Gene for improving aluminum resistance of plants and application thereof

Abstract

The invention discloses a gene for improving an aluminum-resistant function of a plant and application of the gene, and belongs to the technical field of plant breeding. The invention discloses a stylosanthes guianensis SgTMEM208 gene, and in order to further explore the aluminum-resistant function of the SgTMEM208 gene, a 525bp SgTMEM208 gene full-length sequence is cloned in the invention. The SgTMEM208 gene is subjected to heterologous expression in Yunnan black beans, and the result of overexpression of the SgTMEM208 gene improves the citric acid secretion amount and the aluminum resistance of the Yunnan black beans under aluminum stress. Therefore, the SgTMEM208 gene participates in citric acid secretion and antioxidant enzyme regulation and control, and is a plant aluminum-resistant gene. Therefore, the gene bank of the aluminum toxicity resistance of the plant is enriched, and the gene has great application value in cultivation of plant varieties with high aluminum toxicity resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant breeding, and particularly relates to a gene for improving the aluminum tolerance function of plants and application thereof. BACKGROUND

[0002] In recent years, the livestock industry has rapidly developed and expanded, and the demand for high-quality legume forage has gradually increased. The cross-regional transportation cost of forage is high, and planting and utilizing high-quality legume forage in the south can not only strengthen agricultural ecological construction, reduce costs and increase efficiency, but also improve the agricultural structure and realize the virtuous cycle of grass and livestock. However, acid soils are widely distributed in southern China, and a large amount of free aluminum ions (Al 3+ ) exist in acid soils, which have aluminum toxicity effects on plants and inhibit the growth of most high-quality forages. Traditional lime soil improvement methods are costly and ineffective, so current research mainly focuses on studying aluminum-tolerant plants, exploring aluminum-tolerant genes, and cultivating new acid aluminum-tolerant forage varieties to meet the needs of planting in the south.

[0003] Stylosanthes guianensis is an important legume forage in tropical and subtropical regions, and has strong tolerance to acid soil (pH 4.0-5.5) and can still grow normally in red soil. Therefore, in-depth research on the aluminum tolerance function of Stylosanthes guianensis and the molecular mechanism of aluminum toxicity resistance of Stylosanthes guianensis can provide more reference for the cultivation of aluminum-tolerant high-quality forage varieties. SUMMARY

[0004] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide a gene for improving the aluminum tolerance function of plants and application thereof, so as to improve the aluminum tolerance function of plants.

[0005] The technical scheme for solving the above-mentioned technical problems of the present application is as follows: a gene for improving the aluminum tolerance function of plants is provided, and the gene is named Sg T MEM208 . Sg T MEM208 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO. 1.

[0006] Further, Sg T MEM208 the amino acid sequence of the encoded protein of the gene is shown in SEQ ID NO. 2.

[0007] The present application provides the application of the above-mentioned gene in improving the aluminum toxicity stress tolerance of plants.

[0008] The present application provides a method for improving the aluminum toxicity stress tolerance of plants, which utilizes transgenic technology to introduce the Sg T MEM208 gene into the genome of other plants to be enhanced, and through the heterologous expression of the Sg T MEM208 gene, the other plants to be enhanced obtain the Sg T MEM208The gene and the protein it encodes possess the function of resisting aluminum toxicity stress.

[0009] Furthermore, specifically including the following steps: utilizing amplified Sg T MEM208 Genes were constructed to create plant expression vectors, which were then transferred into other plants to be enhanced via Agrobacterium-mediated transformation, resulting in transgenic plants with improved tolerance to aluminum toxicity stress.

[0010] This invention also provides a reagent for improving plant tolerance to aluminum toxicity stress, the reagent comprising the above-mentioned... Sg T MEM208 Genes or the above Sg T MEM208 The protein encoded by the gene.

