CN111826383A - Application of Danbo black soybean superoxide dismutase gene in improving plant aluminum tolerance - Google Patents

Abstract

The invention discloses an application of a Danbo black soybean superoxide dismutase gene GmSOD in improving plant aluminum tolerance, wherein the GmSOD gene is recombined into a plant expression vector, wild tobacco is converted, and tobacco with the converted GmSOD gene is obtained by screening; the experimental result shows that the relative root length reduction amplitude of the transgenic tobacco is smaller than that of WT under the same concentration stress and the same time; the transgenic tobacco root tip shows obvious reduction when the aluminum stress is as high as 400 mu M; root tip H⁺The activity of the pump is higher than that of WT type tobacco after being stressed for 24 hours at different aluminum concentrations, and the capability of secreting hydrogen is strong; the aluminum stress can obviously enhance the PMH of the root tip of the transgenic tobacco⁺-APase activity and induction of root tip thereofThe secretion of citric acid; the result shows that the aluminum resistance of the tobacco with the GmSOD gene is enhanced compared with that of the wild tobacco.

Description

Application of Danbo black soybean superoxide dismutase gene in improving plant aluminum tolerance

Technical Field

The invention relates to the technical field related to molecular biology and genetic engineering, in particular to application of a Danbo black soybean superoxide dismutase gene GmSOD in improving plant aluminum tolerance.

Background

Aluminum is one of the major factors limiting plant growth in acid soils. Currently, about 50% of cultivated lands worldwide are affected by aluminum poisoning of acid soils. When the pH of the soil is lower than 5, Al³⁺Released into the soil, mainly with Al (OH)²⁺、Al(OH)³⁺And Al (H)₂O)³⁺Three soluble forms exist, causing toxic effects on plants. With the increase of acid rain in the global range, the soil acidification is obviously increased, and the toxic effect on crops is enhanced. The aluminum toxicity mainly affects the root system elongation of the plant and further affects the absorption of the plant to nutrient components, and the root tip is a main organ of the plant for the feeling and the response of the plant to the aluminum stress. The main influence of aluminum poisoning on the root system is that the main root is shortened and thickened, the root tip is enlarged and yellowed, lateral root hairs are reduced, the root cap falls off, the biological oxidation capability of the root system is obviously reduced, and the like. In addition to aluminum poisoning affecting plant root tip growth, plant stem growth is also inhibited. After aluminum poisoning, some plant leaves have the phenomena of phosphorus deficiency, purple red veins, leaf senescence, yellowing and necrosis of leaf tips, curling young leaves and the like. The traditional method for improving acid soil is to use lime and a complexing agent, but the effect is not ideal. Therefore, the transgenic aluminum-resistant plant variety is cultivated by utilizing the genetic engineering technology to improve the aluminum toxicity capacity of the plant in acid-resistant soil and improve the continuous productivity of the soil.

When plants are subjected to abiotic stress, SOD is the first defense line for eliminating active oxygen accumulated in vivo and reaction products thereof, and can catalyze O in all subcellular compartments, such as mitochondria, chloroplasts, peroxidized objects, cell nucleus, cytoplasm and apoplast₂ ^.-Conversion to H₂O₂And O₂. SOD can be divided into Fe-SOD, Cu/Zn-SOD and Mn-SOD according to the metal auxiliary factor of the active site. The Fe-SOD is mainly distributed in chloroplasts,a small amount is distributed in apoplast and mitochondrial peroxisomes; Cu/Zn-SOD is distributed in chloroplast, cytosol and peroxisome; Mn-SOD is mainly localized in mitochondria. Mn-SOD and Fe-SOD were reported to be older than Cu/Zn-SOD, since they are thought to be produced by the same homologous enzyme. All types of SOD are nuclear-encoded and are regulated by various abiotic stress factors. Under the stress of adverse environments such as low temperature, ozone, water shortage, salt stress, aluminum stress and the like, the SOD gene is introduced into the plant, so that the ROS can be eliminated by the plant more effectively. Overexpression of SOD genes in plants can increase stress tolerance. SOD is the key enzyme for protecting plants from adversity and oxygen toxicity. SOD activity is closely related to the structure, growth and development and functional stability of a plant cell membrane system. The improvement of antioxidant enzyme activity in plants can enhance the resistance of plants to various oxidative stresses, and various types of exogenous SOD genes are transformed into plants. The results show that the SOD overexpression can enhance the resistance of the plants to environmental stress to different degrees, but researches show that the SOD overexpression can not improve the oxidation resistance of the plants. The MnSOD tobacco is over-expressed, so that the cell membrane protection effect and the tolerance of oxygen stress are improved; over-expressing FeSOD maize also produced similar effects, but did not improve tolerance to salt and cold stress. Although the overexpression of SOD in plants can improve the tolerance to oxidative stress of transgenic plants to some extent, the improvement of the resistance is very limited. In general, the elimination of active oxygen involves a series of enzymatic reactions and cellular metabolic processes, and the enhancement of only one of these enzymes does not significantly improve the antioxidant capacity of the body. At present, the influence of SOD overexpression on the stress resistance of plants is difficult to draw conclusions at present.

At present, no report about the effect of the Danbo black soybean superoxide dismutase gene GmSOD in the aluminum stress process is found.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a new application of the Danbo black soybean superoxide dismutase gene GmSOD, namely the Danbo black soybean superoxide dismutase gene GmSOD is applied to improving the tolerance of plant aluminum, and the GenBank accession number of the Danbo black soybean superoxide dismutase gene GmSOD is as follows: m64267.1. The GmSOD gene is used for constructing a transgenic plant with enhanced aluminum tolerance, can be used for further research on the gene, and can also be planted in strong acid soil aluminum poisoning soil.

In order to achieve the above object of the present invention, the technical solution of the present invention is as follows:

1. the following experiments were performed by selecting the model plant tobacco and soybean superoxide dismutase gene GmSOD as experimental material:

the method comprises the steps of adopting Gateway technology to construct a prokaryotic expression vector pGEX-4T-1-GmSOD of the GmSOD gene, cloning the full length of the GmSOD gene from the root of the Salvia miltiorrhiza Bunge black soybean, successfully constructing the GmSOD prokaryotic expression vector, inducing expression protein in BL21, and purifying to obtain the GmSOD protein. The enzyme characteristic analysis shows that the optimal temperature of the GmSOD is 60 ℃, and the thermal stability is good; the optimum pH value is 7.5, the pH value is more stable in the range of 5.5-9, the acid-base stability is better, but the enzyme activity is obviously inhibited and is easy to inactivate when the pH value is less than 5.5 or more than 9; 1mmol/L K⁺、Mg²⁺、Ca²⁺And Al³⁺Activates enzyme activity and activates action K⁺>Mg²⁺>Ca²⁺>Al³⁺The enzyme activity can be inhibited when the concentration of the metal ions is 3 mmol/L; 3mmol/L Cu²⁺And Mn²⁺All inhibit enzyme activity, but to a different extent, Cu²⁺The inhibition effect is stronger; na (Na)⁺Has less influence on enzyme activity.

