SgSnakin-1 gene involved in adaptation of stellaria dichotoma to aluminum stress and application thereof

Abstract

The application discloses a gene SgSnakin-1 participating in aluminum toxicity tolerance of Stenocarpus sinatus and application thereof. The application provides the gene SgSnakin-1 participating in aluminum toxicity stress adaptation of Stenocarpus sinatus, the nucleotide sequence of which is shown as SEQ ID NO:1, and the amino acid sequence of the coded protein is shown as SEQ ID NO:2. The application shows that the heterologous expression of the SgSnakin-1 gene promotes the growth of the Arabidopsis thaliana root system under aluminum toxicity stress through the construction of a transgenic Arabidopsis thaliana expression system, and the overexpression of the SgSnakin-1 significantly reduces the aluminum content in the Arabidopsis thaliana root and the accumulation of root tip ROS under aluminum toxicity stress, thereby improving the aluminum toxicity tolerance of the root system and changing the root tip active oxygen level caused by aluminum stress. It is shown that the SgSnakin-1 gene is an important aluminum toxicity tolerance gene, which enriches the gene library of plant aluminum toxicity tolerance and plays an important role in the adaptation of plants to acid aluminum stress.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering. More particularly, it relates to an important gene SgSnakin-1 involved in the adaptation of Stizolobium gracile to aluminum toxicity stress and application thereof. BACKGROUND

[0002] Aluminum is the most abundant metal element in the earth's crust. In neutral or alkaline soils, aluminum is usually combined with organic complexes, minerals or colloids to form stable complexes. However, in acid soils with pH < 5.0, aluminum is released from the complex and exists in the form of Al 3+ When aluminum ions enter the root tips, they first inhibit the growth of the root system and then cause toxic damage to the growth and development of plants. At the cellular level, reactive oxygen species (ROS) produced by aluminum toxicity cause lipid peroxidation of the plasma membrane, leading to cell metabolic disorder. On the other hand, ROS binds to purine and pyrimidine in DNA, causing chromosomal aberration and cell pathological death, and inhibiting cell mitosis. Therefore, enhancing the tolerance of plants to aluminum toxicity in acid soils is one of the important ways to alleviate the development of sustainable agriculture.

[0003] Stylosanthes guianensis is a high-quality forage and green manure crop, which is originated from tropical regions and is known as a pioneer crop in acid soils. In addition to its tolerance to low phosphorus and manganese toxicity in acid soils in tropical regions, Stylosanthes guianensis also has good adaptability to aluminum toxicity. The mechanisms of aluminum tolerance in Stylosanthes guianensis mainly focus on the secretion of organic acids and the protection of root tip border cells. For example, the concentration of malate in the roots of aluminum-tolerant genotype Stylosanthes guianensis TPRC2001-1 was 5.8 times higher than that of aluminum-sensitive genotype Fine-stem after 72 h of aluminum stress treatment. Overexpression of malate synthesis gene SgME1 in Arabidopsis and Phaseolus vulgaris hairy roots promoted the synthesis of malate, ultimately enhancing the aluminum tolerance of Arabidopsis and Phaseolus vulgaris hairy roots (Sun L, Liang C, Chen Z, Liu P, Tian J, Liu G, Liao H. Superior aluminium (Al) tolerance of Stylosanthes is achieved mainly by malate synthesis through an Al-enhanced malic enzyme, SgME1. New Phytol. 2014 Apr;202(1):209-219.). In addition to the secretion of malate, the secretion of citrate is also one of the mechanisms of aluminum tolerance in Stylosanthes guianensis. Studies have shown that with the increase of aluminum stress concentration and treatment time, the secretion of citrate from the roots of Stylosanthes guianensis also increases, and the citrate secretion rate of aluminum-tolerant genotypes is significantly higher than that of aluminum-sensitive genotypes (Li X F, Zuo F H, Ling G Z, et al. Secretion of citrate from roots in response to aluminum and low phosphorus stresses in Stylosanthes [J]. Plant and Soil, 2009, 325(1):219-229.; Jiang C, Liu L, Li X, Han R, Wei Y, Yu Y. Insights into aluminum-tolerance pathways in Stylosanthes as revealed by RNA-Seq analysis. Sci Rep. 2018 Apr 17;8(1):6072.). However, whether there are other aluminum tolerance pathways in Stylosanthes guianensis needs further exploration, and there are few reports on the analysis of aluminum tolerance genes in Stylosanthes guianensis.Therefore, in order to further explore the aluminum stress related mechanism of St Johnswort, exploring the related genes from the molecular level not only helps to study the molecular mechanism of aluminum stress tolerance of St Johnswort, but also helps to cultivate new varieties resistant to aluminum stress, which has important value and significance. SUMMARY

[0004] The application provides a gene SgSnakin-1 involved in aluminum tolerance of St Johnswort and application thereof, and the gene SgSnakin-1 involved in aluminum stress adaptation of St Johnswort is cloned for the first time, and function identification is performed on the gene, so as to provide more theoretical support for the aluminum stress adaptation mechanism of St Johnswort.

