CN115894647A - Alfalfa MsWRKY41 transcription factor and application thereof - Google Patents
Alfalfa MsWRKY41 transcription factor and application thereof Download PDFInfo
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Abstract
The invention discloses an alfalfa MsWRKY41 transcription factor and application thereof, and relates to the field of plant genetic engineering, wherein the amino acid sequence of a transcription factor protein is shown as SEQ ID NO. 1; the nucleic acid sequence of the coding gene is shown in SEQ ID NO. 2. Constructing a recombinant cloning vector containing the MsWRKY41 transcription factor gene, a plant expression vector containing the MsWRKY41 transcription factor gene and a host cell transformed by genetic engineering. The WRKY transcription factor of the alfalfa is provided, 1 gene related to chloroplast development and aluminum toxicity resistance is identified through a reverse genetics means, genetic transformation is carried out in the alfalfa, the biological function of the alfalfa is analyzed, and gene resources are provided for cultivating alfalfa varieties with high photosynthetic efficiency and aluminum toxicity resistance.
Description
Technical Field
The invention relates to the field of plant genetic engineering, in particular to an alfalfa WRKY transcription factor and application thereof in chloroplast development regulation and control and improvement of aluminum toxicity resistance of plants.
Background
Alfalfa (Medicago sativa L.) is a perennial legume, has good palatability, high protein content, and good reputation of the king of pasture, and is a high-quality and high-efficiency development life line of the milk industry. Chloroplasts are important sites for plant photosynthesis and are critical to maintaining normal plant growth and development. Al in acid soil (pH less than or equal to 5) 3+ Or Al [ OH ]] 4 - The compound has toxic action on plants, and severely inhibits the growth of the plants in acid soil.
Chloroplast development is controlled by the nucleus and chloroplast genome, and the nuclear gene encodes chloroplast proteins, which account for about 96%. During chloroplast development, the expression of chloroplast proteins encoded by nuclear genes is precisely regulated by different transcription factors. The WRKY transcription factor is one of the largest transcription factor families in plants and is widely involved in the growth and development of the plants and the response process of biotic and abiotic stresses. The WRKY transcription factor contains 1 or 2 conserved DNA binding motifs (WRKYGQK) and 1 zinc finger motif (CX) 4–5 CX 22–23 HXH or CX 7 CX 23 HXC), a core acting element W-box and PRE4 element (TGCGCTT), sucrose response element SURE (TAAAGATTACTAATAGGAA), SURE-like element, etc., which can specifically recognize a downstream target gene promoter.
At present, only a few studies find that WRKY transcription factors participate in regulating chloroplast development and aluminum toxicity resistance. Wherein, arabidopsis WRKY13, WRKY40 and WRKY57 can be combined with W-box elements in LHCB2.4 and HHEMA1 promoters, and the 2 genes are marker genes of chloroplast function and stress response. WRKY40 can also interact with the C-terminal of chloroplast protein magnesium protoporphyrin IX chelate enzyme H subunit (CHLH/ABAR) to jointly regulate and control the response of plants to ABA. When plants respond to aluminum toxicity stress, WRKY transcription factors are regulated and controlled by pathway relations such as AtWRKY46-AtALMT1, osWRKY22-OsFRDL4 and AtWRKY47-AtXTH17/AtELP, and the like, so that the secretion and cell wall characteristics of malic acid and citric acid in root systems are influenced.
Alfalfa is an autotetraploid for perennial and cross pollination, has a complex genetic background, and still faces great challenges in variety breeding and gene resource mining through a traditional breeding mode.
Therefore, the technical personnel in the field are dedicated to developing the alfalfa WRKY transcription factor, and through a reverse genetics means, the candidate gene is subjected to genetic transformation in alfalfa and the biological function of the alfalfa is analyzed, so that theoretical reference and practical guidance significance are provided for cultivating the alfalfa varieties with high photosynthetic efficiency and aluminum toxicity resistance.
Disclosure of Invention
In view of the above defects in the prior art, the technical problem to be solved by the present invention is an alfalfa WRKY transcription factor protein, wherein 1 gene related to chloroplast development and aluminum toxicity resistance is identified, genetic transformation is performed in alfalfa to analyze the biological function of alfalfa, and gene resources are provided for cultivating alfalfa varieties with high photosynthetic efficiency and aluminum toxicity resistance.