The GmSOD gene plant expression vector pK2WG-35S-GmSOD is successfully constructed by using Gateway technology, and tobacco is transfected by agrobacterium pMP90 to obtain tobacco with transferred GmSOD gene. Tobacco was grown in 0, 50, 100, 200 and 400. mu. mol/L aluminum solution for 7 days, wild type tobacco root tip H₂O₂The content and the Malondialdehyde (MDA) content are both obviously higher than those of the transgenic tobacco, which shows that the aluminum resistance of the transgenic tobacco is stronger than that of the wild tobacco.

2. Plant expression vector pK2WG-35S-GmSOD gene constructed by Gateway technology

Extracting soybean total RNA by using a TRIzol Reagent according to the instruction; and reverse transcription is carried out to obtain the cDNA of the GmSOD. Searching a CDS sequence (M64267.1) of Glycine max SOD at NCBI, and designing double enzyme cutting sites (BamH I and Salt I) and gene specific primers of the CDS sequence through DNAman software;

upstream primer 5' -ATGGCCTCATTGGGTGGGTTA (containing BamH I cleavage site)

The downstream primer 3' -GTCCGACTCATGCACTGGTAATTAAAGCC (containing a SaltI cleavage site);

performing RT-PCR amplification by using a GmSOD gene primer to obtain a GmSOD coding region full-length DNA fragment; connecting to pMD18-T vector by enzyme digestion, transforming Escherichia coli DH5 alpha by heat shock, and obtaining TA clone containing correct sequence by resistance screening and sequencing; then enzyme digestion connection is carried out to connect the fragment to a pENTR-2B carrier, and an entry carrier pENTR-GmSOD is obtained through resistance screening and sequencing; recombining the GmSOD to a target vector pK2GW7 by LR reaction according to the instruction of an LR reaction kit LR Clonase TM plus Enzyme Mix (purchased from Invitrogen corporation in America), and obtaining a plant expression vector pK-35S-GmSOD; transferring pK-35S-GmSOD into agrobacterium by an electrical transformation method, transforming wild tobacco by agrobacterium with correct screening and detection through a leaf disc transformation method to obtain a regenerated plant, and screening and detecting the regenerated plant;

3. detecting the genome, mRNA level, protein expression level and GmSOD activity of the transgenic plants obtained by resistance screening to obtain a plurality of GmSOD gene tobaccos, selecting GmSOD7 transgenic tobaccos with the highest POD activity (ST7), and performing propagation;

4. culturing wild tobacco and GmSOD7 tobacco in Hoagland nutrient solution for two weeks, selecting robust plants with consistent growth vigor, and culturing with 0.5mmol/L CaCl₂The Hoagland nutrient solution (pH4.3) is pretreated overnight, and then 0, 50, 100, 200 and 400 mu mol/L AlCl is added₃Treating, collecting root tips, quickly freezing by liquid nitrogen, and freezing at-80 ℃ for later use to detect various aluminum resistance indexes; 6 replicates per group with no AlCl addition₃(0. mu. mol/L) treated as a control group.

According to the invention, the GmSOD gene is transferred into the tobacco, so that the resistance of the tobacco under aluminum stress is improved, materials are provided for further research on the function of the gene, the transgenic tobacco can be planted on the acidic soil with toxic aluminum, and ideas are provided for researching other related genes.

The invention has the beneficial effects that: the tobacco with the GmSOD gene is easy to store germplasm resources in a vegetative propagation mode, can obtain seeds through cultivation for storage, and is easy to popularize for planting. In addition, the functions of the GmSOD gene and related regulation and control mechanisms thereof can be further researched by comparing the structures and physiological and biochemical indexes of the GmSOD tobacco and the wild tobacco.

Drawings

FIG. 1 shows the construction of recombinant plasmid pGEX-4T-1-GmSOD vector and the result of BL21 transformation detection, wherein A is amplification of GmSOD gene of Danbo black soybean, B is verification of pMD-18T-GmSOD vector, C is verification of pGEX-4T-1-GmSOD vector, and D is BL21 transformation detection;

FIG. 2 is a graph showing the results of the induced expression and purification of the recombinant protein pGEX-4T-1-GmSOD; wherein, A is the result of induced expression, B is the result of soluble expression analysis, and C is the result of purification;

FIG. 3 shows the temperature measurement of the optimal enzymatic reaction of GmSOD, wherein A is the effect of temperature on the activity of GmSOD, and B is the thermal stability analysis of GmSOD;

FIG. 4 shows the effect of pH on the enzymatic activity of GmSOD (panel A) and the pH stability analysis of GmSOD (panel B);

FIG. 5 is the identification of tobacco over-expressing GmSOD gene, in which the A picture is the PCR result of tobacco gene group over-expressing GmSOD gene, and the B picture is the RT-PCR detection result; m in the figure is MarkeIII; PC: taking pK-35S-GmSOD plasmid as a PCR template; transferring the wild tobacco with the empty vector by NC; WT: wild-type tobacco; ST1-ST8 transgenic tobacco lines;

FIG. 6 is a graph of RRG changes in WT and ST7 root tips at 1 day (A), 2 days (B), 3 days (C), 4 days (D) of stress at different aluminum concentrations;

FIG. 7 is a graph of H of WT (A) and ST7 root tips (B) stressed at different times with different aluminum concentrations₂O₂Content (c);

FIG. 8 is the MDA content of WT (A) and ST7 root tips (B) stressed at different times with different aluminum concentrations;

FIG. 9 shows soluble protein content of WT (A) and ST7 root tips (B) stressed at different times with different aluminum concentrations;

FIG. 10 shows the SOD activity changes of WT (panel AC) and ST7 root tips (panel BD) stressed at different times with different aluminum concentrations and the gene expression levels thereof;

FIG. 11 shows POD activity changes and gene expression levels of WT (panel AC) and ST7 root tips (panel BD) stressed at different times with different aluminum concentrations;

FIG. 12 shows the CAT activity changes and gene expression levels of WT (FIG. AC) and ST7 root tips (FIG. BD) stressed at different times with different aluminum concentrations;

FIG. 13 shows the change in APX activity and gene expression level of WT (panel AC) and ST7 root tips (panel BD) stressed at different times with different aluminum concentrations;

FIG. 14 is PM H of WT (panel AC) and ST7 root tips (panel BD) stressed at different times for different aluminum concentrations⁺-ATPase enzymatic activity and its gene expression;

FIG. 15H of WT (A) and ST7 root tips (B) stressed for 12H at different Al concentrations⁺Pump activity;

FIG. 16 is PM H of WT and ST7 root tips stressed with 100(A), 200. mu. mol/L (B) aluminum concentration for 12H and 24H⁺-ATPase and 14-3-3 protein interaction level gel imager results (panel AB) and relative quantification analysis (panel C);

FIG. 17 shows the citric acid secretion of WT and ST7 root tips stressed for 1, 2, 3, 4 days with different aluminum concentrations;

FIG. 18 shows PM H of WT and ST7 root tips stressed for 24, 48 hours with different aluminum concentrations⁺Correlation analysis of APase activity and citric acid secretion.