[0005] A first object of the application is to provide a gene SgSnakin-1 involved in aluminum stress adaptation of St Johnswort and a coding protein thereof.

[0006] A second object of the application is to provide a recombinant expression vector.

[0007] A third object of the application is to provide a genetically engineered bacterium.

[0008] A fourth object of the application is to provide application of the gene SgSnakin-1 or the coding protein.

[0009] A fifth object of the application is to provide a method for improving aluminum tolerance of plants.

[0010] The above objects of the application are achieved by the following technical solutions.

[0011] The application clones a gene related to aluminum stress from St Johnswort, and the expression level of the gene is significantly up-regulated when the root tip of St Johnswort is subjected to aluminum stress. After sequencing and sequence alignment, the gene is attributed to the Snakin / GASA gene family, and is a cysteine-rich peptide gene SgSnakin-1 in St Johnswort. The cDNA sequence of the gene is shown as SEQ ID NO:1, the sequence length is 297bp, and the amino acid sequence of the protein of the coding gene SgSnakin-1 is shown as SEQ ID NO:2.

[0012] The application obtains that SgSnakin-1 plays an important function in the aluminum stress adaptation of St Johnswort by heterologous expression transformation system, and shows that SgSnakin-1 is expressed in leaves and roots of plants, and the expression level is significantly up-regulated under aluminum stress, and the up-regulation degree in root tips is particularly significant, the expression of SgSnakin-1 can promote the growth of Arabidopsis plants under aluminum stress, reduce the aluminum concentration in roots, reduce the amount of aluminum ions in Arabidopsis roots, and reduce the superoxide free radical level and H2O2 level of Arabidopsis root tips; it is shown that the mechanism of SgSnakin-1 gene is to reduce the absorption of aluminum ions by root tips, and reduce the ROS accumulated by aluminum toxicity, specifically to reduce the aluminum ion content in roots and the ROS accumulated by aluminum toxicity, regulate the redox homeostasis of root tips, thereby improving the aluminum toxicity tolerance of plants.

[0013] The application provides a recombinant expression vector containing SgSnakin-1, a gene of St Johnswort for adapting to aluminum stress.

[0014] The application provides a genetically engineered bacterium containing the recombinant expression vector.

[0015] In addition, the following applications of the gene SgSnakin-1 or the encoded protein provided by the application are also within the protection scope of the application:

[0016] In improving the aluminum stress resistance of plants.

[0017] In reducing the aluminum content in plant roots or in preparing a preparation for reducing the aluminum content in plant roots.

[0018] In cultivating transgenic plants resistant to aluminum stress or having high aluminum toxicity tolerance.

[0019] In promoting the growth of plants under aluminum stress or in preparing a preparation for promoting the growth of plants under aluminum stress.

[0020] In reducing the active oxygen level of plants under aluminum stress or in preparing an active oxygen inhibitor.

[0021] The application also provides a method for improving the aluminum toxicity tolerance of plants, and overexpressing the gene SgSnakin-1 in plants.

[0022] The application has the following beneficial effects:

[0023] The application first clones a cysteine-rich peptide gene SgSnakin-1 whose expression is significantly up-regulated under aluminum stress in Stylosanthes guianensis. The research of the application shows that the SgSnakin-1 gene is expressed in the leaves and roots of the plant and is significantly up-regulated in the roots of the plant after aluminum stress; through the transgenic Arabidopsis expression system construction, it is shown that the expression of the SgSnakin-1 gene can promote the growth of the roots under aluminum stress, the aluminum concentration in the roots of the overexpression strain of the SgSnakin-1 gene is obviously decreased, the aluminum ion content in the roots of the Arabidopsis is reduced, and the overexpression of the SgSnakin-1 also reduces the superoxide free radical level and the H2O2 level of the root tip of the Arabidopsis, which shows that the SgSnakin-1 can regulate the change of the active oxygen level of the root tip caused by aluminum stress. It is shown that the SgSnakin-1 gene is an important aluminum toxicity tolerance gene, the gene is also the first reported cysteine-rich peptide gene related to the aluminum toxicity tolerance of the plant, the gene enriches the gene library of the aluminum toxicity tolerance of the plant, and has wide application prospects in the preparation of the preparation for promoting the adaptation of the plant to aluminum stress and the construction of the transgenic plant. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Expression pattern analysis of SgSnakin-1 in the root tip of Stylosanthes guianensis under aluminum stress (A is different time, B is different concentration, and the * mark indicates that there is a significant difference between the expression levels of the aluminum treatment (+Al) and the control (-Al) treatment, wherein * represents a significant level P<0.05, ** represents a significant level 0.05