In order to realize the purpose, the invention provides an alfalfa MsWRKY41 transcription factor protein, and the amino acid sequence is shown in SEQ ID No. 1.
Further, the protein also includes a protein obtained by adding 1 to 20 amino acids or less to the C-terminal and/or N-terminal of the amino acid sequence shown in SEQ ID NO. 1.
Further, an alfalfa WRKY transcription factor protein, comprising the following proteins (a), (b) or (c):
(a) A protein consisting of an amino acid sequence shown in SEQ ID No. 1;
(b) And (b) protein which is derived from (a) and has the characteristics of WRKY transcription factor, wherein 1-28 amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID No. 1.
(c) An amino acid sequence having at least 92% homology with the amino acid sequence shown by SEQ ID No. 1.
Furthermore, the alfalfa WRKY transcription factor protein is artificially modified and has an amino acid sequence with the identity of more than or equal to 92 percent compared with the amino acid sequence of SEQ ID NO. 1.
Further, the amino acid sequence comprises a typical WRKY structural domain and a C2HC type zinc finger structure, belonging to the III subfamily in the WRKY transcription factor family.
In a second aspect of the invention, a coding gene of the alfalfa MsWRKY41 transcription factor protein is provided, and the nucleic acid sequence is shown in SEQ ID No. 2.
Further, the nucleic acid sequence is obtained by cloning from alfalfa and/or artificial synthesis methods.
Furthermore, the alfalfa WRKY gene nucleic acid sequence is derived from alfalfa 'WL525' variety and named as MsWRKY41.
Further, the nucleic acid sequence is:
(d) The base sequence is shown as 1 st to 1002 th sites of SEQ ID NO. 2;
or (e) a sequence having at least 93.4% homology to the nucleic acid shown in positions 1 to 1002 of SEQ ID NO. 2;
or (f) a sequence capable of hybridizing with the nucleic acid shown in positions 1-1002 of SEQ ID NO. 2.
Furthermore, the nucleic acid sequence is a DNA sequence which is artificially modified and has identity more than or equal to 93.4% with the nucleic acid sequence of SEQ ID NO. 2.
Further, the nucleic acid sequence is specifically a sequence formed by deletion, insertion and/or substitution of 1-66 nucleotides in the nucleic acid sequence shown in 1 st-1002 th site of SEQ ID NO.2, or addition of 60 nucleotides at the 5 'and/or 3' end.
In a third aspect of the invention, the invention further provides a recombinant cloning vector containing the MsWRKY41 transcription factor gene.
In the fourth aspect of the invention, the invention also provides a plant expression vector containing the MsWRKY41 transcription factor gene.
Further, when the gene of the present invention is used to construct a plant expression vector, any one of an enhancer promoter and an inducible promoter may be added in front of the transcription initiation nucleotide.
In the fifth aspect of the invention, the invention also provides a genetically engineered host cell, wherein the host cell contains the MsWRKY41 transcription factor gene or a recombinant cloning vector and an expression vector containing the constructed MsWRKY41 transcription factor gene.
Further, the host cell is an E.coli cell or an Agrobacterium cell.
Further, the expression vector carrying the MsWRKY41 of the present invention can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and culture the transformed plant tissues into plants.
Further, the host to be transformed may be either a monocotyledonous plant or a dicotyledonous plant.
The sixth aspect of the invention also provides application of the alfalfa MsWRKY41 transcription factor protein in regulating and controlling alfalfa chloroplast development or improving alfalfa aluminum toxicity stress resistance.
The seventh aspect of the invention also provides application of the encoding gene of the alfalfa MsWRKY41 transcription factor protein in regulating and controlling alfalfa chloroplast development or improving alfalfa aluminum toxicity stress resistance.
The eighth aspect of the invention also provides the application of the recombinant cloning vector in the genetic engineering for regulating and controlling the development of plant chloroplasts and improving the aluminum toxicity resistance of plants.
In the ninth aspect, the invention also provides the application of the plant expression vector in the gene engineering for regulating and controlling the development of plant chloroplasts and improving the aluminum toxicity resistance of plants.