[0011] The present invention has the following beneficial effects: The present invention discovers a kind of Stylosanthes pubescens. Sg T MEM208 Genes, for further exploration Sg T MEM208 This invention cloned a 525bp gene to enhance aluminum tolerance. Sg T MEM208 Full-length gene sequence. And will... Sg T MEM208 Genes were heterologously expressed in Yunnan black soybeans, resulting in overexpression. Sg T MEM208 The gene increased citric acid secretion and aluminum tolerance in Yunnan black soybeans under aluminum stress. This indicates that... Sg T MEM208 These genes are involved in the regulation of citric acid secretion and antioxidant enzymes, and are considered to be aluminum-tolerant genes in plants. Therefore, this invention enriches the gene pool of aluminum-tolerant plants and has significant application value in cultivating plant varieties with high aluminum tolerance. Attached Figure Description

[0012] Figure 1 The spectrum of the pCAMBIA1300-MCS-35S-mCherry vector; Figure 2 for Sg T MEM208 Electrophoresis diagram of amplification products; Figure 3 for Sg T MEM208 An amino acid composition diagram; Figure 4 for Sg T MEM208 Predicted map of protein phosphorylation sites; Figure 5 for Sg T MEM208 Predicted diagram of protein transmembrane domains and tertiary structures; Figure 6 for Sg T MEM208 Protein signal peptide prediction graph; Figure 7 for Sg T MEM208 Relative expression levels of genes under different ion treatments; Figure 8 For the transfer Sg T MEM208 Overexpression and control diagram; Figure 9 forSg T MEM208 Root length diagrams after overexpression and empty aluminum treatment; Figure 10 for Sg T MEM208 Overexpression and empty vector root tip staining and citric acid secretion diagram; Figure 11 A graph showing the aluminum content and chromium azurite S staining in the root tip; Figure 12 This refers to key antioxidant enzyme indicators that are overexpressed and unexpressed. Detailed Implementation

[0013] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0014] Example 1: Sg T MEM208 Cloning of genes (1) RNA extraction and cDNA synthesis: ① Thoroughly grind 100 mg of liquid nitrogen-frozen Stylosanthes kirilowii, add 1 mL of Trizol reagent to form a homogenate, and let stand for 5 min to ensure complete lysis. Then, add 0.2 mL of chloroform, vortex for 15 s, and let stand for another 2 min. Centrifuge the sample at 12,000 g for 15 min (4℃), collect the supernatant, add 0.5 mL of isopropanol and mix gently, and let stand at room temperature for 10 min. Then, centrifuge again at 12,000 g for 10 min (4℃), discard the supernatant, and wash the precipitate with 1 mL of 75 wt% ethanol. After centrifugation at 7500 g for 5 min (4℃), discard the ethanol, air dry the precipitate, dissolve it in 20 μL of DEPC water, incubate at 65℃ for 15 min, extract RNA, and perform agarose gel electrophoresis to determine its quality.

[0015] ②Take 200 μg of RNA, add DEPC water to a total volume of 100 μL, heat at 65°C for 2 min, then immediately place in ice bath to cool. Prepare 200 μL of magnetic bead suspension, place on a magnetic stand for 30 s, discard the supernatant, add 100 μL of Binding Buffer to resuspend the magnetic beads, again place on a magnetic stand for 30 s, discard the supernatant. Mix the pretreated magnetic microspheres (100 μL of binding buffer system) with the sample to be extracted with RNA thoroughly, shake on a vortex shaker at a frequency of 15 Hz for 4 min, and use poly(dT)25 oligonucleotide to realize targeted binding with the polyA tail of mRNA through specific base complementary pairing. Then use gradient magnetic separation technology, remove the liquid phase component when the liquid phase transmittance is ≥95%. Then centrifuge twice with 200 μL of pre-cooled deproteinization eluent (800g, 1 min). The final product is recovered by thermal dissociation, 10 μL of low ionic strength Tris-HCl buffer (pH=7.5) is added to a constant temperature metal bath at 80°C for 120 s, then magnetic phase separation is performed immediately, and the high-purity mRNA solution collected can be stored at -80°C for a long time.

[0016] ③cDNA synthesis uses All-In-One 5x RT MasterMix for reverse transcription, and the specific reaction system and steps are shown in Table 1. In order to ensure the accuracy of the experimental results, all operations need to be carried out on an ice box to maintain a low temperature environment.

