP sequences have similar expression patterns under salt and drought stress, and it is speculated that MsRGP and MsRGP may have some genetic redundancy in responding to salt and drought stress. By analyzing transcriptome data under saline-alkali stress, only MsRGP1 genes were found to be differentially expressed. Therefore, msRGP1 is presumed to be involved in responses to salt, drought and ABA stress. Therefore, msRGP1 was selected for subsequent verification and detection of gene function.
Example 2MsRGP preparation of Arabidopsis thaliana overexpressed by Gene
2.1 Construction of MsRGP1 Gene overexpression vector
To verify MsRGP gene function, the coding region (CDS) sequence of MsRGP1 was cloned in alfalfa and a pFGC-MsRGP1-eYFP overexpression vector was constructed.
2.1.1 Experimental methods
(1) MsRGP1 amplification of the coding region sequence
The RNA of alfalfa SY4D was extracted, and specific steps are described in the specification (FastPure Universal Plant Total RNA Isolation Kit). Reverse transcription is carried out on the RNA to obtain the cDNA product of the alfalfa. Primers were designed by PRIMER PRIMER 5.0.0 software (Table 2), amplified and sequenced using primer pair MsRGP1, and the correct product was selected by alignment to give the fragment of interest.
TABLE 2 primer information
(2) Construction of overexpression vector
The plasmid of the pFGC-eYFP over-expression vector was subjected to single cleavage using BamHI restriction enzymes. And (3) connecting the target fragment with the overexpression vector after enzyme digestion by using a seamless cloning kit, wherein the specific steps are carried out according to the specification. After completion of the ligation reaction, the ligation product was transferred into DH5a chemocompetent cells. After the transformation is completed, the bacterial liquid is coated on an LB culture medium containing Kan (50 mg/L), and the bacterial liquid is inverted and cultured in an incubator at 37 ℃ for 24-36 hours. Monoclonal were selected for bacterial liquid PCR identification and sequencing, using primers as shown in table 2. Activating and preserving the bacterial liquid with correct sequencing. Extracting MsRGP a plasmid of an over-expression vector, and transferring the plasmid into EHA105 agrobacterium competent cells, wherein the specific steps are as follows:
① And taking agrobacterium tumefaciens competence stored at-80 ℃ and inserting the agrobacterium tumefaciens competence into ice when the agrobacterium tumefaciens competence is partially melted at room temperature or at palm of a hand for a while and is in an ice water mixed state.
② Every 100 mu l of competent plasmid DNA is added with 0.01-1 mu g, the mixture is slightly stirred at the bottom of the tube and mixed uniformly, and then the mixture is placed on ice for 5min, liquid nitrogen 5min, 37 ℃ water bath 5min and ice bath 5min in sequence.
③ And adding 700 mu l of LB liquid medium, and carrying out shaking culture for 2-3 hours at the temperature of 28 ℃.
④ Collecting bacteria by centrifugation at 6000 rpm and 1:1 min, collecting about 100 μl of supernatant, gently blowing and beating the re-suspended bacteria mass, coating on LB culture medium containing Kan antibiotics (50 mg/L), and culturing in 28 deg.C incubator for 2-3 d
⑤ And (5) performing bacterial liquid PCR identification on the grown monoclonal.
Adding the pFGC-MsRGP-eYFP over-expressed agrobacterium liquid into an LB liquid culture medium containing Kan (50 mg/L) and Rif (50 mg/L) for 28 ℃ activation, mixing the liquid with glycerol 1:1 for preserving bacteria after 36-48 hours until the liquid is turbid, freezing the liquid by using liquid nitrogen, and then placing the liquid at-80 ℃ for preservation.
(3) Infection and subcellular localization of tobacco leaves
The pFGC-MsRGP-eYFP agrobacterium liquid and pFGC-eYFP empty-load agrobacterium liquid which are constructed are activated in LB liquid culture medium containing Rif and Kan, so that the OD 600 of the bacterial liquid reaches 0.5-1.5, supernatant is removed by centrifugation, suspension cells are resuspended by using 10 mM MgCl 2 suspension containing 120 mu M AS, so that the suspension OD 600 is about 0.6, and the culture is carried out in dark at 25 ℃ for 2h.