In the preferred embodiment 1 of the present invention, the cloning and sequence analysis process of the MsWRKY41 gene is described in detail;
in another preferred embodiment 2 of the present invention, the tissue expression pattern and response pattern to aluminum stress of the MsWRKY41 gene are specified;
in another preferred embodiment 3 of the invention, the subcellular localization process of MsWRKY41 in tobacco epidermal cells is detailed;
in another preferred embodiment 4 of the present invention, the process of obtaining the alfalfa transformed with the MsWRKY41 gene is described in detail;
in another preferred embodiment 5 of the invention, the leaf phenotype and chlorophyll content of the MsWRKY41 transgenic alfalfa are specified;
in another preferred embodiment 6 of the invention, the chloroplast ultrastructure of the MsWRKY41 transgenic alfalfa is detailed;
in another preferred embodiment 7 of the present invention, the aluminum toxicity resistance analysis process of MsWRKY41 transgenic alfalfa is detailed.
The beneficial technical effects of the invention are as follows:
1. the invention clones an MsWRKY41 transcription factor gene related to alfalfa development and chloroplast stress resistance, is induced and expressed by Al, is highly expressed in leaves, and the encoded protein is positioned in cell nucleus. MsWRKY41 can regulate and control the chloroplast development of alfalfa, and can improve the aluminum toxicity resistance of alfalfa.
2. The invention provides a basis for effective application of the alfalfa, and has important significance for improving photosynthesis and stress resistance of plants, particularly for cultivating the alfalfa variety with aluminum toxicity resistance.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a diagram illustrating the result of PCR amplification of the MsWRKY41 gene according to a preferred embodiment of the present invention;
FIG. 2 is a diagram showing a response pattern of the MsWRKY41 gene to aluminum stress according to a preferred embodiment of the present invention;
FIG. 3 is a diagram showing a tissue expression pattern of the MsWRKY41 gene according to a preferred embodiment of the present invention;
FIG. 4 is a schematic diagram of the subcellular localization of MsWRKY41 in tobacco epidermal cells according to a preferred embodiment of the present invention;
FIG. 5 is a graph of leaf phenotype and chlorophyll content of wild-type and MsWRKY41 transgenic alfalfa of a preferred embodiment 5 of the present invention;
FIG. 6 is a chloroplast ultrastructure of wild type and MsWRKY41 transgenic alfalfa according to a preferred embodiment 6 of the present invention;
FIG. 7 is a physiological phenotype graph of wild-type and MsWRKY41 transgenic alfalfa under aluminum stress according to a preferred embodiment of the present invention 7;
FIG. 8 is a graph showing the aluminum ion content of wild-type and MsWRKY41 transgenic alfalfa under aluminum stress according to a preferred embodiment 7 of the present invention.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
Example 1: msWRKY41 gene cloning and sequence analysis
Extracting total RNA of alfalfa and synthesizing cDNA: total RNA from alfalfa 'WL525' leaves was extracted using the EasyPure Plant RNA Kit (from all-purpose gold), and cDNA was synthesized by reverse transcription using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (from all-purpose gold).
Design and synthesis of primers: according to gene chip data of Medicago sativa L under Al and contrast treatment, WRKY gene segments with differential expression are selected, comparison is carried out in NCBI, and WRKY genes with higher homology with the genes in Medicago truncatula are found. Designing a primer according to the gene sequence, and carrying out homologous cloning by taking cDNA of alfalfa 'WL525' as a template, wherein the sequences of an upstream primer and a downstream primer are shown as follows: msWRKY41-F:5 'ATGGAAGACTACCAAGAGAAAACG-3', msWRKY41-R:5 'TTAAGAAGAGAAATATTCAGGGGTATT-3'. Performing PCR amplification by using the cDNA as a template according to the following reaction system and conditions: mu.L of a 20. Mu.L system containing 10. Mu.L of 2 XEX Taq super PCR Mix (purchased from TaKaRa), 0.8. Mu.L of each of 10. Mu.M primer F and primer R, 1. Mu.L of cDNA, and 20. Mu.L of deionized water. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min; 30 cycles of 94 ℃ 30s,57 ℃ 30s,72 ℃ 1min; extension at 72 ℃ for 7min. The amplified fragment is recovered and linked with a cloning vector pMD18-T (purchased from TaKaRa), and is subjected to sequencing by a biological manufacturer after identification, and the sequence result is shown as SEQ ID NO. 2.
