CN114940997A

CN114940997A - Application of GmBBE-like43 gene in regulation and control of plant adaptation to low phosphate and aluminum stress and growth promotion

Info

Publication number: CN114940997A
Application number: CN202210609252.9A
Authority: CN
Inventors: 梁翠月; 陈倩倩; 田江; 李季肤; 刘国选; 陆星; 陈康
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-08-26
Anticipated expiration: 2042-05-31
Also published as: CN114940997B

Abstract

The invention discloses application of a GmBBE-like43 gene in regulation and control of low phosphorus and aluminum stress of plants and growth promotion. The research of the invention shows that the cell wall protein GmBBE-like43 can be up-regulated and expressed when the soybean root system is induced by aluminum stress and low phosphorus stress; under the conditions of phosphorus treatment and aluminum treatment with different concentrations, the expression of the excess GmBBE-like43 obviously promotes the growth of transgenic soybean in vitro hair roots and arabidopsis; the GmBBE-like43 gene has the function of positively regulating and controlling the root system of soybean or arabidopsis thaliana to adapt to low phosphorus stress and aluminum toxicity so as to promote the growth of the root system; meanwhile, the GmBBE-like43 gene has the function of regulating and controlling the growth of root systems of Arabidopsis. Therefore, the GmBBE-like43 has an important effect on the adaptability of the plants to low phosphorus and aluminum stress, and can be used for regulating and controlling the adaptability of the plants to the low phosphorus and aluminum stress of acid soil through a transgenic technology.

Description

Application of GmBBE-like43 gene in regulating and controlling adaptation of plants to low phosphate and aluminum stress and growth promotion

Technical Field

The present invention belongs to the field of gene engineering technology. More particularly, relates to the application of GmBBE-like43 in regulating and controlling the adaptation of plants to low phosphorus and aluminum stress and promoting growth.

Background

The acid soil is widely distributed in the world, the total area of the acid soil accounts for 30-40% of the cultivated land area in the world, and more than 50% of potential cultivated lands are the acid soil and are mainly distributed in tropical, subtropical and temperate regions. The effect of acid soil on the growth inhibition of crops is not only expressed in H ⁺ Too high a concentration is detrimental to the crop itself and often manifests itself as a deficiency in available phosphorus and a synergistic inhibition of plant growth and yield by aluminum poisoning. Therefore, increasing the tolerance of the crop itself to low phosphorus stress and aluminum toxicity in acid soils by means of genetic improvement is considered to be an important approach for the development of sustainable agriculture.

In the long-term evolution process, plants form a series of cooperative adaptive mechanisms of morphology, physiology and molecules, and overcome the barrier factors of effective phosphorus deficiency, aluminum toxicity and the like existing in acid soil. Mechanisms by which plants adapt to aluminum poisoning are reported to include mainly internal tolerance and efflux mechanisms (e.g., secretion of organic acids); the mechanism adapting to low phosphorus stress mainly comprises changing the morphological configuration of roots, increasing the secretion of root organic acids and improving the enzyme activity of purple acid phosphatase, inducing the expression of high-affinity phosphorus transporters and forming symbiosis with rhizosphere microorganisms such as mycorrhizal fungi, etc. (Raghothama, 1999; Vance et al, 2003; Liang et al, 2014). Because the lack of available phosphorus and the aluminum toxicity exist in the acid soil at the same time, the fact that plants possibly have a common adaptation mechanism is suggested, and the lack of available phosphorus and the aluminum toxicity are overcome. Early studies found thatThere are 28 BBE-Like proteins in Arabidopsis, of which the bovine carposine peroxidase AtBBE-Like15 is involved in the synthesis of plant cell wall lignin (Daniel et al, 2015). Later studies reported that 4 peroxidase proteins AtBBE-Like1/2/20/21 in Arabidopsis oxidized oligogalacturonan OGs to produce H ₂ O ₂ And in turn designated OGOX4/3/1/2(Benedetti et al, 2015).

The soybean is an important economic crop, has a very important position in agricultural production in China, and is a leguminous crop which is used for both grain, oil and feed. Currently, in the report of soybean berberine family related protein (BBE), the specific function of the protein is not studied. Although 2 aluminum stress responsive BBE-Like proteins and 1 low-phosphorus stress responsive BBE-Like proteins are found in the soybean BBE-Like protein family in the existing research (Wu et al, 2018; ZHao et al, 2020), no research analysis reports the specific functions and effects of the BBE-Like protein family genes in soybean; also, no key genes have been reported for soybean genotypes in southern acid soils.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the problems and provides application of a soybean GmBBE-like43 gene in regulating and controlling the adaptation of plants to low phosphorus and aluminum stress and promoting growth.

The first purpose of the invention is to provide the GmBBE-like43 gene and the application of the protein thereof.

It is a second object of the invention to provide a product for promoting plant growth and/or increasing plant tolerance to low phosphorus and/or aluminium toxicity stress.

It is a third object of the present invention to provide a method for promoting plant growth and/or increasing plant tolerance to low phosphorus and/or aluminum toxicity stress.

The above purpose of the invention is realized by the following technical scheme:

according to the invention, a cell wall protein GmBBE-like43 with a protein level significantly up-regulated by low-phosphorus stress is found in proteomic analysis results of soybean roots treated by high-low phosphorus, a gene GmBBE-like43 is selected as a candidate gene for research, and then qRT-PCR verification shows that the gene transcription level of the GmBBE-like43 is also significantly up-regulated by low-phosphorus stress and is also significantly up-regulated by aluminum stress. Subsequently, the specific functions of the GmBBE-like43 gene in the process of soybean synergistic response to acid soil low-phosphorus stress and aluminum poisoning are analyzed and researched.

The invention clones a gene GmBBE-like43 which is coordinately regulated and controlled by exogenous phosphorus and aluminum by a real-time fluorescent quantitative PCR and homologous cloning method. Then soybean in vitro hair root transformation and arabidopsis transgenic technology are used for obtaining soybean in vitro hair root and arabidopsis transgenic plant materials which are excessive and inhibit the expression of GmBBE-like43, and the result shows that the GmBBE-like43 gene has the function of regulating and controlling soybean root systems to adapt to low phosphorus stress and aluminum toxicity so as to promote the growth of the root systems; the GmBBE-like43 gene has the functions of regulating and controlling the growth of root systems of arabidopsis thaliana and regulating and controlling the root systems of arabidopsis thaliana to adapt to low phosphorus stress and aluminum toxicity so as to promote the growth of the root systems.

Therefore, the invention provides application of the GmBBE-like43 gene shown in SEQ ID NO.1 or the GmBBE-like43 protein shown in SEQ ID NO. 2 or an expression promoter thereof in positively regulating and controlling the low-phosphorus and/or aluminum toxicity stress resistance and/or root growth of a plant root system, promoting the root growth of a plant, improving the low-phosphorus and/or aluminum toxicity stress resistance of the plant root system, cultivating a low-phosphorus and/or aluminum toxicity resistant plant, preparing a plant growth promoter or improving the adaptability of the plant to acid soil and/or preparing an acid soil growth promoter.

