CN114940997B - Application of GmBBE-like43 gene in regulating and controlling plant to adapt to low-phosphorus and acid aluminum stress and promote growth - Google Patents
Application of GmBBE-like43 gene in regulating and controlling plant to adapt to low-phosphorus and acid aluminum stress and promote growth Download PDFInfo
- Publication number
- CN114940997B CN114940997B CN202210609252.9A CN202210609252A CN114940997B CN 114940997 B CN114940997 B CN 114940997B CN 202210609252 A CN202210609252 A CN 202210609252A CN 114940997 B CN114940997 B CN 114940997B
- Authority
- CN
- China
- Prior art keywords
- gmbbe
- like43
- soybean
- gene
- phosphorus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The invention discloses application of GmBBE-like43 gene in regulating and controlling plant adaptation to low phosphorus and acid aluminum stress and growth promotion. The research of the invention shows that the cell wall protein GmBBE-like43 is induced to up-regulate expression under the stress of aluminum and low phosphorus in soybean root system; under the conditions of phosphorus treatment and aluminum treatment with different concentrations, the excessive GmBBE-like43 expression obviously promotes the growth of transgenic soybean in-vitro hairy roots and arabidopsis thaliana; the GmBBE-like43 gene has the function of positively regulating and controlling the soybean or Arabidopsis root system 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 root growth of arabidopsis thaliana. Therefore, the GmBBE-like43 plays an important role in adapting plants to low-phosphorus and acid aluminum stress, and can be used for regulating and controlling the adaptation capacity of the plants to the low-phosphorus and acid aluminum stress of acid soil through a transgenic technology.
Description
Technical Field
The invention belongs to the technical field of genetic engineering. More particularly, the application of GmBBE-like43 in regulating and controlling plants to adapt to low-phosphorus and acid aluminum stress and growth promotion.
Background
The acid soil is widely distributed in the world, the total area of the acid soil in the world accounts for 30-40% of the cultivated land area of the world, and more than 50% of the potential cultivated lands are the acid soil and are mainly distributed in tropical, subtropical and temperate regions. The effect of acid soil on crop growth inhibition is not only shown in H + Too high a concentration jeopardizes the crop itself and often presents a synergistic inhibition of plant growth and yield by the lack of available phosphorus and aluminum toxicity. Thus, by means of genetic improvement, increasing the tolerance of crops themselves to low phosphorus stress and aluminum toxicity in acid soil is considered an important approach to developing sustainable agriculture.
In the long-term evolution process, plants form a series of morphological, physiological and molecular cooperative adaptability mechanisms, and the barrier factors such as available phosphorus deficiency, aluminum toxicity and the like in acid soil are overcome. It is reported that plants adapt to mechanisms of aluminum toxicity, mainly including internal tolerance and efflux mechanisms (e.g., secretion of organic acids); mechanisms for adaptation to low phosphorus stress mainly involve changing the morphological configuration of roots, increasing the secretion of root organic acids and increasing the enzymatic activity of purple acid phosphatases, inducing expression of high affinity phosphorus transporters and forming symbiosis with rhizosphere microorganisms such as mycorrhizal fungi, etc. (ragkothama, 1999; vance et al, 2003; liang et al, 2014). Since the lack of available phosphorus and aluminum toxicity exist in acid soil at the same time, it is suggested that plants may have a common adaptation mechanism to overcome the lack of available phosphorus and aluminum toxicity. Early studies found 28 BBE-keys proteins in arabidopsis, with the amomum fruit alkali peroxidase AtBBE-Like15 involved in the synthesis of plant cell wall lignin (Daniel et al 2015). Later studies reported that there are 4 peroxisome proteins in Arabidopsis AtBBE-Like1/2/20/21 that can oxidizeOligogalacturonate OGs to produce H 2 O 2 And are named OGOX4/3/1/2 (Benedetti et al 2015) in sequence.
The soybean is an important economic crop and has very important position in agricultural production in China, and is a traditional leguminous crop for grain, oil and feed. The specific function of the protein is not studied in the current report of berberine family related protein (binding berberine family protein-related, BBE) of soybean. Although 2 BBE-keys proteins with aluminum stress response and 1 BBE-Like protein with low phosphorus stress response were found in the soybean BBE-keys protein family in the prior study (Wu et al, 2018; zhao et al, 2020), the specific function and role of this BBE-keys protein family gene in soybean has not been reported by research analysis so far; the key genes associated with soybean genotypes in southern acid soils have not been reported.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the problems and provide the application of the soybean GmBBE-like43 gene in regulating and controlling plants to adapt to low phosphorus and acid aluminum stress and promoting growth.
The first object of the present invention is to provide the application of the GmBBE-like43 gene and the protein thereof.
It is a second object of the present invention to provide a product that promotes plant growth and/or increases plant tolerance to low phosphorus and/or aluminium-toxic 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 aluminium-toxic stress.
The above object of the present invention is achieved by the following technical scheme:
according to the invention, a cell wall protein GmBBE-like43 which is obviously up-regulated under low phosphorus stress at the protein level is found in a proteomic analysis result of a soybean root system treated with high and low phosphorus, a GmBBE-like43 gene is selected as a candidate gene for research, and then qRT-PCR verification shows that the GmBBE-like43 is also obviously up-regulated under low phosphorus stress at the gene transcription level and is also obviously up-regulated under aluminum stress. Then, the specific functions of the GmBBE-like43 gene in the process of the soybean synergistic response to the acid soil low-phosphorus stress and the aluminum toxicity are analyzed and studied.
The invention clones a gene GmBBE-like43 synergistically regulated and controlled by exogenous phosphorus and aluminum by a real-time fluorescence quantitative PCR and homologous cloning method. The soybean in-vitro hairy root and arabidopsis transgenic plant material which is excessive and inhibits the expression of GmBBE-like43 is obtained by the soybean in-vitro hairy root transformation and the arabidopsis transgenic technology, and the result shows that the GmBBE-like43 gene has the function of regulating and controlling the soybean root system to adapt to low phosphorus stress and aluminum toxicity so as to promote the growth of the root system; the GmBBE-like43 gene has the functions of regulating and controlling the growth of the root system of the Arabidopsis, and regulating and controlling the adaptation of the root system of the Arabidopsis to low-phosphorus stress and aluminum toxicity so as to promote the growth of the root system.
