CN112813079A

CN112813079A - Danbo black soybean GmFER84 gene and application thereof in soybean aluminum stress improvement

Info

Publication number: CN112813079A
Application number: CN202110383238.7A
Authority: CN
Inventors: 林涛; 玉永雄; 胡艳; 蒋曹德; 刘卢生
Original assignee: Guang'an Feed Industry Management Station; Southwest University
Current assignee: Guang'an Feed Industry Management Station; Southwest University
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2021-05-18
Anticipated expiration: 2041-04-09
Also published as: CN112813079B

Abstract

The invention discloses an application of a Danbo black soybean GmFER84 gene in soybean aluminum stress improvement. The nucleotide sequence of the GmFER84 gene is shown in SEQ ID NO. 1. The overexpression of the GmFER84 gene can obviously improve the aluminum stress resistance of soybeans, and can be applied to the improvement of soybean germplasm resources and the screening of varieties.

Description

Danbo black soybean GmFER84 gene and application thereof in soybean aluminum stress improvement

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a Danbo black soybean GmFER84 gene and application thereof in soybean aluminum stress improvement.

Background

Acid soil and aluminum poison seriously affect 30 percent of arable area worldwide, and are one of the main factors restricting grain production. Typical acid soil such as red earth, red earth and the like widely distributed in southern areas of China is about 2 million hectares, and the production of crops is severely restricted.

Soybeans are traditional crops in China and are important grain and economic crops. The soybean planting range is wide, and the soybean planting method can be divided into a northern soybean planting area, a Huang-Huai-Hai soybean planting area and a southern soybean planting area according to regions. Most of soil in south China belongs to acidic red loam, so that aluminum toxicity stress is easy to occur, and the problems of slow growth and development of crops, reduction in quality and yield and the like are caused.

Plants have evolved a variety of mechanisms of acid-aluminum resistance stress, and the physiological basis of plant aluminum resistance in acid soil is generally considered to be in vitro rejection and in vivo chelation. Wherein, when plants are subjected to abiotic stress such as non-metal poisoning, the precise regulation of genes at the transcription level and the post-transcription level is involved, and the stress mainly comprises AP2/EREBP, MYB, bHLH and C₂H₂Zinc finger transcription factor family. However, no report related to the GmFER84 gene in improvement of soybean aluminum stress is found at present.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of the Danbo black soybean GmFER84 gene in soybean aluminum stress improvement, the gene overexpression can obviously improve the aluminum stress resistance of soybeans, and the gene can be applied to soybean germplasm resource improvement and variety screening.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

an application of a Danbo black soybean GmFER84 gene in soybean aluminum stress improvement, wherein a nucleotide sequence of the GmFER84 gene is shown as SEQ ID No. 1.

Furthermore, the amino acid sequence coded by the GmFER84 gene is shown as SEQ ID NO. 2.

An agent for improving the aluminum resistance of soybeans, which can promote the expression of GmFER84 gene.

A kit for screening aluminum-resistant soybeans comprises primers for amplifying the GmFER84 gene.

A plasmid contains a GmFER84 gene shown as SEQ ID NO. 1.

A recombinant expression vector comprises a GmFER84 gene shown as SEQ ID NO. 1.

An engineering bacterium comprises a GmFER84 gene shown as SEQ ID NO. 1.

The GmFER84 gene is applied to soybean germplasm resource improvement and variety screening.

The invention has the beneficial effects that:

the aluminum stress resistance of the soybeans can be remarkably improved by overexpression of the GmFER84 gene, and the method can be applied to soybean germplasm resource improvement and variety screening.