The PCR amplification result of the MsWRKY41 gene sequence is shown in figure 1, a band between 1000bp and 2000bp is amplified through PCR by taking the DNA standard molecular weight as reference, and the MsWRKY41 gene sequence with the full length of 1002bp is identified through sequencing, encodes a protein consisting of 333 amino acid residues, and comprises a conserved WRKY binding domain and a C2HC zinc finger structure.
Example 2: tissue expression mode and response mode to aluminum stress of MsWRKY41 gene
Culturing and treating alfalfa plants: removing the coating of the seeds, cleaning, uniformly distributing the seeds in a growth disc filled with filter paper, keeping the seeds moist, and selecting the seedlings with consistent growth vigor to transplant the seedlings into 1/2Hoagland nutrient solution for water culture when the seedlings germinate and grow out the first true leaves. The formula of the Hoagland culture solution is as follows: ca (NO) 3 ) 2 ·4H 2 O 0.62g/L、KNO 3 0.34g/L、KH 2 PO 4 0.06g/L、NH 4 NO 3 0.053g/L、MgSO 4 0.24g/L、MgCl 2 0.67mg/L、H 3 BO 3 0.38mg/L、MnSO 4 0.2mg/L、ZnSO 4 ·7H 2 O 0.29mg/L、CuSO 4 0.01mg/L、FeSO 4 ·7H 2 O 0.02785g/L、EDTA-Na 2 0.0373g/L, pH 5.8), 25 ℃,16h light/8 h dark condition culture.
Sampling different tissues: culturing the alfalfa seedlings for two weeks, and respectively taking terminal buds, stem nodes, internodes, leaves, axillary buds, cotyledons, roots and root tips of the alfalfa seedlings;
al treatment and sampling: adding 0 and 100 mu M AlCl into the nutrient solution respectively 3 Underground and above ground parts were taken at 0, 0.5, 1, 3, 6, 9, 12, 24h. The prepared sample is wrapped by tin foil paper, is quickly placed in liquid nitrogen, and is stored in an ultra-low temperature refrigerator at minus 80 ℃.
The total RNA extraction and cDNA synthesis were performed as in example 1. Designing a real-time fluorescent quantitative PCR primer according to the cDNA sequence of MsWRKY41, wherein the sequences of an upstream primer and a downstream primer are shown as follows: msWRKY41-qRTF:5 'AGGGTCTTTGGATGATGAA-3'; msWRKY41-qRTR:5 'and GTGTCTTCCTGTAAGTAAC-3'. The constitutive expression gene EF-alpha of alfalfa is used as an internal reference gene, and the sequences of upstream and downstream primers are shown as follows. MsEF- α -qRTF:5 'GCACCAGTGCTCGATTGC-3'; msEF-alpha-qRTR: 5 'TCGCCTGTCAATCTTGGTAACAA-3'. And (3) carrying out real-time fluorescence quantitative PCR by using the cDNA of the sample as a template by using a Bio-rad real-time quantitative PCR instrument. The reaction system contained 10. Mu.L of 2 XSSYBR qPCR Supermix (from gold), 0.4. Mu.L of each Primer F/R, 2. Mu.L of cDNA, and a total volume of water of 20. Mu.L. The reaction program is 94 ℃ for 30s;95 ℃ 5s,57 ℃ 15s,72 ℃ 15s,40 cycles. Each treatment was performed in 3 biological replicates and 3 technical replicates. By using 2 -ΔΔCT Analytical data, statistical analysis by SPSS14.0, and mapping by EXCEL.
The qRT-PCR result shows that MsWRKY41 can be induced to express by Al. The response pattern of the MsWRKY41 gene to aluminum stress is shown in FIG. 2, wherein part a in FIG. 2 represents the relative expression level values of underground parts (roots) corresponding to different treatment times, and part b represents the relative expression level values of aerial parts corresponding to different treatment times; treatment time MsWRKY41 was up-regulated in both roots and overground parts under Al treatment compared to control (0 h) and peaked at 0.5h and 24h, respectively, 14 times and 2.5 times that of control.