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 positive regulation of low phosphorus and/or aluminum toxicity stress resistance and/or root growth of plant roots, promotion of plant root growth, improvement of low phosphorus and/or aluminum toxicity stress resistance of plant roots, cultivation of low phosphorus and/or aluminum toxicity resistant plants, preparation of plant promoters or improvement of plant adaptability to acid soil and/or preparation of acid soil promoters.
The invention provides a product for promoting plant growth and/or improving plant tolerance to low phosphorus and/or aluminum toxicity stress, which contains 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 is characterized in that the expression level or the protein activity of GmBBE-like43 genes in plants is positively regulated by a gene editing technology to promote plant root growth and/or improve plant tolerance to low-phosphorus and/or aluminum-toxicity stress.
Preferably, the plant root growth is promoted and/or the plant tolerance to low phosphorus and/or aluminium toxicity stress is increased by over-expression of the GmBBE-like43 gene in the plant.
Preferably, an expression vector for over-expressing GmBBE-like43 genes is constructed, and plants are transformed to obtain transgenic plants for promoting plant root growth and/or improving the tolerance of plants to low-phosphorus and/or aluminum-toxicity stress.
The invention has the following beneficial effects:
the invention discloses an application of soybean GmBBE-like43 gene in regulating and controlling plants to adapt to low phosphorus and acid aluminum stress and promote growth. The research of the invention shows that the GmBBE-like43 gene is up-regulated by aluminum stress induction and low-phosphorus stress expression, and the expression quantity is obviously increased along with the extension of phosphorus treatment time. Under the treatment conditions of different phosphorus concentrations, the expression of the excessive GmBBE-like43 obviously increases the biomass of the transgenic plant; meanwhile, under the aluminum treatment condition, the expression of the excessive GmBBE-like43 obviously increases the growth rate of the transgenic plant, which indicates that the GmBBE-like43 positively regulates and controls the capability of the plant root system to adapt to low phosphorus and acid aluminum stress. Therefore, the GmBBE-like43 plays an important role in adapting plants to low-phosphorus and acid aluminum stress, and can be used for regulating and controlling the adaptation capacity of the plants to the low-phosphorus and acid aluminum stress of acid soil through a transgenic technology.
Drawings
FIG. 1 shows a table pattern analysis of soybean GmBBE-like43 (A: effect of low phosphorus treatment time on expression pattern of GmBBE-like43 in soybean root system; B: effect of aluminum treatment time on expression pattern of GmBBE-like43 in soybean root tip; data are mean value of 3 replicates and standard error, 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 shows the tissue localization and subcellular localization analysis of GmBBE-like43 (A: the histochemical localization analysis of GmBBE-like43 in Arabidopsis thaliana, the scale of the upper image of the first row is 2mm, the scale of the last two rows of root system images is 0.5mm; B: the subcellular localization analysis result of GmBBE-like43 fusion GFP protein in tobacco leaves; C: the subcellular localization analysis result of GmBBE-like43 fusion GFP protein in kidney beans Mao Genzhong; the first row of tobacco or kidney bean subcellular localization images (35S: GFP) with transformed empty carriers; the second row of GmBBE-like43 fusion GFP protein in tobacco leaves or kidney bean roots; the images of FIG. B are respectively green fluorescent channels (GFP), light mirror channels (bright field) and overlapped images (fusion) under a laser confocal microscope; the images of FIG. C are respectively green channels (GFP), red channels (20 μm) under a fluorescent confocal microscope; the images of the fluorescent microscope (20 μm) are respectively observed after the overlapping images of the images of FIG. B and C;
FIG. 3 is an analysis of the expression pattern of GmBBE-like43 in empty control (OX-CK/RNAi-CK) and transgenic hair root (OX/RNAi) under aluminum treatment conditions as compared to the effect of excessive or inhibition of GmBBE-like43 expression on transgenic soybean ex vivo hair root growth (A, B: gmBBE-like43 expression pattern in empty control (OX-CK/RNAi-CK) and transgenic hair root (OX/RNAi), C: phenotype of empty control (OX-CK/RNAi-CK) and transgenic hair root (OX/RNAi) under aluminum treatment conditions, scale = 2cm; D, F: the amount of growth of empty control (OX-CK/RNAi-CK) and transgenic hair root (OX/RNAi), E, G: relative growth rate of empty control (OX-CK/RNAi-CK) and transgenic hair root (OX/RNAi), asterisk: significant difference (Student's t-test), P <0.05, P <0.01, P <0.001 >;
FIG. 4 is an analysis of the expression pattern of GmBBE-like43 in empty control (OX-CK/RNAi-CK) and transgenic hair root (OX/RNAi) under phosphorus-rich and phosphorus-poor conditions (A, B: the effect of GmBBE-like43 expression on transgenic soybean ex vivo hair root growth (A, B: the phenotype of empty control (OX-CK/RNAi-CK) and transgenic hair root (OX/RNAi) under phosphorus-rich and phosphorus-poor conditions, scale = 2cm; D, F: the dry weight of empty control (OX-CK/RNAi-CK) and transgenic hair root (OX/RNAi-CK), E, G: the total root length of empty control (OX-CK/RNAi-CK) and transgenic hair root (OX/RNAi-CK), asterisks indicate the significant difference between empty control (OX-CK/RNAi-CK) and transgenic hair root (OX/RNAi) (Student's t-test), P0.05, P <0.01, P < 0.001;
FIG. 5 shows the effect of over-expression GmBBE-like43 on transgenic Arabidopsis growth (A: the relative growth rates of root systems of wild-type (WT) and transgenic Arabidopsis plants (OX 1, OX 2) in the case of wild-type (WT) and transgenic Arabidopsis plants (OX 1, OX 2) on the case of aluminum treatment, B: phenotype of wild-type (WT) and transgenic Arabidopsis plants (OX 1, OX 2) on the scale of 0.5cm, C: root system growth of wild-type (WT) and transgenic Arabidopsis plants (OX 1, OX 2) on the case of aluminum treatment, D: root system relative growth rates of wild-type (WT) and transgenic Arabidopsis plants (OX 1, OX 2) on the case of aluminum treatment, E: phenotype of wild-type (WT) and transgenic Arabidopsis plants (OX 1, OX 2) on the case of high and low phosphorus treatment, F: wild-type (WT) and transgenic Arabidopsis plants (OX 1, OX 2) on the scale of 0.5cm, D: fresh root system weight of wild-type (WT) and transgenic Arabidopsis plants (OX 1, OX 2) on the case of low phosphorus treatment, D: wild-type (OX 1, OX 2) on the case of low phosphorus treatment, P2) on the wild-type (P) and P2) of the wild-type strain (W) on the wild-type (OX 1, P) and P2) strain (P) on the case of P2) is significantly different from the wild type (P) type) and P) strain (P) and P-type strain (P) type).