The invention provides a product for promoting plant growth and/or improving plant tolerance to low-phosphorus and/or aluminum toxicity stress, which contains a GmBBE-like43 protein expression promoter.

The invention provides a method for promoting plant growth and/or improving plant tolerance to low-phosphorus and/or aluminum toxicity stress, which promotes plant root growth and/or improves plant tolerance to low-phosphorus and/or aluminum toxicity stress by positively regulating the expression level or protein activity of GmBBE-like43 gene in a plant through a gene editing technology.

Preferably, the plant root growth is promoted and/or the plant tolerance to low phosphorus and/or aluminum toxicity stress is increased by over-expressing the GmBBE-like43 gene in the plant.

Preferably, an expression vector for over-expressing the GmBBE-like43 gene is constructed, and a plant is transformed to obtain a transgenic plant for promoting the growth of a plant root system and/or improving the tolerance of the plant to low-phosphorus and/or aluminum toxicity stress.

The invention has the following beneficial effects:

the invention discloses application of a soybean GmBBE-like43 gene in regulating and controlling plant adaptation to low phosphorus and aluminum stress and growth promotion. The research of the invention shows that the GmBBE-like43 gene is up-regulated by the induction of aluminum stress and the expression of low phosphorus stress, and the expression quantity of the GmBBE-like43 gene is obviously increased along with the prolonging of the phosphorus treatment time. Under the treatment conditions of different phosphorus concentrations, the biomass of the transgenic plant is obviously increased by the expression of the excess GmBBE-like 43; meanwhile, under the condition of aluminum treatment, the growth rate of transgenic plants is obviously increased by the expression of excessive GmBBE-like43, which shows that the GmBBE-like43 can regulate and control the capability of plant roots to adapt to low-phosphorus and aluminum stress. Therefore, the GmBBE-like43 has an important effect on the adaptability of the plants to low phosphorus and aluminum stress, and can be used for regulating and controlling the adaptability of the plants to the low phosphorus and aluminum stress of acid soil through a transgenic technology.

Drawings

FIG. 1 is a table of pattern analysis of soybean GmBBE-like43 (A: the effect of low phosphorus treatment time on the expression pattern of GmBBE-like43 in soybean root system; B: the effect of aluminum treatment time on the expression pattern of GmBBE-like43 in soybean root tip; data are mean and standard error of 3 replicates; asterisks indicate significant difference between control (+ P/-Al) and treatment (-P/+ Al) (Student's t-test); P <0.05,; P <0.01,; P < 0.001);

FIG. 2 is the tissue localization and subcellular localization analysis of GmBBE-like43 (A: histochemical localization analysis of GmBBE-like43 in Arabidopsis, the scale of the first row of overground part pictures is 2mm, the scale of the last two rows of root system pictures is 0.5 mm; B: the subcellular localization analysis result of GmBBE-like43 fusion GFP protein in tobacco leaf blade; C: the subcellular localization analysis result of GmBBE-like43 fusion GFP protein in kidney bean root; the first row in FIGS. B and C is the subcellular localization map of tobacco or kidney bean with transformed empty vector (35S:: GFP), the second row is the subcellular localization map of GmBBE-like43 fusion GFP in tobacco leaf blade or kidney bean root (35S:: GmBBE-like43-GFP), the pictures in FIG. B are respectively the green fluorescence channel (GFP), light lens channel (bright field) and the picture after refocusing (fusion) under a laser confocal microscope, the pictures in the graph C are respectively observed in a green fluorescence channel (GFP), a red fluorescence channel (PI staining) and a superposed picture (fusion) under a laser confocal microscope, and the ruler is 20 mu m;

FIG. 3 shows the effect of excess or inhibition of GmBBE-like43 expression on the growth of transgenic soybean in vitro hairy roots under aluminum-treated conditions (A, B: analysis of expression patterns of GmBBE-like43 in empty-load control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi); C: phenotype of empty-load control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi) under aluminum-treated conditions, scale ═ 2 cm; D, F: amount of growth of empty-load control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi); E, G: relative growth rate of empty-load control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi); asterisks indicate significant difference between empty-load control (OX-CK/RNAi-CK) and transgenic hairy roots (OX/RNAi); s t-test; P < 0.05), **: p <0.01, x: p < 0.001);

FIG. 4 shows the effect of excess or inhibition of GmBBE-like43 expression on the growth of transgenic soybean in vitro hair roots under high and low phosphorus conditions (A, B: analysis of expression patterns of GmBBE-like43 in the no-load control (OX-CK/RNAi-CK) and transgenic hair roots (OX/RNAi); C: phenotype of no-load control (OX-CK/RNAi-CK) and transgenic hair roots (OX/RNAi) under high and low phosphorus treatment conditions, scale ═ 2 cm; D, F: dry weight of no-load control (OX-CK/RNAi-CK) and transgenic hair roots (OX/RNAi); E, G: total root length of no-load control (OX-CK/RNAi-CK) and transgenic hair roots (OX/RNAi); asterisk indicates significant difference between no-load control (OX-CK/RNAi-CK) and transgenic hair roots (OX/RNAi); Student's t-test), *: p <0.05, x: p <0.01, x: p < 0.001);

FIG. 5 is an analysis of the expression pattern of over-expressed GmBBE-like43 on the growth of transgenic Arabidopsis (A: analysis of the expression pattern of GmBBE-like43 in wild-type (WT) and transgenic Arabidopsis plants (OX1, OX 2; B: phenotype of wild-type (WT) and transgenic Arabidopsis lines (OX1, OX2) under aluminum-treated conditions, scale in the figure 0.5 cm; C: amount of root growth of wild-type (WT) and transgenic Arabidopsis lines (OX1, OX2) under aluminum-treated conditions; D: relative rate of root growth of wild-type (WT) and transgenic Arabidopsis lines (OX1, OX2) under aluminum-treated conditions; E: phenotype of wild-type (WT) and transgenic Arabidopsis lines (OX1, OX2) under high-low-phosphorus-treated conditions, scale in the figure 1 cm; F: weight of wild-type (WT) and transgenic lines (OX1, OX2) under high-phosphorus-treated lines; G: wild-type (WT) and transgenic Arabidopsis lines (OX 16), OX2) major root length under high and low phosphorous treatment; asterisks indicate significant differences (Student's t-test) between Wild Type (WT) and transgenic arabidopsis strains (OX1, OX2) as: p <0.05, x: p <0.01, x: p < 0.001).

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 analysis of expression Pattern of GmBBE-like43 Gene

According to the invention, a cell wall protein GmBBE-like43 with the protein level significantly up-regulated by low-phosphorus stress is found in the proteomics analysis result of soybean roots treated by high-low phosphorus, so that a gene GmBBE-like43 is selected as a candidate gene for research, and then qRT-PCR verification shows that the gene transcription level of the gene GmBBE-like43 is also significantly up-regulated by low-phosphorus stress and is also significantly up-regulated by aluminum stress. Subsequently, the specific functions of the GmBBE-like43 gene in the process of soybean synergistic response to acid soil low-phosphorus stress and aluminum poisoning are analyzed and researched.