Selecting a tobacco plant with good growth vigor of one month, sucking the heavy suspension by using a 1 mL injector, injecting the lower epidermis of the tobacco leaf, and marking. And (3) carrying out dark treatment at 25 ℃ for 10-14 hours after infection, recovering normal photoperiod culture for 2-3 d, sampling tobacco leaves marked with injection positions, manufacturing a glass slide, and photographing and recording.
2.1.2 Experimental results and analysis
(1) MsRGP1 CDS cloning of 1
The extracted alfalfa RNA is subjected to quality detection. The RNA concentration of the extracted alfalfa was about 200 ng/. Mu.l, and the gel electrophoresis was performed to detect two clear bands, which indicated that the RNA was not degraded and could be used in the subsequent experiments (FIG. 4).
The MsRGP gene CDS was amplified using alfalfa cDNA as template, and the PCR products were gel-electrophoretically detected to conform to the expected band size (FIG. 5).
Sequencing gave a MsRGP gene CDS length 1074 bp, consistent with the sequence alignment obtained with the transcriptome, indicating successful cloning of the MsRGP CDS. Translation of the MsRGP CDS sequence into an amino acid sequence, aligned with the amino acid sequence of alfalfa MtRGP, revealed an amino acid sequence similarity of MsRGP and MtRGP1 of about 98.61% (FIG. 7).
(2) MsRGP1 construction of an overexpression vector
The CDS of the amplified MsRGP gene was ligated to the pFGC-eYFP vector by a seamless cloning method and PCR was performed, and the result showed successful ligation of the vector (FIG. 6), and the resulting recombinant vector was designated pFGC-MsRGP1-eYFP. The promoter of the vector is cauliflower mosaic virus 35S (CaMV 35S), has the eYFP marker gene and has Kan and PPT resistance.
In this example, subcellular localization was performed using the overexpression vector pFGC-MsRGP-eYFP with the promoter CaMV 35S. The CaMV35S strong promoter can improve the expression level of YFP fusion protein and does not influence subcellular localization results. By performing subcellular localization of MsRGP protein in tobacco leaves, msRGP protein is localized in the cytoplasm and nucleus.
2.2 MsRGP1 acquisition of transgenic Arabidopsis thaliana
2.2.1 Experimental method
(1) Planting of Arabidopsis thaliana
Taking a proper amount of seeds of wild arabidopsis thaliana, placing the seeds in a centrifuge tube sterilized by 1.5 mL, washing the seeds with dd H 2 O, and then placing the seeds in 75% alcohol for sterilization for 1-2 min; the washing is performed once again with dd H 2 O; adding 10% NaClO solution to soak for 7-10 min; dd H 2 O is washed for 5 to 8 times and then is kept stand for 30 to min. Uniformly spreading Arabidopsis seeds on a 1/2MS culture medium; after vernalization at 4 ℃ for 2-3 d, placing the culture in a 24 ℃ illumination incubator for culture, growing 4 true leaves, and then transferring the leaves into soil (vermiculite: nutrient soil=20:1).
(2) Pattern dipping method for infecting arabidopsis thaliana
Taking out agrobacterium transformed with MsRGP1 over-expression vector, adding 100 mu L of the bacterial liquid into 10 mL liquid LB medium containing Kan (50 mg/L) and Rif (50 mg/L), and placing in a shaking table at 28 ℃ for 200 rpm culture for 2-3 d; adding 1-2 mL of activated bacterial liquid into 200 mL liquid LB culture medium containing Kan (50 mg/L) and Rif (50 mg/L), and placing in a shaking table at 28 ℃ for shaking 200 rpm until the bacterial liquid OD 600 is 1.2-2.0; centrifuge 15: 15 min at 6500 rpm ℃and discard the supernatant. Resuspension to OD 600 of 1 using a 5% sucrose solution; activating the heavy suspension at 28 ℃ for 30 min; adding 0.03% of activator sliwet-77; immersing an arabidopsis inflorescence into the bacterial liquid for 15-30 sec, and culturing normally after culturing in darkness for one day; and (5) carrying out secondary infection after 7-8 d.
(3) Screening and identification of positive plants
Harvesting mature seeds after infection of arabidopsis thaliana; uniformly spreading the sterilized seeds on a 1/2MS culture medium containing PPT (7.5 mg/L), and carrying out illumination culture at 24 ℃ after vernalization for two days; seedlings with 4 true leaves were transferred to soil (vermiculite: nutrient soil=20:1).