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 analysis of expression Pattern of GmBBE-like43 Gene
According to the invention, a proteomic analysis result of soybean root system treated with high and low phosphorus shows that a cell wall protein GmBBE-like43 which is obviously up-regulated under low phosphorus stress at a protein level is obtained, so that a GmBBE-like43 gene is selected as a candidate gene for research, and then qRT-PCR verification shows that the GmBBE-like43 is also obviously up-regulated under low phosphorus stress at a gene transcription level and is also obviously up-regulated under aluminum stress. Then, the specific functions of the GmBBE-like43 gene in the process of the soybean synergistic response to the acid soil low-phosphorus stress and the aluminum toxicity are analyzed and studied.
1. Effect of low phosphorus stress on expression pattern of GmBBE-like43 in soybean root system
The method comprises the steps of selecting seeds with undamaged seed coats and uniform size by adopting a roll paper seedling raising method, sterilizing for 12 hours by using chlorine generated by reacting 100ml of sodium hypochlorite with 4.2ml of hydrochloric acid, and blowing for 1 hour on an ultra-clean workbench for later use. Cutting square filter paper of 20×20cm, preparing 1/4 soybean total nutrient solution with pH of 5.8 and sterile water, and sterilizing.
When the paper is rolled, a preservative film is paved on a test table, the filter paper is soaked by the prepared 1/4 soybean full nutrient solution, 7 sterilized beans are placed at a position about 1cm away from one side of the filter paper, the umbilicus of the beans is downward, and the first beans are rolled to the tail end. The rolled filter paper is put into a 500mL beaker filled with 1/4 soybean full nutrient solution with one end without beans facing downwards, and the upper filter paper with the beans is wrapped by a preservative film. Placing the beaker into an incubator at 24-26 ℃, culturing in dark for 1d, and culturing in light/dark (12 h/12 h) for 3-4 d until radicle is 5-6 cm.
Seedlings of consistent growth were selected and transferred to +P (250. Mu.M KH) 2 PO 4 ) and-P (5. Mu.M KH) 2 PO 4 ) In the nutrient solution, 4 replicates were treated each, 8 seedlings each. The pH of the nutrient solution is adjusted to about 5.8 every two days, the nutrient solution is changed every week, root samples are respectively harvested at 3d, 6d, 9d and 12d, and the root samples are stored in a refrigerator at the temperature of minus 80 ℃ for standby after liquid nitrogen freezing.
2. Effect of aluminium treatment on expression pattern of GmBBE-like43 in soybean root tip
Culturing for 3-4 d to radicle 5-6 cm by using roll paper seedling method, selecting seedlings with consistent growth, and transferring to-Al (pH 4.2,0.5mM CaCl) 2 ) And +Al (pH 4.2, 50. Mu.M AlCl) 3 ，0.5mM CaCl 2 ) And (3) processing in the solution, respectively harvesting soybean root tip (0-2 cm) samples after 12, 24, 48, 72 and 96 hours, rapidly freezing with liquid nitrogen, and storing in a refrigerator at-80 ℃ for later use.
3. Real-time fluorescent quantitative PCR (qRT-PCR) analysis
Total RNA was extracted from the above treated plant samples using TRIzol kit (Invitrogen, USA), respectively. RNA treated with DNase I was reverse transcribed into cDNA using MMLV-reverse transcription kit (Promega, USA). qRT-PCR analysis was then performed using SYBR (Promega, USA) kit. After completion of reverse transcription, the sample was diluted 15-fold and subjected to real-Time fluorescent quantitative PCR analysis by Applied Biosystems StepOnePlus Real-Time PCR system.
Standard curve preparation: firstly, 1-2 mu L of sample is sucked into a new PCR centrifuge tube from cDNA stock solution of each sample, then the mixed solution is diluted by 10 times to form a first standard sample S1, the first standard sample is diluted by 10 times to form a second standard sample S2, and the like, namely S1, S2, S3, S4 and S5 are diluted into 5 concentration gradient standard samples.
The quantitative PCR primer adopts GmBBE-like43 gene quantitative primer and housekeeping gene primer of soybean as internal reference gene as follows:
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: into each 20. Mu.l reaction system, 10. Mu.l SYBR Premix Ex Taq (2X), 0.5. Mu.l forward/reverse primer, 7. Mu.l ddH were added 2 O and 2 microliters of template. The amount of reaction required was calculated, and the reagents except cDNA were mixed well, and 18. Mu.L of each tube was dispensed, and 2. Mu.L of cDNA template was added to give a final volume of 20. Mu.L.
The quantitative PCR reaction procedure was: the PCR reaction (denaturation at 95℃for 30 seconds, renaturation at 60℃for 15 seconds, extension at 72℃for 30 seconds) was performed for 40 cycles at 95℃for 30 seconds. The detection result was used to calculate the expression level of each sample by Real-Time Analysis Software of the Rotor-Gene.
As a result, as shown in FIG. 1, in 96h of aluminum treatment, the expression of GmBBE-like43 was up-regulated by aluminum stress induction, and the expression amount thereof showed a tendency of increasing and then decreasing with the increase of aluminum stress time, wherein the expression amount of GmBBE-like43 was highest at 12h, followed by 3.8 times and 25.5 times of that of control treatment at 24h, respectively (FIG. 1A). In addition, under the high-low phosphorus treatment conditions, the low phosphorus treatment 3d had no obvious effect on the expression level of GmBBE-like43 compared with the high phosphorus treatment. However, after 6d of low phosphorus treatment, the expression level of the gene is obviously up-regulated by low phosphorus stress, and along with the extension of the phosphorus treatment time, the expression level is obviously increased. That is, the expression levels of the 6 th, 9 th and 12d low-phosphorus treatments were 2, 3.1 and 5.6 times that of the control treatments, respectively, as compared with the normal phosphorus conditions (FIG. 1B).