[0017] Table 1 Reagent system

[0018] Reaction conditions: 37°C, 15 min; 60°C, 10 min; 4°C, save.

[0019] (2) Target sequence amplification and recovery: ① Target sequence amplification: According to the yeast sequencing and full-length transcriptome alignment results, the full-length CDS sequence is obtained Sg T MEM208 , which is used for primer design, and Taq Master Mix premix enzyme is used for PCR amplification. The reagent system and amplification system are shown in Table 2.

[0020] Sg T MEM208 -F: 5'-ATGGCGAATCAAGGCGCTA-3' (SEQ ID NO. 3); Sg T MEM208 -R: 5'-CTATCTGGTTCTTGTCTTGACGA-3' (SEQ ID NO. 4).

[0021] Table 2 Reagent system

[0022] ② Perform the PCR reaction under the following conditions: 94℃, 90s; (94℃, 20s, 60℃, 20s, 72℃, 60s) for 31 cycles; 72℃, 5min; 4℃, store.

[0023] ③PCR products were detected by 10% agarose gel electrophoresis. The electrophoresis conditions were: voltage 200V, current = 200A, power 50W, reaction time 18min. The bands were observed using a UV light in a developer and the target bands were recovered for subsequent experiments and sequencing verification.

[0024] ④ Gel extraction (using OMEGO gel extraction kit): Cut an agarose gel block containing the target fragment using a sterile scalpel and transfer it to a 2 mL centrifuge tube; add Extraction Buffer at a ratio of 1:3 (g / v), sol in a 65°C metal bath, vortex every 2 min until completely dissolved (approximately 10 min); then transfer the sol to an adsorption column, centrifuge at 12,000 g for 1 min at room temperature, discard the filtrate, add 300 μL Binding Buffer, centrifuge at 12,000 g for 1 min; add 700 μL Wash Buffer, centrifuge at 12,000 g for 1 min; centrifuge an empty column for 2 min to remove residual ethanol. Then transfer the adsorption column to a new 1.5 mL centrifuge tube for preheating elution: preheat the adsorption column in a 65°C metal bath for 1 min, add 40 μL Elution Buffer, incubate at room temperature for 1 min, then centrifuge at 12,000 g for 2 min to collect DNA.

[0025] (3) qRT-PCR: using BlasTaq TM Polymerase, according to Sg T MEM 208 Sequence and internal reference gene Sg EF1 -a The designed primers were used for amplification. The sample addition process needed to be carried out at low temperature on ice. The reaction system is shown in Table 3.

[0026] q- Sg T MEM208 -F: 5'-GGCTCCGCCAAGTCATCATA-3' (SEQ ID NO.5); q- Sg T MEM208 -R: 5'-TAGCCCAATCCAATGCTTCCA-3' (SEQ ID NO. 6); Sg EF1 -α-F;5'-TGGGAGGTATTGACAAGCGT-3' (SEQ ID NO.7); Sg EF1 -α-R: 5'-CGGGAGCATCAATGACAGTG-3' (SEQ ID NO. 8).

[0027] Table 3 Reagent System

[0028] Reaction program: 95℃, 3 min; (95℃, 15 s; 60℃, 1 min;) for 40 cycles.

[0029] Example 2: Sg T MEM208 Carrier construction The vector was constructed using homologous recombination, and the constitutive overexpression vector with the 35S promoter was pCAMBIA1300-MCS-35S-mCherry (vector map shown). Figure 1 Using vector primers and partial CDS sequence of the target gene, amplification of [amplification] gene containing [amplification] gene can be designed. Sg T MEM208 After supplementing the reaction reagent system according to Table 4, the gene overexpression vector was subjected to PCR reaction at 37℃ for 5 min and 65℃ for 10 min.

[0030] Table 4 Reagent System

[0031] The fragments were PCR-treated at 50℃ for 60 min. The homologous recombination reagent system is shown in Table 5.