Drawings

FIG. 1 is a GmFER84 protein evolutionary tree analysis;

FIG. 2 shows the results of predictive analysis of the primary structure and protein function of GmFER84 transcription factor;

FIG. 3 shows a comparison of GmFER84 homologous sequences;

FIG. 4 shows the subcellular localization of GmFER84 protein;

FIG. 5 is a screening of transgenic Danbo black soybean positive hairy roots;

FIG. 6 shows the study on the aluminum resistance of Danbo black soybean by overexpressing GmFER84 gene.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

Example 1 cloning of GmFER84 Gene

1. Cultivation and treatment of plant material

(1) Black soybean seeds were sterilized with 1% NaClO for 20min, washed 3 times with double distilled water and incubated on wet filter paper. After germination, seedlings were transplanted into 1/2 Hoagland nutrient solution (pH 6.0) for continued culture, with the nutrient solution being changed every 2 days. 200 μmol/(m) at 27 ℃/22 ℃ (day/night)²S) cultivation under constant light conditions.

(2) Taking 20 seedlings with 2 weeks of age and consistent growth, adding 0.5mmol/L CaCl₂Preculture overnight in solution (pH 4.5) and then separately with 0 (containing 0.5mmol/L CaCl)₂pH 4.5) and 50. mu. mol/L AlCl₃(containing 0.5mmol/L of CaCl)₂pH 4.5) for 72h, 3 biological replicates each, 0 μ M (1), 0 μ M (2), 0 μ M (3) for the control group, 50 μ M (1), 50 μ M (2), 50 μ M (3) for the aluminum treated group, and the treated root tips were flash frozen in liquid nitrogen and stored in a freezer at-80 ℃.

(3) Taking 2-week-old seedlings with consistent growth vigor, and adding CaCl at a concentration of 0.5mmol/L₂Preculture overnight in solution (pH 4.5) in 50. mu. mol/L AlCl₃(containing 0.5mmol/L of CaCl)₂pH 4.5) treating in the treating solution for 0, 3, 6, 9, 12, 24, 48 and 72 hours respectively, taking root tips, quickly freezing by liquid nitrogen, and storing at-80 ℃ for later use; taking 0, 50 mu mol/L AlCl₃Processing the black soybean roots for 24h respectively, quickly freezing the black soybean roots by liquid nitrogen, and storing the processed black soybean roots at the temperature of minus 80 ℃ for later use.

2. Extraction of RNA and synthesis of cDNA by reverse transcription

RNA extraction was performed according to RNAiso kit instructions. Each RNA concentration was adjusted to about 500 ng/. mu.L for reverse transcription, see Prime Script^TMreagent Kit with gDNA Eraser (Perfect Real Time) Kit instructions.

3. Amplification and recovery of sequences of interest

According to a gene sequence provided on a soybean genome website, a GmFER84 full-length primer FL-GmFER84-F/R is designed, and amplification of a target gene is carried out by using a high-fidelity enzyme premix, wherein a reaction system is shown in a table 1:

TABLE 1 PCR reaction System

The PCR reaction procedure was as follows:

at 95 ℃ for 30 s; 94 ℃, 10s, 56 ℃, 10s, 72 ℃, 30s, 30 cycles; 72 ℃ for 3 min.

After the PCR product is detected by 1% agarose gel electrophoresis, the product is recovered by using an OMEGA gel recovery kit.

Example 2 bioinformatic analysis

1. At 50. mu. mol/L AlCl₃(containing 500. mu. mol/L CaCl)₂) The treated root tip of the Danbo black soybean is taken as a material, RNA is extracted, cDNA obtained by reverse transcription is taken as a template, a specific primer is designed by the full-length sequence of soybean genome FER84, RT-PCR is carried out, and the full-length CDS sequence of the gene is obtained by amplification. The sequencing result shows that the size of the target fragment is 969bp, and the sequence of the GmFER84 gene of the Danbo black soybean is completely consistent with the comparison sequence of genomic Phytozome and NCBI (https:// blast.ncbi.n.m.nih.gov/blast.cgi) by comparing with the full-length sequence of genomic FER84, thereby indicating that the GmFER84 gene is conserved in soybean. The GmFER84 CDS sequence was translated into a protein sequence, and GmFER84 was known to encode 322 amino acids.