Extracting Arabidopsis DNA and carrying out PCR verification, wherein the primer information is shown in Table 3; RNA of positive plants was extracted and reverse transcribed, and then subjected to qRT-PCR assay for expression analysis, and primer information is shown in the primer information Table 4-1. Selecting 2-3 strains with highest expression quantity, propagating and harvesting seeds of T 3 generations for later experiments.
TABLE 3 primer information
2.2.1 Results and analysis
PFGC-MsRGP-YFP over-expression vector was transferred into Arabidopsis and resistant plants were selected by 1/2MS medium containing PPT (FIG. 8). Specific primers 35S-F and MsRGP-R were used to identify plants that were resistant. 7 MsRGP transgenic Arabidopsis positive lines were obtained by DNA level identification (FIG. 9). The qRT-PCR test was performed on these 7 positive lines to determine the amount of expression, and the two lines with the highest amount of expression (OE 17 and OE 18) were selected for subsequent testing (FIG. 10).
EXAMPLE 3 screening of Arabidopsis mutants
3.1 Method:
Arabidopsis thaliana homologous genes AtRGP1 and AtRGP2 of MsRGP are obtained through Tair online websites (https:// www.Arabidopsis.org/index. Jsp) searching, and the gene numbers are At3g02230 and At5g15650 respectively. Mutant strains atrgp (N664336) and atrgp (N656992) of the two genes were found on-line website Arashare (https:// www.arashare.cn/index /). Extracting the Arabidopsis mutant DNA for identification.
The identification method comprises the steps of double-primer identification: n664336 was amplified by PCR using AtRGP1-F and AtRGP1-R, T-DNA-F and AtRGP1-R, the former was a homozygous mutant when the latter had no band and the band was correct in size, and N656992 was amplified by PCR using AtRGP2-F and AtRGP2-R, T-DNA-F and AtRGP2-R in the same manner as described above (see Table 3).
3.2 Results and analysis
To perform bidirectional validation of the gene function of MsRGP1, mutant lines AtRGP and AtRGP were screened for the homologous genes AtRGP1 and AtRGP2 of MsRGP1 in Arabidopsis. PCR was performed on atrgp and atrgp2 to finally obtain atrgp1 and atrgp Arabidopsis homozygous mutant lines (see FIG. 11).
Example 4 stress treatment methods and phenotypic analysis of transgenic and mutant Arabidopsis thaliana
4.1 Experimental methods
The following experiments were performed using the MsRGP1 over-expression strain and the two mutant strains obtained in examples 2 and 3 as materials:
(1) Evaluation method for influence of saline-alkali, drought and ABA stress treatment on seed germination
Salt treatment (125 mM and 150 mM NaCl), alkali treatment (5 mM NaHCO3), drought treatment (300 mM and 400 mM mannitol), ABA treatment (1. Mu.M ABA) and blank control were performed with the wild type Arabidopsis seeds, two MsRGP over-expressed strains and two mutant strains as materials, each group treated with three biological replicates. Transferring into 24 deg.C illumination incubator for culturing, performing biological statistics on germination rate, survival rate and green leaf rate, observing phenotype difference, and recording.
(2) Method for evaluating influence of saline-alkali, drought and ABA stress treatment on root growth
The wild type arabidopsis thaliana, two MsRGP1 over-expressed strains and two mutant strains which are 7 d post-germination and consistent in growth vigor are used as materials, and salt treatment (125 mM NaCl), alkali treatment (5 mM NaHCO3), drought treatment (300 mM mannitol) and ABA treatment (30 mu M ABA) and blank control are carried out, wherein each group of treatment is subjected to three biological replicates. Transferring into 24 ℃ illumination incubator for continuous vertical culture, performing biological statistics on root length at 12 d hours after seed germination, observing phenotype difference and recording.
(3) Evaluation method for influence of drought stress on seedling growth of arabidopsis thaliana
After culturing the wild type arabidopsis seed, the two MsRGP over-expressed lines and the two mutant lines in 1/2MS medium for 7: 7 d, germinated arabidopsis was transferred to vermiculite. After culturing about 14 d in vermiculite, the arabidopsis thaliana is treated, which comprises: the blank and drought treatments (natural drought) were not less than 3 biological replicates per treatment group. Drought treatment methods are stopping watering until phenotypic differences or wilting occurs, rehydrating at wilting and later observing survival and performing biological statistics.