[0032] Table 5 Reagent System

[0033] Will Sg T MEM208 The overexpression vector was used to transform E. coli, and after PCR verification of the colonies, the plasmid was extracted and then sent for sequencing.

[0034] Example 3: Positive screening of Agrobacterium rhizogenes in Yunnan black soybeans and identification of aluminum tolerance and antioxidant capacity Yunnan black soybean is a variety sensitive to acidic soils; tests in acidic soils showed that it failed to form root nodules and exhibited poor growth. On the other hand, genetic transformation technology for *Stylosanthes spp.* is not yet mature, making it difficult to achieve [the desired results]. Sg T MEM208 Gene knockout. For further exploration... Sg T MEM208 This study will investigate the aluminum tolerance function of genes. Sg T MEM208 Heterologous expression was performed in Yunnan black soybeans.

[0035] (1) Transformation of K599 Agrobacterium: ① Take out a tube of K599 Agrobacterium competent cells (100 μL), transfer 50 μL of it to another 1.5 mL centrifuge tube for subsequent centrifugation and balancing. Place the two centrifuge tubes on ice to thaw slowly, when they are close to thawing, add 500 ng (1 μL) of pCAMBIA1300-SgTMEM208-mCherry recombinant plasmid respectively. Gently stir the bottom of the centrifuge tube with your fingers to mix it.

[0036] ② Place the above mixture on ice for 5 min in ice bath, then quickly transfer it to liquid nitrogen for 5 min. Then, transfer it to a water bath preheated to 37℃ for heat shock treatment for 5 min, and then quickly transfer it back to ice for 5 min.

[0037] ③ Add 700 μL of TY culture solution without antibiotics to the mixture, incubate in a constant temperature shaking incubator at 28℃, 200 rpm for 4 h. Then, centrifuge at 5000 rpm for 1 min at room temperature, discard most of the supernatant, and reserve about 150 μL of supernatant for resuspending K599 Agrobacterium competent cells. Take 100 μL of resuspended bacteria and spread it on TY medium containing 50 μg / mL kanamycin, then incubate it in a constant temperature incubator at 28℃ for 2 days.

[0038] (2) Positive colony screening: use a pipette tip to pick up a single colony, and make a mark on the back of the culture plate with a marker pen. Mix the colony into the PCR mixture for colony PCR amplification. The mixture system and PCR program are shown in Table 6 and Table 7. After colony PCR, measure 50 mL of 1×TAE solution, add 0.5 g of agarose, heat to boiling in a microwave oven, then add 5 μL of Gold View, shake well and pour the plate. Take 5 μL of PCR product and mix it with 1 μL of 6×Loading Buffer and point into the electrophoresis well of agarose gel, 200 V electrophoresis for 20 min. Turn on the ultraviolet observation instrument to observe. After screening the positive single colony, pick the single colony into 50 mL of TY culture solution (Km 50 μg / mL), 28℃, 200 rpm overnight shaking, the next day take 1 mL of bacterial solution and spread it again on solid TY medium (Km 50 μg / mL) for culture, which is used for hypocotyl infection of hot research No. 2 column flower grass.

[0039] Table 6 Reagent system

[0040] Table 7 PCR program

[0041] (3) K599 Agrobacterium infection of Yunnan black soybean: ①The seeds of Yunnan black soybean for K599 Agrobacterium infection need to be soaked in water at 85°C for 2 min in advance, then the seeds are placed in the vermiculite soil for culture, the seeds are evenly spread on the soil, the amount and frequency of watering in the early stage should be appropriately increased, a layer of thin soil is covered on the top after the seeds are spread, and the seeds are kept in a humid and dark environment by timely watering, which is helpful for germination. When the cotyledon is slightly open and breaks the soil, the next step of the experiment can be started.

[0042] ②The lower hypocotyl of Yunnan black soybean seed is obliquely cut at an angle of 45° using clean and sterile scissors or surgical knife.