Example 2GmBBE-like43 histochemical localization and subcellular localization analysis
1. GmBBE-like43 histochemical localization analysis
(1) According to the gene sequence of GmBBE-like43, a specific primer pGmBBE-like43 is designed, namely GUS-F (SEQ ID NO. 7): 5'-CGGAATTCCACGATGGAGTGCAAAAGCAT-3', pGmB BE-like 43:GUS-R (SEQ ID NO. 8): 5'-AAGGATCCTTTGGCTTATCCCAATGATGAG-3'. The root DNA of soybean genotype YC03-3 is used as a template for PCR amplification, and a 2000bp sequence on the initiation codon of GmBBE-like43 is amplified. The reaction conditions are that the reaction is carried out at 98 ℃ for 5min, the reaction is carried out at 98 ℃ for 30s, the reaction is carried out at 58 ℃ for 30s, the reaction is carried out at 72 ℃ for 1-3 min (determined according to the size of the fragment), the process is circulated for 30 times, and the reaction is carried out at 72 ℃ for 10min.
(2) And (3) constructing a carrier: the PCR amplified product is recovered and purified by gel electrophoresis and a kit, and after the purified PCR product is obtained, the homologous recombination kit is used
II the PCR product was subjected to a recombinant ligation reaction with pTF102 vector cut by the restriction enzymes EcoRI and BamHI double cleavage method. The reaction system was 20. Mu.L, containing 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. The reagent mixture is reacted for 30min at 37 ℃, the recombinant plasmid is transformed into escherichia coli and sequenced, and the plant expression vector of the soybean pGmBBE-like 43:GUS fusion gene is obtained. Finally, the target vector is transformed by GV3101 agrobacterium, positive clones are detected and bacterial liquid is preserved at-80 ℃.
(3) Obtaining transgenic Arabidopsis thaliana: inflorescence infection is adopted. The method mainly comprises the following steps: GUS vector plasmid is transferred into agrobacterium GV3101, positive clone is selected and cultured in 5mL YEP culture solution (containing spectacular and rifampicin) at 28 deg.C overnight; then transferring into 100mL YEP culture solution to enlarge and culture until reaching OD 600 1.6 to 2.0; then centrifuging at 6000rpm for 10min, collecting thalli after discarding supernatant, re-suspending thalli by 5% sucrose water or 1/2MS culture solution with equal volume, adding 0.005-0.02% Silwet L-77 to prepare a conversion solution; the transformation method comprises soaking Arabidopsis thaliana (plant is kept moist by watering the plant day before transformation) in the transformation solution for 1min, taking out, and slightly filtering with filter paperAnd (3) wiping off redundant conversion liquid, covering a preservative film to keep plants moist, covering dark culture for 18h by using a black bag, and culturing under normal culture conditions until seed collection (the seed collection is carried out once every 1 turnover during the culture period, and the total conversion is carried out for 3 times).
Harvesting T from transformed Arabidopsis thaliana 0 After seed generation, about 100 mu L of seeds are taken for seed reproduction identification. The method comprises the following specific steps: the whole operation is completed in an ultra-clean workbench, firstly, 70% ethanol is used for rinsing for 1min, ethanol is centrifugally absorbed, and then the ultra-clean workbench is cleaned for 1 time by sterilized secondary water; pre-washing for 1 time by using 10% sodium hypochlorite, centrifugally absorbing and removing the sodium hypochlorite, vibrating and rinsing for 5 minutes by using 1mL of 10% sodium hypochlorite, centrifugally absorbing and removing the sodium hypochlorite, and rinsing for 5-6 times by using sterile water; finally, re-suspending the seeds with sterile water, uniformly broadcasting the seeds on an MS culture medium containing herbicide, breaking dormancy after low-temperature treatment for 1d at 4 ℃, and moving the seeds 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 transferred into a matrix for continuous growth, a few leaves are taken out for DNA extraction after the plants grow up, and PCR identification is carried out on positive plants.
(4) GUS staining of transgenic Arabidopsis: after the homozygous transgenic arabidopsis thaliana is subjected to high-low phosphorus treatment for 6d, the treated arabidopsis thaliana is firstly taken out, washed 3 times with secondary water, then fixed in 90% acetone for 30min, washed 3 times with prepared washing liquid, and respectively placed in GUS staining solution (0.1M Na 2 HPO 4 /NaH 2 PO 4 pH 7.2,1mM X-Gluc), vacuum-pumping 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 stained, the hair roots were transferred into 75% (v/v) ethanol for preservation, and the root system was observed for GUS staining under a stereoscopic microscope (Leica, germany) and photographed.
As a result, as shown in FIG. 2A, the GmBBE-like43 promoter fused GUS reporter gene was expressed in a significantly higher level in Arabidopsis than in the control treatment, and was mainly up-regulated in the old leaves, lateral roots and main root tips of Arabidopsis, regardless of the low-phosphorus or aluminum treatment conditions.
2. GmBBE-like43 subcellular localization analysis
(1) Specific primer GmBBE-like43-GFP-F (SEQ ID NO. 9) was designed: 5'-CTCTAGCGCTACCGGTATGGGAGTCCTTTCTTCTCA-3', gmBBE-like43-GFP-R (SEQ ID NO. 10): 5'-CATGGTGGCGACCGGTCGACCCTTCCTATATGACAGCG-3'. The full length of ORF of the soybean GmBBE-like43 gene is amplified by taking root cDNA of soybean genotype YC03-3 as a template. The reaction conditions are that the reaction is carried out for 5min at 98 ℃, the denaturation is carried out for 30s at 98 ℃, the annealing is carried out for 30s at 58 ℃, the renaturation is carried out for 1-3 min at 72 ℃, the process is circulated for 30 times, and the extension is carried out for 10min at 72 ℃.
(2) The PCR amplified product is recovered and purified by gel electrophoresis using a kit, and after the purified PCR product is obtained, the homologous recombination kit is used
II, carrying out recombinant ligation reaction on the PCR product and the linearized vector pEGAD after being digested 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 recombinant ligase Exnase II, and 4. Mu.L of reaction buffer. The reagent mixture was reacted at 37℃for 30min, the recombinant plasmid was transformed into E.coli and sequenced, and the plasmid was extracted after no error. The 35S GmBBE-like43-GFP plasmid is transformed into agrobacterium GV3101 and agrobacterium rhizogenes K599, and the obtained product is detected and stored for standby.