4.2 Results and analysis
4.2.1 Effect of saline-alkali, drought and ABA stress on Arabidopsis seed germination
(1) Effect of salt stress on Arabidopsis seed germination
MsRGP1 transgenic Arabidopsis thaliana (OE 17 and OE 18), arabidopsis thaliana mutants (atrgp 1 and atrgp) and wild type Arabidopsis thaliana were plated on 1/2MS medium containing NaCl (125 mM and 150 mM) for cultivation, and the results showed that:
Under the treatments of 125 mM and 150 mM NaCl, the germination rates of OE17 and OE18 are significantly lower than that of WT, especially at 2-4 d. Whereas atrgp and atrgp2 have a significantly higher germination rate than WT (P < 0.05) (fig. 12). The viability of the 5 strains was biologically counted at 18 d and as a result, it was found that there was no difference in the viability of the 5 strains in the blank. At 125 mM NaCl treatment, the survival rates for both OE17 and OE18 were significantly lower than for WT, while atrgp1 survived significantly higher than for WT (P < 0.05). At 150 mM NaCl treatment, the survival rate of OE17 was significantly lower than that of WT, while that of arabidopsis mutant atrgp1 was significantly higher than WT (P < 0.05) (fig. 13).
These results indicate that over-expression MsRGP under salt stress reduces salt tolerance of arabidopsis thaliana and is consistent with the result that OE17 is expressed higher than OE18, whereas salt tolerance of arabidopsis mutants atrgp1 and atrgp is higher than wild type.
(2) Effects of drought stress on Arabidopsis seed germination
MsRGP1 transgenic Arabidopsis thaliana (OE 17 and OE 18), arabidopsis thaliana mutants (atrgp 1 and atrgp) and wild type Arabidopsis thaliana were plated onto 1/2MS medium containing different concentrations of mannitol (300 mM and 400 mM) for cultivation and observation. The germination rates of the 5 Arabidopsis lines were consistent with 0mM mannitol treatment. Under 300 mM and 400 mM mannitol treatments, the germination rates of both OE17 and OE18 were significantly lower than WT (P < 0.05), while the germination rate of arabidopsis mutant atrgp was significantly higher than WT (P < 0.05) (fig. 14). At the same time, the viability of the 5 lines was biologically counted at 18 d and found to be no difference in viability of the 5 lines with 0mM mannitol treatment, but the viability of OE17 and OE18 was significantly lower than WT with 300 mM and 400 mM mannitol treatments, the viability of atrgp1 was significantly higher than WT, and the viability of atrgp2 was significantly higher than WT with 400 mM mannitol treatment (P < 0.05) (fig. 15).
These results indicate MsRGP that MsRGP responds to osmotic stress and reduces the germination rate of arabidopsis under drought stress. Thus overexpression MsRGP1 reduced drought tolerance in arabidopsis, whereas arabidopsis mutants atrgp and atrgp2 were more tolerant to drought than wild-type arabidopsis.
(3) Effect of alkali stress on Arabidopsis seed germination
MsRGP1 transgenic Arabidopsis thaliana (OE 17 and OE 18), arabidopsis thaliana mutants (atrgp 1 and atrgp 2), and wild type Arabidopsis thaliana were plated into 1/2MS medium containing 5mM NaHCO 3 for cultivation. The germination rates of the 5 lines were consistent at 0mM NaHCO 3 treatments. Under 5mM NaHCO 3 treatment, the germination rates of OE17 and OE18 were significantly lower than WT at 2-3 d after incubation, while atrgp1 was significantly higher than WT (P < 0.05).
These results indicate that over-expression MsRGP1 reduced the tolerance of the plants to alkali stress and that the tolerance of the arabidopsis mutant atrgp1 to alkali stress was higher than the wild type.