[0025] Figure 2 Subcellular localization and chemical tissue localization analysis of SgSnakin-1 (A: the left column is the empty control of the GFP fluorescent protein, the right column is the fluorescent signal distribution of the SgSnakin-1 fusion GFP, the first row is the GFP green fluorescent channel, the second row is the bright field channel, and the third row is the fusion of the fluorescent channel and the bright field channel, and the scale in the figure is 20 μm; B: the left column is the GUS staining under the control treatment, and the right column is the GUS staining under the aluminum treatment, the upper, middle and lower three parts are the leaf part, the root break and the root tip of the Arabidopsis, and the scale is 0.1 mm);

[0026] Figure 3Effect of overexpression of SgSnakin-1 gene on root growth of Arabidopsis under aluminum stress (A is the semi-quantitative detection result of SgSnakin-1 overexpression Arabidopsis, M is a nucleic acid fragment size marker, WT is wild-type Arabidopsis, OX is the overexpression strain, AtEFa is an Arabidopsis internal reference gene; B is the effect of aluminum stress on the growth of SgSnakin-1 overexpression transgenic Arabidopsis plants, the scale in the figure is 0.5 cm; C is the effect of overexpression of SgSnakin-1 on the root elongation of Arabidopsis under aluminum stress, the * in the figure indicates that the root elongation of the overexpression Arabidopsis plant is significantly different from that of the wild type, * indicates a significant level of P < 0.05, ** indicates a significant level of 0.05 < P < 0.01, and *** indicates a significant level of P < 0.001);

[0027] Figure 4 Effect of overexpression of SgSnakin-1 gene on aluminum content in the roots of Arabidopsis under aluminum stress (A is the effect of overexpression of SgSnakin-1 on the aluminum content in the root tips of Arabidopsis, the scale in the figure is 0.5 mm; B is the aluminum concentration determination result of the root system of Arabidopsis);

[0028] Figure 5 Effect of overexpression of SgSnakin-1 gene on ROS in the root tips of Arabidopsis under aluminum stress (A is the NBT staining result, the scale in the figure is 100 μm; B is the DAB staining result, the scale in the figure is 50 μm). DETAILED DESCRIPTION

[0029] The present application will be further described below in conjunction with the drawings of the specification and specific embodiments, but the embodiments do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0030] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0031] Example 1 Expression pattern analysis of SgSnakin-1 gene in root tips of Stylosanthes guianensis in response to aluminum toxicity stress

[0032] The present application clones a cysteine-rich peptide gene in Stylosanthes guianensis, sequences and performs sequence alignment, and attributes the gene to the Snakin / GASA gene family, which is a cysteine-rich peptide gene SgSnakin-1 in Stylosanthes guianensis, the cDNA sequence of which is shown as SEQ ID NO: 1, and the sequence length is 297 bp, and the amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO: 2.

[0033] 1. Germination of Stylosanthes guianensis seeds

[0034] Seed hulling, 2 ml centrifuge tube, 0.5 ml, 1 ml deionized water, 65 °C oven, 10 min, softening seed coat, transfer to clean bench, remove deionized water, 1 ml 75% alcohol, 30 s, remove alcohol and wash 3-4 times with sterile water, 1 ml 10% (v / v) sodium hypochlorite solution, shake for a few seconds, stand for 5 min, remove sodium hypochlorite and wash 3-4 times with sterile water, arrange seeds in solid MS germination medium (pH = 5.8) with blue gun head, 3 rows per tube, hypocotyls downward, seal with breathable medical tape, 23 °C, 120 μmol·m-2s-1 light intensity, 16 h light per day, 60% relative humidity, constant temperature incubator. -2 ·S -1

