Application of gene GmPP2-10 in promoting growth of plant in low-phosphorus stress environment

Abstract

The invention 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. Researches show that the expression of the GmPP2-10 gene is increased under the induction of low phosphorus stress, and under different phosphorus concentration treatment conditions, the over-expression of the GmPP2-10 can increase the biomass of a transgenic plant and promote the growth of the plant under the low phosphorus condition; meanwhile, overexpression of GmPP2-10 can improve the tolerance of plants to low-phosphorus stress, and reduce the inhibition effect of low phosphorus on plant root growth. Therefore, the GmPP2-10 plays an important role in adapting to the low-phosphorus stress of the plant, and the adaptive capacity of the plant to the low-phosphorus stress of the acid soil can be improved by transferring the GmPP2-10 into the plant through a transgenic technology.

Description

Technical Field

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

[0002] Soybeans are rich in protein and fat and are important raw materials for making foods such as soybean oil and food seasonings. In recent years, with the development of the market, soybeans in China are difficult to be self-sufficient and need to rely on a large amount of imports to meet the domestic market demand. The latest data from China Grain and Oils Information Network shows that in recent years, China's annual soybean import volume has exceeded 95 million tons, while the total annual domestic soybean production is about 20 million tons. The supply-demand contradiction of domestic soybeans has reached a new high point, and increasing domestic soybean production has become a major demand in the Chinese market. At present, most of China's soybean production comes from the Northeast region, but the soybean planting area in the Northeast has been continuously declining, while the demand for soybeans has increased significantly, resulting in a shortage of domestic soybean production in short supply. Therefore, vigorously developing soybean planting is an important measure to solve the shortage of soybean supply in China.

[0003] Soybeans are phosphorus-loving plants with a relatively high demand for phosphorus, especially in the seedling stage and the flowering and pod-setting stage, they require sufficient phosphorus supply. Phosphorus (P) is one of the essential macronutrients for plant growth and development and plays an important role in plant growth and development. For example, biological processes such as substance synthesis, energy transfer, and signal transduction all require the participation of phosphorus. In acidic soils, phosphorus will be fixed by a large amount of iron, aluminum, calcium and other ions, and the content of phosphorus that can be absorbed by plants is limited, making it difficult to meet the growth needs of plants. Under low-phosphorus stress, plants mainly regulate root architecture and increase root exudates to activate and utilize insoluble phosphorus; at the same time, enhancing the expression of phosphorus transporter genes can also improve the phosphorus absorption efficiency. Among them, the response of root morphological architecture to low-phosphorus stress is mainly achieved through the regulation of the expression of a variety of hormones and a series of genes.

[0004] In order to adapt to the phosphorus-deficient soil environment, plants have evolved a complete set of fine morphological changes and physiological and biochemical adaptation mechanisms to enhance the utilization of soil phosphorus. Although in recent years, botanists have made some important progress in the study of the mechanism of plants' response to phosphorus starvation and have identified hundreds of related genes, the number of signal pathways involved in plant phosphorus starvation regulation is numerous and complex and changeable. Therefore, people's 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, in-depth study of the adaptation mechanism and molecular mechanism of plant soybean genes under phosphorus-deficient conditions, so as to cultivate new soybean varieties tolerant to phosphorus starvation and improve the absorption and utilization efficiency of phosphorus by soybeans, is of great significance for protecting the environment and promoting the sustainable development of agriculture. Summary of the Invention

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

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

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

[0008] The fourth object of the present invention is to provide a preparation for improving the low-phosphorus stress tolerance ability of plants. The active ingredient of the preparation contains the soybean low-phosphorus tolerance gene GmPP2-10 or the protein encoded by the soybean low-phosphorus tolerance gene GmPP2-10. The nucleotide sequence of the soybean low-phosphorus tolerance gene GmPP2-10 is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0009] The fifth object of the present invention is to provide a method for promoting the growth of plant roots. The method is to overexpress the soybean low-phosphorus tolerance gene GmPP2-10 in plants. The nucleotide sequence of the soybean low-phosphorus tolerance gene GmPP2-10 is shown in SEQ ID NO.1, or the soybean low-phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown in SEQ ID NO.2.