Detailed Description

The invention is explained in more detail below with reference to examples and figures, without limiting the scope of the invention. In the examples, the procedures were carried out in accordance with the usual procedures unless otherwise specified, and the reagents used were either conventional commercial reagents or reagents prepared in accordance with the conventional procedures unless otherwise specified

Example 1: prokaryotic expression vector construction of GmSOD gene and protein expression purification thereof

Extracting total RNA of root tips of the salvia miltiorrhiza bunge black soybeans by using TRIZOL Reagent (Invitrogen), and operating according to the instruction; using M-MLV Reve25. mu.L of RNA was reverse transcribed into cDNA using rse Transcriptase (Fermentas corporation) reverse transcription kit; the target fragment was amplified by RT-PCR using 1. mu.L of cDNA as a template. The CDS sequence of Glycine max SOD was searched at NCBI (M64267.1), and its double cleavage sites (BamHI and SaltI) and gene specific primers (F-ATGGCCTCATTGGGTGGGTTA; R-TCATGCACTGGTAATTAAAGCC) were designed by DNAman software. PCR amplification is carried out by using a designed specific primer, a GmSOD gene segment is recovered by glue (figure 1A), and the GmSOD gene segment is stored at the temperature of-20 ℃ for standby. Recovering the GmSOD gene from the gel, performing TA cloning with pMD18-T to obtain a pMD18-GmSOD vector, thermally shocking to transform DH5 alpha, coating and inoculating in an ampicillin (Amp) solid culture medium, and culturing overnight in a constant temperature box at 37 ℃. And (3) selecting a single bacterial colony in a liquid culture medium for screening culture in the next day, taking 1 mu L of bacterial liquid as a DNA template for PCR detection after the bacterial liquid is turbid (figure 1B), and sending the successfully detected bacterial liquid extracted plasmid to a company for sequencing. The sequencing results were aligned with GmSOD using DNAman, and the correctly aligned pMD18-GmSOD plasmid and pGEX-4T-1 vector were double digested using BamH I and Salt I, respectively (25. mu.L plasmid, 5. mu.L BamH I, 5. mu.L Salt I, 10. mu.L 10 XBuffer, 5. mu.L ddH)₂O), observing the target fragment by gel electrophoresis, and carrying out gel recovery on the target gene fragment GmSOD and the cut pGEX-4T-1 vector fragment. Recovering the obtained fragments, and connecting the fragments in a metal bath at 16 ℃ overnight to obtain pGEX-4T-GmSOD plasmids; pGEX-4T-GmSOD plasmid is transferred into DH5 alpha through heat shock, coated in Amp solid culture medium, cultured overnight in a thermostat at 37 ℃, single colony is selected to be cultured in Amp liquid for a large amount of proliferation culture, 1 mu L culture solution is taken for PCR detection, and plasmid double enzyme digestion detection is extracted after the detection is successful (figure 1C).

The prokaryotic expression vector pGEX-4T-1-GmSOD plasmid successfully detected by double enzyme digestion is transferred into escherichia coli BL21 protein expression bacteria through heat shock transformation, is coated in an Amp-containing solid culture medium and is cultured overnight in a thermostat at 37 ℃, a single bacterial colony is selected and screened and cultured in an Amp liquid culture medium, and the culture medium for normal growth of the bacterial body is subjected to bacterial liquid PCR detection (figure 1D). Inoculating the successfully detected bacterial liquid into 25mL LB liquid culture medium, culturing in a shaking table (37 ℃, 220rpm) until the OD value of the bacterial liquid is 0.5, and then adding IPTG to make the final concentration respectively 0.5mmol/L and 1 mmol/L; respectively carrying out induction expression on the culture solution added with the inducer in shaking tables at 16 ℃, 28 ℃ and 37 ℃, taking 2mL of bacterial solution after induction for 0, 2, 4 and 6 hours, respectively, centrifuging for 1min at 12000rpm, collecting thalli, adding 2mL of PBS (phosphate buffer solution) for heavy suspension, taking 20 mu L of the thalli after heavy suspension, adding 20 mu L of 2 x protein electrophoresis sample loading buffer solution, boiling for 10min in boiling water, cooling, and carrying out polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis to analyze protein expression (figure 2A); the GmSOD protein is only expressed at 28 ℃ and the expression level of the protein is maximum at 0.5mmol/L IPTG and 6 h.

The GmSOD gene recombinant expression strain is induced for 6 hours in a large quantity under the conditions of 28 ℃ and 0.5mmol/L IPTG, the thalli sediment is repeatedly washed three times by 4ml LTBS, 2ml LPBS is added to resuspend the thalli sediment, ultrasonic crushing is carried out for 15min, the supernatant and the sediment are obtained by centrifugation, and SDS-PAGE analysis is carried out on the supernatant to find that the expression protein exists (figure 2B). Under the optimal condition, inducing the strain to express protein in a large amount; centrifuging at 4 deg.C and 12000rpm for 15min to collect thallus precipitate, adding PBS for resuspension, placing the resuspended thallus into a 50mLEP tube, and placing the tube in ice to break cells for 15min by ultrasound; after centrifugation, the supernatant was collected, and the supernatant containing a large amount of the desired expression protein was applied to a GST column to obtain a purified SOD fusion protein, and 20. mu.L of the supernatant was subjected to 12% SDS-PAGE (FIG. 2C).

Example 2: determination of enzymatic Activity of superoxide dismutase

Measuring the activity of the superoxide dismutase by an NTB reduction method; 3mL of a reaction mixture (5mmol/L of pH7.8 phosphate buffer, 13. mu. mol/L methionine solution, 63. mu. mol/L nitroblue tetrazolium solution, 1.3. mu. mol/L riboflavin, 0.1mmol/L EDTA-Na₂Solution) is added with 20 mul of supernatant enzyme solution, a control test tube is replaced by PBS, the control test tube is placed in a dark place, and the other tubes react for 20-30min under 4000lx sunlight; after the reaction, a control tube was used as a blank, and the absorbance was measured at 560 nm. Calculating the formula: the enzyme amount required for inhibiting NBT photochemical reduction by 50% is taken as an enzyme activity unit (U).