[0035] 2. Hydroponic aluminum treatment of Amaranthus retroflexus seedlings

[0036] After 2 days of seed germination, select seedlings with uniform growth, remove root tip exfoliation for use. Prepare 0.5 mM / L CaCl2 solution as a control without aluminum (-Al), and aluminum treatment (+Al) solution requires additional AlCl3 preparation of the required concentration, adjust pH to 4.5, fill the control and aluminum treatment solutions into blue bread boxes with a volume of 4 liters, cut black plastic floating plates that can cover the size of the box, use a sharp tool to punch a small hole with a diameter of 1 cm on the floating plate, cover the floating plate on the bread box, wrap the hypocotyls of Amaranthus retroflexus seedlings with a clean sponge, and insert them into the small hole on the plastic floating plate to fix them. The roots of Amaranthus retroflexus seedlings are completely immersed in the treatment solution, and different time (3, 6, 9, 12, 24 h) and different concentration (0, 10, 50, 100 μM / L) aluminum treatments are carried out. The AlCl3 concentration used for different time treatments is 10 μM / L, and the time for different concentration treatments is 12 h.

[0037] 3. Real-time fluorescent quantitative PCR (qRT-PCR) analysis

[0038] ​Root tips of about 1.0 g were collected at corresponding time points, frozen in liquid nitrogen and stored at -80 °C until use. Total RNA was extracted from the samples using TRIzol reagent (Invitrogen, USA). 2 μg of DNase I-treated RNA was used to synthesize the first strand of cDNA using M-MLV reverse transcriptase (Promega, USA). SYBR Premix Ex Taq (Promega, USA) was used for qRT-PCR. The first strand of cDNA was diluted 10-20 times with Mili-Q water to be used as the template for quantitative PCR. The cDNA stock solution of multiple samples was mixed to prepare the first point of the standard curve, i.e., standard 1. 2 μL of standard 1 was added to 18 μL of Mili-Q water to prepare standard 2, which was diluted 10 times to prepare the other 5 points of the standard curve, i.e., standard 3, standard 4, standard 5, standard 6 and standard 7. The reaction mixture was prepared as follows: 10 μL of SYBR Premix Ex Taq (2x), 0.5 μL of forward / reverse primers, 7 μL of ddH2O and 2 μL of template were added to 20 μL of the reaction system. The required amount of the reaction was calculated, and the reagents except for the cDNA were mixed and aliquoted into 18 μL per quantitative PCR tube. Then, 2 μL of the cDNA template was added. The qRT-PCR reaction program was set as follows: 95 °C for 30 s for pre-denaturation, 40 cycles of PCR reaction (95 °C for 30 s, 60 °C for 15 s, 72 °C for 30 s), and the expression amount of each sample was calculated using the Real-Time Analysis Software of Rotor-Gene. Four biological replicates were set for each sample. The primers used for the quantitative PCR reaction are shown as follows:

[0039] SgSnakin-1 quantitative primer SgSnakin-1-RT-F (SEQ ID NO: 3):

[0040] 5'-CAAACGCAGGGTTCTCTTCA-3'

[0041] SgSnakin-1 quantitative primer SgSnakin-1-RT-R (SEQ ID NO: 4):

[0042] 5'-AAACTTCTTGGTTACCGGCG-3'

[0043] Reference gene SgEF1-a quantitative primer SgEF1-a-F (SEQ ID NO: 5):

[0044] 5'-CACTTCAGGACGTGTACAAGATC-3'

[0045] Internal reference gene SgEF1-α quantitative primer SgEF1-α-R (SEQ ID NO: 6):

[0046] 5'-CTTGGAGAGCTTCATGGTGCA-3'

[0047] The results are as follows Figure 1 As shown, in Figure 1 A shows that compared with the control, the expression level of SgSnakin-1 in the root tip of Stylosanthes was significantly upregulated at 3, 6, 9, 12 and 24 h after aluminum treatment, and the upregulation was particularly significant at 9 and 12 h after aluminum treatment. Figure 1 In B, with the increase of aluminum concentration, the expression level of SgSnakin-1 in the root tip of Stylosanthes showed an upward trend and was significantly higher than that of 0 μmol·L -1 Compared with 10, 50 and 100 μmol·L -1 The expression level of SgSnakin-1 increased significantly under aluminum treatment, suggesting that SgSnakin-1 may play an important role in the adaptation of Stylosanthes to aluminum toxicity.

