CN108410880B

CN108410880B - Danbo black soybean citric acid transport protein gene and application thereof

Info

Publication number: CN108410880B
Application number: CN201810075636.0A
Authority: CN
Inventors: 李昆志; 郝竞一; 吴远双; 陈丽梅; 徐慧妮
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2018-01-26
Filing date: 2018-01-26
Publication date: 2021-10-22
Anticipated expiration: 2038-01-26
Also published as: CN108410880A

Abstract

The invention discloses a Danbo black soybean citric acid transport protein geneGmMATE75The nucleotide sequence is shown as SEQ ID NO. 1, the coding sequence is shown as SEQ ID NO. 2, and the Danbo black soybean citric acid transport protein gene is applied to improving the aluminum resistance of plants; experiments prove that the Danbo black soybean citric acid transport protein geneGmMATE75Has the function of improving the aluminum resistance of tobacco plants;GmMATE75the gene has application value in the aluminum toxicity stress resistance of acid soil plants, and can be used in the fields of plant aluminum-resistant molecular mechanism research and crop aluminum toxicity-resistant molecular breeding.

Description

Danbo black soybean citric acid transport protein gene and application thereof

Technical Field

The invention relates to the technical field related to molecular biology and genetic engineering, and relates to a Danbo black soybean citric acid transport protein geneGmMATE75And application thereof in improving the aluminum resistance of plants.

Background

Aluminum stress is one of the most important factors affecting crop growth on acid soils. Aluminum is widely distributed in soil as one of the most abundant mineral elements in earth crust. Generally, aluminum does not have a major effect on the growth of crops, but as the pH decreases, aluminum is present in the soil in a toxic ionic form. When Al is contained in acid soil³⁺When the concentration of the compound (A) reaches micromolar concentration, the absorption of water and mineral nutrients by crops can be influenced in a mode of inhibiting the growth of roots of the crops, so that the growth of the crops is inhibited, and the large-area yield reduction of the crops on the acid soil is caused.

The traditional method for neutralizing acid soil by applying lime can relieve the acidity of soil to a certain extent, thereby reducing aluminum toxicity. However, the method has high cost, cannot be implemented in a large range, and can only neutralize the acidity of surface soil to play a temporary effect, but cannot fundamentally solve the aluminum toxicity problem. Therefore, the method has important theoretical value and practical significance for analyzing the aluminum toxicity mechanism on the acid soil at the molecular level and cultivating the aluminum toxicity resistant crops by using the related technology of genetic engineering.

Most of aluminum-resistant monocotyledons and dicotyledons can be released through root systemsThe organic acid is used for coping with aluminum stress; the organic acid secreted by the plant root can react with Al in apoplast³⁺Binding to a chelated state and discharging the chelated state, thereby reducing or eliminating the toxicity of Al; aluminum poison tolerant lines of the same species usually secrete more organic acids than do aluminum poison sensitive lines.

The citrate transporter, namely MATE protein belongs to a large gene family in prokaryotes and eukaryotes, and secondary metabolites and toxins are transported through an ionic electrochemical gradient. The SbMATE gene of sorghum corresponds to the major QTL site Alt for resisting aluminum toxicity_SBThe first MATE family anti-aluminum-toxin gene obtained by map-based cloning can cause Al when over-expressed in Arabidopsis thaliana³⁺Activated citrate efflux is accompanied by an increase in aluminium toxicity resistance. In addition, HvAACT1 also encodes MATE protein, expressed in the apical part of barley with anti-aluminum-toxin genotype, and Al³⁺Can activate the discharged citric acid of HvAACT1, and the anti-aluminum toxicity capability of 10 different barley strains is positively correlated with the secretion of the citric acid of the root system, which shows that HvAACT1 is used as Al³⁺The activated citrate transporter plays an important role in the resistance of barley to aluminum toxicity. The expression of the sequence expression tag of a MATE gene in wheat is related to the phenotype of citric acid secretion of the segregating population, which indicates that a similar mechanism for resisting aluminum toxicity stress by secreting citric acid also exists in wheat.

Disclosure of Invention

The invention aims to provide a Danbo black soybean citric acid transport protein geneGmMATE75The nucleotide sequence is shown as SEQ ID NO. 1, the full length of the gene is 1288bp, and the coding sequence is shown as SEQ ID NO. 2.