(3) GmBBE-like43 subcellular localization analysis
The transient expression subcellular localization analysis of tobacco epidermal cells is carried out by introducing 35S:: gmBBE-like43-GFP or pEGAD empty vector (35S: GFP) into Agrobacterium strain GV3101 by Agrobacterium transformation. GV3101 containing the vector after successful transformation was inoculated into YEP medium, shake-cultured at 28℃for 16h, centrifuged (5000 rpm,10 min), and soaked with a solution (containing 10mM MgCl) 2 10mM MES and 10mM acetosyringone, ph=5.6) was resuspended to OD 600 The spectrophotometry value of (2) is 0.4-0.5, the bacterial suspension is kept stand for 3 hours at 43 ℃ in dark, and then the bacterial suspension is infiltrated into the lower epidermis of the leaf blade of the tobacco with the age of 5-6 weeks through a syringe. After normal incubation of the transformed tobacco for 3d, the distribution of the fluorescent signal in the tobacco epidermal cells was observed with a laser copolymerization scanning microscope (Zeiss LSM780, germany). For the localization of the transgenic kidney beans Mao Genya cells, 35S:: gmBBE-like43-GFP or pEGAD were empty (35S:: GFP)) Agrobacterium rhizogenes strain K599 was introduced for transformation of the green roots of Phaseolus vulgaris. Specific methods refer to the kidney bean hairy root transformation system established in the laboratory. The constructed transgenic material was observed for the localization of GFP signal in cells under a laser copolymerization scanning microscope (Zeiss LSM780, germany).
As a result, as shown in FIG. 2, after the 35S:: gmBBE-like43-GFP vector was transferred into the epidermal cells of tobacco leaf to be transiently expressed, no-load control (35S:: GFP) had green fluorescent signals of GFP in the nucleus, cytoplasm and plasma membrane, whereas 35S:: gmBBE-like43-GFP had strong GFP fluorescence at the cell edge and overlapped with the cell contour, indicating that GmBBE-like43 might be localized on the cell wall (FIG. 2B).
To further verify subcellular localization of GmBBE-like43, 35S:: gmBBE-like43-GFP vector was introduced into Agrobacterium rhizogenes K599, and transgenic hairy roots stably expressed by 35S:: gmBBE-like43-GFP fusion protein were obtained by Agrobacterium rhizogenes-mediated soybean cotyledon node in vitro hairy root transformation. The results were similar to tobacco epidermal cells, and GFP fluorescence signal detection revealed that GFP fluorescence from empty transgenic control green bean roots was spread throughout the cells. And 35S, GFP fluorescence of GmBBE-like43-GFP transgenic green bean roots is only at the cell edge and fused with red fluorescence excited by PI dye (FIG. 2C). Thus, gmBBE-like43 may be localized to the cell wall.
Example 3 Effect of GmBBE-like43 expression on in vitro Soy root growth after phosphorus treatment under different conditions
1. GmBBE-like43 functional analysis vector construction
(1) Construction of overexpression vector (OX-GmBBE-like 43-pTF101 s)
The soybean genotype YC03-3 root cDNA is used as a template, a GmBBE-like43 specific primer is designed, and an upstream specific primer 5'-GTACCCGGGGATCCTCTAGAATGGGAGTCCTTTCTTCTCA-3' (SEQ ID NO: 11) and a downstream specific primer 5'-GCCTGCAGGTCGACTCTAGACTAACCCTTCCTATATGACAGCG-3' (SEQ ID NO: 12) are used for amplifying a GmBBE-like43 coding region fragment. The PCR reaction system is as follows: a total of 50. Mu.L of system containing 43. Mu.L of Master mix (2X), 0.5. Mu.L of each of the gene forward/reverse primers, 2. Mu.L of cDNA template, 1. Mu.L of dNTPs (10. Mu.M) and 1. Mu.L of high-fidelity enzymeThe balance is water. The PCR procedure was: pre-denaturation at 98 ℃ for 5min, denaturation at 98 ℃ for 30s, annealing at 58 ℃ for 30s, renaturation at 72 ℃ for 1-3 min, and extension at 72 ℃ for 10min. After the PCR fragment is recovered and sequenced without error, the amplified CDS fragment of GmBBE-like43 is subjected to homologous recombination kit
II kits is connected to a linearized vector pTF101S which is digested by XbaI, the obtained recombinant vector is transformed into escherichia coli, sequencing verification is carried out, and after the sequencing result is correct, the recombinant plasmid 35S, gmBBE-like43, is transferred into agrobacterium rhizogenes K599 and agrobacterium GV3101 for standby.
(2) Construction of interferometric expression vectors (RNAi-GmBBE-like 43-pFGC 5941)
Two sections of specific primers RNAi-GmBBE-like43-pFGC5941-F/R (SEQ ID NO: 13-16) are designed by taking root cDNA of YC03-3 as a template, and the specific sequences are shown 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-pFGC 5941-BamHI-F (SEQ ID NO: 15):
5’-ATTTGCAGGTATTTGGATCCCAAAGCTAGCTCCACCACCA-3’；
primer RNAi-GmBBE-like43-pFGC 5941-BamHI-R (SEQ ID NO: 16):
5’-TCTAGACTCACCTAGGATCCGGTGTGTCTTACGTGGCAGA-3’。
amplifying an open reading frame sequence of about 300bp of GmBBE-like43, and then carrying out restriction enzyme digestion on a target vector by using restriction enzyme Asc I to obtain a linearization binary vector pFGC5941; the 15 bp-20 bp sequence at the tail end of the linearization vector is used as a homologous sequence, and is respectively added to the 5' end of a gene specific forward/reverse amplification primer sequence, so that the primer pair is used for amplification to obtain an insert fragment with the homologous sequence, and the concentration of the recovered product is determined. Following the ligation procedure of one-step cloning (Nuo-uzan Co.), recombination reactions: mixing linearization vector and insert according to a ratio of 1:2, reacting for 1h at 37 ℃ under the catalysis of Exnase II to complete recombination reaction, then cutting a binary vector pFGC5941 of the recombined product by a restriction enzyme BamH I, recombining the amplified second fragment sequence according to a one-step cloning connection method (Nuo Wei Zan Co.), absorbing about 3 mu L of recombined product (according to the product concentration) after the recombination reaction is finished, adding the recombined product into 50 mu L of escherichia coli Trans T1 competence for transformation, selecting monoclonal for later positive screening, and carrying out sequencing verification after detection. And (3) carrying out positive monoclonal amplification culture, preserving bacterial liquid and carrying out plasmid extraction to obtain the soybean GmBBE-like43 interference expression vector. And (3) carrying out K599 agrobacterium transformation on the target vector, detecting positive clones and preserving bacterial liquid at-80 ℃.