[0048] Example 2 Subcellular Localization and Histochemical Localization and Analysis of SgSnakin-1

[0049] 1. Subcellular localization analysis of SgSnakin-1

[0050] Specific primers were designed: SgSnakin-1-GFP-F (SEQ ID NO: 7): 5'-CTCTAGCGCTACC GGTATGGGTTTCTCTAAGGCTCTAGTT-3'; SgSnakin-1-GFP-R (SEQ ID NO: 8): 5'-CATGGTGGCGACCGGTGCAGGGCACTTGCGCTTG-3'. The full-length ORF of the SgSnakin-1 gene was amplified using Stylosanthes root cDNA as a template. PCR reaction conditions were: 98°C pre-denaturation for 5 minutes, 98°C denaturation for 30 seconds, 58°C annealing for 30 seconds, and 72°C annealing for 1 minute, followed by 30 cycles of extension at 72°C for 10 minutes. The amplified product was purified by gel electrophoresis and recovered using a kit.

[0051] Using a homologous recombination kit II Recombination and ligation reaction was performed between PCR product and linearized vector pEGAD digested by Age I. The reaction system was 20 μL, including PCR product 6 μL, pEGAD linearized vector plasmid 8 μL, recombination ligase Exnase II 2 μL, reaction buffer 4 μL, and was placed at 37 °C for 30 min. The recombination plasmid was transformed into E. coli and sequenced, and the plasmid was extracted after no error. The 35S:SgSnakin-1-GFP plasmid was transformed into Agrobacterium GV3101, and was detected and saved for later use after no error.

[0052] The Agrobacterium strain GV3101 containing 35S:GmBBE-like43-GFP or pEGAD empty (35S:GFP) was inoculated into YEP liquid medium by Agrobacterium transformation method, and was cultured at 28 °C for 16 h. After centrifugation (6000 rpm, 10 min), the bacterial suspension was resuspended to OD 600 of 0.4-0.5 with infiltration solution (containing 10 mM MgCl2, 10 mM MES and 10 mM acetosyringone, pH = 5.6). The bacterial suspension was placed at 28 °C in the dark for 2 h, and the bacterial solution was transformed into the lower epidermis of 4-5 week old tobacco leaves by injection. After 2 d of normal culture of the transformed tobacco, the distribution of fluorescence signal in the tobacco epidermal cells was observed by laser confocal scanning microscope (Zeiss LSM780, Germany).

[0053] The results are shown in Figure 2 A. The subcellular localization results of SgSnakin-1 showed that SgSnakin-1 was mainly located in the cell wall.

[0054] 2. Chemical tissue localization analysis of SgSnakin-1

[0055] The specific primers pSgSnakin-1:GUS-F (SEQ ID NO: 9): 5'-CTATGACATGATTACGAATTCCAATGCACAAGCTTCCATTGC-3' and pSgSnakin-1:GUS-R (SEQ ID NO: 10): 5'-GACTGACCTACCCGGGGATCCTTGATTGCCAAGTGCAGCA-3' were designed. The sequence of about 2800 bp upstream of the start codon of SgSnakin-1 was amplified using root system DNA of Ipomoea nil as template. The PCR reaction conditions were pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 3 min, which was repeated for 30 times, and extension at 72 °C for 10 min. The amplified product was recovered and purified by using a kit after gel electrophoresis.

[0056] The homologous recombination kit II Recombination and ligation reaction was performed between PCR product and pTF102 vector digested by restriction enzymes EcoR I and BamH I. The reaction system was 20 μL, including 6 μL of PCR product, 8 μL of linearized vector plasmid, 2 μL of recombination enzyme Exnase II, and 4 μL of reaction buffer, and the reaction was performed at 37 °C for 30 min. The recombinant plasmid was transformed into E. coli and sequenced to obtain the plant expression vector of pSgSnakin-1:GUS fusion gene of Nicotiana attenuata. Finally, the target vector was transformed into GV3101 Agrobacterium, and the positive clones were detected and stored at -80 °C for standby use.

[0057] The transgenic Arabidopsis was obtained by inflorescence infection method: Agrobacterium GV3101 containing pSgSnakin-1:GUS vector plasmid was cultured at 28 °C overnight, transferred into 100 mL YEP culture solution for expansion culture until OD 600 was 1.6-2.0; then centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the bacterial body was resuspended with an equal volume of 5% sucrose solution or 1 / 2 MS culture solution to prepare the transformation solution with 0.005-0.02% Silwet L-77; during transformation, the Arabidopsis (watered the day before transformation to keep the plant moist) inflorescence was immersed in the transformation solution for 1 min, and then the excess transformation solution was gently wiped off with a paper towel, and the plant was covered with plastic wrap to keep it moist. The plant was first covered with a black bag for dark culture for 18 h, and then transferred to normal culture conditions for culture until seed harvesting (the plant was transformed once every week during culture, and the transformation was performed for a total of 3 times).