(4) Influence of ABA stress on Arabidopsis seed germination
The results show that there is no difference in the wild type, over-expressed strain and mutant under 0. Mu.M ABA treatment. Under 1. Mu.M ABA treatment, the germination rates of both OE17 and OE18 were significantly higher than that of WT, whereas the germination rates of both mutant strains were significantly lower than that of wild-type (P < 0.05). At the same time, the green leaf rates of the Arabidopsis and WT were calculated for overexpression at 10 d and for the Arabidopsis mutant and WT at 20 d. Under the blank control, there was no difference in green leaf rate between the overexpression of Arabidopsis thaliana and WT, and the Arabidopsis thaliana mutant and WT. Under ABA stress, the green leaf rates of both OE17 and OE18 were significantly higher than WT, while the greening rates of both mutant lines were significantly lower than wild type (P < 0.05) (fig. 16).
The above results demonstrate MsRGP that MsRGP responds to ABA stress and that it is possible to reduce the tolerance of arabidopsis to salt and drought stress by ABA signaling pathway.
4.2.2 Effects of saline-alkali, drought and ABA stress on Arabidopsis seedlings
The following are obtained from the experimental results of fig. 17:
(1) At 125 mM NaCl, it was observed that both OE17 and OE18 lines exhibited plant death, which was not present in WT and mutant lines. In addition, both over-expressed lines had root lengths lower than that of the WT and mutant lines, while both mutant lines had root lengths significantly higher than that of the WT (P < 0.05). The fresh weight of both mutant lines was significantly higher than WT (P < 0.05). The MsRGP s1 showed a negative regulation phenomenon in response to salt stress, and reduced salt tolerance of the plants in the arabidopsis seedling stage.
(2) Under 5mM NaHCO 3 alkali stress, leaves of Arabidopsis seedlings under alkali stress were observed to show that OE17 and OE18 had a higher degree of leaf green loss than WT and mutant strains. The root length and fresh weight of WT, overexpressed arabidopsis and mutants were not significantly different (P < 0.05). It was revealed that the response to alkali stress at the Arabidopsis thaliana seedling stage MsRGP1 was not obvious.
(3) The results under drought stress are consistent with those of salt stress. The root length of both mutant lines was significantly higher than that of WT by biological statistics under 300 mM mannitol treatment, whereas that of MsRGP1 transgenic arabidopsis was lower than WT (P < 0.05). It is shown that overexpression MsRGP < 1 > reduces drought tolerance of plants and that the resistance of Arabidopsis mutants to drought stress is higher than that of wild type.
(4) The root system of the mutant strain was shorter under 30 μm ABA treatment. After biological analysis, the root length of OE17 and OE18 was found to be significantly higher than that of WT, whereas the root length of both mutant lines was significantly lower than that of WT (P < 0.05). This suggests MsRGP that 1 responds to ABA stress and increases the tolerance of the plant to ABA stress.
The above results indicate that over-expression MsRGP1 reduces the tolerance to salt and alkaline and drought stress in arabidopsis seedling stage and enhances the tolerance to exogenous ABA stress, the tolerance of arabidopsis mutant to salt and alkaline and drought stress is higher than that of wild type arabidopsis, and the tolerance to exogenous ABA stress is lower than that of wild type arabidopsis.
4.2.3 Effect of drought stress on Arabidopsis seedling
Drought treatment was performed on 5 strains of Arabidopsis thaliana (WT, OE17, OE18, rgp1 and rgp 2), and leaves of WT, OE17, OE18 appeared to lose green, wrinkled and withered yellow during drought treatment 12 d, where the extent of losing green of OE17 was most severe, while leaves of atrgp and atrgp strains remained fresh green, and no loss of green appeared. Rehydration was performed at drought treatment 20 d, and after rehydration, plants were again observed at 2: 2d, and atrgp and atrgp lines were found to grow better and leaves green compared to WT, OE17 and OE 18. While OE17 showed substantially no viable plants after rehydration, the leaves of the plants exhibited a withered and yellow state as a whole (FIG. 18).
Biological statistics show that the survival rate of WT after rehydration was about 88.9%, whereas the survival rate of OE17 was as low as 14.8%, the survival rate of OE18 was about 63%, and that the survival rate of both over-expressed strains was significantly lower than that of WT (P < 0.05), and that of both atrgp1 and atrgp was about 96.3%. This demonstrates MsRGP that 1 reduced drought tolerance in plants, consistent with the results of previous experiments with arabidopsis seed germination and arabidopsis seedling development under drought stress (fig. 19).