2. Acquisition of transgenic Material
Adopts agrobacterium rhizogenes mediated soybean cotyledon 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, sterilizing the surfaces of the soybean seeds in chlorine for 12-14 hours, and then placing the seeds in an ultra-clean workbench for blowing for 30 minutes to remove redundant chlorine; then sowing on germination medium, and culturing for 4 days under 28 ℃ illumination condition.
(2) Preparation of bacterial liquid: agrobacterium rhizogenes K599 containing OX-GmbBE-like43-pTF101s or RNAi-GmbBE-like43-pFGC5941 plasmid was streaked on a plate, and the monoclonal was picked up and incubated at 28℃at 200rpm/min for 12h to OD 600 About 1.0.
(3) Cotyledonary node infection and co-cultivation: the germinated seeds were cut off with a scalpel at the 0.5cm lower hypocotyl area of the leaf, then the seeds were cut vertically along the ridge with a scalpel, and the seed shoots were cut off. In addition, the bacterial solution was dipped with a scalpel, and a plurality of wounds were cut perpendicular to the cotyledonary node and hypocotyl. Moving the cut external body horizontally upwards into a culture dish containing wet filter paper; afterwards, the dishes were sealed with a preservative film, transferred to an incubator (24 ℃), and cultured for 5 days under light. The co-cultured explants were transferred to medium containing herbicide and carbenicillin for 14d growth.
3. Transgenic soybean hairy root positive identification
After extracting the roots of the control group and transgenic hair root RNA to be detected, reversing cDNA, detecting the expression level of the transgenic line which overexpresses GmBBE-like43 by using a fluorescent quantitative PCR primer of GmBBE-like43 in example 1, designing primers GmBBE-like43-RT2-F (SEQ ID NO: 17) and GmBBE-like43-RT2-R (SEQ ID NO: 18) by taking housekeeping gene EF 1-alpha (Glyma 17g 23900) as an internal reference gene, and detecting the expression level of the transgenic line which interferes 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 roots
(1) Aluminum treatment of transgenic soybean roots
Selecting transgenic hair roots with fresh growth, good state and consistent growth, photographing, and transferring to 100 μm AlCl or not 3 (filtration followed by addition to the sterilized and cooled broth) 1/4MS liquid medium (pH 4.5, no KH addition) 2 PO 4 (ii) in (a). Harvest 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 a result, as shown in FIG. 3, quantitative PCR analysis revealed that the expression level of GmBBE-like43 in the in vitro hairy roots of the over-expressed GmBBE-like43 (OX) soybean was increased by about 10-fold and 4-fold, respectively, and the expression level in the in vitro hairy roots of soybean interfering with the expression (RNAi) of GmBBE-like43 was reduced by about 78% and 89%, respectively, as compared with the empty vector control (OX-CK/RNAi-CK) under the condition of no or aluminum addition (FIG. 3B). MS culture experiment results show that under normal treatment conditions, excessive or inhibition of GmBBE-like43 expression has no obvious effect on soybean root growth. Whereas over-expression of GmBBE-like43 (OX) significantly promoted growth of the in vitro hairy roots of transgenic soybeans under aluminum treatment conditions, the root growth and relative growth rate increased by about 67% and 77% respectively compared to the empty vector control (OX-CK) (fig. 3C-E). In contrast, interfering with GmBBE-like43 (RNAi) expression significantly inhibited the growth of the transgenic soybean in vitro hairy roots under aluminum treatment conditions, in particular with a reduction in root growth and relative growth rate of about 31% and 22%, respectively, compared to the empty vector control (RNAi-CK) (fig. 3F and G).
(2) High-low phosphorus treatment of transgenic soybean roots
Selecting transgenic hair roots with fresh growth, good condition, similar root system morphology and weight of about 0.1g, and transferring to a transgenic plant containing high phosphorus (+ P:1250 μm KH 2 PO 4 ) Or low phosphorus (-P: 10. Mu.M KH) 2 PO 4 ) Is grown 14d in MS solid medium. The dry weight of the hair roots and the total root length were collected and measured.
As a result, as shown in FIG. 4, quantitative PCR analysis revealed that the expression level of GmBBE-like43 in the in vitro hairy roots of soybean overexpressing GmBBE-like43 (OX) was increased by about 1.7-fold and 1.8-fold, respectively, and the expression level in the in vitro hairy roots of soybean interfering with GmBBE-like43 expression (RNAi) was decreased by about 80% and 77%, respectively, under high and low phosphorus treatment conditions, as compared with the empty vector control (OX-CK/RNAi-CK) (FIG. 4A). MS culture experimental results show that the in vitro hairy root growth of transgenic soybean is obviously promoted by the over-expression GmBBE-like43 (OX) under the high-phosphorus or low-phosphorus treatment condition, the dry weight of the root system is respectively 1.6 times and 3.4 times that of the control (OX-CK), and meanwhile, the total root length of the in vitro hairy root of the soybean with the over-expression GmBBE-like43 (OX) under the high-phosphorus or low-phosphorus treatment condition is respectively 1.6 times and 2.3 times that of the control (OX-CK) (fig. 4C-E). In contrast, interference of GmBBE-like43 expression in soybean in vitro hair roots under high and low phosphorus treatment conditions reduced dry weight by about 43% and 38% respectively compared to the control, while their total root length and total root length were reduced by about 59% and 35% respectively compared to the control (fig. 4F and G).
Example 4 effect of overexpression of GmBBE-like43 on Arabidopsis growth
1. Obtaining transgenic Arabidopsis thaliana
The constructed over-expression vector (OX-GmBBE-like 43-pTF101 s) plasmid in example 3 is transferred into agrobacterium GV3101, then the inflorescence dip-dyeing method and herbicide screening in example 2 are adopted to obtain T3 generation transgenic arabidopsis seeds, and finally different transgenic arabidopsis lines with high expression quantity of GmBBE-like43 are confirmed by quantitative PCR for subsequent gene function research. The Arabidopsis thaliana GmBBE-like43 quantitative primer is: gmBBE-like43-RT3-F (SEQ ID NO: 19) and GmBBE-like43-RT3-R (SEQ ID NO: 20). The Arabidopsis thaliana housekeeping gene EF 1-alpha is taken as an internal reference gene, and the Arabidopsis thaliana housekeeping gene adopts EF 1-alpha quantitative primers (SEQ ID NO: 21-22), and the specific primer sequences are shown as follows:
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 thaliana
Two transgenic strains of Arabidopsis (OX 1 and OX 2) overexpressing GmBBE-like43 were obtained by an Arabidopsis heterologous gene expression transformation system. Appropriate amounts of wild type and 2 filled seeds overexpressing GmBBE-like 43-positive Arabidopsis lines OX1 and OX2 were selected. After disinfection treatment, the seedlings are firstly sown in a normal MS culture medium, after about 3-4 days, the root system of the arabidopsis grows to about 1cm, the seedlings which are orderly and consistent are selected for photographing, and then the seedlings are moved to the position without containing aluminum or containing aluminum (5 mu M AlCl) 3 ) 1/5Hoagland nutrient solution (pH 4.5, no KH added) 2 PO 4 ，0.5mM CaCl 2 ) Is cultured. Culture conditions: room temperature 23 deg.c and light intensity 120 mu mol.m -2 ·S -1 The light time per day was 16 hours. The seedlings were harvested 48 hours after transplanting, photographs of the seedlings were taken, and root length was measured using image J.