2. The physicochemical property prediction is carried out through the ProtParam (https:// web. ExPaSy. org/ProtParam /) function of the ExPasy online software, protein signal peptide is predicted by SingalP online software (http:// www.cbs.dtu.dk/services/SignalP /), nuclear localization signal prediction is carried out by NetNES 1.1 online software (http:// www.cbs.dtu.dk/services/NetNES /), transmembrane domain prediction is carried out by TMHMM Server v.2.0 online software (http:// www.cbs.dtu.dk/services/TMHMM-2.0/), functional domain analysis is carried out by CDD online software (https:// www.ncbi.nlm.nih.gov/Structure/CDD/wrpsb. cgi), the secondary structure of the polypeptide is predicted by SOPMA (https:// npsa-prabi. ibcp.fr/cgi-bin/npsa _ Automat.plpage ═ npsa _ SOPMA. htmL), and the three-dimensional structure of the polypeptide is analyzed and predicted in Phyre2 online software (http:// www.sbg.bio.ic.ac.uk/Phyre2/htmL/page ═ cgiid ═ index). The amino acid sequence of GmFER84 was aligned by BLAST, and the sequences with higher homology were selected and aligned, and a phylogenetic tree was constructed using MEGA7.0 (FIG. 1).

3. The GmFER84 CDS sequence was translated into a protein sequence, which was known to encode 322 amino acids. The molecular weight of 36147.61ku of the GmFER84 protein is obtained by predicting the ProtParam function of ExPasy software, and the molecular formula of the protein is C₁₅₈₅H₂₄₆₉N₄₃₅O₅₀₁S₁₆Total atomic number 5006, Theoretical isoelectric point (Theoretical pI) 5.09. The fat index (Aliphatic index) was 76.06, The average total hydrophilic coefficient (GRAVY) was-0.436, The N-terminus of The protein was The estimated half-life of The protein (The estimated half-life of The protein) 30 hours in vitro in mammalian reticulocytes, 20 hours in yeast, and 10 hours in E.coli. The Extinction coefficient (Extinction coefficients) of the protein is M^-1cm^-1Measured in units of 280 nm of water. Assuming that all pairs of cysteine (cys) residues form cystine, the extinction coefficient of the cys residue of the protein is 23630, and Abs 0.1% (═ 1g/l) is 0.654. Assuming that all residual cys residues were reduced, the protein cys residue had an extinction coefficient 23380, and an Abs 0.1% (═ 1g/l) of 0.647.

In the amino acid composition, asparagine Asn (N), Leu (L) account for the highest percentage, 9.6%, 9.3%, and tryptophan Trp (W) account for the lowest percentage, 0.3%. The positive charge residue (Arg + Lys)) of the GmFER84 protein is 30, the negative charge residue (Total number of negative charge residues (Asp + Glu)) is 41, and the N end of the protein sequence is methionine M (Met); the Instability index (Instability index) was 38.34(<40), which is a stable protein.

The GmFER84 transcription factor protein signal peptide is predicted by SingalP online software, and the result shows that the nuclear localization signals of various amino acids of GmFER84 are weak and do not reach the threshold value. Transmembrane domain prediction was performed using TMHMMs Server v.2.0 online software and results showed that GmFER84 transcription factor did not have a transmembrane domain.