1. Influence of low phosphorus stress on expression pattern of GmBBE-like43 in soybean root system

Selecting seeds with undamaged seed coats and uniform size by adopting a paper roll seedling method, disinfecting the seeds for 12 hours by using chlorine generated by adding 4.2ml hydrochloric acid into 100ml sodium hypochlorite, and blowing the seeds for 1 hour on a superclean bench for later use. Cutting into 20 × 20cm square filter paper, preparing 1/4 soybean total nutrient solution with pH of 5.8 and sterile water, and sterilizing.

During the cultivation in roll paper, the preservative film was spread on the test table, the filter paper was soaked with the prepared 1/4 soybean complete nutrient solution, 7 sterilized beans were placed at a distance of about 1cm from one side of the filter paper with the umbilicus facing downward, from the first bean roll to the end. The rolled filter paper is placed in a 500mL beaker filled with 1/4 soybean complete nutrient solution with the end without the beans facing downwards, and the filter paper at the upper end with the beans is wrapped by a preservative film. Putting the beaker into an incubator for 24-26 ℃, performing dark culture for 1 day, and performing light/dark (12h/12h) culture for 3-4 days until the radicle is 5-6 cm.

Seedlings with consistent growth were picked and transferred to + P (250. mu.M KH) ₂ PO ₄ ) and-P (5. mu.M KH) ₂ PO ₄ ) In the nutrient solution, each treatment was repeated 4 times, and each treatment was repeated 8 seedlings. Adjusting the pH of the nutrient solution to about 5.8 every two days, changing the nutrient solution once a week, respectively harvesting root system samples at 3d, 6d, 9d and 12d, freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C for use.

2. Effect of aluminum treatment on expression pattern of GmBBE-like43 at soybean root tip

Culturing for 3-4 days by adopting a paper seedling culture method until radicles are 5-6 cm, selecting seedlings with consistent growth, and respectively transferring the seedlings to-Al (pH 4.2 and 0.5mM CaCl) ₂ ) And + Al (pH 4.2, 50. mu.M AlCl) ₃ ，0.5mM CaCl ₂ ) Treating in the solution, respectively harvesting soybean root tips (0-2 cm) after treating for 12 hours, 24 hours, 48 hours, 72 hours and 96 hours, quickly freezing by using liquid nitrogen, and storing in a refrigerator at the temperature of-80 ℃ for later use.

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

Total RNA of the above-treated plant samples was extracted using TRIzol kit (Invitrogen, usa), respectively. The RNA treated with DNase I was synthesized as cDNA by reverse transcription using MMLV-reverse transcription kit (Promega, USA). The qRT-PCR analysis was then performed using SYBR (Promega, USA) kit. After completion of reverse transcription, the samples were diluted 15-fold and subjected to Real-Time fluorescent quantitative PCR analysis by Applied Biosystems StepOnePlus Real-Time PCR system.

Preparation of a standard curve: firstly, sucking 1-2 mu L of sample from cDNA stock solution of each sample to a new PCR centrifuge tube, then diluting the mixed solution by 10 times to obtain a first standard sample S1, diluting the first standard sample by 10 times to obtain a second standard sample S2, and diluting by analogy to obtain 5 concentration gradient standard samples, namely S1, S2, S3, S4 and S5.

Quantitative PCR primers were as follows using GmBBE-like43 gene quantitative primers and housekeeping gene primers whose reference gene was soybean:

GmBBE-like43–RT1-F(SEQ ID NO.3)：5’-CGTGAACCATTCTGAGCCTTC-3’；

GmBBE-like43–RT1-R(SEQ ID NO.4)：5’-AATGGAACCTCTGCCACGTAAG-3’；

EF1-α-F(SEQ ID NO.5)：5’-TGCAAAGGAGGCTGCTAACT-3’；

EF1-α-R(SEQ ID NO.6)：5’-CAGCATCACCGTTCTTCAAA-3’。

preparing a reaction mixed solution: to 20. mu.l of the reaction system, 10. mu.l of SYBR Premix Ex Taq (2X), 0.5. mu.l of forward/reverse primer, and 7. mu.l of ddH were added ₂ O and 2. mu.l template. The amount of the reaction required was calculated by mixing the reagents except for cDNA, and dispensing 18. mu.l/tube, followed by 2. mu.l of cDNA template to a final volume of 20. mu.l.

The quantitative PCR reaction procedure was: pre-denaturation at 95 ℃ for 30s, PCR reaction (denaturation at 95 ℃ for 30s, renaturation at 60 ℃ for 15 s, extension at 72 ℃ for 30 s) for 40 cycles. The detection result was obtained by calculating the expression level of each sample by Real-Time Analysis Software of Rotor-Gene.

As shown in FIG. 1, within 96h of aluminum treatment, the expression of GmBBE-like43 is up-regulated by aluminum stress, and the expression level of the GmBBE-like43 is increased and decreased with the increase of the aluminum stress time, wherein the expression level of the GmBBE-like43 is highest at 12h and is next at 24h, which is respectively 3.8 times and 25.5 times of that of the control treatment (FIG. 1A). In addition, the low phosphorus treatment 3d had no significant effect on the expression level of GmBBE-like43 compared to the high phosphorus treatment under the high and low phosphorus treatment conditions. However, after 6d of low-phosphorus treatment, the expression level of the gene is obviously up-regulated by low-phosphorus stress, and the expression quantity of the gene is obviously increased along with the prolonging of the phosphorus treatment time. That is, the low phosphorus treatment of 6 th, 9 th and 12 th d expressed 2, 3.1 th and 5.6 th times as much as the control treatment, respectively, as compared with the normal phosphorus condition (FIG. 1B).

Example 2GmBBE-like43 histochemical localization and subcellular localization analysis

1. GmBBE-like43 histochemical localization analysis

(1) According to the sequence of the GmBBE-like43 gene, a specific primer pGmBBE-like43 is designed, wherein the specific primer is GUS-F (SEQ ID NO. 7): 5'-CGGAATTCCACGATGGAGTGCAAAAGCAT-3', pGmB BE-like43 GUS-R (SEQ ID NO. 8): 5'-AAGGATCCTTTGGCTTATCCCAATGATGAG-3' are provided. Taking root DNA of soybean genotype YC03-3 as a template, carrying out PCR amplification, and amplifying a 2000bp sequence on an initiation codon of GmBBE-like 43. The reaction conditions are pre-denaturation at 98 ℃ for 5min, denaturation at 98 ℃ for 30s, annealing at 58 ℃ for 30s and renaturation at 72 ℃ for 1-3 min (determined according to the size of the fragment), the process is circulated for 30 times, and extension is carried out at 72 ℃ for 10 min.