[0043] ③Prepare positive K599 Agrobacterium colonies and bacterial solution in advance, dilute the bacterial solution to OD600≈0.8 using magnesium sulfate (1 mol / L), evenly place the cut explants in a large culture dish, pour the prepared bacterial solution for immersion, and place the culture dish in a shaker for 30 min. After the immersion is completed, use a pipette tip or other sterile slender tool to pick single colonies and gently smear them on the wound of the immersed explant, so as to wrap the entire wound with Agrobacterium.

[0044] ④Evenly place the explants obtained in step ③ in a large culture dish lined with moist germination paper (ensure sufficient moisture and avoid watering), and pay attention to the spacing not being too dense.

[0045] ⑤After placing the culture dish (do not open the culture dish to expose the callus to air), keep the inside of the culture dish moist, and when the callus forms and shows a tendency to root, appropriate watering can be performed.

[0046] ⑥After 14 days, hairy roots will grow from the wound of Yunnan black soybean explants. Open the cover on the culture dish and use a fluorescence hand-held excitation light source LUYOR-3415RG to irradiate the positive plants under green laser illumination at 540 nm. Then, the plants with bright red fluorescence (M-Cherry) are subjected to PCR verification, and the positive plants are transferred to water for culture after screening.

[0047] Example 4: Phenotype identification of Yunnan black soybean (1) Root elongation of Yunnan black soybean: take wild-type Yunnan black soybean with empty transfection and 3 groups of Yunnan black soybean overexpressing Sg T MEM208 genes with consistent growth (each group contains 10 positive plants), pre-culture in 0.5 mmol / L CaCl2 for 24 h, then add 50 μmol / L AlCl3 solution for treatment for 24 h, measure the relative root elongation of Heyuan No. 2 column-flowered grass in each comparison group, and the calculation formula is as follows: RRE (%) =

[0048] (2) Chromium azure S and Evans blue staining: After the plant treatment was completed, the plant roots were gently rinsed three times with deionized water. Then, the plant roots were immersed in Evans blue and chromium azure S staining solutions respectively in the dark for 20 minutes. After immersion, the roots were gently rinsed with deionized water to ensure that no fading occurred after multiple rinses. The roots were then observed and photographed under a stereomicroscope.

[0049] (3) Determination of aluminum content in the roots of Yunnan black soybeans: 100 μmol / L AlCl3 solution was used to treat Yunnan black soybeans that had been converted to empty vector and three groups of overexpression plants with similar growth. Sg T MEM208 0.5g of root tissue samples from Yunnan black soybeans were placed in Erlenmeyer flasks, along with 10mL of HNO3 and 0.5mL of H2SO4. The mixture was heated on a hot plate (100℃ for 1 hour, 150℃ for 1 hour, 180℃ for 2 hours, finally reaching 200℃) until white fumes appeared at the mouth of the flask and the nitric acid-sulfuric acid digest turned colorless and transparent or pale yellow. After digestion, the mixture was cooled to room temperature and then transferred to a volumetric flask and diluted to 25mL with ordinary water. The digested samples were atomized using a graphite furnace, and the absorbance was measured at 257.4nm using a UV spectrophotometer.

[0050] (4) Determination of citric acid secretion in the root tips of Yunnan black soybeans: Three groups of Yunnan black soybeans transfected with empty vector and those with similar growth were selected for overexpression. Sg T MEM208 Yunnan black soybeans (each group containing 15 positive Yunnan black soybean plants) were pre-cultured in 0.5 mmol / L CaCl2 for 24 h, followed by treatment with 100 μmol / L AlCl3 solution for 24 h. The citric acid secretion from the roots of each group of Yunnan black soybeans was measured using a UV spectrophotometer and a citric acid assay kit (Biobox, Beijing). The citric acid secretion was calculated as follows: Citric acid content (μmol / g) =

[0051] (5) Determination of antioxidant capacity of Yunnan black soybean roots: ① Malondialdehyde (MDA): Plant tissue treatment, reagent addition order, and dosage were performed according to the instructions of the Malondialdehyde content detection kit (Bobox, Beijing). The mixture was placed in a 100℃ water bath for 60 min (tightly sealed to prevent moisture loss), cooled on ice, and centrifuged at 10000g for 10 min using a benchtop centrifuge. Calculations were made: ΔA532 = A532 (measured) - A532 (blank), ΔA600 = A600 (measured) - A600 (blank), ΔA = ΔA532 - ΔA600. MDA calculation formula: MDA (nmol / g) = 32.258 × ΔA ÷ sample mass.