The tissue expression mode of the MsWRKY41 gene is shown in FIG. 3, the relative expression quantity of the MsWRKY41 gene in the terminal bud is used as a control, the expression quantity of the MsWRKY41 gene in the leaf is the highest, and the mature leaf is higher than the young leaf and the old leaf at the lower part and is up to 110 times of that of the control; the expression level of MsWRKY41 in leaf stalk, cotyledon, stem node and stem is second to lowest, and the expression level in root, root tip and terminal bud is lowest.
Example 3: subcellular localization of MsWRKY41 in tobacco epidermal cells
Construction of plant expression vectors: introducing an enzyme digestion site into the cloned MsWRKY41 gene to link to a pMD18-T vector, and then carrying out double enzyme digestion. After the gel is recovered, the gel is linked with a linearized pHB-YFP vector with the same restriction enzyme cutting sites, escherichia coli is transformed, plasmid is extracted after PCR verification of bacterial liquid, and agrobacterium-infected GV3101 is transformed.
Culturing tobacco seedlings: uniformly scattering tobacco seeds in a matrix of 1.
Preparing agrobacterium: the Agrobacterium strains containing pHB-MsWRKY41-YFP and empty were streaked for activation, and single clones were picked up in YEB liquid medium supplemented with the antibiotics Kan50 and Rif100 and grown at 28 ℃ at 200rpm to OD600=1.2. After that, the cells were centrifuged at 6000rpm for 10min, collected and resuspended to OD600=0.6 with MS liquid medium for further use.
Transient transformation of tobacco leaves: and (4) sucking the resuspended bacterial liquid by using a 1mL disposable syringe, selecting tobacco leaves with consistent growth states for injection, and marking. And observing under a laser confocal microscope after culturing for 48h in a dark environment. Wherein the subcellular localization of MsWRKY41 in tobacco epidermal cells is shown in FIG. 4, the upper part of the graph shows the imaging result of the unloaded control 35S:: YFP, and the lower part shows the imaging result of 35S:: msWRKY 41-YFP; as can be seen in the unloaded control 35S, YFP detected fluorescent signals in cytoplasm, membrane and nucleus; msWRKY41-YFP can detect fluorescence signals only in the nucleus, therefore, msWRKY41 is localized in the nucleus.
Example 4: obtaining of alfalfa transformed by MsWRKY41 gene
Preparing agrobacterium: pHB-MsWRKY41-Flag and pHellsgate-MsWRKY41RNAi agrobacterium strains stored in a refrigerator at the temperature of-80 ℃ are respectively streaked and activated on YEB solid culture media containing Kan50+ Rif100 and Spe50+ Rif100, after culture is carried out for 48 hours at the temperature of 28 ℃, monoclones are picked into YEB liquid culture media containing the same antibiotics, and the YEB liquid culture media are placed on a shaker at the temperature of 28 ℃ at the speed of 200rpm and cultured until OD600=0.8 for infection.
Preparing an explant: the explant takes alfalfa WL525 wild type strain (WL 525-7) leaves with differentiation capacity as materials. The leaf-containing culture flask was charged with 30% sodium hypochlorite solution (0.1% Tween 20), and surface-sterilized by placing on a shaker at 100rpm for 8-10 min. After which it was rinsed three times with sterile distilled water.
And (3) agrobacterium infection: and (3) centrifuging the agrobacterium liquid prepared in the first step at 6000rpm for 10min, discarding the supernatant, and suspending the supernatant by using a heavy suspension until the OD600=0.2-0.4. Pouring into a sterile culture bottle, placing sterilized sterile leaf, screwing the bottle cap, placing in a dry bottle, and vacuumizing for 10min. Then placing the culture bottle in an ultrasonic cleaner for ultrasonic treatment at 40kHz and 20 ℃ for 3min, taking out and vacuumizing for 10min.
Callus induction and differentiation: after the vacuum pumping, the leaves are clamped by a pair of tweezers and spread in a plurality of layers of sterile paper for covering, after about 30min in an ultra-clean workbench, the residual agrobacterium on the surfaces of the leaves is sucked dry, transferred to a co-culture medium and cultured for 5 days in a dark place. Thereafter, the leaves were cut into 1 × 1cm2 leaf disks with sterile scissors or a scalpel, placed on the selection medium, placed under light to induce callus and selected for 4 weeks, during which subculture was performed every 2 weeks. When more callus is differentiated, transferring the positive callus to a regeneration culture medium, and after 4 weeks, transferring the callus with differentiated bud points to a stem elongation culture medium. Transferring the leaves to a rooting culture medium after the leaves are differentiated. After the root system grows out for two weeks, the root system is transplanted into a sterile matrix and cultured and propagated in a growth chamber.