The invention obtains the citrate transporter gene from the aluminum-resistant Danbo black soybeanGmMATE75The complete segment of the gene is specifically amplified by PCR; recovering and purifying a GmMATE75 gene fragment, connecting the gene fragment to a pMD18-T vector, transforming Escherichia coli DH5 alpha, extracting a plasmid, and performing enzyme digestion detection to obtain a recombinant vector pMD18-T-GmMATE 75; transforming escherichia coli DH5 alpha by enzyme cutting pMD18-T-GmMATE75 vector and connecting with vector pENTR-2B, and extracting plasmid to obtain entry clone vectorpENTR-2B-GmMATE 75; the GmMATE75 gene is subcloned into a plant expression vector pK2GW7 through LR reaction of Gateway technology to obtain a plant expression vector pK2-35S-GmMATE75, and the vector contains an enhanced promoter and can excessively express a target gene in a receptor plant.

By utilizing an agrobacterium tumefaciens mediated method, firstly, a pK2-35S-GmMATE75 vector is transferred into agrobacterium competent cells, then a target gene is transferred into a receptor tobacco plant by a dip-dyeing method, a transgenic plant which excessively expresses the gene is obtained by utilizing a plant tissue culture method, and whether the gene has the characteristic of improving the aluminum toxicity resistance of the plant is verified by further experiments; the result shows that the tobacco over expressing the gene has stronger aluminum toxicity resistance compared with wild tobacco.

The gene provided by the invention can improve the resistance of plants to aluminum stress, and can be used for research on the molecular mechanism of the aluminum stress resistance of the plants and the field of molecular breeding of the aluminum stress resistance plants.

Drawings

FIG. 1 is an electrophoretogram of a GmMATE75 gene fragment amplified from aluminum-treated Danbo black soybean, wherein M is DNA Marker, and 1 is a GmMATE75 gene fragment;

FIG. 2 is a graph showing the root relative elongation measurement results of tobacco type GmMATE75 under aluminum stress;

FIG. 3 is a schematic diagram showing the measurement results of soluble sugar content in cells of GmMATE75 type tobacco after aluminum treatment, wherein A is the polysaccharide content in leaves, and B is the polysaccharide content in roots;

FIG. 4 is a graph showing the measurement results of hydrogen peroxide content in cells of GmMATE75 type tobacco after aluminum treatment, wherein A is the hydrogen peroxide content in leaves, and B is the hydrogen peroxide content in roots.

Detailed Description

The test methods in the examples described below were all conventional methods unless otherwise specified, and the reagents used were all conventional commercially available reagents and reagents prepared according to conventional methods unless otherwise specified.

Example 1: construction of GmMATE75 transgenic tobacco line

Collecting semen Sojae Atricolor liquidCulturing seedling with 50 μ M AlCl (pH4.5)₃Treating the solution for 24h, taking leaves of the leaves, extracting total RNA by a guanidinium isothiocyanate method, and synthesizing a first cDNA chain by using reverse transcriptase M-MLV (promega) and the total RNA as a template, wherein a reaction system and an operation process are as follows: taking 5 μ g of Total RNA, adding 50ng oligo (dT), 2 μ L dNTP (2.5 mM each) and DEPC water in turn to make the reaction volume be 14.5 μ L; after uniformly mixing, heating and denaturing at 70 ℃ for 5 min, then rapidly cooling on ice for 5 min, then sequentially adding 4 mu L of 5 XFirst-stand buffer, 0.5 mu L of RNase (200U) and 1 mu L M-MLV (200U), uniformly mixing and centrifuging for a short time, carrying out warm bath at 42 ℃ for 1.5 h, taking out, heating at 70 ℃ for 10 min, and stopping reaction; the first strand cDNA is synthesized and stored at-20 deg.C for further use.