Example 5MsRGP preparation of transgenic alfalfa overexpressed in Gene and stress-resistance analysis
5.1 Experimental method
5.1.1 Alfalfa genetic transformation Process and acquisition of Positive plants
(1) Adding 100 mu L of pFGC-MsRGP-eYFP agrobacterium into 10 mL liquid LB medium containing Kan (50 mg/L) and Rif (50 mg/L), and placing in a shaking table at 28 ℃ for 200 rpm activation for 2-3 d until bacterial liquid is turbid and orange; 100mL liquid LB containing Kan (50 mg/L) and Rif (50 mg/L) is added into a 250 mL sterilization conical flask, 1-2 mL of activated bacterial liquid is added, and the mixture is placed in a shaking table at 28 ℃ and is shaken until the OD600 of the bacterial liquid is 0.6-0.8.
(2) Adding 100 mL bacterial liquid into two sterilized 50 mL centrifuge tubes, centrifuging at 4 ℃ and 6500 Xg for 15min, discarding supernatant, adding SH3a liquid culture medium for resuspension, enabling the OD600 of the resuspension to be between 0.2 and 0.3, and transferring to a sterilized tissue culture bottle.
(3) 10-15 Pieces of mature complex leaves of alfalfa are taken. And cleaning the blade under a sterile state, cleaning the blade by using dd H 2 O, cleaning the blade by using 75% alcohol for about 1-min, cleaning the blade by using dd H 2 O for 1-3 times, soaking the blade into 15-20% sodium hypochlorite solution added with a drop of Tween 20 (Tween 20), putting the blade into a shaking table for 50 rpm for 6-10 min, and finally cleaning the blade by using dd H 2 O for 5-8 times.
(4) Transferring the cleaned leaves into the heavy suspension, and vacuumizing for 5-10 min. And then putting the mixture into an ultrasonic instrument containing ice-water mixture for ultrasonic treatment for about 5-15 sec, wherein the specific time is determined according to the state of the blade. Vacuumizing for 5-10 min again. The leaves were transferred to sterilized filter paper to draw excess water, and then transferred to SH3a co-culture solid medium. And then placing the mixture in a 24 ℃ incubator for dark culture for 22-30 hours.
(5) Transferring the co-cultured leaves to SH3a selective medium, and continuously culturing in a dark incubator at 24 ℃ until the medium is replaced every 12-16 d until the expanded callus is formed for 2-3 months.
(6) Transferring the expanded callus to MSBK culture medium, culturing at 24 ℃ under 16: 16 h light/8: 8h dark, and replacing the culture medium every 14-20 d, wherein the culture medium is about 30: 30 d.
(7) The callus is transferred to SH9a culture medium after green buds are generated, the culture medium is replaced every 20-30 d, and the callus is placed in the SH9a culture medium without PPT for culture after about 60-d d until regenerated plants are grown.
(8) And transferring the regenerated plants to soil for culture, and ensuring humidity and weak light in initial culture.
(9) Leaves of alfalfa regenerated plants were sampled and DNA extracted and identified using specific primers 35S-F2 and MsRGP1-R1 (Table 4), with positive plants of the correct band size.
(10) Sampling leaves of positive plants, extracting RNA of the positive plants, carrying out reverse transcription, and verifying the expression quantity by using a reverse transcription product through a qRT-PCR test. Ms-actin-F and Ms-actin-R are internal reference gene primers, Q-MsRGP-F and Q-MsRGP-R are primers for detecting target gene expression quantity. The detailed procedure of qRT-PCR was followed as described, three replicates were set per sample and the data was analyzed using the 2-fatter CT method. Results are presented as mean ± Standard Deviation (SD). qRT-PCR primer information is listed in the table (Table 4). And selecting 2-4 strains with highest expression quantity for culture and carrying out cuttage with wild type in the same period for subsequent experiments.
5.1.2 Functional analysis of MsRGP1 transgenic alfalfa under salt stress and drought stress
And when the wild alfalfa and the transgenic alfalfa are subjected to cuttage for about 1-2 months, selecting alfalfa with consistent growth vigor after cuttage, and transferring the alfalfa into new soil (vermiculite) for salt stress and drought stress treatment.