Nuclear localization signal prediction is carried out on GmFER84 protein through NetNES 1.1 online software, and the result shows that the GmFER84 nuclear localization signal is probably located at 130-205 amino acids at the N end (FIG. 2A), but the signal is weak and does not reach the threshold value. The GmFER84 transcription factor protein signal peptide is analyzed by SingalP online software, and the result shows that the probability of the GmFER84 protein to generate the signal peptide is extremely small and is only 0.08% (see figure 2B). TMHMMS Server v.2.0 on-line software analyzed the transmembrane region of GmFER84, and showed that GmFER84 did not transmembrane (FIG. 2C). Functional domain analysis of the GmFER84 protein was performed on CDD online software, and it was found that a transcription regulator (transcription regulators) bHLH-SF (basic Helix Loop Helix) subfamily conserved domain (Access: cl00081) exists at the 130-205 amino acids of the N-terminal of GmFER84, the E value is 3.11E-33 (FIG. 2D), and the bHLH protein is a transcription regulator found in organisms from yeast to human. Members of the bHLH superfamily have two highly conserved and functionally distinct regions. The base portion is located at the amino terminus of the bHLH and the DNA can be bound to a six nucleotide sequence called the E-box (CANNTG). Different bHLH protein families recognize different E-box consensus sequences. At the carboxy terminus of this region is a region of HLH, which interacts with other proteins to form homodimers and heterodimers. bHLH proteins function as a diverse group of regulatory factors because they recognize different DNA sequences and dimerize with different proteins.

4. When compared by using BLAST of NCBI database to compare with GmFER84 amino acid sequence and searching homologous sequences of other species of GmFER84, the result of amino acid primary structure comparison analysis shows that GmFER84 has high conservation (the amino acid sequence number of GmFER84 is shown in figures 3A-E, XP _ 003540203.1), has a typical bHLH structural domain (HLH domain: SM000353), and has a 50aa sequence DNA binding domain at the C end of the amino acid sequence which is highly similar (LISENRRRGRMKENKLYALRSLVPNITKMDKASIIGDAVSYVHDLQAQAR K).

Example 3 GmFER84 Gene subcellular localization analysis

1. Gene synthesis

The GmFER84-GFP gene was cloned by gene synthesis into pHBT-GFP-NOS vector.

2. Concentration of plasmid mininote assay

(1) Inoculating the correctly synthesized bacterial liquid pHBT-FER84-GFP and pHBT-NLS-mCherry into a test tube filled with 5mL LB culture medium (resistance is Amp), and culturing at 37 ℃ and 220rpm overnight;

(2) taking 5mL of bacterial liquid, filling the bacterial liquid into 1.5mL of EP tubes in batches, and centrifuging the bacterial liquid for 2min at the room temperature at 11,000 rpm;

(3) carefully remove the supernatant and blot the residual liquid on the vessel wall with a clean paper towel. Adding 250 mu L of the solution I, blowing and beating for several times, and fully suspending and precipitating by using a vortex oscillator;

(4) adding 250 mu L of the solution II, and slightly turning and mixing the solution II back and forth for 4-6 times until the lysate is clarified;

(5) adding 350. mu.L of solution III, and gently inverting for several times until a flocculent precipitate is formed;

(6) centrifuging at room temperature for 10min to obtain compact genome DNA and clastic mass;

(7) carefully aspirating the supernatant, loading onto a small adsorption column equipped with centrifuge tubes, capping, and centrifuging at 11,000rpm at room temperature for 1 min;

(8) sucking the penetrated liquid to a small absorption column with an assembled centrifuge tube, covering the absorption column, centrifuging at room temperature of 11,000rpm for 1min, and discarding the filtrate;

(9) adding 500 μ L HBC buffer solution into the absorption column, covering, centrifuging at room temperature of 11,000rpm for 1min, and discarding filtrate;

(10) adding 700 μ L of DNA Wash Buffer diluted with ethanol, covering, centrifuging at 11,000rpm for 1min at room temperature, and discarding the filtrate;

(11) repeating the step 10;

(12) centrifuging at room temperature of 11,000rpm for 2min, placing the DNA adsorption column in a DNA collection tube, and drying at room temperature for 10 min;

(13) add 50. mu.L ddH₂0 (having been subjected to a water bath at 65 ℃) in a DNA adsorption column and centrifuged at 11,000rpm for 2min at room temperature;

(14) sucking the filtrate into a DNA adsorption column, and centrifuging at room temperature of 11,000rpm for 2 min;

(15) 1 μ L of each was taken to extract the concentration and quality of plasmid.