Performing PCR amplification on a GmMATE75 gene by using the synthesized cDNA as a template and a GmMATE75 specific primer;

specific primers were designed as follows:

MA 75-F: CGGGATCCCGATGGACGAGAATAGAAGTTCCAAC, GGATCC is BamH I restriction enzyme site;

MA 75-R: CCGCTCGAGCGGTCATTTGCAGTGTCCTTGTTGCTG, CTCGAG is XhoI restriction site;

and (3) PCR reaction conditions: 4 min at 95 ℃; 30 cycles of 95 ℃ for 30s, 54 ℃ for 30s, and 72 ℃ for 1 min; 10 min at 72 ℃; the reaction system (20. mu.L) was 1. mu.L of cDNA, 2. mu.L of 10 × Advantage 2 PCR Buffer, 1.8. mu.L of 50 × dNTP Mix (10 mM each), 0.2. mu.L of forward primer (10. mu.M), 0.2. mu.L of reverse primer (10. mu.M), 0.2. mu.L of Advantage 2 PCR Polymerase Mix, 14.6. mu.L of PCR-Grade water; after the PCR was completed, 5. mu.L of the resulting product was subjected to agarose gel electrophoresis to examine the specificity and size of the amplified product, which was consistent with the size of the target gene (see FIG. 1).

The DNA fragment is obtained by an agarose gel recovery method and is connected to a pMD18T cloning vector, the used kit is a pMD18-T vector kit (Dalianbao biology), and the reaction system and the operation process are as follows: 1.5 mu L of PCR product is taken, 1 mu L of pMD18-T vector (50 ng/. mu.L) and 2.5 mu L of 2 × Ligation solution I are sequentially added, mixed evenly and placed at 16 ℃ for overnight reaction; transferring the ligation product into escherichia coli DH5 alpha by adopting a heat shock transformation method; positive clones were selected using LB solid medium containing ampicillin (Amp), and plasmids were extracted after colony PCR detection was correct.

Extracting plasmids by using a SanPrep column type plasmid DNA small extraction kit (Shanghai worker), and detecting the integrity and concentration of the extracted plasmids by taking 1 mu L of the extracted plasmids for agarose gel electrophoresis; plasmid pMD-18T-GmMATE75And pENTR-2B for double enzyme digestion (100 mu L system), the reaction system and the operation process are as follows: taking 20. mu.L of pMD-18T-GmMATE75And pENTR-2B plasmid, adding 10 mul enzyme cutting buffer solution, 5 mul and 60 mul ddH of corresponding incision enzyme respectively₂O, mixing uniformly, centrifuging for a short time, and reacting at 37 ℃ overnight; all the products of the digestion are spotted in agarose gel for electrophoresis, and thenGmMATE75The fragment and pENTR-2B carrier fragment are respectively subjected to gel recovery, and a SanPrep column type DNA gel recovery kit (Shanghai Biotechnology) is used in the whole process; taking 1 microliter of the recovered product, detecting the size and concentration of the recovered fragment by agarose gel electrophoresis, and storing at-20 ℃ for later use.

The recovered DNA was purified by using T4 DNA Ligase (TaKaRa)GmMATE75The fragment and pENTR-2B vector fragment were ligated, and the reaction system (20. mu.L) and the procedure were as follows: taking 10 μ LGmMATE75 The DNA fragment was sequentially added with 2. mu.L of pENTR-2B vector DNA, 2. mu.L of 10 XT 4 DNA Ligase Buffer, 1. mu. L T4 DNA Ligase, and 5. mu.L of ddH₂O, mixing uniformly, centrifuging for a short time, and then carrying out water bath at 16 ℃ for overnight reaction; then transferring the ligation product into Escherichia coli DH5 alpha by using a heat shock transformation method, and screening positive clones by using a solid culture medium containing 50mg/L kanamycin (Km); and selecting single colony shake bacteria, detecting by PCR, and selecting a sample with correct detection for sequencing.

And (3) carrying out plasmid extraction on the bacterial liquid with correct sequencing, carrying out LR reaction on the bacterial liquid and an over-expression vector pK2GW7, obtaining a plasmid of a recombinant vector pK2-35S-GmMATE75 after reaction, and converting E.coliDH5 alpha, culturing with solid culture medium containing 100mg/L spectinomycin, screening positive clone and carrying out colony PCR detection; the plasmid was extracted to transform Agrobacterium pMP90 competent cells.