(2) Vector construction: subjecting the PCR amplified product to gel electrophoresis, recovering and purifying by using a kit, obtaining a purified PCR product, and then using a homologous recombination kit

II, carrying out recombination ligation reaction on the PCR product and pTF102 vector cut by double digestion method of restriction enzymes EcoR I and BamH I. The reaction system is 20. mu.L, and comprises 6. mu.L of PCR product, 8. mu.L of linearized vector plasmid, 2. mu.L of recombinant ligase Exnase II and 4. mu.L of reaction buffer. And (3) reacting the reagent mixture at 37 ℃ for 30min, transforming the recombinant plasmid into escherichia coli, and sequencing to obtain a plant expression vector of the soybean pGmBBE-like43:: GUS fusion gene. Finally, the target vector is transformed by GV3101 agrobacterium, positive clone is detected, and the bacterial liquid is preserved at-80 ℃.

(3) Obtaining of transgenic arabidopsis thaliana: adopting inflorescence infection method. The method mainly comprises the following steps: transferring GUS vector plasmid into agrobacterium GV3101, selecting positive clone in 5mL YEP culture solution (containing spectinotropic and rifampicin), and culturing at 28 deg.C overnight; then transferring the culture medium into 100mL YEP culture solution for amplification culture until the culture medium is OD ₆₀₀ 1.6 to 2.0; then, the mixture is centrifuged at 6000rpm for 10min, and the supernatant is discarded for collectionCollecting thalli, resuspending the thalli by using 5% of sucrose water or 1/2MS culture solution with the same volume, and adding 0.005-0.02% of Silwet L-77 to prepare a transformation solution; during transformation, the arabidopsis (watering the day before transformation to keep the plant moist) is completely immersed into transformation liquid for 1min, the arabidopsis is taken out, redundant transformation liquid is wiped off by using filter paper slightly, then a preservative film is covered to keep the plant moist, and the plant is firstly covered by a black bag for dark culture for 18h and then is transferred to normal culture conditions for culture until seeds are harvested (the transformation is carried out for 3 times at intervals of 1 turnover during culture).

T harvest from transformed Arabidopsis thaliana ₀ After the generation of seeds, about 100. mu.L of seeds were used for seed reproduction and identification. The method comprises the following specific steps: the whole operation is finished in a super clean bench, rinsing with 70% ethanol for 1min, centrifuging to remove ethanol, and cleaning with sterilized secondary water for 1 time; pre-washing for 1 time by using 10% sodium hypochlorite, centrifugally absorbing the sodium hypochlorite, washing for 5min by using 1mL of 10% sodium hypochlorite in a vibration mode, centrifugally absorbing the sodium hypochlorite, and washing for 5-6 times by using sterile water; finally, resuspending the seeds with sterile water, uniformly scattering the seeds on an MS culture medium containing herbicide, breaking dormancy after low-temperature treatment at 4 ℃ for 1d, and transferring into a plant illumination incubator with a photoperiod of 16h/8h (illumination/darkness) and a temperature of 22 ℃/20 ℃ (day/night); after about 2 weeks, the normally surviving T1 generation seedlings are transplanted into a matrix to continue growing, a few leaves are picked after the plants grow up to extract DNA, and PCR identification is carried out on positive plants.

(4) GUS staining of transgenic arabidopsis thaliana: after the homozygous transgenic arabidopsis thaliana is subjected to high-low phosphorus treatment for 6 days, the treated arabidopsis thaliana is taken out firstly, washed by secondary water for 3 times, fixed in 90% acetone for 30min, washed by prepared washing liquid for 3 times and then respectively placed in GUS staining solution (0.1M Na) ₂ HPO ₄ /NaH ₂ PO ₄ pH 7.2, 1mM X-Gluc), vacuumizing for 20min, transferring to a constant temperature incubator at 37 ℃, and dyeing for 6-8 h in a dark place. After the root system was colored, the hair root was transferred to 75% (v/v) ethanol and stored, and the staining of GUS in the root system was observed under a stereoscopic microscope (Leica, Germany) and photographed.

As a result, as shown in FIG. 2A, the expression level of the GmBBE-like43 promoter fused with the GUS reporter gene in Arabidopsis thaliana was significantly higher than that of the control treatment, and was up-regulated mainly in the old leaves, lateral roots and main root tips of Arabidopsis thaliana, both under the low-phosphorus and aluminum treatment conditions.

2. GmBBE-like43 subcellular localization analysis

(1) Designing a specific primer GmBBE-like43-GFP-F (SEQ ID NO. 9): 5'-CTCTAGCGCTACCGGTATGGGAGTCCTTTCTTCTCA-3', GmBBE-like43-GFP-R (SEQ ID NO. 10): 5'-CATGGTGGCGACCGGTCGACCCTTCCTATATGACAGCG-3' are provided. The full length of the ORF of the soybean GmBBE-like43 gene is amplified by taking the root cDNA of the soybean genotype YC03-3 as a template. The reaction conditions are pre-denaturation at 98 ℃ for 5min, denaturation at 98 ℃ for 30s, annealing at 58 ℃ for 30s and renaturation at 72 ℃ for 1-3 min, the process is circulated for 30 times, and extension at 72 ℃ is 10 min.

(2) Performing gel electrophoresis on the PCR amplified product, recovering and purifying by using a kit, obtaining a purified PCR product, and then using a homologous recombination kit

II, carrying out recombination and connection reaction on the PCR product and a linearized vector pEGAD cut by Age I. The reaction system was 20. mu.L, containing 6. mu.L of PCR product, 8. mu.L of pEGAD linearized vector plasmid, 2. mu.L of Exnase II with recombinant ligase, 4. mu.L of reaction buffer. The reagent mixture is reacted at 37 ℃ for 30min, the recombinant plasmid is transformed into escherichia coli and sequenced, and the plasmid is extracted after no error. And transforming the 35S plasmid GmBBE-like43-GFP into agrobacterium GV3101 and agrobacterium rhizogenes K599, and storing for later use after no error is detected.

(3) GmBBE-like43 subcellular localization analysis

Transient expression of tobacco epidermal cells in subcellular localization analysis was carried out by Agrobacterium transformation, 35S:: GmBBE-like43-GFP or pEGAD (35S: GFP) was introduced into Agrobacterium strain GV 3101. GV3101 containing vector after successful transformation was inoculated into YEP medium, cultured with shaking at 28 ℃ for 16 hours, centrifuged (5000rpm, 10min), and soaked in a solution containing 10mM MgCl ₂ 10mM MES and 10mM acetosyringone, pH 5.6) resuspended to OD ₆₀₀ The spectrophotometric value of (2) is 0.4-0.5, and the thallus suspension is kept stand for 3 hours in a dark place at 43 ℃, and then the lower epidermis of the leaf of 5-6 weeks old tobacco is co-transformed by a method of penetrating bacterial liquid into an injector. Rotating shaftAfter the transformed tobacco was cultured normally for 3 days, the distribution of the fluorescent signal in tobacco epidermal cells was observed by a laser copolymerization scanning microscope (Zeiss LSM780, Germany). For the subcellular localization of the transgenic green bean roots, 35S:GmBBE-like 43-GFP or pEGAD empty load (35S:: GFP) is introduced into the Agrobacterium rhizogenes strain K599 for transforming the green bean roots. The specific method refers to a kidney bean hairy root transformation system established in the laboratory. The constructed transgenic material is observed in a laser copolymerization scanning microscope (Zeiss LSM780, Germany) to locate the GFP signal in the cells.