[0052] ② Peroxidase (POD): Plant tissue treatment, reagent addition order, and dosage were performed according to the instructions of the peroxidase activity assay kit (Biobox, Beijing). Record the absorbance value A1 at 470nm for 30 seconds, and record the absorbance value A2 after 90 seconds. Calculate the result: ΔA = A2 - A1. POD calculation formula: POD (U / g) = 7133 × ΔA ÷ sample mass.

[0053] ③ Superoxide dismutase (SOD): Plant tissue treatment, reagent addition order, and dosage were performed according to the instructions of the Superoxide Dismutase Activity Assay Kit (Biobox, Beijing). The absorbance (A) of each tube was measured at 450 nm. ΔAmeasured = Ameasured - Acontrol, ΔAblank = A1blank - A2blank. The inhibition percentage was calculated as: Inhibition percentage = (ΔAblank - ΔAmeasured) ÷ ΔAblank × 100%. SOD calculation formula: SOD activity (U / g) = 10 × Inhibition percentage ÷ (1 - Inhibition percentage) ÷ Sample mass × Dilution factor.

[0054] ④ Catalase (CAT): Plant tissue treatment, reagent addition order, and dosage were performed according to the instructions of the Catalase Activity Assay Kit (Biobox, Beijing). The initial absorbance A1 of the mixture was measured at 240 nm wavelength. After waiting 1 minute, the absorbance A2 was read. Calculate ΔA = A1 - A2. The formula for calculating catalase (CAT) is: CAT (U / g) = 678 × ΔA ÷ sample mass.

[0055] Results and Analysis 1. Sg T MEM208 Gene cloning and sequence analysis (1) According to Sg T MEM208 Primers were designed based on the complete CDS sequence of the gene for PCR amplification, yielding a bright band longer than 500 bp (see [link]). Figure 2 The amplified products were sequenced and compared with the target gene CDS. The results showed that... Sg T MEM208 There is an open reading frame (ORF) of 525bp in length.

[0056] (2) Using the online prediction website ProtParam to predict the outcome of the prediction. Sg T MEM208 The physicochemical properties and structure of the protein were analyzed. The molecular formula of the protein is C0. 917 H 1425 N 239 O 247 S 10 It contains 174 amino acids and has a molecular weight of 20.07 kDa. Figure 3 It can be seen that, Sg T MEM208The number of lysine Lys (K) in the encoded protein is 16, accounting for 9.2% of the total, the number of glycine Gly (G) and glutamic acid Ile (I) is 14, each accounting for 8.0%, the content of asparagine Asn (N), cysteine Cys (C) and tryptophan Trp (W) is the least, which is 3, accounting for 1.7% of the total, and there is no pyrrolysine Pyl (O) and selenocysteine Sec (U). The theoretical value of protein isoelectric point (PI) is 9.45; the half-life of mammalian reticulocyte in vitro is 30h, and the half-life in yeast cells and Escherichia coli is more than 20h and 10h respectively; in the prediction of protein stability, the calculation result of instability index (II) of SgTMEM208 is 40.99, so it is identified as an unstable protein, which shows that the protein is unstable when it is affected by environmental conditions and other factors in vitro; the average value of hydrophilicity (GRAVY) is -0.207; the total number of negatively charged residues (Asp + Glu) is 17, and the total number of positively charged residues (Arg + Lys) is 26. The protein phosphorylation site is predicted by NetPhos, and the result is shown in Figure 4 . Sg T MEM208 There are 17 positive phosphorylation sites, of which 6 are serine residue (Ser) phosphorylation modification sites, 6 are threonine residue (Thr) phosphorylation modification sites, and 5 are tyrosine residue (Tyr) phosphorylation modification sites.