The recipient plant used in this experiment was Wild Type (WT) tobacco: (A)Nicotiana L) Obtained byThe preparation method comprises soaking tobacco seed in 75% ethanol for 30s, washing with sterile water, and adding 0.1% HgCl₂Soaking for 8min, washing with sterile water for several times, sowing on MS culture medium, dark culturing at 28 deg.C for 6 d, germinating, transferring to light incubator (25 deg.C, 16h/d light), and subculturing with MS culture medium once a month. And (3) taking WT tobacco leaves, and transforming the tobacco leaves by using an agrobacterium-mediated leaf disc transformation method. The transformed leaf discs were placed on a co-culture solid medium MS1 and were cultured for 24 hours in the absence of light. The leaves were then transferred to shoot induction solid medium MS4 to induce germination, and the selection marker was kanamycin. After about 1 month, sprouts grew out. Cutting buds 1-2cm in length, and culturing in MS culture medium until they grow into complete tobacco seedling. Taking leaves from the cultured tobacco, extracting total DNA, taking the total DNA as a template, carrying out PCR identification by using a specific primer of GmMATE75, identifying successfully transformed tobacco plants, continuously culturing the successfully transformed tobacco and carrying out subsequent experiments.

Example 2: evaluation of aluminum resistance of GmMATE75 transgenic tobacco plants

This experiment was carried out to evaluate the resistance of tobacco to aluminium mainly from 3 aspects, the relative root elongation under aluminium treatment, the soluble sugar content in plant cells after aluminium treatment and hydrogen peroxide (H)₂O₂) And (4) content. Since the main harm of aluminum toxicity to plants under acidic conditions is to inhibit the growth of roots, the determination of relative root elongation is indicative of the extent of damage to plants by aluminum toxicity. Soluble sugars are used as solutes that relieve osmotic pressure in plant cells, and when plant cells are damaged, soluble sugars are produced to relieve cell damage. The extent of cell damage and the amount of soluble sugars produced can be considered to be positively correlated before the upper limit of cellular tolerance is reached. H accumulation in plant tissues₂O₂Is formed by the oxidation reaction of super-cation anions catalyzed by some oxidase (mainly superoxide dismutase SOD), and the content of the super-cation anions can indirectly reflect the damage degree of plant cell membrane lipids.

(1) Tobacco relative root elongation determination

Taking GmMATE75 transgenic tobacco (MAC) with young root length of about 1cm, washing off the solid from root with waterThe culture medium is cultured in tap water for 3 days, and then added with 0.5mM CaCl with pH of 4.5₂Pretreating in the solution for 12 h; then measuring the root length of tobacco, and adding AlCl into the culture solution₃To make the concentrations 0. mu.M, 50. mu.M, 100. mu.M, 200. mu.M and 400. mu.M, respectively; the tobacco root length was then determined at 24h, 48h and 72h, respectively, with WT type tobacco as control and three replicates per set of experiments.

Root elongation (cm) = root length determination at this time-root length determination at last time;

relative root elongation (%) = (root elongation/0. mu.M aluminum chloride-treated root elongation)

100%；

According to the measurement result, the relative root elongation of the tobacco is in a descending trend along with the increase of the aluminum treatment time and the increase of the treatment concentration; however, the relative root elongation of the GmMATE75 transgenic tobacco was significantly higher than that of the corresponding WT tobacco (fig. 2) at any time period and treatment concentration, and thus it was found that the roots of the GmMATE75 tobacco exhibited higher resistance to aluminum stress than the roots of the WT tobacco.

(2) Tobacco cell soluble sugar content determination

The tobacco of GmMATE75 type which is about 1 month after rooting after identification is taken and put into clear water for water culture for one week, and then 0.5mM CaCl with pH4.5 is added₂Pretreating the solution for 12h, and adding AlCl at 0 μ M, 50 μ M, 100 μ M, 200 μ M and 400 μ M respectively₃After 24, 48 and 72H treatment, root and leaf samples were taken, 1g of each sample was added with liquid nitrogen for grinding, 1.5 mL of 1M Tris-HCl (pH 7.4) was added, transferred to an EP tube, centrifuged at 12000 rpm for 15 min, and the supernatant was transferred to a new EP tube for soluble sugars and H₂O₂Measuring the content; 3 samples of each were assayed, with WT tobacco as a control.