(1) Optimum temperature and thermal stability of superoxide dismutase

Measuring SOD enzyme activity at 10, 20, 30, 40, 50, 60, 70, 80 and 90 deg.C under pH7.5, and determining optimum temperature; storing the enzyme solution at 10, 20, 30, 40, 50, 60, 70, 80 and 90 deg.C for 60min, rapidly cooling to measure enzyme activity, and determining enzyme stability in the temperature range; the untreated enzyme activity was 100%;

the determination result shows that the enzyme activity of the GmSOD is rapidly increased within the range of 15-60 ℃; after the temperature is higher than 60 ℃, the enzyme activity is rapidly reduced along with the temperature rise, which shows that the enzyme has stronger heat resistance and shows that the high temperature has an inhibition effect on the enzyme activity; the GmSOD enzyme has the maximum relative enzyme activity at 60 ℃, and the temperature can be considered as the optimal temperature of the enzyme. The activity of the GmSOD enzyme is kept above 60 percent within the range of 0-80 ℃, and the thermal stability of the enzyme activity is the best at 60-70 ℃, and is all above 80 percent; after the temperature is over 70 ℃, the enzyme activity stability is gradually reduced; the thermal stability of the GmSOD enzyme is obviously reduced above 80 ℃, which shows that higher temperature has larger influence on the enzyme activity (figure 3).

(2) Optimum pH and pH stability of superoxide dismutase

Reaction solution systems (50mmol/L phosphate buffer) with different pH values are prepared, and enzyme solution is added to determine the enzyme activity. Dissolving a certain amount of GmSOD enzyme solution in buffer solutions with different pH values of 4-9.5, placing the buffer solutions at 4 ℃ for 2 hours, and adding reaction liquid to determine the enzyme activity;

the determination result shows that the activity of the GmSOD enzyme changes rapidly when the pH is within the range of 3.5-5; the pH value rises slowly at 5-7.5, and the enzyme activity reaches the highest at 7.5; when the pH value is within the range of 7.5-10, the activity of the GmSOD enzyme is rapidly reduced. The SOD is incubated for 2 hours in different pH systems, and the relative enzyme activity reaches more than 60 percent and is relatively stable within the pH range of 5.5-9.0. Outside this pH range, the relative activity of the enzyme decreases. Within the pH range of 6.5-7.5, the relative enzyme activity can reach about 80% of the original activity, which shows that the GmSOD has good alkali tolerance; the relative enzyme activity of the GmSOD reaches about 40% when the pH of the GmSOD is 3.5 compared with that of the GmSOD and when the pH of the GmSOD is 9.5 compared with that of the GmSOD, which shows that the enzyme activity of the GmSOD is greatly influenced by acid or alkali (figure 4).

(3) Effect of Metal ions on enzymatic Activity of superoxide dismutase

Respectively adding 3mmol/L and 9mmol/L Na into the reaction system⁺、Cu²⁺、Mn²⁺、K⁺、Mg²⁺、Ca²⁺、Al³⁺Metal ions at optimum temperatureMeasuring the enzyme activity of the GmSOD at the pH value and the optimal temperature; the enzyme activity without treatment was 100%. The measurement result shows that K⁺、Mg²⁺、Ca²⁺、Al³⁺Low concentrations activate enzyme activity and activation K⁺>Mg²⁺>Ca²⁺>Al³⁺，3mmol/L K⁺The enzyme activity of the GmSOD can be improved by 48 percent; high concentrations inhibit enzyme activity; cu²⁺、Mn²⁺All inhibit enzyme activity, but to a different extent, Cu²⁺The inhibition effect is stronger; 3mmol/L and 9mmol/L Cu²⁺When treating enzyme, the enzyme activity is within 20 percent and is in an inactivated state; na (Na)⁺The influence on the enzyme activity is small; the metal ions have strong affinity with the enzyme activity products in different ways, resulting in the change of the enzyme activity; the results show that different metal ions have different influences on the activity of the GmSOD; the experimental indexes are determined repeatedly for three times, the experimental data are statistically analyzed by Excel 2010, and the results are shown in the following table:

example 3: construction of overexpression GmSOD transgenic tobacco strain and identification of overexpression GmSOD gene tobacco

The invention constructs a eukaryotic target expression vector pK-35S-GmSOD by Gateway technology; and (3) amplifying a target fragment (749bp) of the GmSOD gene by RT-PCR. pMD18T and the target fragment are cloned by T/A to obtain pMD18T-GmSOD plasmid, and positive clone is sequenced to detect whether exogenous gene GmSOD has mutation. And detecting the successful and non-mutated pMD18T-GmSOD plasmid and pENTR plasmid, respectively recovering a target fragment GmSOD and a cut pENTR by double-enzyme gel cutting through EcoR I and BamH I, connecting the two fragments over night to form a gate-in cloning vector pENTR-GmSOD, and observing whether the GmSOD gene is successfully cloned on the pENTR vector by gel electrophoresis after double enzyme cutting. After successful detection, the plant binary expression vector pK2GW7.0 and the entry clone vector pENTR-GmSOD are subjected to LR reaction under the action of LR Mix Enzyme to form the plant expression vector pK-35S-GmSOD for short.

Transferring a plant expression vector pK-35S-GmSOD into agrobacterium pMP90 by an electrical transformation method, obtaining positive clones by screening and culturing in a spectinomycin (Spe) solid culture medium, selecting a single bacterium, screening and culturing in an LB liquid culture medium containing the Spe, detecting the positive clones with exogenous plant expression vector plasmids by bacterial liquid PCR, transfecting tobacco by an expression vector pK-35S-GmSOD agrobacterium containing target genes by a leaf disc transformation method, and obtaining transgenic tobacco by screening in a Kan resistance culture medium.