As shown in FIG. 2, after the 35S: (35S:: GFP) GmBBE-like43-GFP vector is transferred into the epidermal cells of tobacco leaves for transient expression, a green fluorescence signal of GFP exists in the cell nucleus, cytoplasm and plasma membrane of an empty load control (35S:: GFP), and the 35S: (35S:: GmBBE-like43-GFP) has strong GFP fluorescence at the cell edge and is coincided with the cell outline, which indicates that the GmBBE-like43 can be located on the cell wall (FIG. 2B).

In order to further verify the subcellular localization of the GmBBE-like43, a 35S-GmBBE-like 43-GFP vector is introduced into agrobacterium rhizogenes K599, and then a transgenic hairy root stably expressed by the 35S-GmBBE-like 43-GFP fusion protein is obtained by adopting an agrobacterium rhizogenes-mediated soybean cotyledonary node in vitro hairy root transformation method. The result is similar to that of tobacco epidermal cells, and GFP fluorescence signal detection finds that GFP fluorescence of unloaded transgenic control phaseolus vulgaris roots is distributed throughout the cells. In contrast, 35S, the GFP fluorescence of the GmBBE-like43-GFP transgenic green bean roots was only at the cell edge and fused to the red fluorescence excited by the PI dye (FIG. 2C). Therefore, GmBBE-like43 may be located on the cell wall.

Example 3 Effect of GmBBE-like43 expression on growth of Soybean root in vitro after phosphorus treatment under various conditions

1. GmBBE-like43 functional analysis vector construction

(1) Construction of overexpression vector (OX-GmBBE-like43-pTF101s)

Soybean genotype YC03-3 root system cDNA is taken as a template to design a GmBBE-like43 specific primer, and an upstream specific primer 5'-GTACCCGGGGATCCTCTAGAATGGGAGTCCTTTCTTCTCA-3' (SEQ ID NO:11) and a downstream specific primer 5 ' -GCCTGCAGGTCGACTCTAGACTAACC are usedCTTCCTATATGACAGCG-3' (SEQ ID NO:12) amplified a fragment of the coding region of GmBBE-like 43. The PCR reaction system is as follows: a total of 50. mu.L of a system containing 43. mu.L of Master mix (2X), 0.5. mu.L of each gene forward/reverse primer, 2. mu.L of cDNA template, 1. mu.L of dNTPs (10. mu.M) and 1. mu.L of Hi-Fi enzyme, the remainder being made up with water. The PCR procedure was: pre-denaturation at 98 ℃ for 5min, denaturation at 98 ℃ for 30s, annealing at 58 ℃ for 30s, and renaturation at 72 ℃ for 1-3 min, wherein the process is circulated for 30 times, and extension at 72 ℃ is 10 min. After the PCR fragment is recovered and sequenced without errors, the CDS fragment of GmBBE-like43 obtained by amplification is subjected to homologous recombination by a kit

II kits are connected to a linearized vector pTF101S digested by Xba I, the obtained recombinant vector is transformed into escherichia coli and is sequenced and verified, and after the sequencing result is correct, a recombinant plasmid 35S is extracted, wherein GmBBE-like43 is transformed into agrobacterium rhizogenes K599 and agrobacterium GV3101 for later use.

(2) Construction of interferometric expression vector (RNAi-GmBBE-like43-pFGC5941)

A root cDNA of YC03-3 is taken as a template, two sections of specific primers RNAi-GmBBE-like43-pFGC5941-F/R (SEQ ID NO: 13-16) are designed, and the specific sequences are as follows:

primer RNAi-GmBBE-like43-pFGC5941-Asc I-F (SEQ ID NO: 13):

5’-ACAATTACCATGGGGCGCGCCGGTGTGTCTTACGTGGCAGA-3’；

primer RNAi-GmBBE-like43-pFGC5941-Asc I-R (SEQ ID NO: 14):

5’-AAATCATCGATTGGGCGCGCCCAAAGCTAGCTCCACCACCA-3’；

primer RNAi-GmBBE-like43-pFGC5941-BamH I-F (SEQ ID NO: 15):

5’-ATTTGCAGGTATTTGGATCCCAAAGCTAGCTCCACCACCA-3’；

primer RNAi-GmBBE-like43-pFGC5941-BamH I-R (SEQ ID NO: 16):

5’-TCTAGACTCACCTAGGATCCGGTGTGTCTTACGTGGCAGA-3’。

amplifying an open reading frame sequence of about 300bp of GmBBE-like43, and then carrying out enzyme digestion on a target vector by using a restriction enzyme Asc I to obtain a linear binary vector pFGC 5941; taking the 15 bp-20 bp sequence at the tail end of the linearized vector as a homologous sequence, respectively adding the homologous sequence to the 5' end of a gene specificity forward/reverse amplification primer sequence, amplifying by the primer pair to obtain an insert with the homologous sequence, and determining the concentration of a recovered product. According to the ligation method of one-step cloning (Novozan Co.), recombination reaction: mixing the linearized vector and the insert in a ratio of 1:2, reacting at 37 ℃ for 1h under the catalysis of Exnase II to complete a recombination reaction, then digesting a recombinant product by a restriction enzyme BamH I to obtain a binary vector pFGC5941, recombining an amplified second fragment sequence according to a one-step cloning connection method (Novozam company), sucking about 3 mu L of the recombinant product (determined according to the product concentration) after the recombination reaction is finished, adding the recombinant product into 50 mu L of Trans E.coli T1 competence for transformation, selecting a single clone for later-stage positive screening, and performing sequencing verification after detection. And (3) carrying out amplification culture on the positive monoclonal, preserving bacterial liquid and carrying out plasmid extraction to obtain a soybean GmBBE-like43 interference expression vector. And (3) carrying out K599 agrobacterium transformation on the target vector, detecting positive clone and storing the bacterial liquid at-80 ℃.

2. Obtaining transgenic Material

Adopting an agrobacterium rhizogenes mediated soybean cotyledonary node in vitro hairy root transformation method. The method mainly comprises the following steps:

(1) seed sterilization and germination: selecting soybean seeds with undamaged seed coats and consistent sizes, carrying out surface disinfection in chlorine gas for 12-14 h, and then placing the seeds on an ultra-clean workbench to blow for 30min to remove redundant chlorine gas; then, the seeds were sown on a germination medium and cultured at 28 ℃ for 4 days under light conditions.