The genetic transformation method comprises the following culture medium formula references: chunxiang Fu, timothy Hernandez, chuanen Zhou et al Agrobacterium Protocols, springer New York,2015.
Screening and identifying transgenic alfalfa: approximately 0.2g of leaves from wild-type alfalfa and transgenic lines were taken, ground well in liquid nitrogen, and subjected to hygromycin and kanamycin gene validation by extracting gDNA using a Plant genomic DNA extraction kit (purchased from whole-Plant gold). And selecting positive strains for further screening.
Example 5: leaf phenotype and chlorophyll content of MsWRKY41 transgenic alfalfa
And (3) selecting wild plants and transgenic plants which grow consistently, taking stem nodes at the same positions, removing redundant leaves, only keeping the leaves with the top ends completely opened, dipping rooting powder at the cut at the bottom ends, and carrying out cuttage. After about one month, the color of the leaves was observed, and the chlorophyll content was measured. Determination of chlorophyll content: the same position of the blade, weighing fresh weight of about 0.2g, quickly placed in 8mL 95% ethanol 10mL centrifuge tube, room temperature in the dark under 150rpm shake 24h. After the leaves are decolored and cleaned, the leaves are inverted up and down and mixed evenly, diluted by 95 percent ethanol for one time, and the absorbance values of the leaves under 665nm, 649nm and 470nm are respectively measured in an enzyme-linked immunosorbent assay. The concentration was calculated according to the following formula:
ca (chlorophyll a) =13.95 × a665-6.8 × a649
Cb (chlorophyll b) =24.96 a649-7.32 a665
Cx.c (carotenoids) = (1000 × a470-2.05 × ca-114.8 × cb)/248
Total Chl (Total chlorophyll) = (Ca + Cb) × V × N/W/1000
V is the volume of the extract, N is the dilution factor, and W is the fresh weight of the tissue. And converting the chlorophyll content into mg/g according to the formula and the weighed fresh weight.
Leaf phenotype and chlorophyll content of wild type and MsWRKY41 transgenic alfalfa are shown in a graph 5, a part in the graph shows that under the normal growth condition, an MsWRKY41 interference strain (41-RNAi) shows a phenotype of leaf whitening or variegated, the growth speed is slow, and the leaf color of an overexpression MsWRKY41 (41-OE) strain is darker; the sections b-e in the figure show the chlorophyll content results, and compared with the Wild Type (WT), the chlorophyll content, chlorophyll a, chlorophyll b and carotenoid content of the leaves of the over-expression strain are obviously increased, but the content of the leaves of the RNAi strain is obviously reduced.
Example 6: chloroplast ultrastructure of MsWRKY41 transgenic alfalfa
Cutting leaf tissue about 1mm 2 After fixation with 2.5% glutaraldehyde for at least 6h, it was placed in a refrigerator at 4 ℃ overnight. Waste liquid0.8mL of 0.1M PB was added and the mixture was placed in a refrigerator at 4 ℃ for 10min and the above procedure was repeated 4 times. Adding 0.2mL of the post-fixing agent, and standing in a refrigerator at 4 ℃ for 1.5-2h. The washing was performed sequentially with 0.1M PB and pure water for 10min each 2 times. Dehydrating with 50%, 70% and 90% ethanol for 15min each time. Sequentially adding 90% ethanol: 90% acetone (1). The samples were placed in a4 ℃ freezer during each interval. Dehydrating with 100% acetone at room temperature for 10min for 3 times. Sequentially using acetone: epoxy resin (1: the epoxy resin (1: the epoxy resin (1. The pure resin was then replaced twice with 3h intervals. After the resin is replaced for the second time, embedding is carried out, and the resin is placed in an oven at 60 ℃ for polymerization. The sample was cut into 70nm thick sections on a copper grid using an ultra-microtome and stained. And then observing the ultrastructure by using a 120kv transmission electron microscope.