Extracting plant genome from the transgenic tobacco with long roots in the resistant culture medium by a CTAB method, and detecting the extracted genome DNA by gel electrophoresis. Carrying out PCR reaction by taking the extracted plant genome DNA as a template, and detecting a PCR product on 1% agarose gel electrophoresis; detecting plants with exogenous GmSOD genes inserted in the genome by genome PCR, and analyzing the transcription level of the plants by RT-PCR; extracting total RNA from the transgenic strain by a TRIzol method, detecting the concentration and the quality of the RNA by a microplate reader and 1.5 percent agarose gel electrophoresis, and then reversely transcribing 25 mu L of RNA to synthesize first strand cDNA; taking 1 microliter of cDNA as a template, taking 18sRNA of tobacco as an internal reference to perform RT-PCR analysis, and detecting an RT-PCR amplification product by agarose gel electrophoresis;

transforming tobacco with pK-35S-GmSOD to obtain 8 transgenic strains with kanamycin resistance, and detecting the insertion condition of an exogenous gene by using genome DNA as a template and using a specific primer of a GmSOD gene; after PCR reaction of the genome, the following results were obtained (FIG. 5A); the results indicated that the GmSOD gene was inserted into the genome of 4 strains (ST1, ST3, ST4, ST 7); the RT-PCR analysis results (FIG. 5B) show that the exogenous SOD genes in the 4 transgenic plants can be transcribed normally, and the transcription levels of the transgenic tobacco strains are similar.

The results of DNA and RNA detection and analysis show that the exogenous GmSOD gene is accurately inserted into the tobacco genome, and the exogenous GmSOD gene can be correctly transcribed under the 35S promoter; in the experiment, a transgenic tobacco strain 7 with a higher transcription level is selected as a subsequent transgenic tobacco test material.

Example 4: aluminum stress treatment of GmSOD transgenic tobacco plants

Taking out normal tobacco from solid culture medium, cleaning root, and placing inCulturing in MS liquid culture medium diluted by 10 times; selecting wild type and transgenic tobacco seedlings which grow consistently after 2 weeks of water culture, and adding 0.5mmol/L CaCl₂(pH4.3) after an overnight pretreatment, the cells were incubated with 0, 50, 100, 200 and 400. mu. mol/L AlCl₃After 0, 12, 24, 48, 96h of treatment in solution, 3 replicates of each treatment were run, with no aluminum treatment as a control. Sucking water on the root tip by using water absorption paper, collecting the root tip with the diameter of 0-10mm, and storing the root tip at the temperature of-80 ℃ as a subsequent experimental material;

1. influence of different aluminum stress concentrations and time on relative growth amount (RRG) of tobacco root tips and detection of citric acid secretion amount of tobacco root tips

In the experiment, a transgenic tobacco strain 7 with a higher transcription level is selected as a transgenic tobacco test material; selecting plants with the same growth vigor for aluminum stress treatment, recording the root length before treatment, and using CaC1 with concentration of 0.5mmol/L₂(pH4.3) light pretreatment overnight, then respectively placed in 100, 200, 400. mu. mol/L AlCl₃(pH4.3) culturing in a thermostatic chamber at 25 ℃ for 1d, 2d, 3d and 4d, recording the length of the treated roots, collecting treated water samples, and treating each group for 6 times, wherein the treated water samples are used as a control without aluminum; relative root elongation (RRG) is the amount of root growth after aluminum treatment/the amount of root growth without aluminum treatment × 100%;

the inhibition of aluminium toxicity on root elongation is the most typical toxic phenomenon, and the aluminium tolerance of two tobaccos is analyzed by measuring the relative root growth; change of RRG after different aluminum stresses of WT and ST7 root tips for different time; when the aluminum stress is carried out for 1 day at 50 mu mol/L, the RRG of the WT root is reduced by 19 percent, and the ST7 is reduced by 9 percent, but the relative root length between the two is not significantly different; the difference between WT and ST7 root tips was evident at 2 days and 3 days of treatment. By day 4, RRG at the WT root tip was reduced to 65.5%, and RRG at the ST7 root tip was reduced to 73%, with no significant difference; the change in WT and ST7 root tips was similar to 50. mu. mol/L aluminum stress at different times for 100. mu. mol/L aluminum stress. When 200 mu mol/L aluminum is stressed for 1 day, RRG of WT roots is reduced by 37.5%, ST7 is reduced by 17%, and relative root length change between the two is not significantly different. After 2 days of treatment, the RRG of WT roots was reduced by 47.5%, ST7 by 22.5%, and there was no significant difference in the relative root length change between the two. At 4 days of treatment, there was no significant difference in the reduction of WT and ST7 root tips, indicating that the duration of aluminum stress was too long and that both tips were strongly inhibited by aluminum stress. WT and ST7 root tip RRG differed significantly when treated with 400. mu. mol/L aluminum stress for 1 and 2 days, indicating that WT with weaker aluminum resistance has suffered complete irreversible oxidative damage under high aluminum stress. When the aluminum with the concentration of 400 mu mol/L is stressed for 3 and 4 days, the RRG of WT is reduced to 27 percent, the RRG of ST7 is reduced to 39 percent, and the reduction amplitude of the RRG between the WT and ST7 point aluminum resistance mechanisms is obvious and has no obvious difference, which shows that the WT and ST7 point aluminum resistance mechanisms are invalid and present similar aluminum toxicity status quo under the condition of high-concentration stress for a long time. Under aluminum stress, both tobacco RRG show a tendency to decline. However, the magnitude of the decrease in ST7 relative to root length was smaller than that of WT under the same concentration stress for the same time, indicating that ST7 root tip was more aluminum resistant than the WT root tip. The results show that ST7 is more aluminum resistant than WT (FIG. 6);

measuring the secretion of the citric acid at the root tip according to methods of Yang and the like; aluminium-induced root citric acid secretion is widely regarded as one of the important aluminium-resistant mechanisms of plants; when no aluminum stress exists, the secretion amount of the citric acid of WT and ST7 root tips has no obvious change; at the same time of aluminum stress, the secretory volume of citric acid at the root tip of ST7 is significantly higher than that of WT root tip. The secretion of citric acid at the tip of WT and ST7 roots firstly rises and then falls along with the increase of the aluminum stress concentration, and both reach the peak value when the aluminum stress is 50 mu M; as the stress time increases, the secretion amount also decreases; the results are shown in FIG. 17, and indicate that low-concentration aluminum stress induces the secretion of citric acid by the root tip of a plant, and enhances the aluminum stress capability of the root tip.