[0058] The transformed Arabidopsis was used for T0 generation seed harvesting, and about 100 μL of seeds were used for propagation and identification. The seeds were rinsed with 70% ethanol for 1 min, centrifuged to remove ethanol, and then washed once with sterilized secondary water; then prewashed once with 10% sodium hypochlorite, centrifuged to remove sodium hypochlorite, and then shaken and rinsed with 1 mL of 10% sodium hypochlorite for 5 min, centrifuged to remove sodium hypochlorite, and then rinsed with sterile water for 5-6 times; finally, the seeds were resuspended with sterile water, and the seeds were uniformly sowed on MS medium containing herbicide, and the whole operation was completed in a clean bench. The seeds were treated at low temperature of 4 °C for 1 d to break dormancy, and then transferred to a plant illumination incubator with a light cycle of 16 h / 8 h (light / dark) and a temperature of 22 °C / 20 °C (day / night); after about 2 weeks, the normally surviving T1 generation seedlings were transferred to the substrate for continuous growth, and then a little leaf was taken for DNA extraction, and the positive plants were identified by PCR.

[0059] The positive transgenic Arabidopsis was treated with aluminum, and the specific steps of aluminum treatment were the same as in Example 1. The treated Arabidopsis seedlings were taken out, washed with secondary water for 3 times, fixed with 90% acetone for 30 min, then washed with prepared washing solution for 3 times, and then placed in GUS staining solution (0.1M Na2HPO4 / NaH2PO4, pH 7.2, 1mM X-Gluc) for 20 min vacuum extraction, and then transferred to a 37°C constant temperature incubator for dark staining for 12 h. The colored root system was moved into 75% (v / v) ethanol for preservation, and the GUS staining of the root system was observed under a stereomicroscope (Leica, Germany) and photographed.

[0060] The results are shown in Figure 4B, and the staining results of the SgSnakin-1 promoter fused with GUS in Arabidopsis showed that SgSnakin-1 was expressed in the leaves and roots of the plants, and more importantly, the expression level was significantly up-regulated under aluminum stress, and the up-regulation degree in the root tip was particularly significant. Figure 2

[0061] Example 3 Effect of overexpression of SgSnakin-1 on the growth of Arabidopsis plants under aluminum stress

[0062] The cDNA of the root tip of Stylosanthes guianensis was used as a template to design the specific upstream primer 5'-GTACCCGGGGATCCTCTAGAATGGGTTTCTCTAAGGCTCTAGTT-3' (SEQ ID NO: 11) and the specific downstream primer 5'-GCCTGCAGGTCGACTCTAGATTAAGGGCACTTGCGCTTG-3' (SEQ ID NO: 12) to amplify a 297 bp fragment of the ORF of SgSnakin-1 gene. The recovered fragment was ligated to the linearized vector pTF101s digested with Xbal I, and transformed into E. coli Trelief TM5α (Kangke Biology, Guangzhou). After sequencing analysis, the target plasmid was transformed into Agrobacterium GV3101, and used for Arabidopsis plant transformation after positive detection.

[0063] The overexpression transgenic Arabidopsis plants were obtained by inflorescence dip method, and the operation was the same as the inflorescence dip steps of Arabidopsis in Example 2.

[0064] ​The breeding and identification method of transgenic Arabidopsis is the same as that of Example 2. Three times of breeding and identification are carried out in succession until the homozygous T3 generation of transgenic Arabidopsis seed is obtained, which is harvested and used in subsequent experiments. The SgSnakin-1 detection primers used in the semi-quantitative PCR are shown in SEQ ID NO: 3 and SEQ ID NO: 4, wherein the AtEFα detection primers of Arabidopsis are AtEF1-α-F (SEQ ID NO: 13): 5'-GTCGATTCTGGAAAGTCGACC-3' and AtEF1-α-R (SEQ ID NO: 14): 5'-AATGTCAATGGTGATACCACGC-3', and the specific reaction program is set as: 95℃ pre-denaturation for 30s, PCR reaction (95℃ denaturation for 30s, 60℃ recombination for 15s, 72℃ extension for 30s) for 28 cycles.