3. Protoplast preparation process

(1) Taking the uninjured and undressed arabidopsis leaves, cleaning with clear water, cutting into strips with the width of 1mm, and placing in 0.4M mannitol solution (maintaining osmotic pressure);

(2) taking out the strips, and placing in enzymolysis solution (adding enzyme into small conical flask to dissolve cell wall);

(3) carrying out enzymolysis at 25 ℃ and 40-50 rpm in the dark (time is variable, 0.5-3 h), and carrying out microscopic examination;

(4) filtering with a double-layer filter screen, filtering into a 10mL glass centrifuge tube, washing with a W5 dissolving solution, and filtering;

(5) centrifuging at 700rpm for 10min, and discarding the supernatant; the precipitate was gently washed with pre-cooled W5 lysate, 4mL, 100g (700rpm) and centrifuged for 1 min;

(6) the supernatant was discarded, and the pellet was gently washed with ice-cold W5 solution (4 mL tube) and left on ice for 30min (protoplast recovery);

(7) centrifugation was carried out at 23 ℃ and 100g (700rpm) for 1min, the supernatant was discarded, and the pellet was resuspended in 0.5mL of Mmg solution (23 ℃ for each step).

4. Protoplast transformation and visualization

(1) Taking 10 mu g of pHBT-FER84-GFP and 10 mu g of pHBT-NLS-mCherry plasmid into a 1.5mL EP tube, adding 100 mu L of the protoplast in the step 7, and mixing the protoplast and the protoplast gently by using a spearhead;

(2) add 110. mu.L PEG/Ca²⁺Mixing the solution (the volume of the solution is equal to that of the plasmid and the protoplast) gently and uniformly, and standing for 10-20 min;

(3) 440 μ L W5 solution (volume PEG/Ca) was added²⁺4 times of the PEG), reversing the direction and mixing evenly, centrifuging for 1min at 23 ℃ and 700rpm, discarding the supernatant and removing the PEG;

(4) adding 100 mu L W5 solution, mixing uniformly, adding 900 mu L W5 solution, mixing uniformly; placing the mixture in low light at 23 ℃, and incubating for 12-18 h;

(5) sampling observation shows that FER84 protein is localized in cell nucleus (see FIG. 4).

The transcription factor plays a role in transcriptional regulation in the nucleus, and in order to verify whether the protein coded by the GmFER84 gene is located in the nucleus, GmFER84 and an eGFP reporter gene are expressed in a fusion mode, the positioning of the GmFER84 and the eGFP reporter gene in root cells is observed by transforming Arabidopsis, and as a result, as shown in FIG. 4, the GmFER84 transcription factor only can see a fluorescence signal in the nucleus.

Example 4 transformation of Danbo black Soybean hairy root and Positive hairy root screening

1. Construction of recombinant plasmids

Designing an amplification primer eGFP-GmFER84-F/R of GmFER84, introducing Xba I and Sma I enzyme cutting sites into the upstream and downstream primers, constructing a pBI121-GmFER84-eGFP over-expression vector, and amplifying a target gene by using a high-fidelity enzyme premix.

TABLE 2 cleavage reaction system for recovery product of target fragment

Incubate at 37 ℃ for 2h, and inactivate the enzyme at 65 ℃ for 10 min.

Detecting the enzyme digestion product by using 1% agarose gel electrophoresis, recovering the target fragment and connecting the target fragment with the pBI121 plasmid, wherein the connection reaction system of the target fragment and the pBI121 plasmid is as follows:

TABLE 3 connection System

2. Recombinant plasmid transformed agrobacterium

GV3101 (or K599) agrobacterium-sensitive cells are transformed by a freeze-thaw method, and the specific steps are as follows:

(1) sucking 2 μ L of recombinant plasmid, adding into 50 μ L GV3101 (or K599) competent cells, and mixing gently;