[0010] The sixth object of the present invention is to provide an application of a recombinant expression vector in increasing the root length of plants and improving the low-phosphorus tolerance ability of transgenic plants under low-phosphorus conditions. The recombinant expression vector contains the soybean low-phosphorus tolerance gene GmPP2-10. The nucleotide sequence of the soybean low-phosphorus tolerance gene GmPP2-10 is shown in SEQ ID NO.1, or the soybean low-phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown in SEQ ID NO.2.

[0011] Objective seven of the present invention is to provide an application of a genetically engineered bacterium in increasing the root length of plants under low phosphorus conditions and improving the low phosphorus tolerance of transgenic plants.

[0012] The above objective is achieved through the following technical solutions:

[0013] In the present invention, the GmPP2-10 gene of the PP2 family was cloned from soybean by real-time fluorescence quantitative PCR and homologous cloning methods, and it was found that when GmPP2-10 was under low phosphorus stress at the gene transcription level, its expression level was significantly up-regulated. In this application, Arabidopsis transgenic lines with overexpression of GmPP2-10 were obtained through Arabidopsis transgenic technology, and then the physiological indexes of Arabidopsis wild-type lines and transgenic lines were compared through experiments under high phosphorus (HP) and low phosphorus (LP) conditions. The experimental results showed that the GmPP2-10 gene has the function of regulating the adaptation of soybean roots to low phosphorus stress; at the same time, it can reduce the inhibitory effect of low phosphorus stress on the growth of plant roots.

[0014] In summary, the beneficial effects of the present invention are as follows:

[0015] An important gene GmPP2-10 for plant low phosphorus tolerance and its application are disclosed. For the first time in the present invention, a GmPP2-10 gene of the PP2 family was cloned from soybean and studied. The study showed that the expression of the GmPP2-10 gene was up-regulated under low phosphorus stress induction, and its expression level increased significantly with the extension of phosphorus treatment time; under different phosphorus concentration treatment conditions, overexpression of GmPP2-10 could significantly increase the biomass of transgenic plants and promote the growth of plants under low phosphorus conditions; at the same time, overexpression of GmPP2-10 could improve the tolerance of plants to low phosphorus stress and reduce the inhibitory effect of low phosphorus on the growth of plant roots;

[0016] In addition, this application discloses the application of the soybean GmPP2-10 gene in regulating plant adaptation to low phosphorus stress and promoting growth, reflecting that the PP2 gene of soybean is involved in the relevant mechanism of plant regulation of soil phosphorus stress, and GmPP2-10 positively regulates the ability of plant roots to adapt to low phosphorus;

[0017] Through experimental research, this application fully proves the important role played by the GmPP2-20 gene in the process of plant adaptation to low phosphorus stress. By implanting the GmPP2-20 gene into plants through transgenic technology, the ability of plants to adapt to low phosphorus stress in acidic soil can be significantly improved.

[0018] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. And in order to make the above and other purposes, features and advantages of the present invention more obvious and understandable, the following preferred embodiments are specifically given and described in detail in conjunction with the accompanying drawings as follows. Brief Description of the Drawings

[0019] Figure 1 For the analysis of the relative expression level of soybean GmPP2-10;

[0020] Figure 2 For the screening and identification of positive Arabidopsis seedlings. Part a shows the identification of positive Arabidopsis seedlings, and part b shows the detection of the expression level of GmPP2-10 in different lines of positive Arabidopsis seedlings;

[0021] Figure 3 For the phenotypes of soybean lines in the control group (CK: pTF101 empty vector), overexpression (OE: pTF101-GmPP2-10) soybean lines, and GmPP2-10 gene-edited (Cas9) soybean lines under normal phosphorus and low phosphorus concentration treatments;

[0022] Figure 4 For the comparison charts of the relative expression level, primary root length, root fresh weight, and root phosphorus content of soybean lines in the control group (CK: pTF101 empty vector), overexpression (OE: pTF101-GmPP2-10) soybean lines, and GmPP2-10 gene-edited (Cas9) soybean lines under high and low phosphorus treatments;

[0023] Figure 5 For the effects of different phosphorus concentrations (1000 μΜ, 100 μΜ, 10 μΜ) on the growth of GmPP2-10 overexpressing Arabidopsis plants;

[0024] Figure 6 For the subcellular localization analysis of GmPP2-10;

[0025] Figure 7 For the phylogenetic tree of the GmPP2 gene family. Detailed Embodiments

[0026] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field, and the reagents and materials used in the experiments are all commercially available.