(2) Preparing a bacterial liquid: carrying out streak culture on agrobacterium rhizogenes K599 containing OX-GmBBE-like43-pTF101s or RNAi-GmBBE-like43-pFGC5941 plasmid on a plate, picking out a single clone, placing at 28 ℃, culturing at 200rpm/min for 12h to OD ₆₀₀ Is about 1.0.

(3) Infection and co-culture of cotyledonary nodes: the germinated seeds were cut with a scalpel in the hypocotyl area saved 0.5cm from the cotyledons, and then the seeds were dissected vertically along the ridge with a scalpel, and the sprouts of the seeds were cut off. In addition, the sections were dipped with a scalpel and multiple wounds were cut perpendicular to the cotyledonary node and hypocotyl. Horizontally moving the cut external body upwards to a culture dish containing wet filter paper; then, the dish was sealed with a preservative film, and the plate was transferred to an incubator (24 ℃ C.) and cultured for 5 days under light. The co-cultured explants were transferred to medium containing herbicide and carbenicillin for growth for 14 d.

3. Positive identification of transgenic soybean hairy root

After extracting hair roots of a control group and transgenic hair root RNA to be detected, reversing cDNA, detecting the expression quantity of a transgenic line excessively expressing GmBBE-like43 by using a GmBBE-like43 fluorescent quantitative PCR primer in example 1, designing a primer GmBBE-like43-RT2-F (SEQ ID NO:17) and a GmBBE-like43-RT2-R (SEQ ID NO:18) by using a housekeeping gene EF 1-alpha (Glyma17g23900) as an internal reference gene, amplifying, and detecting the expression quantity of the transgenic line interfering with the expression of the GmBBE-like43, wherein the specific sequences are as follows:

primer GmBBE-like43-RT2-F (SEQ ID NO: 17):

5’-GGACGTTGTGAACGGTACACG-3’；

primer GmBBE-like43-RT2-R (SEQ ID NO: 18):

5’-AGGATGCAATGTCTATGTTGTCC-3’。

4. functional verification of transgenic soybean hairy root

(1) Aluminum treatment of transgenic soybean hairy roots

Selecting transgenic hair roots with fresh growth, good state and consistent growth, taking a picture, and respectively transferring to the step of adding or not adding 100 mu M AlCl ₃ 1/4MS liquid culture medium (pH 4.5, without KH) added to the sterilized and cooled culture solution ₂ PO ₄ ,) in (b). Harvesting was performed after 48 hours of treatment at 100rpm/min in a shaker at 28 ℃, photographs of the root system were taken, and root length was measured using software Image J.

As shown in FIG. 3, quantitative PCR analysis showed that GmBBE-like43 was expressed in soybean excised hair roots overexpressing GmBBE-like43(OX) by about 10-fold and 4-fold respectively under the conditions of no or aluminum treatment, as compared to the empty vector control (OX-CK/RNAi-CK) (FIG. 3A), while the expression in soybean excised hair roots interfering with the expression of GmBBE-like43(RNAi) was reduced by about 78% and 89%, respectively (FIG. 3B). MS culture experiment results show that under normal treatment conditions, the excessive or inhibited expression of GmBBE-like43 has no obvious influence on the growth of soybean hair roots. Whereas under aluminum treatment conditions, overexpression of GmBBE-like43(OX) significantly promoted growth of transgenic soybean in vitro hair roots, with root growth and relative growth rate increased by about 67% and 77%, respectively, compared to the empty vector control (OX-CK) (FIGS. 3C-E). In contrast, under aluminum treatment conditions, interfering with GmBBE-like43(RNAi) expression significantly inhibited the growth of transgenic soybean roots ex vivo, as evidenced by a reduction in root growth and relative growth rate of about 31% and 22%, respectively, as compared to the empty vector control (RNAi-CK) (fig. 3F and G).

(2) High-low phosphorus treatment of transgenic soybean hairy roots

Selecting transgenic hairy root with fresh growth vigor, good state, similar root system form and weight of about 0.1g, transferring to high phosphorus (+ P: 1250. mu.M KH) containing ₂ PO ₄ ) Or low phosphorus (-P: 10. mu.M KH) ₂ PO ₄ ) The MS solid medium of (1) was grown for 14 d. The dry weight of the hair root and the total root length were harvested and measured.

As shown in FIG. 4, quantitative PCR analysis showed that under the high and low phosphorus treatment conditions, the expression level of GmBBE-like43 in soybean excised hair roots overexpressing GmBBE-like43(OX) was increased by about 1.7-fold and 1.8-fold, respectively, compared to the empty vector control (OX-CK/RNAi-CK) (FIG. 4A), while the expression level in soybean excised hair roots interfering with the expression of GmBBE-like43(RNAi) was decreased by about 80% and 77%, respectively (FIG. 4B). MS culture experiment results show that under the conditions of high-phosphorus treatment and low-phosphorus treatment, the transgenic soybean in vitro hairy root is remarkably promoted to grow by excessively expressing GmBBE-like43(OX), the dry weight of the root system is 1.6 times and 3.4 times of that of a control (OX-CK), and meanwhile, the total root length of the transgenic soybean in vitro hairy root excessively expressing GmBBE-like43(OX) under the conditions of high-phosphorus treatment and low-phosphorus treatment is 1.6 times and 2.3 times of that of the control (OX-CK) (fig. 4C-E). In contrast, the dry weight of soybean excised hair roots related to the expression of GmBBE-like43 under high and low phosphorus treatment conditions was reduced by about 43% and 38%, respectively, while the total root length was reduced by about 59% and 35%, respectively, compared to the control (fig. 4F and G).

Example 4 Effect of overexpression of GmBBE-like43 on growth of Arabidopsis

1. Acquisition of transgenic Arabidopsis

The plasmid of the overexpression vector (OX-GmBBE-like43-pTF101s) constructed in the embodiment 3 is transferred into agrobacterium GV3101, then a T3 generation transgenic arabidopsis seeds are obtained by adopting an inflorescence dip-dyeing method and herbicide screening in the embodiment 2, and finally different transgenic arabidopsis strains with high expression of GmBBE-like43 are obtained by quantitative PCR confirmation and used for subsequent gene function research. The quantitative primers of the Arabidopsis GmBBE-like43 are as follows: GmBBE-like43-RT3-F (SEQ ID NO:19) and GmBBE-like43-RT3-R (SEQ ID NO: 20). Taking an Arabidopsis housekeeping gene EF 1-alpha as an internal reference gene, adopting EF 1-alpha quantitative primers (SEQ ID NO: 21-22) for the Arabidopsis housekeeping gene, and specifically adopting the following primer sequences:

GmBBE-like43–RT3-F(SEQ ID NO:19)：

5’-CTCCTTTCCCTCATCGAGCTG-3’；

GmBBE-like43–RT3-R(SEQ ID NO:20)：

5’-TCCATAAACTCTCCCTTCAGCG-3’；

EF1-α-F(SEQ ID NO:21)：

5’-GTCGATTCTGGAAAGTCGACC-3’