(2) immediately placing on ice for 5min, freezing in liquid nitrogen for 5min, rapidly moving to 37 deg.C water bath for 5min, and ice-cooling for 5 min;

(3) adding LB (or TY) liquid culture medium without antibiotics into a centrifuge tube, and carrying out shaking culture at the temperature of 28 ℃ and the rpm of 180 for 2-3 h;

(4) centrifuging at 6000g for 5min at room temperature, removing part of supernatant, and uniformly spreading the remaining 100 μ L of resuspended thallus on solid culture medium containing 25mg/L of Rif +50mg/L of Km (GV3101) or 50mg/L of Str +50mg/L of Km (K599);

(5) performing inverted plate culture at 28 ℃ for 2-3 days, and selecting a single colony for PCR detection;

(6) and performing amplification culture on the positive clone with correct PCR detection, uniformly mixing the positive clone with 50% glycerol at a ratio of 1: 1, quickly freezing the mixture for 2-5 min by using liquid nitrogen, and storing the mixture at-80 ℃ for later use.

3. The method for obtaining soybean transgenic hairy roots by infecting hypocotyls of Danbo black soybean with agrobacterium rhizogenes comprises the following steps:

(1) sterilizing the surface of soybean, planting in sterile soil, and transforming when cotyledon is not unfolded after 5 days;

(2) culturing K599 target colonies containing target recombinant plasmids in 10mL TY (containing 50mg/L Str and 50mg/L Km) culture solution at 28 ℃ and 180rpm for 16-24 h;

(3) taking 1mL of viable bacteria liquid to be cultured in 100mL of TY (containing 50mg/L Str and 50mg/L Km) culture solution at the temperature of 28 ℃ and at the speed of 180rpm until the OD600 value of the bacteria liquid is 0.8-1.2;

(4) centrifuging at room temperature of 6000g for 10min, discarding the supernatant, suspending the Agrobacterium tumefaciens precipitate with 0.1mol/L MgSO4 of corresponding volume, repeating the steps, and adjusting the OD600 value of the bacterial liquid to about 0.8;

(5) shearing hypocotyls 1cm below cotyledonary node of soybean, immediately placing in prepared dye-invasion solution, and slightly oscillating for 1h by decolorizing shaking table;

(6) sucking off redundant bacteria liquid, placing the explant in a moisture-preserving container paved with wet filter paper for culturing, replacing the filter paper every other day, and screening plants containing positive hairy roots after 3 weeks.

Screening soybean hairy root with LUYOR-3415RG, making positive hairy root capable of showing red fluorescence under 520nm light source (figure 5), extracting DNA and RNA from partial root, and further detecting to determine its positivity.

Example 5 detection of aluminum resistance of transgenic Danbo black soybean and analysis of expression of related genes

1. Wild type soybean (WT) and GmFER84 overexpressing Danbo black soybean (OE) were cultured in different aluminum concentration media for 24h, respectively.

(1) The aluminum content in the root tips was measured and the results are shown in FIG. 6A:

under 50 mu mol/L aluminum treatment, the aluminum content of the root tips of the WT and EV groups of the Danbo black soybeans is obviously higher than that of OE-1, OE-2 and OE-3 strains (the difference is obvious), and the fact that the GmFER84 is over-expressed in the Danbo black soybeans can obviously reduce the absorption of the Danbo black soybeans to aluminum.

(2) The citric acid content in the root tips was measured and the results are shown in fig. 6B:

under 50 mu mol/L aluminum treatment, the citric acid content of the root tips of the WT and EV groups of the Danbo black soybeans is obviously higher than that of OE-1, OE-2 and OE-3 strains (marked by a mark), which shows that the GmFER84 overexpression in the Danbo black soybeans can obviously promote the generation of the citric acid in the Danbo black soybeans and reduce the aluminum content.