As can be seen from FIG. 5A, the expression level of GmBBE-like43 in transgenic Arabidopsis was significantly increased as compared with WT. Meanwhile, over-expression of GmBBE-like43 significantly promoted the growth of transgenic Arabidopsis root system, both under the treatment conditions of adding aluminum and not adding aluminum (FIG. 5B). Under normal treatment conditions, root growth of both transgenic arabidopsis lines OX1 and OX2 over-expressing GmBBE-like43 increased by about 35% compared to WT, whereas under aluminum treatment conditions root growth of transgenic lines OX1 and OX2 increased by 72% and 133% compared to WT, respectively (fig. 5C). Meanwhile, the root relative growth rates of transgenic lines OX1 and OX2 overexpressing GmBBE-like43 increased by about 27% and 72%, respectively, compared to WT (fig. 5D).
(2) High-low phosphorus treatment of transgenic arabidopsis thaliana
Appropriate amounts of wild type and 2 filled seeds were selected which overexpressed the GmBBE-like43 positive lines OX1 and OX 2. After disinfection treatment, the seedlings are firstly sown in a normal MS culture medium, after about 3-4 d, the root system of the arabidopsis grows to about 1cm, the seedlings which are orderly and consistent are selected, the seedlings are moved into square dishes of the 1/2MS culture medium which is correspondingly treated, and two phosphorus level treatments are arranged: normal phosphorus supply treatment (1250. Mu.M KH) 2 PO 4 ) And low phosphorus treatment (6.25. Mu.M KH) 2 PO 4 ) One replicate per square dish, 5 replicates per treatment. And (5) collecting samples on the 9 th day after treatment, and measuring the fresh weight and the main root length of the plants.
As a result, as shown in FIG. 5, over-expression of GmBBE-like43 significantly promoted root growth of transgenic Arabidopsis, both under high-phosphorus and low-phosphorus treatment conditions (FIG. 5E). Under normal phosphorus treatment conditions, the fresh root weights of transgenic lines OX1 and OX2 over-expressing GmBBE-like43 were increased by about 23% compared to WT, whereas under low phosphorus treatment conditions the fresh root weights of transgenic lines OX1 and OX2 were increased by 20% and 47% compared to WT, respectively (fig. 5F). Meanwhile, under normal phosphorus treatment conditions, the major root length of transgenic lines OX1 and OX2 over-expressing GmBBE-like43 was increased by about 9% as compared with WT, while under low phosphorus treatment conditions, the major root length of transgenic lines OX1 and OX2 was increased by 17% and 23% as compared with WT, respectively (fig. 5G).
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
Application of <120> GmBBE-like43 gene in regulating and controlling plant to adapt to low-phosphorus and acid aluminum stress and promoting growth
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1608
<212> DNA
<213> GmBBE-like43 Gene (SIPOSEQUENCELISTER 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 (SIPOSEQUENCELISTER 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 (6)
1. SEQ ID NO.1GmBBE-like43Application of gene or GmBBE-like43 protein shown in SEQ ID NO. 2 in positively regulating and controlling growth of soybean root system under low phosphorus-resistant condition.
2. SEQ ID NO.1GmBBE-like43Genes or SApplication of GmBBE-like43 protein shown in EQ ID NO. 2 in positively regulating and controlling growth of soybean root system under the condition of resisting aluminum toxicity stress.
3. SEQ ID NO.1GmBBE-like43Application of the gene or GmBBE-like43 protein shown in SEQ ID NO. 2 in improving the growth capacity of soybean root systems under the condition of low phosphorus or aluminum toxicity stress.
4. SEQ ID NO.1GmBBE-like43Application of gene or GmBBE-like43 protein shown in SEQ ID NO. 2 in cultivation of low phosphorus resistant or aluminum toxicity resistant soybean.
5. A method for promoting soybean growth and/or improving soybean tolerance to low phosphorus or aluminum toxicity stress is characterized by positive regulation of soybean by gene editing technologyGmBBE-like43The gene expression level or the protein activity is used for promoting the growth of soybean root systems, so that the tolerance of the soybean to low phosphorus or aluminum toxicity stress is improved;GmBBE-like43the sequence of the gene is shown as SEQ ID NO.1, and the sequence of the GmBBE-like43 protein is shown as SEQ ID NO. 2.