(1) The salt stress treatment method comprises the following steps: salt treatment (300 mM NaCl Hoagland nutrient solution) was performed using three lines of WT, OE26 and OE27, each line being set up in four replicates. The phenotype was observed and recorded by photographing starting from the first day of treatment.
(2) The drought treatment method comprises the following steps: drought treatment (natural drought) was performed using three lines of WT, OE26 and OE27, each line being set up with four replicates. The phenotype was observed and recorded by photographing starting from the first day of treatment.
TABLE 4 primer information
5.2 Experimental results and analysis
5.2.1 Obtaining of MsRGP1 transgenic alfalfa
Finally obtaining 42 regenerated plants by a alfalfa genetic transformation method. The PCR identification shows that the total number of positive plants is 23, and the positive rate is about 54.76%. Subsequently, positive plants were subjected to expression level verification by qRT-PCR technique (fig. 20), OE26 and OE27 were selected for subsequent experiments, and cut.
5.2.2 Functional analysis of MsRGP transgenic alfalfa under salt stress and drought stress
Two MsRGP over-expressed alfalfa lines (OE 26 and OE 27) and wild-type alfalfa (WT) were selected for cutting, about 45: 45 d after cutting, and MsRGP transgenic alfalfa and wild-type alfalfa with consistent growth vigor were selected for salt treatment and drought treatment.
OE26 and OE27 were not phenotypically different from WT prior to salt stress treatment. After treatment of 10 d with 300 mM NaCl aqueous solution, OE27 had limited growth and development during salt treatment and had lodging, leaf wilting, few leaves of OE26 had wrinkling, and WT had not been significantly altered. At salt treatment 15 d, the stems and leaves of OE27 lose moisture and wither and leave the leaves, the leaves of OE26 begin to wilt over a large area, and WT remains unchanged. At salt treatment 20 d, OE27 was completely deactivated, OE26 was lodged and leaves and stems were essentially withered, while WT only a few leaves were wilted (fig. 21). These results indicate MsRGP that MsRGP reduces alfalfa tolerance to salt stress.
About 45 d after cutting, msRGP1 transgenic alfalfa (OE 26 and OE 27) and WT with consistent growth vigor were selected for drought treatment. Before drought treatment, WT, OE26 and OE27 had no phenotypic differences. After drought treatment 15 d, no significant changes were made to WT, whereas leaves of OE26 and OE27 showed a phenomenon of loss of water and wilting, with leaves of OE27 being relatively heavy. During drought treatment 17 d, the leaves of OE27 substantially lost moisture and withered and fallen off, the degree of wrinkling of the OE26 leaves was increased but withered, and only a small portion of the leaves of WT had wilted. At drought treatment 20 d, both OE26 and OE27 leaves had completely lost moisture and leaf atrophy, wilting and water loss of WT leaves, and a relatively light degree of drought stress (FIG. 22). The results indicate MsRGP that alfalfa reduces drought stress tolerance.
It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.
Claims (3)
1. An application of MsRGP protein in improving drought resistance and/or salt tolerance of plants, wherein the plants are alfalfa or arabidopsis thaliana, and the application is characterized in that the amino acid sequence of the MsRGP protein is shown in SEQ ID NO:1, wherein the drought resistance and salt tolerance of the plant are improved by down regulating MsRGP gene expression level in the plant or knocking out MsRGP gene, and the nucleotide sequence of MsRGP gene is shown as SEQ ID NO: 2.
Use of the msrgp1 gene for improving drought and/or salt tolerance in plants, said plants being alfalfa or arabidopsis thaliana, characterized in that the nucleotide sequence of said MsRGP gene is as set forth in SEQ ID NO:2, by down regulating MsRGP gene expression level in plant or knocking out MsRGP gene, drought resistance and salt tolerance of plant are improved.
3. A method for producing a transgenic plant, characterized in that the method is (1) or (2) below:
(1) By reducing the expression of MsRGP gene in wild type plant, obtaining plant with stronger salt stress tolerance and drought resistance than wild type plant;
(2) By promoting the expression of MsRGP gene in wild type plant, obtaining plant with salt stress tolerance and drought resistance lower than wild type plant,
The plant is alfalfa or Arabidopsis thaliana, and the nucleotide sequence of the MsRGP gene is shown in SEQ ID NO: 2.
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