[0057] (3) Using TMHMM2.0 and DeepTMHMM transmembrane structure prediction, it is found that Sg T MEM208 the protein contains 4 transmembrane helices (see Figure 5 A), which is similar to the characteristics of traditional TMEM family, and using Cell-PLoc online prediction website of subcellular localization, it is found that Sg T MEM208 the protein is located in the cytoplasmic membrane. Using AlphaFold 3 to simulate the tertiary structure of Sg T MEM208 the protein, it is found that the black reticular structure is the cytoplasmic membrane, which proves that the protein indeed has 4 transmembrane helices embedded in the cytoplasmic membrane (see Figure 5 B). Using SignalP online prediction, Figure 6 it is shown that the protein does not have signal peptide (Sec / SPI), lipoprotein signal peptide (Sec / SPII), TAT signal peptide (Tat / SPI), TAT lipoprotein signal peptide (Tat / SPII), Pilin-like signal peptide (Sec / SPIII); using CDD to predict the conserved domain of the protein, it is found that Sg T MEM208 the N-terminal 1-152 amino acids of the protein exist SND2 domain.

[0058] 2、 Sg T MEM208 aluminum tolerance function (1) Different Al3+ Changes in SgTMEM208 expression levels after treatment: by Figure 7 It can be seen that, Sg T MEM208 The expression level of [a substance] increased with increasing aluminum treatment concentration (P<0.05); in Na [a substance]... 2+ Let's deal with it. Sg T MEM208 The expression of [a specific substance] was significantly upregulated, but its expression level was significantly lower than that of [another substance]. 3+ Treatment (P<0.05); and Sg T MEM208 The expression in K + Cu 2+ There was no change in the control group under the treatment (P>0.05).

[0059] (2) Overexpression Sg T MEM208 Effects of aluminum tolerance on Yunnan black soybeans: This invention utilizes the overexpression of aluminum in this variety. Sg T MEM208 To explore the aluminum tolerance function of this gene, the mCherry gene was used to emit red light at 540 nm. Positive hair roots were screened using a LUYOR-3415RG handheld fluorescent protein excitation light source. Root length and other parameters of the overexpressing positive hair roots were then measured. Figure 8 It can be seen that overexpression Sg T MEM208 The plants that were transformed with the empty vector emitted a reddish glow, while the plants transformed with the empty vector appeared dull and did not emit light. Further testing was conducted... Sg T MEM208 To investigate the effect of overexpression on aluminum tolerance in Yunnan black soybean, unexpressed (EV) and overexpressed (OE) plants of similar size and at the same growth stage were treated with aluminum. Previous laboratory studies showed that Yunnan black soybean exhibited significant aluminum resistance at a concentration of 50 μM / L, therefore, this study used an aluminum concentration of 50 μM / L for treatment. After 24 h of treatment with 50 μM / L AlCl3, the relative root elongation of the overexpressed plants was significantly higher than that of the control group (P<0.05, see...). Figure 9 ).

[0060] (3) Evans blue staining was used to examine the root tip damage of the plants. Figure 10 As shown in Figure A, compared with the EV group, the root tip damage of Yunnan black soybeans in the OE group was significantly less after treatment with 50 μmol / L aluminum (P<0.05). The citrate secretion levels before and after treatment in the overexpression and empty vector groups are shown in Figure A. Figure 10 B. There was no significant difference between the OE group and the EV group under the 0 treatment (P>0.05), but the amount of citric acid secreted by the root tips of soybeans in the OE group was significantly higher than that in the EV group after treatment with 50 μmol / L (P<0.05).