(3) The citrate synthase content in the root tip was measured, and the results are shown in FIG. 6C:

under 50 mu mol/L aluminum treatment, the content of the citrate synthase in the root tips of the WT and EV group of the Danbo black soybeans is obviously higher than that of OE-1, OE-2 and OE-3 strains (marked by a mark), which shows that the overexpression of GmFER84 in the Danbo black soybeans can obviously promote the generation of the citrate synthase in the Danbo black soybeans and the generation of citric acid.

Sequence listing

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<120> Danbo black soybean GmFER84 gene and application thereof in improvement of soybean aluminum stress

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Met Asn Thr Met Asp Val His Gln Asp Thr Leu Thr Tyr Met Asn Asp

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20 25 30

Asn Leu Phe Arg Gly Glu Asn Glu Asp Ala Asn Cys Asp His Phe Gly

35 40 45

Ser Asp Leu Ile Asn Asp Cys Phe Ala Asn Asn Gln Gln Gln Leu Leu

50 55 60

Ser Cys Pro Ala Asn Pro Phe Asp Gln Asn Asn Asn Asn Asn Ala Val

65 70 75 80

Asn Val Tyr Asp Pro Ser Ser Thr Phe Ser Ser Phe Ser Tyr Tyr Asp

85 90 95

Arg Glu Leu Lys Gly Glu Gly Gly Glu Glu Leu Asp Glu Glu His Ser

100 105 110

Ser Gly Thr Met Thr Thr Thr Thr Asn Asn Ala Val Gly Lys Pro Lys

115 120 125

Val Lys Thr Asp Met Ser Lys Thr Leu Ile Ser Glu Arg Arg Arg Arg

130 135 140

Gly Arg Met Lys Glu Lys Leu Tyr Ala Leu Arg Ser Leu Val Pro Asn

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Ile Thr Lys Met Asp Lys Ala Ser Ile Ile Gly Asp Ala Ala Ser Tyr

165 170 175

Val His Asp Leu Gln Ala Arg Ala Arg Lys Leu Lys Ala Glu Val Ala

180 185 190

Gly Leu Glu Ala Ser Leu Leu Val Ser Glu Asn Tyr Gln Gly Ser Ile

195 200 205

Asn Tyr Pro Lys Asn Val Gln Val Ala Arg Asn Ile Gly His Pro Ile

210 215 220

Cys Lys Lys Ile Met Gln Met Glu Met Phe Gln Val Glu Glu Arg Gly

225 230 235 240

Tyr Tyr Ala Lys Ile Met Cys Asn Lys Val Gln Gly Leu Ala Ala Ser

245 250 255

Leu Tyr Arg Ala Leu Glu Ser Leu Ala Gly Phe Asn Val Gln Asn Ser

260 265 270

Asn Leu Ala Thr Val Asp Asp Ser Phe Leu Leu Thr Phe Thr Leu Asn

275 280 285

Val Lys Gly Thr Glu Pro Glu Ile Asn Leu Pro Asn Leu Lys Leu Trp

290 295 300

Val Thr Ala Ala Leu Leu Asn Gln Gly Phe Glu Phe Val Ala Ser Phe

305 310 315 320

Pro Ala

Claims

1. An application of a Danbo black soybean GmFER84 gene in soybean aluminum stress improvement is characterized in that a nucleotide sequence of the GmFER84 gene is shown as SEQ ID No. 1.

2. An agent for improving the aluminum resistance of soybeans, which is characterized by being capable of promoting the expression of a GmFER84 gene.

3. A kit for screening aluminum-resistant soybeans, which comprises a primer for amplifying the GmFER84 gene of claim 1.

4. A plasmid, which is characterized by comprising a GmFER84 gene shown as SEQ ID NO. 1.

5. A recombinant expression vector is characterized by comprising a GmFER84 gene shown as SEQ ID NO. 1.

6. An engineering bacterium, which is characterized by comprising a GmFER84 gene shown as SEQ ID NO. 1.

7. The application of the GmFER84 gene in the improvement of soybean germplasm resources and in the screening of varieties in claim 1.