EF1-α-R(SEQ ID NO:22)：

5’-AATGTCAATGGTGATACCACGC-3’。

2. functional verification of transgenic lines

(1) Aluminum treatment of transgenic Arabidopsis

Two transgenic lines of Arabidopsis (OX1 and OX2) which overexpress GmBBE-like43 were obtained by an Arabidopsis heterologous gene expression transformation system. Selecting a proper amount of wild type and 2 filled seeds which excessively express GmBBE-like43 positive Arabidopsis strains OX1 and OX 2. After disinfection treatment, the seedlings are firstly sowed in a normal MS culture medium, the root systems of the arabidopsis thaliana grow to about 1cm after about 3-4 days, the orderly and consistent seedlings are selected for photographing, and then the seedlings are moved to a place without or containing aluminum (5 mu M AlCl) ₃ ) 1/5Hoagland nutrient solution (pH 4.5, without KH) ₂ PO ₄ ，0.5mM CaCl ₂ ) Culturing in the medium. The culture conditions are as follows: room temperature 23 deg.c, light intensity 120 micro mol/m ^-2 ·S ^-1 The light irradiation time was 16 hours per day. Harvesting was performed 48 hours after seedling transplantation, photographs of the seedlings were taken, and the root length was measured using image J.

As seen from FIG. 5A, the expression level of GmBBE-like43 in transgenic Arabidopsis was significantly higher than that in WT. Meanwhile, under the treatment conditions of adding aluminum or not adding aluminum, the excessive expression of GmBBE-like43 obviously promotes the growth of the root system of the transgenic Arabidopsis (FIG. 5B). Under normal treatment conditions, the root growth of transgenic arabidopsis lines OX1 and OX2, which overexpress GmBBE-like43, were both increased by about 35% compared to WT, while under aluminum treatment conditions, the root growth of transgenic lines OX1 and OX2 were increased by 72% and 133%, respectively, compared to WT (fig. 5C). Meanwhile, the root relative growth rates of transgenic lines OX1 and OX2 overexpressing GmBBE-like43 were increased by about 27% and 72%, respectively, compared to WT (fig. 5D).

(2) High and low phosphorus treatment of transgenic arabidopsis thaliana

Selecting a proper amount of wild type and 2 filled seeds over-expressing GmBBE-like43 positive strains OX1 and OX 2. After the disinfection treatment, firstly sowing in a normal MS culture medium, after about 3-4 d, growing the root system of arabidopsis thaliana to about 1cm, selecting the uniform seedling, moving the seedling to a square dish of 1/2MS culture medium for corresponding treatment, and setting two phosphorus level treatments: normal phosphorus supply treatment (1250. mu.M KH) ₂ PO ₄ ) And low phosphorous treatment (6.25. mu.M KH) ₂ PO ₄ ) One for each square dish, 5 replicates for each treatment. And collecting samples on the 9 th day after treatment, and measuring the fresh weight and the main root length of the plants.

As shown in FIG. 5, overexpression of GmBBE-like43 significantly promoted root growth in transgenic Arabidopsis thaliana, whether under high-phosphorus or low-phosphorus treatment conditions (FIG. 5E). Root fresh weights of transgenic lines OX1 and OX2, which overexpressed GmBBE-like43, were increased by about 23% compared to WT under normal phosphorus treatment conditions, while root fresh weights of transgenic lines OX1 and OX2 were increased by 20% and 47%, respectively, compared to WT under low phosphorus treatment conditions (FIG. 5F). Meanwhile, under normal phosphorus treatment conditions, the major root length of transgenic lines OX1 and OX2, which overexpress GmBBE-like43, were both increased by about 9% compared to WT, while under low phosphorus treatment conditions, the major root length of transgenic lines OX1 and OX2 were increased by 17% and 23% compared to WT, respectively (FIG. 5G).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

Application of <120> GmBBE-like43 gene in regulation and control of plant adaptation to low phosphate and aluminum stress and growth promotion

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1608

<212> DNA

<213> GmBBE-like43 gene (SIPOS sequenceListing 1.0)

<400> 1

atgggagtcc tttcttctca tagaatacaa ctattattat ttcccattgt tgtgttgctt 60

tggtcagctt cagctgcaaa ttcagctaac aacacttttc ttcattgcct cgtgaaccat 120

tctgagcctt ctcaccccat aacttcagca attttcacac caaacaacac ctcattctct 180

tcagtattgg aagcctacat tagaaacctt cgtttcaaca cctccacaac ccgtaagcca 240

ttcctcataa taactgcact tcatgtatcc cacatacaag catccattat ttgtgctcaa 300

aaacacaact tgcaaatgaa aatccgaagt ggtggccatg actatgaggg tgtgtcttac 360

gtggcagagg ttccattctt catccttgac atgttcaatc taagaaccat tgaggttgac 420

ataggcactg aaaccgcttg ggtccaagct ggtgcaacac ttggtgaagt ttattataga 480

attgctgaga agagtaaaac ccatgctttc ccagcagggg tttgtcacac agttggggtg 540

ggaggacaca taagtggtgg tggctatggc aacatgatga gaaaatatgg tctctcagtg 600

gataatgtta ttgatgcaca aatggttgat gttcaaggta gattgcttga tagaaaatcc 660

atgggtgaag atctcttttg ggccatcaca ggtggtggtg gagctagctt tggtgttgtt 720

cttgcctaca aaataaagct agttcgagtt ccagaaattg tcactgtttt ccaagttggg 780

agaaccttag agcaaaatgc cactgatata gtttacaatt ggcagcatgt tgcaccaact 840

atcgacaacg atcttttcct tagggttatc ttggacgttg tgaacggtac acgaaatgga 900

acaaagactg taagagctag gttcatagct ctattccttg gtgactccaa aagccttgtt 960

tctctcttga atgacaagtt tcctcaattg ggtttgaagc aatctgattg catcgaaacg 1020

agctggcttc gatctgtgct gttttgggac aacatagaca ttgcatcctc acttgacatt 1080

ttgcttgaga gacaaccacg atcactcaac tacttgaaaa ggaaatctga ctatgtgaag 1140

aaaccgattt ccatagaggg ttttgaaggg atttggaaga agatgattga gttggaggat 1200

acactatttc aattcaatcc ttatggcgga agaatggctg agattccttc aacagcatct 1260

cctttccctc atcgagctgg gaacctatgg aagatccaat accaagcgaa ttggaataag 1320

ccagggaaag aggtagcaga tcactacata aacttgacaa gaaaacttca caagttcatg 1380

actccttttg tctccaagaa ccctagagag gctttctaca attataagga ccttgacttg 1440

gggattaacc acaatggtaa aaacagctac gctgaaggga gagtttatgg agtggagtat 1500

ttcaaggata acttcgacag gttggttcaa ataaagacca aggttgatcc ccataatttc 1560

tttaggaacg aacaaagcat ccctacgctg tcatatagga agggttag 1608

<210> 2

<211> 535

<212> PRT

<213> GmBBE-like43 protein (SIPOSequenceListing 1.0)