The content of soluble sugar is determined by anthrone colorimetry in [ mu ] mol.g^-1FW denotes. Adding 20 mu L of supernatant into 1 mL of 1 mg/mL^-1Adding 180 mu L ddH into the anthrone solution₂After O, cooling to room temperature after boiling water bath for 10 min;calculating the soluble sugar content after measuring the absorbance at 625 nm and entering a standard curve y =0.485x +0.0118 (the standard curve is measured by a glucose standard solution);

according to the measurement result, the soluble sugar of the tobacco after the aluminum treatment is mainly generated in leaf cells; by contrast, the production of polysaccharides in the root and leaf cells of WT type tobacco was higher than those of the leaf and root cells of GmMATE75 type tobacco after being subjected to aluminum stress (fig. 3), and thus it was found that GmMATE75 type tobacco was less affected by aluminum toxicity than WT type tobacco.

(3) Tobacco cell H₂O₂Determination of content

Measurement of H Using the sample extract obtained in step (2)₂O₂Content (c); h₂O₂The method adopts a xylenol orange method to carry out determination, and the determination is carried out according to mu mol/g^-1FW denotes. By ddH₂O water preparation reagent A (3.3 mM FeSO)₄, 3.3 mM (NH4)₂SO4, 412.5 mM H₂SO₄) And reagent B (165 μ M xylenol orange, 165 mM sorbitol). The reagent A and the reagent B are mixed according to the proportion of 1:10 before use to form a working reagent. The working reagent and H₂O₂Mixing the solutions at a ratio of 2:1, performing metal bath at 30 deg.C for 30 min, and measuring OD₅₆₀Substitution of values into the standard curve y =0.26544x-0.0523 calculates H₂O₂Content variation (in H)₂O₂Standard solution preparation standard curve).

As is clear from the results, H was produced in the root cells of the aluminum-treated tobacco₂O₂More; h in WT-type tobacco cells can be known by comparison₂O₂The content of the tobacco was significantly higher than that of the tobacco GmMATE75 (fig. 4), so that it was found that the tobacco GmMATE75 had less damage to cell membrane lipids under aluminum stress compared to the WT type tobacco.

Sequence listing

<110> university of Kunming science

<120> Danbo black soybean citric acid transport protein gene and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Danbo black soybean (Glycine max)

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ttcaaaggtg caaggaatgt tttcaagctg gatgctctat ctcgggagat attagggatt 120

gcactcccct cagcactggc cgtttctgct gatccaattg cttctctcat agacacagca 180

ttcataggcc gtttaggacc ggtggaactt gcagctgctg gagtttccat ttctttgttt 240

aaccaagctt cgaggattac catattccct ctggtcaaca ttaccacttc ctttgtggct 300

gaagaagata ccattcaaaa actgaacacc aaagcagctg aaaatggtaa tagtaaggcc 360

aagttcggcg aaacaattat gccagaggat catatgcttc aagacatgga aaaaggtacc 420

cccaaagtga tgaatactga cgctccaaca gaatttaggg aagaaaaaga tgaatcaaag 480

gaatataatg ctaccggcaa caatgacaca aatattggag atggagccaa tacaatatgc 540

aagttttcat cggttactag cagtaagaag agtaaggaca aagttggaaa gaaaaagcga 600

cttattgctt cagcatcaac agcactactt tttggcacaa tccttggtct cattcaagct 660

gcagttctta tatttgcaac caaacctctg ttaggtgtta tgggtgtcaa acgagattct 720

cctatgctaa aacctgcaga gagctactta agattgagat cgttcggggc accagcagta 780

cttctctcct tggccatgca aggcatcttt cgagggttca aggatacaac aactccttta 840

tatgtcatcg tttcgggata tgcattgaat gtcatattgg acccaatttt tattttcaca 900

ttgaagttag gcatcaaagg tgcagccatt gcacatgttc tctcccagta catgatggca 960

ttcactctct tattgatatt aatgaaaaaa gtgcatctcc tacctccaag aataaaggat 1020

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gttgctgttc aggcaattct agcttgttct tttactgaga aagactataa gaaagcaaca 1260

gcagcagcaa caaggacact gcaaatga 1288

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<213> Danbo black soybean (Glycine max)