2. Root tip of tobacco H₂O₂Content, MAD content and soluble protein content determination

H₂O₂Content determination: reagent A (3.3mM (NH) was prepared first₄)₂SO4；3.3mmol/L FeSO₄；412.5mmol/LH₂SO₄) With reagent B (165mmol/L sorbitol; 165. mu. mol/L xylenol orange). Before use, the reagent A and the reagent B are mixed according to the proportion of 1:10 and are prepared as before use. The working reagent and H₂O₂Mixing the solutions to be tested at a ratio of 2:1, developing in water bath at 30 deg.C for 30min, and ultraviolet irradiatingReading the OD560 value on a spectrophotometer; calculating the hydrogen peroxide content with reference to a standard curve (fig. 7);

and (3) determining the content of MAD: 1mL of supernatant enzyme solution was added to 1mL of ddH as a control₂O, adding 3mL of 0.67% thiobarbituric acid, sealing the opening of the boiling water bath for 15min, cooling and centrifuging, and taking the supernatant to read the light absorption values at 532nm, 600nm and 450nm for calculation. CMDA (. mu.M): 6.45X (A)₅₃₂-A₆₀₀)-0.56×A₄₅₀(ii) a MDA (μmoL g-1FW) ═ cxv/W (fig. 8);

H₂O₂is the most important active oxygen component in the plant body, and the content of the active oxygen component can reflect the oxidative damage level in the plant body; MDA is one of the products of peroxidation of plant cell membrane lipid and can be used as one of important physiological indexes of oxidative damage. H of WT and ST7 root tips stressed by different aluminum concentrations for different time₂O₂Content variation and variation in MDA content. WT and ST7 transgenic tobacco had identical H in the absence of aluminum stress₂O₂And MDA content are kept low; ST7 root-tipped H under the same concentration and time of aluminum stress₂O₂And the MDA content is increased by less amount than that of the WT root tip. WT and ST7 root tips H with increasing aluminum stress concentration and time₂O₂And the MDA content is also increasing. Treating with 50 μmol/L aluminum stress for 0-48H, WT root tip H₂O₂And MDA content change is not significantly different, and the content of the MDA content change does not exceed twice of the CK value, and the MDA content change is kept at a lower content level. The content of WT root tips has no obvious change when the aluminum stress of 100 mu mol/L is treated for 6 hours and 12 hours; the content is obviously enhanced after treatment for 24-96 h. Under the stress of 200 and 400 mu mol/L aluminum, the root tip of the WT is at a higher H₂O₂And MDA content level, indicating severe oxidation and damage to tobacco root cells. ST7 root tip H under 50, 100, 200. mu. mol/L aluminum stress₂O₂No significant difference with MDA content, H₂O₂The contents are all 0.6 mu mol g^-1With FW, the MDA content is substantially less than 50. mu. mol g^-1FW. Under the stress of 400 mu M aluminum concentration, the content change of the ST7 root tips is obviously increased along with the increase of time. Treating 24-96H, ST7 root tip H₂O₂The content is 1.0 mu mol g^-1Above FW, MThe content of DA is as high as 93.29 mu mol g^-1FW, which is about 8 times the CK value, indicates that the tobacco root tips have been subjected to irreversible aluminum poisoning under high aluminum stress. The results indicate that when the tobacco root tip is subjected to aluminum stress, the oxidative stress in the plant body is increased with the time and concentration of the stress. The root tip of wild tobacco has become seriously oxidized and damaged under the aluminum stress of 200 mu M, but the root tip of transgenic plants is oxidized and damaged under the aluminum stress of 400 mu mol/L similarly. The results show that ST7 has stronger aluminum resistance than WT plants;

determination of soluble protein content: 20 μ L of supernatant was taken and 1mL ddH was added to the control₂O, adding 3mL Coomassie brilliant blue G-250 solution (1000mL system: 100mg G-250 is dissolved in 50mL 90% ethanol, adding 100mL 85% phosphoric acid, constant volume), standing for 2min, and reading OD on ultraviolet spectrophotometer₅₉₅Values, soluble protein content calculated from the standard curve (figure 9);

soluble protein content of WT and ST7 root tips after different aluminum stresses for different time periods. With the increase of the aluminum stress concentration, the content of soluble protein of the WT or ST7 root tips is increased and then decreased. WT or ST7 root tip soluble protein content was maintained at a relatively low level under 0. mu.M aluminum stress. WT was also peaked in ST7 root tip soluble protein content under 50 μ M aluminum stress, and ST7 root tip soluble protein content was approximately 1.5 times that of WT under the same stress time. Under 100 mu M aluminum stress, the soluble protein content of ST7 root tips is still higher than that of WT root tips, and the soluble protein content is higher than that of CK. Under the stress of 200-400 mu M aluminum, the content of the soluble protein in the WT root tip is lower than that in the CK group, which indicates that the abiotic stress degree of the WT root tip is stronger, and the content of the soluble protein in the root tip is as low as 0.73 mu mol g^-1FW, about 4.57 times less compared to CK values. Under 400 μ M aluminum stress, the soluble protein of ST7 root tip was significantly reduced, indicating that ST7 root tip was severely aluminum stressed. The above results indicate that changes in soluble protein content are an important component of tobacco response to aluminum stress.

3. Detection of tobacco root tip antioxidant enzyme activity and SOD, POD, CAT, APX antioxidant enzyme genes and PM H in tobacco root tip⁺-ATPaExpression of se Gene

Weighing 0.2g of tobacco root tip, adding 2mL of protein extract (50mmol/L PBS (pH7.8); 0.2mmol/L EDTA; 0.06mmol/L PVP), grinding, and centrifuging at 1200rpm for 20min at normal temperature to obtain supernatant as enzyme solution;

SOD enzyme activity determination: 3mL of the reaction mixture (5mmol/L of pH7.8 phosphate buffer, 13. mu. mol/L methionine solution, 63. mu. mol/L nitroblue tetrazolium solution, 1.3. mu. mol/L riboflavin, 0.1mmol/L EDTA-Na₂Solution) is added with 20 mu L of supernatant enzyme solution, a contrast test tube is replaced by phosphate buffer solution, the contrast test tube is placed in the dark, the rest tubes react for 20-30min under 4000lx sunlight, after the reaction is finished, the contrast tube is used as a blank, the light absorption value is measured at 560nm, and the enzyme quantity required for inhibiting 50 percent of NBT photochemical reduction is used as an enzyme activity unit (U); the specific activity of SOD is the catalytic activity of SOD enzyme contained in each milligram of protein, and the result is shown in FIG. 10 AB;