<400> 2

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

1 5 10 15

Val Val Leu Leu Trp Ser Ala Ser Ala Ala Asn Ser Ala Asn Asn Thr

20 25 30

Phe Leu His Cys Leu Val Asn His Ser Glu Pro Ser His Pro Ile Thr

35 40 45

Ser Ala Ile Phe Thr Pro Asn Asn Thr Ser Phe Ser Ser Val Leu Glu

50 55 60

Ala Tyr Ile Arg Asn Leu Arg Phe Asn Thr Ser Thr Thr Arg Lys Pro

65 70 75 80

Phe Leu Ile Ile Thr Ala Leu His Val Ser His Ile Gln Ala Ser Ile

85 90 95

Ile Cys Ala Gln Lys His Asn Leu Gln Met Lys Ile Arg Ser Gly Gly

100 105 110

His Asp Tyr Glu Gly Val Ser Tyr Val Ala Glu Val Pro Phe Phe Ile

115 120 125

Leu Asp Met Phe Asn Leu Arg Thr Ile Glu Val Asp Ile Gly Thr Glu

130 135 140

Thr Ala Trp Val Gln Ala Gly Ala Thr Leu Gly Glu Val Tyr Tyr Arg

145 150 155 160

Ile Ala Glu Lys Ser Lys Thr His Ala Phe Pro Ala Gly Val Cys His

165 170 175

Thr Val Gly Val Gly Gly His Ile Ser Gly Gly Gly Tyr Gly Asn Met

180 185 190

Met Arg Lys Tyr Gly Leu Ser Val Asp Asn Val Ile Asp Ala Gln Met

195 200 205

Val Asp Val Gln Gly Arg Leu Leu Asp Arg Lys Ser Met Gly Glu Asp

210 215 220

Leu Phe Trp Ala Ile Thr Gly Gly Gly Gly Ala Ser Phe Gly Val Val

225 230 235 240

Leu Ala Tyr Lys Ile Lys Leu Val Arg Val Pro Glu Ile Val Thr Val

245 250 255

Phe Gln Val Gly Arg Thr Leu Glu Gln Asn Ala Thr Asp Ile Val Tyr

260 265 270

Asn Trp Gln His Val Ala Pro Thr Ile Asp Asn Asp Leu Phe Leu Arg

275 280 285

Val Ile Leu Asp Val Val Asn Gly Thr Arg Asn Gly Thr Lys Thr Val

290 295 300

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

305 310 315 320

Ser Leu Leu Asn Asp Lys Phe Pro Gln Leu Gly Leu Lys Gln Ser Asp

325 330 335

Cys Ile Glu Thr Ser Trp Leu Arg Ser Val Leu Phe Trp Asp Asn Ile

340 345 350

Asp Ile Ala Ser Ser Leu Asp Ile Leu Leu Glu Arg Gln Pro Arg Ser

355 360 365

Leu Asn Tyr Leu Lys Arg Lys Ser Asp Tyr Val Lys Lys Pro Ile Ser

370 375 380

Ile Glu Gly Phe Glu Gly Ile Trp Lys Lys Met Ile Glu Leu Glu Asp

385 390 395 400

Thr Leu Phe Gln Phe Asn Pro Tyr Gly Gly Arg Met Ala Glu Ile Pro

405 410 415

Ser Thr Ala Ser Pro Phe Pro His Arg Ala Gly Asn Leu Trp Lys Ile

420 425 430

Gln Tyr Gln Ala Asn Trp Asn Lys Pro Gly Lys Glu Val Ala Asp His

435 440 445

Tyr Ile Asn Leu Thr Arg Lys Leu His Lys Phe Met Thr Pro Phe Val

450 455 460

Ser Lys Asn Pro Arg Glu Ala Phe Tyr Asn Tyr Lys Asp Leu Asp Leu

465 470 475 480

Gly Ile Asn His Asn Gly Lys Asn Ser Tyr Ala Glu Gly Arg Val Tyr

485 490 495

Gly Val Glu Tyr Phe Lys Asp Asn Phe Asp Arg Leu Val Gln Ile Lys

500 505 510

Thr Lys Val Asp Pro His Asn Phe Phe Arg Asn Glu Gln Ser Ile Pro

515 520 525

Thr Leu Ser Tyr Arg Lys Gly

530 535

Claims

Application of GmBBE-like43 gene shown in SEQ ID NO.1 or GmBBE-like43 protein shown in SEQ ID NO. 2 in positively regulating and controlling low-phosphorus and/or aluminum-toxicity stress resistance and/or root growth of plant roots.
Application of the GmBBE-like43 gene shown in SEQ ID NO.1 or the GmBBE-like43 protein shown in SEQ ID NO. 2 or an expression promoter thereof in promoting plant root growth.
3, application of the GmBBE-like43 gene shown in SEQ ID NO.1 or the GmBBE-like43 protein shown in SEQ ID NO. 2 or an expression promoter thereof in improving the low phosphorus and/or aluminum toxicity stress resistance of a plant root system.
Application of GmBBE-like43 gene shown in SEQ ID NO.1 or GmBBE-like43 protein shown in SEQ ID NO. 2 or expression promoter thereof in cultivating plants resistant to low phosphorus and/or aluminum virus.
Application of GmBBE-like43 gene shown in SEQ ID NO.1 or GmBBE-like43 protein shown in SEQ ID NO. 2 or expression promoter thereof in preparation of plant growth promoting agent.
6, application of a GmBBE-like43 gene shown in SEQ ID NO.1 or a GmBBE-like43 protein shown in SEQ ID NO. 2 or an expression promoter thereof in improving adaptability of plants to acid soil and/or preparing an acid soil growth promoter.
7. A product for promoting plant growth and/or improving plant tolerance to low-phosphorus and/or aluminum toxicity stress, which is characterized by comprising a GmBBE-like43 protein expression promoter.
8. A method for promoting plant growth and/or improving plant tolerance to low-phosphorus and/or aluminum toxicity stress is characterized in that plant root system growth is promoted and/or plant tolerance to low-phosphorus and/or aluminum toxicity stress is improved by positively regulating the expression level or protein activity of GmBBE-like43 gene in a plant through a gene editing technology.
9. The method of claim 8, wherein the GmBBE-like43 gene is overexpressed in the plant to promote plant root growth and/or increase plant tolerance to low phosphorus and/or aluminum toxicity stress.
10. The method of claim 8, wherein an expression vector for over-expressing the GmBBE-like43 gene is constructed, and the plant is transformed to obtain a transgenic plant for promoting the growth of the root system of the plant and/or improving the tolerance of the plant to low-phosphorus and/or aluminum toxicity stress.