[0065] Taking the Columbia wild-type Arabidopsis as a control, the obtained SgSnakin-1 overexpression transgenic Arabidopsis seed is germinated and grown on MS medium for 4 days (the disinfection and culture conditions of the seed are the same as those of the Nicotiana tabacum seed in Example 1), and the wild-type Arabidopsis and the transgenic Arabidopsis with consistent growth are selected, photographed, and the root length before treatment is measured by ImageJ. The +Al (containing 0.5 mmol·L -1 CaCl2) and +Al (containing 0.5 mmol·L -1 CaCl2and 10 μmol·L -1 AlCl3) treatment solutions are prepared, the pH is adjusted to 4.5, the solution is poured into the tip box of a medium transfer gun, the hypocotyl of Arabidopsis is wrapped with a clean sterile sponge, the root is vertically inserted into the treatment solution, and the sponge is fixed in the tip hole. The culture device is placed in a culture room at 23℃ with 16 hours of light per day, and the sample is collected after 3 days of culture. The treated Arabidopsis plants are arranged in order, photographed, and the root length is measured, and the root elongation is calculated.

[0066] The results are shown in Figure 3 , wherein Figure 3 A is the semi-quantitative detection result of the SgSnakin-1 overexpression Arabidopsis line, indicating that the obtained overexpression line is a positive line; Figure 3 B is the effect of aluminum stress on the growth of the SgSnakin-1 overexpression transgenic Arabidopsis plant, and the results show that the overexpression of SgSnakin-1 has no effect on the growth of the Arabidopsis plant under the control condition, and the overexpression of SgSnakin-1 significantly promotes the growth of the Arabidopsis plant under the aluminum stress condition; Figure 3C is the effect of overexpression of SgSnakin-1 on the root elongation of Arabidopsis under aluminum stress. The results show that the root elongation of Arabidopsis overexpressing SgSnakin-1 under aluminum stress is significantly increased compared with the wild type, indicating that overexpression of SgSnakin-1 significantly promotes the growth of Arabidopsis roots.

[0067] Example 4 Effect of overexpression of SgSnakin-1 on the aluminum content of Arabidopsis roots under aluminum stress

[0068] 1, hematoxylin staining

[0069] 0.2 grams of hematoxylin powder and 0.02 grams of potassium iodate (KIO3) powder were weighed and added into 100 mL of deionized water, respectively, and dissolved for more than 2 hours in the dark to prepare a hematoxylin staining solution. An appropriate amount of the staining solution was poured into a glass container, and the Arabidopsis treated under aluminum stress in Example 3 was taken out and immersed in the staining solution. After 1 minute of staining, the Arabidopsis was transferred into deionized water to terminate the reaction, and the floating color was washed away. The distribution of aluminum in the root tip of Arabidopsis was observed under a body lens (LEICA DFC420, Germany) and photographed. -1 After being washed with a CaCl2 solution for 3 times, the Arabidopsis was immersed in the staining solution, and after 1 minute of staining, it was transferred into deionized water to terminate the reaction, and the floating color was washed away. The distribution of aluminum in the root tip of Arabidopsis was observed under a body lens (LEICA DFC420, Germany) and photographed.

[0070] 2, determination of root aluminum concentration

[0071] The inductively coupled plasma atomic emission spectrometer (ICP-AES) 710-ES was used to determine the aluminum concentration. The roots of Arabidopsis treated under aluminum stress in Example 3 were collected and dried in an oven at 65°C. The dry weight of the sample was weighed and recorded, and then carbonized on a hot plate at 170°C for 10 minutes. The sample was then transferred to a muffle furnace at 550°C and ashed for 10 hours. After the sample cooled down, it was carefully taken out and added to 100 mmol / L HCl for 12 hours of leaching. After dilution, the leaching solution was filtered, and the aluminum concentration was measured and calculated. -1 After being washed with a CaCl2 solution for 3 times, the Arabidopsis was immersed in the staining solution, and after 1 minute of staining, it was transferred into deionized water to terminate the reaction, and the floating color was washed away. The distribution of aluminum in the root tip of Arabidopsis was observed under a body lens (LEICA DFC420, Germany) and photographed.