[0061] (4) The aluminum content in the root tips of the treated Yunnan black soybeans was determined, and the cumulative aluminum content in the root tips was visually reflected by chromium azure S staining. The results are as follows: Figure 11As shown, under the condition of 0 μmol / L aluminum treatment, the root tip aluminum content of the EV group is similar to that of the OE group, and there is no significant difference (P>0.05); after 50 μmol / L aluminum treatment, the root tip aluminum content of the overexpression group is significantly lower than that of the empty vector group (P<0.05). Sg T MEM208

[0062] (5) Under aluminum stress, plants accumulate ROS active oxygen substances, causing oxidative damage, so the key enzymes related to oxidative stress of Yunnan black soybean are determined to prove the aluminum tolerance function of the gene. These antioxidant enzymes include MDA, POD, SOD and CAT. As shown in the results, compared with the control empty vector group, the MDA accumulation of the overexpression group is significantly reduced (P<0.05), and the contents of the three antioxidant key enzymes POD, SOD and CAT are significantly increased (P<0.05), which indicates that the overexpression improves the antioxidant capacity of the plant. Figure 12 Sg T MEM208

[0063] In the present application Sg T MEM208 The nucleotide sequence of the coding region and the amino acid sequence of the encoded protein are as follows: (1) The nucleotide sequence of the coding region is: ATGGCGAATCAAGGCGCTAAGAAACGGAAGGAAGAGAACGCTCGTCACATGGCTAGGCTCCGCCAAGTCATCATAGCCTGCAACGTTATCTATGTCTTGGTGAGGATGTTGTTTTTCCATTCCACGATCACTTGGAAGCATTGGATTGGGCTAATTGTGACGTCTCTGGCATATTTTATACCATATAAACAACTTGAGAAGATGGCCACGCCAAGTTATGATGACGATGGTGAACTTTTAGATGGTGGTTTTGATATGACTACTGGTGGAGTTTGTGGCTATTTACATGATATTATCTACATAACATGCTTTGTGCAAGTCATGTCTATCATCTCTGGAAAATTTTGGTACACATATCTTGTGATACCAGCTTTTGGAGCATACCAATCTTTTGGACTCATTAAGGGATTTTTGCCAGGAGGAAGTTCAGAGGAACCATATGAAGATGAAAAGACCCGCAAGAAGAGGGAAAAGATGGAAAAGAAAGCATCCAGGCCTAAGTTCGTCAAGACAAGAACCAGATAG (SEQ ID NO. 1); ​​​(2) Amino acid sequence of the encoded protein: MANQGAKKRKEENARHMARLRQVIIACNVIYVLVRMLFFHSTITWKHWIGLIVTSLAYFIPYKQLEKMATPSYDDDGELLDGGFDMTTGGVCGYLHDIIYITCFVQVMSIISGKFWYTYLVIPAFGAYQSFGLIKGFLPGGSSEEPYEDEKTRKKREKMEKKASRPKFVKTRTR (SEQ ID NO. 2).

[0064] The above merely provides the preferred embodiments of the application, and is not intended to limit the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall fall within the protection scope of the application.

Claims

1. A gene that enhances aluminum tolerance function in plants, characterized in that, The gene is named SgTMEM208 , and the SgTMEM208 nucleotide sequence of the coding region of the gene is shown as SEQ ID NO.

1.

2. The gene of claim 1, wherein The SgTMEM208 The amino acid sequence of the encoded protein of the gene is shown as SEQ ID NO.

2.

3. Use of a gene according to claim 1 or 2 for increasing the tolerance of a plant to aluminium toxicity stress.

4. A method for increasing the tolerance of a plant to aluminum toxicity stress, characterized in that, By using transgenic technology, the SgTMEM208 gene is introduced into the genome of other plants to be enhanced, and the other plants to be enhanced are provided with SgTMEM208 the function of resisting aluminum toxicity stress possessed by the gene and the protein encoded by the gene through SgTMEM208 heterologous expression of the gene.

5. The method of increasing tolerance to aluminum toxicity stress in plants according to claim 4, characterized in that, Specifically comprising the following steps: Using the amplified SgTMEM208 Gene constructs plant expression vectors, by Agrobacterium-mediated method into other plants to enhance the expression of the plant vector into the body, get transgenic plants to improve the tolerance of aluminum toxicity stress.

6. An agent for improving the tolerance of a plant to aluminum toxicity stress, characterized in that, The agent includes the polypeptide of claim 1 SgTMEM208 the gene of claim 2 SgTMEM208 the encoded protein of the gene.

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