POD enzyme activity determination: 3mL of the reaction mixture (0.1M pH6.0 phosphorus buffer; 0.46mM guaiacol, 0.06M H₂O₂) Add 20. mu.L of supernatant enzyme solution to the cuvette, add 0.1M pH7.8 PBS to the control, and record the OD every 30 seconds₄₇₀A value; specific POD Activity (Δ OD470 min)^-1g^-1protein) — total activity/protein content, results are shown in fig. 11 AB;

and (3) CAT enzyme activity determination: 3mL of the reaction solution (9mM H)₂O₂(ii) a 66mM pH7.8 Phosphorus) was added to 100. mu.L of the supernatant enzyme solution and placed in a cuvette, 3.3mM pH7.8 PBS was added to the control, and OD was recorded every 30S₂₄₀The value is obtained. CAT specific Activity (Δ OD)₂₄₀min^-1g^{- 1}protein) — total activity/protein content, results are shown in fig. 12 AB;

and (3) APX enzyme activity determination: is used in H₂O₂In the presence of APX, the enzyme activity is determined by the principle of APX reduction, and when the enzyme activity is determined, 1mL of enzyme reaction solution (50mmol/L, pH7.0 Na)₂HPO₄-NaH₂PO₄Buffer solution, 1mM ascorbic acid, 2.5mM H₂O₂) Adding 50 μ L of the supernatant enzyme solution, and mixing well; read OD every 15s in 3min on UV-visible spectrophotometer₂₉₀A value; the amount of ascorbic acid converted per mg of protein per minute was calculated as an indication of the enzyme activityIn units of μmol. min^-1·mg^-1protien, see fig. 13 AB;

in the invention, a TRIZOL Reagent (Invitrogen) method is adopted to extract RNA in a tender tissue material, 25 microliter of RNA is taken as a template, and first strand cDNA is synthesized by reverse transcriptase catalytic reaction; carrying out PCR reaction with 1 mu L cDNA as a template and a target fragment specific primer of the DNA to be amplified to amplify a target gene fragment of the SOD, and carrying out gel electrophoresis detection;

the gene of which the expression level is not influenced by aluminum stress is taken as an internal reference gene. Design 28sRNA, SOD, POD, CAT, APX and PM H⁺ATPase gene amplification primer sequences (see Table below);

taking 1 uL cDNA as template, amplifying all genes, analyzing the brightness of amplified band by gel electrophoresis, and judging SOD, POD, CAT and PM H in tobacco root tip after aluminum treatment⁺The results of the semi-quantitative PCR are shown in FIG. 10CD, FIG. 11CD, FIG. 12CD, FIG. 13CD and FIG. 14CD, and it can be seen from the figure that both WT with weak aluminum resistance and ST7 with strong aluminum resistance cause serious damage to the expression level of antioxidant enzyme at the root tip of the plant under the stress of high aluminum concentration.

Activity change of tobacco root tip antioxidant enzymes SOD, POD, CAT and APX and gene expression analysis thereof under different aluminum stress concentrations and time

The activity of SOD, POD, CAT and APX of WT and ST7 root tips changes and the gene expression level thereof after different aluminum stresses for different time. With the increase of the aluminum stress concentration, the change trend of the WT and ST7 root tip enzyme activities is increased firstly and then reduced, and the gene expression trends are consistent. The activity of the enzyme at the WT root tip was always lower than that of the SOD at the ST7 root tip at the same stress time at different aluminum stress concentrations. Under the condition of no aluminum stress, the enzyme activities of WT and ST7 root tips are not obviously changed at different times, and the initial activity of the enzyme activity of ST7 root tips is higher than that of WT, which shows that the expression amount of the enzyme gene in ST7 is high. When 50-100 mu M aluminum is stressed, the SOD enzyme activity and gene expression quantity of WT root tips and ST7 root tips are increased and then reduced along with the increase of time, and the peak value is reached in 24 hours, so that the CK value is 2-3 times; POD and CAT enzyme activity and gene expression reach peak values in 48 h; the APX enzyme activity and the gene expression level also show a remarkable increase at 48 h. When 200 mu M aluminum stress is carried out, the four enzyme activities and gene expression levels of the ST7 root tips are consistent with the changes of 50-100 mu M aluminum stress. When 200-400 mu M aluminum stress is applied, the activity of WT root tip enzyme is obviously reduced, and the trend of gene expression amount is consistent with the trend. Under 400 mu M aluminum stress, the enzyme activity and gene expression level of four enzymes at the root tip of ST7 are obviously reduced, and the SOD, POD, CAT and APX are respectively 55.19%, 46.31%, 67.11% and 71.8% of the CK value. The result shows that when the root tip of the tobacco is stressed by aluminum, the activity and the gene expression quantity of the antioxidant enzyme are increased along with the increase of the stress concentration, and the trend that the activity and the gene expression quantity are increased and then reduced appears; the first significant increase occurs as the stress time increases, reaching a maximum value at some time. SOD content and expression activity reach maximum value basically in 24h, while other antioxidant activity enzyme reaches maximum value in 48h and later. When 50 mu M of aluminum is stressed, the enzyme content in the root tips of two tobaccos is obviously increased, and the enzyme expression level is also increased. The WT root tip remained relatively steadily reduced under 100. mu.M stress; the content and the activity of the antioxidant enzyme of the root tip are obviously reduced when the 200-400 mu M aluminum stress is applied, while the root tip of the ST7 tobacco shows obvious reduction when the aluminum stress is as high as 400 mu M; the results show that the ST7 tobacco root tip has better aluminum resistance than the WT tobacco root tip.

Collecting frozen tobacco root tip 0.5g, adding liquid nitrogen, grinding into powder, adding 1.5mL reaction solution (1 mmol/LEDTA; 0.25mmol/L sorbitol; 1mmol/L MgSO 2)₄(ii) a 10mmol/L Tris-HCl), grinding again, transferring into centrifuge tube, centrifuging to obtain supernatant, and reading OD on ultraviolet spectrophotometer₅₉₅A value; 0.5mL of a reaction solution (0.02% Brij 35; 4 mmol/LATP-Na)₂；50mmol/L KCl；1mmol/L(NH₄)₂MoO₄；0mmol/L KNO₃；1mmol/L NaN₂(ii) a 50mmol/L BTP/MES) was added to 0.5g of plasma membrane protein, and after 30min water bath at 30 ℃ 1mL of reaction stop solution (0.7% SDS (w/v); (NH)₄)₂MoO₄(w/v)；2％H₂SO₄(v/v)) and 0.5mL of developing solution 20min, reading OD660 value on an ultraviolet spectrophotometer. Calculating PM H according to the standard curve y-1204 x +1.235⁺-ATPase enzyme activity; under the reaction condition of 30 ℃, the mole number of PM H with 1 unit is released by catalyzing ATP decomposition to release inorganic phosphate per mg of protein within 1min⁺-ATPase activity, results are shown in fig. 14 AB;

variation of different aluminum stress concentrations and time on activity of tobacco root tip PM H + -ATPase and analysis of gene expression quantity thereof