[0072] The results are shown in Figure 4 Figure 4 A is the effect of overexpression of SgSnakin-1 on the aluminum content of Arabidopsis root tips, which shows that the degree of hematoxylin staining of Arabidopsis root tips overexpressing SgSnakin-1 under aluminum stress is significantly lower than that of the wild type; Figure 4 B is the result of determination of the aluminum concentration in the roots of Arabidopsis, which shows that the aluminum concentration in the roots of the overexpression lines is significantly lower than that of the wild type Arabidopsis. This result indicates that SgSnakin-1 can reduce the aluminum ions in the roots of Arabidopsis.

[0073] Example 5 Effect of overexpression of SgSnakin-1 on the ROS level of Arabidopsis root tips under aluminum stress

[0074] 1, NBT staining method to observe superoxide free radicals (O2​- ·)level

[0075] NBT solution was used with 20 mmol·L -1 NaH2PO4 and Na2HPO4 buffer solutions were prepared with a concentration of 200 μmol·L -1 The buffer solution was mainly Na2HPO4, and NaH2PO4 was added to adjust the pH to 6.1. The Arabidopsis seedlings treated with aluminum stress in Example 3 were placed in the above buffer solution and vacuum infiltrated for 1 hour. Then, 200 μmol·L -1 The samples were stained with NBT solution for 5 min and transferred to deionized water to terminate the reaction. Transparent solution was added to prepare slides, and the slides were observed and photographed under a microscope (LEICA DMi8, Germany).

[0076] 2. DAB staining to observe H2O2 levels

[0077] Prepare 50mmol·L -1 Tris-HCl buffer solution, pH 5.0, was used to prepare 0.1 mg mL -1 DAB stain was prepared and dispensed into 2 mL centrifuge tubes. The Arabidopsis seedlings treated with aluminum stress as described in Example 3 were vertically suspended in the stain, ensuring that the entire root was completely immersed in the stain. The plants were then infiltrated under vacuum for 30 minutes and then placed in a light-controlled constant-temperature incubator for 8 hours, until the root tips developed a reddish-brown color. The stained Arabidopsis plants were quickly terminated with a fixative solution consisting of 3:1:1 alcohol:lactic acid:glycerol. The plants were then observed and photographed under a microscope (LEICAD Mi8, Germany).

[0078] The results are as follows Figure 5 As shown by Figure 5 As shown in Figures A and 5B, compared with wild-type Arabidopsis, the NBT and DAB staining levels in the root tips of Arabidopsis plants overexpressing SgSnakin-1 under aluminum stress were significantly reduced, indicating that overexpression of SgSnakin-1 reduced the levels of superoxide radicals and H2O2 in the root tips. This result suggests that SgSnakin-1 can regulate the changes in reactive oxygen species levels in the root tips caused by aluminum stress.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A gene involved in adaptation of Stipa capillata to aluminum stress SgSnakin-1 characterized in that, The nucleotide sequence of the gene SgSnakin-1 is shown as SEQ ID NO:

1.

2. The gene of claim 1 SgSnakin-1 encoding a protein, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, A gene involved in adaptation of ipomoea nil to aluminum stress according to claim 1 SgSnakin-1 .

4. A genetically engineered bacterium, characterized by, The recombinant expression vector of claim 3.

5. The gene of claim 1 SgSnakin-1 or the use of the encoded protein of claim 2 for increasing the resistance of plants to aluminum toxicity stress, characterized in that, The plant is Sg or Arabidopsis.

6. The gene of claim 1 SgSnakin-1 or use of the encoded protein of claim 2 for reducing the aluminum content in the root system of a plant or for the manufacture of a preparation for reducing the aluminum content in the root system of a plant, characterized in that, The plant is Sg or Arabidopsis.

7. The gene of claim 1 SgSnakin-1 or the use of the encoded protein of claim 2 in breeding transgenic plants resistant to aluminum toxicity stress or having high aluminum toxicity tolerance, characterized in that, The plant is Sg or Arabidopsis.

8. The gene of claim 1 SgSnakin-1 or use of the encoded protein of claim 2 for promoting plant growth under aluminum stress or in the preparation of a preparation for promoting plant growth under aluminum toxicity stress, characterized in that, The plant is Sg or Arabidopsis.

9. The gene of claim 1 SgSnakin-1 or use of the encoded protein of claim 2 in reducing the level of active oxygen in plants under aluminum stress or in the preparation of an active oxygen inhibitor of plants under aluminum stress, characterized in that, The plant is Sg or Arabidopsis.

10. A method for increasing the aluminum toxicity tolerance of a plant, comprising, overexpression of the gene according to claim 1 in plants SgSnakin-1 ; the plants are Arabidopsis thaliana or Nicotiana plumbaginifolia.

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