The chloroplast ultrastructure of wild-type and MsWRKY41 transgenic alfalfa is shown in FIG. 6, and the symbols in FIG. 6 have the following meanings: ch: chloroplast; GA: basal-particle thylakoids; ST: a stromal thylakoid; PL: a plastid pellet; VL: a vesicle-like structure.
In the figure, parts a-c represent wild type, parts d-i represent over-expressed plants; the result shows that under the normal growth condition, the chloroplast structure of the wild type and the overexpression plant is complete and is a regular spindle-shaped structure, and the wild type and the overexpression plant have clear basal granule sheet layers, clear starch granules and a small amount of lipid droplets; but the chloroplasts of the overexpression lines contain a greater abundance of thylakoid membranes than the wild type. In the figure, the j-u section shows the leaves of the RNAi interference strain, and two types of chloroplast structures were observed. A chloroplast maintains a substantially spindle-like or oval shape, has no intact thylakoid membrane structure within, and has only a few vacuolated structures and aggregated lipid droplets, as shown in sections j-l and p-r. Another chloroplast maintains a substantially fusiform or oval structure with a large number of substrate sheets inside, but does not substantially form a continuous thylakoid sheet structure, as shown in FIGS. m-o and s-u. The MsWRKY41 plays a key role in the alfalfa maintenance of normal development of chloroplast.
Example 7: aluminum toxicity resistance analysis of MsWRKY41 transgenic alfalfa
Selecting wild plants and transgenic plants which grow consistently, taking stem nodes at the same positions of the plants, removing redundant leaves, only keeping the leaves with the top completely opened, and carrying out water culture after dipping rooting powder at the cut at the bottom. After 10 days of growth, seedlings with consistent growth vigor (before treatment) were selected and respectively set as a control group (0. Mu.M AlCl) 3 ) And treatment group (100. Mu.M AlCl) 3 ) The culture is carried out. After 21 days of treatment, the growth phenotype was observed and samples were taken to determine the Al content of the above-ground and underground parts.
The physiological phenotype of wild-type and MsWRKY41 overexpression alfalfa under aluminum stress is shown in FIG. 7, and the results show that the transgenic strain line is 100 mu M AlCl 3 The inhibition degree of the growth under the treatment is obviously smaller than that of the wild type; the aluminum ion content of wild-type and MsWRKY41 over-expressed alfalfa under aluminum stress is shown in FIG. 8, and the ion content of aerial and underground parts of the wild-type and MsWRKY41 over-expressed alfalfa is also significantly lower than that of the wild-type alfalfa. Therefore, msWRKY41 positively regulates the aluminum toxicity resistance of alfalfa.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (10)
1. The alfalfa MsWRKY41 transcription factor protein is characterized in that the amino acid sequence of the transcription factor protein is shown in SEQ ID No. 1.
2. The alfalfa MsWRKY41 transcription factor protein of claim 1, further comprising a protein obtained by adding up to 1-20 amino acids to the C-terminus and/or the N-terminus of the amino acid sequence shown in SEQ ID No. 1.
3. The encoding gene of alfalfa MsWRKY41 transcription factor protein as claimed in claim 1, wherein the nucleic acid sequence of the gene is shown in SEQ ID No. 2.
4. Constructing a recombinant cloning vector containing the MsWRKY41 transcription factor gene.
5. Constructing a plant expression vector containing the MsWRKY41 transcription factor gene.
6. A genetically engineered host cell, wherein the host cell contains an MsWRKY41 transcription factor gene, or a recombinant cloning vector and an expression vector containing the constructed MsWRKY41 transcription factor gene.
7. The use of the alfalfa MsWRKY41 transcription factor protein of any of claims 1 or 2 to regulate alfalfa chloroplast development or increase alfalfa aluminum tolerance stress.
8. The use of the alfalfa msWRKY41 transcription factor protein coding gene of claim 3 for regulating alfalfa chloroplast development or increasing alfalfa aluminum toxicity stress tolerance.
9. Use of the recombinant cloning vector of claim 4 in genetic engineering to regulate plant chloroplast development and improve aluminum toxicity resistance in plants.
10. The use of the plant expression vector of claim 5 in genetic engineering for regulating plant chloroplast development and increasing aluminum toxicity resistance in plants.
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