PM H + -ATPase is the most abundant protein on the cell membrane and has recently been found to play an important role in responding to a variety of stresses. With the increase of the aluminum stress concentration, the activity of WT and ST7 root tips and the gene expression thereof increased and then decreased, but the ST7 root tips were always higher than the WT root tips. In the absence of aluminum stress, the activity and gene expression of PM H + -ATPase at the WT and ST7 root tips have no obvious change along with the change of time, and the activity of the WT root tip enzyme is about 1.4 mu mol/mg lower than that of the ST7 root tip at the same time^-1protien·min^-1. When 50-100 mu M aluminum is stressed, the activity and gene expression level of WT and ST7 root tip enzymes are higher than CK values, and the activity of the root tip enzymes is obviously improved in 48h and is not obviously different from that of the root tip enzymes in 96 h. When 200 mu M of aluminum is stressed, the activity and the gene expression quantity of the activity of the PM H + -ATPase enzyme at the WT root tip are lower than CK values; the activity of the PM H + -ATPase enzyme of the ST7 root tip is still higher than that of CK, but the activity of the enzyme is obviously reduced when the enzyme is stressed by 200 mu M aluminum, which shows that the activity of the enzyme is greatly reduced when the ST7 root tip is stressed by high-strength aluminum. PM H + -ATPase gene expression of WT root tips and ST7 root tips is weak under 400 mu M aluminum stress; the enzyme activity is obviously lower than the CK value, the WT root tip enzyme activity obviously reduces the enzyme activity to 0.0385 mu mol mg within 96h^-1protien·min^-1ST7 root tip enzyme activity was only eleven fifths of CK (FIG. 14 CD).

4. Determination of H according to the acridine orange method (AO)⁺Pump activity

1.5mL reaction system (250mmol/L sucrose; 1mmol/L Na)₂MoO₄；0.5mmol/L EGTA；12μmol/LAO；300mmol/L KCl；5mmol/L BTP/MES；50mmol/L KNO₃；0.05％Brij58；1mmol/L NaN₃) Adding 50 μ g plasma membrane protein, standing at room temperature for 25min, adding 5mmol/L ATP/BTP to start reaction, reading OD660 value every 20s on ultraviolet spectrophotometer, and measuringSetting for 15 min; the results are shown in FIG. 15;

PM H⁺increase in APase Activity activatable H⁺Pump, promoting H⁺From the endocrine to the extracellular, the electrochemical potential that produces protons provides energy for the transport of secondary substances in plant metabolism. Therefore, we analyzed WT and ST7 root tip H after different aluminum stress treatments⁺Pump Activity Observation root tip H⁺Change in secretion amount. In the absence of aluminum stress, WT root tip H⁺The pump activity was slightly higher than that of ST7 root tip. WT and ST7 root-tip H with increasing aluminum stress concentration⁺The pump activity increases first and then decreases; h of WT root tip⁺The pump activity reaches the maximum value under the aluminum stress of 50 mu mol/L, which is about 1.18 times of the CK value, and the activity is lower than the CK value under the aluminum stress of 100 and 400 mu mol/L, and is reduced to 67.4 percent of the CK value under the aluminum stress of 400 mu mol/L; the activity of the ST7 root tip H + pump reaches the maximum value under 50 mu mol/L aluminum stress, the activity is higher than CK value under 50 and 100 mu mol/L aluminum stress, and the activity is lower than CK value under 200 and 400 mu mol/L aluminum stress.

5、PM H⁺Analysis of the level of interaction of ATPase with 14-3-3 protein

SDS-PAGE (4% gel concentrate and 10% gel isolate) analysis of plasma membrane proteins for the presence of PM H⁺-ATPase protein (97kD) and 14-3-3 protein (around 29 kD) bands. Detecting the interaction of the 14-3-3 protein and PM H + -ATPase by adopting co-immunoprecipitation and Western blotting; mu.g of plasma membrane protein was added to 2. mu.g of 14-3-3 protein-specific antibody, 20. mu.L of agar protein A/G was added, and shaking was carried out overnight at 4 ℃. Centrifuging for 5min the next day, collecting protein precipitate, washing with PBS for 3 times, dissolving the washed precipitate in 40 μ L1 × loading buffer, and performing Western blotting analysis; taking 20 μ L of co-immunoprecipitation binding protein, performing SDS-PAGE analysis, transferring the co-immunoprecipitation protein on white gel to PVDF membrane by semi-dry electrotransfer instrument, non-specifically sealing skimmed milk, and performing PM H analysis⁺-ATPase specific antibody primary antibody incubation, goat anti-rabbit secondary antibody incubation, adding a reaction substrate generating fluorescence, and observing the interaction result by a gel imager, wherein the result is shown in figure 16;

PM H⁺ATPase is the major target protein for the 14-3-3 protein to bind to the plasma membrane, PM H⁺-ATPase Thr via phosphorusAfter acidification, it can enhance the binding with 14-3-3 protein. PM H after 12H and 24H of WT and ST7 root tips treated with 100. mu.M and 200. mu.M aluminum⁺-analysis of gel imager results and relative quantification of the level of the interaction between APase and 14-3-3 protein. The results show that PM H is present in the absence of aluminum stress⁺The level of interaction between-APase and 14-3-3 protein is not very different. The protein interaction level decreased with 200. mu.M aluminum treatment for 12, 24 h. The gel imager has similar color depth, and the protein interaction level cannot be judged, so that the protein interaction level is relatively quantitatively analyzed. The protein interaction level of the ST7 root tip was always higher than that of the WT root tip under aluminum stress. The protein interaction level of the ST7 root tip was higher than that of the WT root tip under the same concentration of aluminum stress. 100 mu M aluminum treatment is carried out for 12h and 24h, and the interaction level of WT root tip protein is 39.2 percent and 38.4 percent of the CK value respectively; the interaction level of ST7 root tip protein is 1.03 times and 1.01 times of CK value respectively. The interaction level of WT root tip protein is reduced in 12h and 24h after 200 mu M aluminum treatment, and is 58% and 30% of CK value respectively; ST7 root tip protein interaction level increased and then decreased, which were 1.32 and 1.05 times higher than CK value, respectively.

6、PM H⁺Correlation analysis of APase Activity and citric acid secretion

Treating PM H of 24H, 48H, WT and ST7 root tips at different aluminum stress concentrations⁺Correlation analysis of APase activity and citric acid secretion as in FIG. 18; PM H of WT and ST7 apices⁺The correlation between the APase activity and the citric acid secretion is positive, and is more obvious at the ST7 root tip, and the R2 reaches 0.92. The result shows that the aluminum stress can remarkably enhance the activity of ST7 root tip PM H + -APase and induce the citric acid secretion of the root tip.

Sequence listing

<110> university of Kunming science

Application of <120> Danbo black soybean superoxide dismutase gene in improving plant aluminum tolerance

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<213> Artificial sequence (Artificial)

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<213> Artificial sequence (Artificial)

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gtccgactca tgcactggta attaaagcc 29

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<213> Artificial sequence (Artificial)

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Claims (1)

1. Application of the Danbo black soybean superoxide dismutase gene GmSOD in improving plant aluminum tolerance.

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