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Met Asp Glu Asn Arg Ser Ser Asn Glu Pro Asn Lys Trp Lys Met Pro

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Leu Phe Val Phe Phe Lys Gly Ala Arg Asn Val Phe Lys Leu Asp Ala

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Leu Ser Arg Glu Ile Leu Gly Ile Ala Leu Pro Ser Ala Leu Ala Val

35 40 45

Ser Ala Asp Pro Ile Ala Ser Leu Ile Asp Thr Ala Phe Ile Gly Arg

50 55 60

Leu Gly Pro Val Glu Leu Ala Ala Ala Gly Val Ser Ile Ser Leu Phe

65 70 75 80

Asn Gln Ala Ser Arg Ile Thr Ile Phe Pro Leu Val Asn Ile Thr Thr

85 90 95

Ser Phe Val Ala Glu Glu Asp Thr Ile Gln Lys Leu Asn Thr Lys Ala

100 105 110

Ala Glu Asn Gly Asn Ser Lys Ala Lys Phe Gly Glu Thr Ile Met Pro

115 120 125

Glu Asp His Met Leu Gln Asp Met Glu Lys Gly Thr Pro Lys Val Met

130 135 140

Asn Thr Asp Ala Pro Thr Glu Phe Arg Glu Glu Lys Asp Glu Ser Lys

145 150 155 160

Glu Tyr Asn Ala Thr Gly Asn Asn Asp Thr Asn Ile Gly Asp Gly Ala

165 170 175

Asn Thr Ile Cys Lys Phe Ser Ser Val Thr Ser Ser Lys Lys Ser Lys

180 185 190

Asp Lys Val Gly Lys Lys Lys Arg Leu Ile Ala Ser Ala Ser Thr Ala

195 200 205

Leu Leu Phe Gly Thr Ile Leu Gly Leu Ile Gln Ala Ala Val Leu Ile

210 215 220

Phe Ala Thr Lys Pro Leu Leu Gly Val Met Gly Val Lys Arg Asp Ser

225 230 235 240

Pro Met Leu Lys Pro Ala Glu Ser Tyr Leu Arg Leu Arg Ser Phe Gly

245 250 255

Ala Pro Ala Val Leu Leu Ser Leu Ala Met Gln Gly Ile Phe Arg Gly

260 265 270

Phe Lys Asp Thr Thr Thr Pro Leu Tyr Val Ile Val Ser Gly Tyr Ala

275 280 285

Leu Asn Val Ile Leu Asp Pro Ile Phe Ile Phe Thr Leu Lys Leu Gly

290 295 300

Ile Lys Gly Ala Ala Ile Ala His Val Leu Ser Gln Tyr Met Met Ala

305 310 315 320

Phe Thr Leu Leu Leu Ile Leu Met Lys Lys Val His Leu Leu Pro Pro

325 330 335

Arg Ile Lys Asp Leu Gln Ile Phe Arg Phe Leu Lys Asn Gly Gly Leu

340 345 350

Leu Met Leu Lys Val Ile Ala Val Thr Phe Cys Val Thr Leu Ala Thr

355 360 365

Ser Leu Ala Ala Arg Leu Gly Ser Ile Pro Met Ala Ala Phe Gln Thr

370 375 380

Cys Leu Gln Val Trp Met Thr Ser Ser Leu Leu Ala Asp Gly Leu Ala

385 390 395 400

Val Ala Val Gln Ala Ile Leu Ala Cys Ser Phe Thr Glu Lys Asp Tyr

405 410 415

Lys Lys Ala Thr Ala Ala Ala Thr Arg Thr Leu Gln Met

420 425

<210> 3

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<212> DNA

<213> Artificial sequence (Artificial)

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cgggatcccg atggacgaga atagaagttc caac 34

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<212> DNA

<213> Artificial sequence (Artificial)

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ccgctcgagc ggtcatttgc agtgtccttg ttgctg 36

Claims

1. Danbo black soybean citric acid transport protein geneGmMATE75The application of the aluminum-resistant agent in improving the aluminum-resistant capability of tobacco is characterized in that: danbo black soybean citric acid transport protein geneGmMATE75The nucleotide sequence of (A) is shown as SEQ ID NO. 1.