6. The method of claim 5, wherein the over-expression is constructedGmBBE-like43The expression vector of the gene is transferred into a soybean plant body to obtain transgenic soybean which promotes the growth of soybean root systems and improves the tolerance of the soybean to low phosphorus or aluminum toxicity stress.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210609252.9A CN114940997B (en) | 2022-05-31 | 2022-05-31 | Application of GmBBE-like43 gene in regulating and controlling plant to adapt to low-phosphorus and acid aluminum stress and promote growth |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210609252.9A CN114940997B (en) | 2022-05-31 | 2022-05-31 | Application of GmBBE-like43 gene in regulating and controlling plant to adapt to low-phosphorus and acid aluminum stress and promote growth |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114940997A CN114940997A (en) | 2022-08-26 |
| CN114940997B true CN114940997B (en) | 2023-06-09 |
Family
ID=82909573
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210609252.9A Active CN114940997B (en) | 2022-05-31 | 2022-05-31 | Application of GmBBE-like43 gene in regulating and controlling plant to adapt to low-phosphorus and acid aluminum stress and promote growth |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114940997B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116200404A (en) * | 2023-04-10 | 2023-06-02 | 华南农业大学 | Soybean asparagine synthetase analogous gene and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6077694A (en) * | 1990-09-21 | 2000-06-20 | The United States Of America As Represented By The Department Of Health And Human Services | Method for over-expression and rapid purification of biosynthetic proteins |
| CN105821060A (en) * | 2016-05-03 | 2016-08-03 | 河南农业大学 | Soybean low-phosphorus-resistance related gene GmACP2, encoded protein and application of GmACP2 |
| CN107435047A (en) * | 2017-08-15 | 2017-12-05 | 华南农业大学 | In a kind of plant phosphorus signal network Tolerant to low P key gene GmPHR25 and its with application |
| CN108728451A (en) * | 2018-06-14 | 2018-11-02 | 福建农林大学 | A kind of transcription factor GmPHR of the special responding low-phosphor of soybeanLPAnd application |
| CN109608530A (en) * | 2019-01-11 | 2019-04-12 | 华南农业大学 | A kind of low-phosphorous responsive genes of soybean and its albumen and application for promoting lateral root to be formed |
| CN110358789A (en) * | 2019-07-11 | 2019-10-22 | 华中农业大学 | A kind of genetically engineered soybean of acidproof Acid-Al stress and its application |
| CN113322261A (en) * | 2021-06-04 | 2021-08-31 | 华南农业大学 | Application of soybean ABC transporter gene GmALS3 in breeding of low-phosphorus-resistant and aluminum-toxicity-resistant plants |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10750711B2 (en) * | 2015-06-08 | 2020-08-25 | Indigo Ag, Inc. | Streptomyces endophyte compositions and methods for improved agronomic traits in plants |
-
2022
- 2022-05-31 CN CN202210609252.9A patent/CN114940997B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6077694A (en) * | 1990-09-21 | 2000-06-20 | The United States Of America As Represented By The Department Of Health And Human Services | Method for over-expression and rapid purification of biosynthetic proteins |
| CN105821060A (en) * | 2016-05-03 | 2016-08-03 | 河南农业大学 | Soybean low-phosphorus-resistance related gene GmACP2, encoded protein and application of GmACP2 |
| CN107435047A (en) * | 2017-08-15 | 2017-12-05 | 华南农业大学 | In a kind of plant phosphorus signal network Tolerant to low P key gene GmPHR25 and its with application |
| CN108728451A (en) * | 2018-06-14 | 2018-11-02 | 福建农林大学 | A kind of transcription factor GmPHR of the special responding low-phosphor of soybeanLPAnd application |
| CN109608530A (en) * | 2019-01-11 | 2019-04-12 | 华南农业大学 | A kind of low-phosphorous responsive genes of soybean and its albumen and application for promoting lateral root to be formed |
| CN110358789A (en) * | 2019-07-11 | 2019-10-22 | 华中农业大学 | A kind of genetically engineered soybean of acidproof Acid-Al stress and its application |
| CN113322261A (en) * | 2021-06-04 | 2021-08-31 | 华南农业大学 | Application of soybean ABC transporter gene GmALS3 in breeding of low-phosphorus-resistant and aluminum-toxicity-resistant plants |
Non-Patent Citations (4)
| Title |
|---|
| Association of extracellular dNTP utilization with a GmPAP1-like protein identified in cell wall proteomic analysis of soybean roots;Weiwei Wu等;《J Exp Bot. 》;第69卷(第23期);第603–617页 * |
| Comparative Analyses Reveal Peroxidases Play Important Roles in Soybean Tolerance to Aluminum Toxicity;Juge Liu等;《Agronomy》;第11卷(第4期);第1-17页 * |
| 丹波黑大豆GmDREPP基因的克隆与耐铝性;魏运民等;《浙江农业学报》(第6期);第7-18页 * |
| 铝胁迫下大豆根系分泌物对根际土壤的影响;金婷婷等;《中国油料作物学报》;第29卷(第1期);第42-48页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114940997A (en) | 2022-08-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6259003B1 (en) | Cotton plant promoters | |
| CN110734916B (en) | Application of OsbHLH98 in regulation of rice leaf included angle | |
| CN112226455B (en) | Rice grain length and grain weight related protein, and coding gene and application thereof | |
| CN106834314B (en) | Millet stress resistance gene SiR L K35, encoding protein and application | |
| CN108624596B (en) | Gene for regulating growth of leguminous root noduleGmSPX5And uses thereof | |
| US20140317782A1 (en) | High temperature resistant plant gene and use thereof | |
| CN114940997B (en) | Application of GmBBE-like43 gene in regulating and controlling plant to adapt to low-phosphorus and acid aluminum stress and promote growth | |
| CN108948169B (en) | Protein and gene for promoting synthesis of cotton fiber green pigment, and coding sequence and application thereof | |
| CN110760526B (en) | Sweet orange CsMYB120 gene and application thereof | |
| CN114277041B (en) | Application of soybean gibberellin 3 beta-hydroxylase encoding gene GmGA3ox1 | |
| CN113528538B (en) | Cucumber CsSTK gene, protein, expression vector and application | |
| CN116120413A (en) | SlHAT5 gene and application thereof in high temperature stress resistance of tomatoes | |
| KR102265780B1 (en) | EFG1 gene for regulating flowering date of plantbody and uses thereof | |
| CN111926035B (en) | Application of two WRKY transcription factor genes in rice and coding protein thereof | |
| CN109971772B (en) | Breeding method of low-temperature-resistant cotton variety | |
| CN110066810B (en) | Tamarix chinensis plasma membrane Na+/H+Antiporter gene and application thereof | |
| KR101161276B1 (en) | Vascular tissue-specific promoter and expression vector comprising the same | |
| KR102000465B1 (en) | Method of improving resistance of Bakanae disease | |
| CN107488223B (en) | Tobacco nicotine content regulating gene Ribosomal L4/L1 and cloning method and application thereof | |
| CN112745377A (en) | Application of potato tonoplast monosaccharide transporter StTMT2 gene in improving plant photosynthetic rate | |
| CN114085854B (en) | Drought-resistant and salt-tolerant gene OsSKL2 for rice and application thereof | |
| CN113913441B (en) | Application of rice nascent polypeptide binding complex alpha subunit NACA gene in osmotic stress resistance of plants | |
| CN113528533B (en) | Limonium bicolor gene LbRSG and application thereof | |
| CN113604475B (en) | Application of cotton GH_D03G1517 gene in promotion of drought resistance and salt tolerance | |
| CN110183524B (en) | Gene GmKRP2a for promoting soybean main root elongation, protein and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |