Use of gene GmPP2-10 in promoting growth of plants in low-phosphorus stress environment

Abstract

The application relates to the technical field of plant genetic engineering, and discloses application of a soybean low-phosphorus-tolerant gene GmPP2-10 in promoting growth of plants in a low-phosphorus stress environment. Research shows that the GmPP2-10 gene is induced to express up-regulation under low-phosphorus stress, under different phosphorus concentration treatment conditions, overexpression of the GmPP2-10 can increase the biomass of the transgenic plants, and promote the growth of the plants under low-phosphorus conditions; meanwhile, overexpression of the GmPP2-10 can improve the tolerance of the plants to the low-phosphorus stress and reduce the inhibiting effect of low phosphorus on the growth of the plant roots. Therefore, the GmPP2-10 has an important role in adaptation of the plants to the low-phosphorus stress, and the GmPP2-10 can be transferred into the plants through a transgenic technology to improve the adaptation of the plants to the low-phosphorus stress in acid soil.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a gene GmPP2-10 in promoting growth of plants in a low-phosphorus stress environment. BACKGROUND

[0002] Soybean contains rich protein and fat, and is an important raw material for making soybean oil and food seasonings. In recent years, with the development of the market, China's soybean is difficult to be self-sufficient, and needs to rely on a large amount of imports to meet the demand of the domestic market. The latest data from China Grain and Oil Information Network shows that in recent years, China's annual soybean imports have exceeded 95 million tons, while the total annual domestic soybean production is about 20 million tons. The contradiction between supply and demand of domestic soybean has reached a new high point, and improving domestic soybean production has become a major demand of the Chinese market. At present, most of China's soybean production comes from the northeast region, but the soybean planting area in the northeast continues to decline, while the demand for soybean has increased significantly, leading to a shortage of domestic soybean production. Therefore, vigorously developing soybean planting is an important measure to solve the shortage of China's soybean supply.

[0003] Soybean is a phosphorus-loving plant and has a high demand for phosphorus, especially during the seedling and flowering and pod setting stages. Phosphorus (P) is one of the essential macronutrients for plant growth and development, and plays an important role in plant growth and development, such as material synthesis, energy transfer, and signal transduction. In acid soils, phosphorus is fixed by a large amount of iron, aluminum, and calcium ions, and the content of phosphorus that can be absorbed by plants is limited, making it difficult to meet the needs of plant growth. Under low-phosphorus stress, plants mainly regulate root architecture and increase root exudates to activate and utilize insoluble phosphorus, and also enhance the expression of phosphorus transporter genes to improve the efficiency of phosphorus uptake. The response of root architecture to low-phosphorus stress is mainly achieved through the expression of a variety of hormones and a series of genes.

[0004] In order to adapt to the soil environment of phosphorus deficiency, plants have evolved a set of sophisticated morphological changes and physiological and biochemical adaptation mechanisms to enhance the utilization of soil phosphorus. Although plant scientists have made some important progress in the study of plant mechanisms for coping with phosphorus starvation in recent years, and have identified hundreds of related genes, the number of signal pathways involved in the regulation of plant phosphorus starvation is numerous and complex, so our understanding of how plants perceive changes in phosphorus concentration in the external environment and how to enhance phosphorus starvation tolerance is still very limited. Therefore, it is of great significance to study the adaptation mechanisms and molecular mechanisms of soybean genes under phosphorus deficiency conditions, to breed new varieties of soybean with phosphorus starvation tolerance, and to improve the absorption and utilization efficiency of soybean for phosphorus, in order to protect the environment and promote sustainable agricultural development. SUMMARY

[0005] To solve the problems in the background art, the first object of the present application is to provide an application of a soybean low-phosphorus-tolerant gene GmPP2-10 in promoting the growth of plants in a low-phosphorus stress environment, wherein the nucleotide sequence of the soybean low-phosphorus-tolerant gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus-tolerant gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO. 2.

[0006] The second object of the present application is to provide an application of a soybean low-phosphorus-tolerant gene GmPP2-10 in improving the low-phosphorus stress tolerance of plant roots, wherein the nucleotide sequence of the soybean low-phosphorus-tolerant gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus-tolerant gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO. 2.

[0007] The third object of the present application is to provide an application of a soybean low-phosphorus-tolerant gene GmPP2-10 in breeding low-phosphorus-tolerant plants, wherein the nucleotide sequence of the soybean low-phosphorus-tolerant gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus-tolerant gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO. 2.

[0008] The fourth object of the present application is to provide a method for promoting the growth of plant roots, which comprises overexpressing a soybean low-phosphorus-tolerant gene GmPP2-10 in plants, wherein the nucleotide sequence of the soybean low-phosphorus-tolerant gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus-tolerant gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO. 2.

[0009] The fifth object of the present application is to provide an application of a recombinant expression vector in increasing the length of plant roots and improving the low-phosphorus tolerance of transgenic plants under low-phosphorus conditions, wherein the recombinant expression vector contains a soybean low-phosphorus-tolerant gene GmPP2-10, and the nucleotide sequence of the soybean low-phosphorus-tolerant gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus-tolerant gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO. 2.

[0010] The sixth object of the present application is to provide an application of a genetically engineered bacterium in increasing the length of plant roots and improving the low-phosphorus tolerance of transgenic plants under low-phosphorus conditions.

[0011] The above objects are achieved by the following technical solutions.

[0012] The application clones a GmPP2-10 gene of a PP2 family in soybean by a real-time fluorescent quantitative PCR and a homologous cloning method, and finds that the expression of the GmPP2-10 gene is significantly up-regulated at a transcription level of a gene under low-phosphorus stress. The application obtains an Arabidopsis thaliana transgenic strain with overexpression of the GmPP2-10 gene by an Arabidopsis thaliana transgenic technology, and compares physiological indexes of a wild-type strain and the transgenic strain of the Arabidopsis thaliana under high-phosphorus (HP) and low-phosphorus (LP) conditions by experiments. The experimental results show that the GmPP2-10 gene has a function of regulating the soybean root system to adapt to low-phosphorus stress, and can reduce the inhibition of low-phosphorus stress on the growth of the plant root system.

[0013] In summary, the application has the following beneficial effects:

[0014] Disclosed are a plant low-phosphorus tolerance important gene GmPP2-10 and application thereof. The application clones a GmPP2-10 gene of a PP2 family in soybean for the first time, and studies the gene. The study shows that the GmPP2-10 gene is up-regulated in expression under low-phosphorus stress, and the expression amount of the gene is obviously increased with the extension of the phosphorus treatment time. Under different phosphorus concentration treatment conditions, overexpression of the GmPP2-10 gene can obviously increase the biomass of a transgenic plant, promote the growth of the plant under low-phosphorus conditions, improve the tolerance of the plant to low-phosphorus stress, and reduce the inhibition of low-phosphorus on the growth of the plant root system.

[0015] In addition, the application discloses application of the soybean GmPP2-10 gene in regulating plant growth promotion under low-phosphorus stress, reflects that the PP2 gene of the soybean participates in a related mechanism of plant regulation of soil phosphorus stress, and the GmPP2-10 gene positively regulates the ability of the plant root system to adapt to low phosphorus.

[0016] The application fully proves, through experimental research, that the GmPP2-20 gene plays an important role in the process that the plant adapts to low-phosphorus stress. The GmPP2-20 gene is implanted into the plant body by a transgenic technology, and the adaptability of the plant to low-phosphorus stress of acid soil can be obviously improved.

[0017] The above description is only a summary of the technical solutions of the application. In order to more clearly understand the technical means of the application, the application can be implemented according to the content of the specification, and in order to make the above and other purposes, characteristics and advantages of the application more obvious and easy to understand, the following preferred embodiments are described in detail below, and the accompanying drawings are described as follows. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The relative expression amount of the soybean GmPP2-10 is analyzed.

[0019] Figure 2Figure for screening and identifying positive seedlings of Arabidopsis, wherein part a is identification of positive seedling plants of Arabidopsis, and part b is expression amount detection of GmPP2-10 in different lines of positive seedling plants of Arabidopsis;

[0020] Figure 3 Phenotypes of a control group (CK: pTF101 empty load) soybean line, an overexpression (OE: pTF101-GmPP2-10) soybean line and a GmPP2-10 gene editing (Cas9) soybean line under normal phosphorus and low phosphorus concentration treatment;

[0021] Figure 4 Comparison figure of relative expression amount, main root length, root fresh weight and root phosphorus content of a control group (CK: pTF101 empty load) soybean line, an overexpression (OE: pTF101-GmPP2-10) soybean line and a GmPP2-10 gene editing (Cas9) soybean line under high and low phosphorus treatment.

[0022] Figure 5 Effect of different phosphorus concentrations (1000 μΜ, 100 μΜ, 10 μΜ) treatment on GmPP2-10 overexpression Arabidopsis plant growth;

[0023] Figure 6 GmPP2-10 subcellular localization analysis;

[0024] Figure 7 GmPP2 gene family evolution tree. DETAILED DESCRIPTION

[0025] The present application will be further described below in combination with the drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field, and the reagents and materials used in the experiment are commercially available.

[0026] The soybean material used in the following experiment is Huachun No. 6, which is from the Guangdong Branch of the National Soybean Improvement Center of South China Agricultural University.

[0027] The present application first clones a GmPP2-10 gene of a PP2 family in soybean, wherein the GmPP2 gene family evolution tree is as shown in Figure 7 The nucleotide sequence of the GmPP2-10 gene is as follows (SEQ ID NO. 1):

[0028] ATGAAGTCCCAAGACTTGCCGGAAGGATGCGTCGCACACATATTGTCGTACATTTGTACTCCTGAAGACATAGTCAGGCTCTCCCTCGTTTCCAAGGCCTTTTATTCTGCAGCTGATTACGACACTGTGTGGGATCGTTTTATACCCTCTGATTTCTCCTCTACCATTTCTCCACTTTCTTCCTCTAACTCTAAGAAGGATCTCTATTTCACTCTCTCCGACCGTCCCACCATCATCGACCAGGGTAGAAAGAGCTTTCAATTGGAAAAGCGAACTGCGAAGAAGTGCTACATGCTTTCCGCTAGGGATATTTCTATTACATGGGCTCCAACGCAGGGAGAGGCTTCTCAATATTGGGAGTGGAAAAGCCTGCCAGAGTCAAGGTTCCAAGAAGTTGCGAGGCTTTACGCTGTGTGCTGGTTTAACATCACTGGGCAGATAAAGACCCGCGTCTTGTCCCCAAATACTCAGTACGCAGCTTTTCTTGTGTTCCAGATGATCGATGCCAGTGGATTTCACCACCATCCTGCGATGTTATCAGTAAGTAATGTTGGAGGCAGTAGAACTTCCAAATATGTTTGTTTGGATCCCAACTTAGAAGATAATGATCTTGATGACAGATTTCGGGGTCTGCAACGTCCTAATGTGAGAAAAGATAAGTGGTTAGAGATTGAGATGGGAGAGTTCTTCAACTCAGGCTTGGAAGAAGATGAAATCTATATGAATGTCAGGGAAACTAGTGATATGTGGAAGCACGGTTTCATTCTTGAAGGAATAGAAGTTAGGCCTAAACATGTTTGA

[0029] The amino acid sequence encoded by the GmPP2-10 gene is as follows (SEQ ID NO. 2):

[0030] MKSQDLPEGCVAHILSYICTPEDIVRLSLVSKAFYSAADYDTVWDRFIPSDFSSTISPLSSSNSKKDLYFTLSDRPTIIDQGRKSFQLEKRTAKKCYMLSARDISITWAPTQGEASQYWEWKSLPESRFQEVARLYAVCWFNITGQIKTRVLSPNTQYAAFLVFQMIDASGFHHHPAMLSVSNVGGSRTSKYVCLDPNLEDNDLDDRFRGLQRPNVRKDKWLEIEMGEFFNSGLEEDEIYMNVRETSDMWKHGFILEGIEVRPKHV

[0031] Example 1: Cloning of GmPP2-10 gene and analysis of tissue expression

[0032] Step 1: According to the sequence of GmPP2-10 gene, specific primers were designed:

[0033] GmPP2-10-F (SEQ ID NO. 3): TCTCCATCCAACAGAAATCAAGTG

[0034] GmPP2-10-R (SEQ ID NO. 4): TTATTCCGGGTCTCCCTCCA.

[0035] Real-time fluorescent quantitative PCR primers used GmPP2-10 gene quantitative primers and internal reference gene Actin as the housekeeping gene primers of soybean, as shown below:

[0036] GmPP2-10-RT-F (SEQ ID NO. 5): RTCTCCTCTACCATTTCTCCACT;

[0037] GmPP2-10-RT-R (SEQ ID NO. 6): CTGGTCGATGATGGTGGGAC;

[0038] Actin-F (SEQ ID NO. 7): GCACCACCGGAGAGAAAATA

[0039] Actin-R (SEQ ID NO. 8): GTGCACAATTGATGGACCAG

[0040] Step 2: Extraction and reverse transcription of RNA

[0041] The soybean seeds of Huachun 6 were pre-cultured, and fresh samples of about 0.1 g of roots, stems, leaves, flowers and young pods were quickly frozen in liquid nitrogen and stored in a refrigerator at-80°C. The RNA of the above samples was extracted, and the cDNA was reversely transcribed by a reverse transcription kit (Vazyme, Nanjing). Real-time fluorescent quantitative PCR and gene fragment cloning were carried out by taking the cDNA as a template to obtain the target gene. The cloning reaction system is shown in Table 1.

[0042] Table 1 Cloning reaction system

[0043]

[0044] Example 2: Construction of vector

[0045] Step 1: Connection of target gene and pLB zero background expression vector

[0046] S1, according to the instruction of Tian Gen zero background rapid ligation kit, the pLB vector and the target gene were connected to obtain the connector one (pLB-GmPP2-10);

[0047] S2, according to the product instruction of Weidi Biology DH5α Chemically Competent Cell, the connector one was transformed into E. coli DH5α competent cells by PCR amplification to obtain an expression vector;

[0048] S3, the E. coli transformed by the connector in S2 was inoculated on a solid culture medium plate and cultured. After the E. coli grew on the plate to form single colonies, the single colonies were inoculated into centrifuge tubes under sterile conditions. Then the centrifuge tubes were placed in a shaker to carry out shake culture, so that the bacteria proliferated in large quantities to obtain the expression vector bacterial liquid;

[0049] S4, PCR detection of the expression vector bacterial liquid was carried out, and the target band was identified by SDS polyacrylamide gel electrophoresis to determine whether the target gene was connected to the expression vector. The correct bacterial liquid was inoculated for overnight shake culture, and the plasmid was extracted and sent for sequencing. The sequencing results were compared by using DNAMAN software. The plasmid extraction was referred to the instruction of Nuowuzan DC201. The PCR amplification reaction system of the bacterial liquid is shown in Table 2.

[0050] Table 2 Bacterial liquid PCR system

[0051]

[0052] Step 2: Construction of super-expression vector

[0053] The expression vector obtained in step 1 was identified by single and double enzyme digestion, and the recombinant fragment containing the GmPP2-10 gene CDS sequence was cloned. Xba I andSac I linearize the vector pTF101, and then connect and transform it with the recombinant fragment to obtain connector two (pTF101-GmPP2-10). After successful transformation, the bacterial liquid of connector two is identified, the bacterial liquid of the correct band is selected, and the bacterial liquid of connector two is sequenced. The bacterial liquid with a successfully aligned sequence is expanded and shaken, and the plasmid is extracted.

[0054] The plasmid with a correct sequence is transformed into Agrobacterium rhizogenes K599 and Agrobacterium GV3101. After successful transformation, the bacterial liquid is identified, the bacterial liquid of the correct band is selected, and the plasmid is extracted to obtain the overexpression vector.

[0055] Step three: construction of a transient expression vector

[0056] Select a vector-specific endonuclease Nco I and Spe I linearize the vector pCAMBIA1300, and then connect and transform it with the recombinant fragment to obtain connector three. After successful transformation, the bacterial liquid of connector three is identified, the bacterial liquid of the correct band is selected, and the bacterial liquid of connector three is sequenced. The bacterial liquid with a successfully aligned sequence is expanded and shaken, and the plasmid is extracted to obtain the transient expression vector.

[0057] Step four: construction of a gene editing vector

[0058] S1. Four target points (guide2, guide4, guide11, and guide16) are designed for the GmPP2-10 gene using the sgRNA website of Huazhong Agricultural University: CRISPR-P 2.0 Chttp: / orispr.hzau. edu Cn / CRISPR2A. The primers of the four target points are as follows:

[0059] guide2

[0060] F (SEQ ID NO. 9): GGATTGTAGAGTTAGAGGAAGAAAG

[0061] R (SEQ ID NO. 10): AAACTAGAGTTAGAGGAAGAAAGCA

[0062] guide4

[0063] F (SEQ ID NO. 11): GGATTGAGTCCCAAGACTTGCCGGA

[0064] R (SEQ ID NO. 12): AAACAGTCCCAAGACTTGCCGGACA

[0065] guide 11

[0066] F (SEQ ID NO. 13): GGATTGGTAATAGAAATATCCCTAG

[0067] R (SEQ ID NO. 14): AAACGTAATAGAAATATCCCTAGCA

[0068] guide 16

[0069] F (SEQ ID NO. 15): GGATTGGTGCTACATGCTTTCCGCT

[0070] R (SEQ ID NO. 16): AAACGTGCTACATGCTTTCCGCTCA

[0071] The length of the primers is 25 bp.

[0072] S2, the synthesized four target primers are annealed, the annealed primers are connected with pUC19 vector, pUC19 vector 1, 3, 5, 6 connection vectors are constructed, then the pUC19 vector 1, 3, 5, 6 connection vectors are respectively transferred into E. coli DH5α, and then the E. coli DH5α is coated on the LB medium added with ampicillin, after the bacteria grow to form single colonies, the single colonies are selected and the M13-26 universal primer is used for single sequencing, and then sequence alignment is carried out, after successful alignment, the E. coli containing the correct recombinant plasmid is expanded and cultured, then the plasmid is extracted by using the plasmid extraction kit and the plasmid concentration is determined.

[0073] S3, the plasmid extracted in the step S2 is connected to the pCas9 vector to obtain the connector four, the connector four is transferred into E. coli DH5α, and then the E. coli DH5α is coated on the LB medium added with ampicillin, after the bacteria grow to form single colonies, the bacterial samples are picked from the culture medium plate, then the bacterial samples are subjected to electrophoresis analysis, and the band at about 1000 bp proves that the vector construction is successful, and the gene editing vector is obtained, wherein the connection system of pUC19 is shown in Table 3.

[0074] Table 3 pUC19 connection system

[0075]

[0076] Example three: soybean genetic transformation experiment

[0077] The Huachun 6 sprouted for about 3 days (d) was taken out, washed with vermiculite, and the parts below the root base were removed. The K599 bacteria liquid containing empty carrier (pTF101), overexpression carrier (pTF101-GmPP2-10) and gene editing carrier (GmPP2-10) with OD of about 0.6-0.8 was injected into the stem about 2 cm below the cotyledon of the seedling by a syringe. The seedling injected with the bacteria liquid was placed on the wet vermiculite with two layers of filter paper on the surface and sealed with plastic wrap. After 1 d of dark culture, 16 h of light was carried out, and 8 h of dark culture was carried out at a temperature of 26°C, and then light culture was carried out at a temperature of 24°C. After successful rooting and root length of 3-5 cm, the seedling was transferred to a nutrient solution containing different phosphorus concentrations for culture. The phenotypic differences of soybean plants with different carriers under low phosphorus (Low phosphate, LP) and normal phosphorus (Normal phosphate, NP) concentration treatments were observed, and the related data were counted and photographed.

[0078] Figure 1 The expression pattern of soybean GmPP2-10 in this embodiment was analyzed, and the expression patterns of soybean GmPP2-10 under normal phosphorus treatment (NP) and low phosphorus treatment (NP) were compared. It can be seen from the comparison experiment that the expression of soybean GmPP2-10 is up-regulated under low phosphorus stress induction.

[0079] As Figure 3 shown, wherein part a and part b are the phenotypes of the control group (CK: pTF101 empty carrier) soybean strain, overexpression (OE: pTF101-GmPP2-10) soybean strain and GmPP2-10 gene editing (Cas9) soybean strain under normal phosphorus and low phosphorus concentration treatment. The scale in the figure is 1 cm.

[0080] Figure 4 The comparison chart of the relative expression amount, main root length, root fresh weight and root phosphorus content of the control group (CK: pTF101 empty carrier) soybean strain, overexpression (OE: pTF101-GmPP2-10) soybean strain and GmPP2-10 gene editing (Cas9) soybean strain under high and low phosphorus treatment. It can be seen from Figure 4 that under normal phosphorus and low phosphorus concentration, the relative expression amount, main root length, fresh weight and root phosphorus content of the transgenic strain are obviously higher than those of the control strain, and there is a significant difference.

[0081] Example 4: Genetic transformation of Arabidopsis thaliana

[0082] Step one: infestation of Arabidopsis thaliana inflorescence

[0083] Wild type Arabidopsis was planted in nutrient soil. The main bud was cut off at the first time of germination, and the side bud was prepared for infection when it was about to bloom. The vector pTF101-GmPP2-10 was transferred into Agrobacterium GV3101. The Agrobacterium successfully transferred with the vector was screened, and the positive bacteria were put into the liquid medium for culture until the OD600 value reached 0.6. Then the positive bacteria were centrifuged, and the bacteria were resuspended with resuspension solution. Then the Arabidopsis flower was soaked in the resuspension solution for about 1 min, and was sealed and dark-cultured for 1 d, and then was normally cultured.

[0084] Step two: positive seedling screening and identification

[0085] The harvested Arabidopsis seeds after infection were planted in nutrient soil. The herbicide was sprayed regularly and repeatedly after the two true leaves were fully grown. Some false positive seedlings were screened out. After the plant grew well, the fresh leaves were selected, and the DNA was extracted with TPS extraction solution for DNA molecular level identification, and 9 independent GmPP2-10 gene overexpression lines (L1-L9) were obtained. This method was used for identification of T3 generation.

[0086] The results are shown in Figure 2 , wherein the substance in lane 1 of part a is a standard reference (DNA Marker), the substance in lane 2 is water, the substance in lane 3 is the DNA sample of wild plants (Wild Type, WT), the substance in lane 4 is the gene overexpression vector plasmid, and lanes 5-14 are DNA samples of positive seedlings.

[0087] The RNA of different Arabidopsis lines was extracted, and the lines with high expression were selected for mass propagation. The results of fluorescence quantitative identification are shown in Figure 2 part b, wherein WT is the wild type line; L1-L9 are transgenic Arabidopsis lines with overexpression of GmPP2-10; the comparison results show that the expression amount of L3, L6 and L7 in Arabidopsis plants is the highest.

[0088] Among them, the Arabidopsis GmPP2-10 quantitative primer is: GmPP2-10-RT-F and GmPP2-10-RT-R. The Arabidopsis housekeeping gene was used as the internal reference gene, and the Tubulin quantitative primer was used for the Arabidopsis housekeeping gene. The specific primer sequences are as follows:

[0089] GmPP2-10-RT-F (SEQ ID NO. 17): RTCTCCTCTACCATTTCTCCACT

[0090] GmPP2-10-RT-R (SEQ ID NO. 18: CTGGTCGATGATGGTGGGAC

[0091] Tubulin-F (SEQ ID NO. 19): ATCGATTCCGTTCTCGATGT;

[0092] Tubulin-R (SEQ ID NO. 20): ATCCAGTTCCTCCTCCCAAC.

[0093] Example Five: Arabidopsis low phosphorus phenotype identification

[0094] Step one: germination culture

[0095] The wild type and transgenic Arabidopsis seeds (OE-3, OE-6, OE-7) were sterilized with 10% sodium hypochlorite for about 10 min, then washed with sterile water for 4-5 times, and then sowed on 1 / 2 MS solid medium (pH 5.8) square plates, sealed with a breathable tape, and placed at 4°C for 3-4 d, and then cultured under normal conditions.

[0096] Step two: phosphorus treatment experiment

[0097] S1, configure the Arabidopsis long-term treatment medium, and adjust the pH of the medium to 4.5, then sterilize the medium. When configuring the medium, different amounts of NH4H2PO4 need to be added to the medium, so that the concentration of Pi in the medium is 1000 μΜ, 100 μΜ, and 10 μΜ, respectively.

[0098] S2, when the germination culture Arabidopsis seedlings grow to about 1 cm, select uniform seedlings and transfer them to different phosphorus concentration media for continuous culture for about 2 weeks. Observe the phenotype differences of wild type and transgenic Arabidopsis under different phosphorus concentrations, count the related data and take photos of the phenotype.

[0099] The experimental results are shown in Table 1: Figure 5 Under normal phosphorus and no phosphorus concentration, there is no obvious difference between wild type and transgenic lines, and under low phosphorus concentration, the length of the main root and the fresh weight of the transgenic lines are significantly higher than those of the wild type lines, and there is a significant difference.

[0100] Example Six: Tobacco subcellular localization analysis

[0101] GV3101-superp19 strains carrying super-1300, super-1300-GmPP2-10 respectively were inoculated into YEP liquid medium added with kan, and cultured at a temperature of 28 ℃ for 12 h. 50 ml of bacterial liquid was taken and centrifuged in a centrifuge at a speed of 12000 rpm for 10 min, and then the collected bacterial body precipitate was resuspended to OD600=0.45-0.55 with tobacco epidermal cell transformation resuspension liquid. It should be noted that the bacterial body suspension needs to be placed in the dark at 22 ℃-24 ℃ for 2-3 h before transforming tobacco leaves.

[0102] Then the resuspension liquid was injected into the leaves of 3-4 week old tobacco. After 3 days of transformation, the distribution of fluorescence in tobacco epidermal cells was observed under the excitation light of 488 nm (GFP) / 587 nm (mChe) wavelength using a laser confocal scanning microscope.

[0103] The results are shown in Figure 6 The figure shows that GFP is the control group; Gmpp2-10 is the experimental group; DAPI represents the nuclear dye channel; eGFP represents the green fluorescent protein signal channel; Bright Field is the bright field image; Merged is the merged field image. The GmPP2-10-GFP fusion protein is only transiently expressed in the nucleus and has a strong fluorescence signal, while the control GFP protein is distributed throughout the cell, and the GmPP2-10 protein is located in the nucleus.

[0104] The data of this example are the average and standard error of 3 replicates, and the asterisk indicates that the difference between the control (CK: pTF101 empty load) and the overexpression (OE: pTF101-GmPP2-10) and gene editing (GmPP2-10) is significant (Student's t-test), *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001).

Claims

1. The use of a soybean low phosphorus tolerance gene GmPP2-10 in promoting the growth of soybean in a low phosphorus stress environment, characterized in that, The nucleotide sequence of the soybean low-phosphorus tolerance gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO.

2.

2. The application of soybean low phosphorus tolerance gene GmPP2-10 in improving the low phosphorus stress tolerance of soybean roots, characterized by, The nucleotide sequence of the soybean low-phosphorus tolerance gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO.

2.

3. The application of soybean low phosphorus tolerance gene GmPP2-10 in breeding soybean with low phosphorus stress tolerance, characterized in that, The nucleotide sequence of the soybean low-phosphorus tolerance gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO.

2.

4. A method of promoting growth of soybean root system, characterized by, The nucleotide sequence of the soybean low-phosphorus tolerance gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO.

2.

5. The use of a recombinant expression vector in increasing the length of soybean root and improving the low phosphorus stress tolerance of transgenic soybean under low phosphorus conditions, characterized in that, The nucleotide sequence of the soybean low-phosphorus tolerance gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO.

2.

6. The use of genetically engineered bacteria in increasing the length of soybean root system and improving the low phosphorus stress tolerance of transgenic soybean under low phosphorus conditions, characterized in that, The nucleotide sequence of the soybean low-phosphorus tolerance gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO.

2. The nucleotide sequence of the soybean low-phosphorus tolerance gene GmPP2-10 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown as SEQ ID NO. 2.

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