[0027] The soybean material used in the following experiments is Huachun 6, which is sourced from the Guangdong Branch of the National Soybean Improvement Center, South China Agricultural University.

[0028] The present invention first cloned a GmPP2-10 gene of the PP2 family in soybean. The phylogenetic tree of the GmPP2 gene family is as Figure 7 shown. The nucleotide sequence of the GmPP2-10 gene is as follows (SEQ ID NO.1):

[0029] ATGAAGTCCCAAGACTTGCCGGAAGGATGCGTCGCACACATATTGTCGTACATTTGTACTCCTGAAGACATAGTCAGGCTCTCCCTCGTTTCCAAGGCCTTTTATTCTGCAGCTGATTACGACACTGTGTGGGATCGTTTTATACCCTCTGATTTCTCCTCTACCATTTCTCCACTTTCTTCCTCTAACTCTAAGAAGGATCTCTATTTCACTCTCTCCGACCGTCCCACCATCATCGACCAGGGTAGAAAGAGCTTTCAATTGGAAAAGCGAACTGCGAAGAAGTGCTACATGCTTTCCGCTAGGGATATTTCTATTACATGGGCTCCAACGCAGGGAGAGGCTTCTCAATATTGGGAGTGGAAAAGCCTGCCAGAGTCAAGGTTCCAAGAAGTTGCGAGGCTTTACGCTGTGTGCTGGTTTAACATCACTGGGCAGATAAAGACCCGCGTCTTGTCCCCAAATACTCAGTACGCAGCTTTTCTTGTGTTCCAGATGATCGATGCCAGTGGATTTCACCACCATCCTGCGATGTTATCAGTAAGTAATGTTGGAGGCAGTAGAACTTCCAAATATGTTTGTTTGGATCCCAACTTAGAAGATAATGATCTTGATGACAGATTTCGGGGTCTGCAACGTCCTAATGTGAGAAAAGATAAGTGGTTAGAGATTGAGATGGGAGAGTTCTTCAACTCAGGCTTGGAAGAAGATGAAATCTATATGAATGTCAGGGAAACTAGTGATATGTGGAAGCACGGTTTCATTCTTGAAGGAATAGAAGTTAGGCCTAAACATGTTTGA

[0030] The amino acid sequence encoded by the GmPP2-10 gene is as follows (SEQ ID NO.2):

[0031] MKSQDLPEGCVAHILSYICTPEDIVRLSLVSKAFYSAADYDTVWDRFIPSDFSSTISPLSSSNSKKDLYFTLSDRPTIIDQGRKSFQLEKRTAKKCYMLSARDISITWAPTQGEASQYWEWKSLPESRFQEVARLYAVCWFNITGQIKTRVLSPNTQYAAFLVFQMIDASGFHHHPAMLSVSNVGGSRTSKYVCLDPNLEDNDLDDRFRGLQRPNVRKDKWLEIEMGEFFNSGLEEDEIYMNVRETSDMWKHGFILEGIEVRPKHV

[0032] Example 1: Cloning of the GmPP2-10 gene and analysis of tissue expression levels

[0033] Step 1: Design specific primers based on the GmPP2-10 gene sequence:

[0034] GmPP2-10-F (SEQ ID NO.3): TCTCCATCCAACAGAAATCAAGTG

[0035] GmPP2-10-R (SEQ ID NO.4): TTATTCCGGGTCTCCCTCCA.

[0036] The primers for real-time fluorescence quantitative PCR are the quantitative primers of the GmPP2-10 gene and the housekeeping gene primer of Actin for soybeans, as shown below:

[0037] GmPP2-10-RT-F (SEQ ID NO.5): RTCTCCTCTACCATTTCTCCACT;

[0038] GmPP2-10-RT-R (SEQ ID NO.6): CTGGTCGATGATGGTGGGAC;

[0039] Actin-F (SEQ ID NO.7): GCACCACCGGAGAGAAAATA

[0040] Actin-R (SEQ ID NO.8): GTGCACAATTGATGGACCAG

[0041] Step 2: RNA extraction and reverse transcription

[0042] Pre-culture Huachun 6 soybean seeds, take about 0.1 g of fresh samples of roots, stems, leaves, flowers and young pods, freeze them quickly in liquid nitrogen, and store them in a refrigerator at -80 °C. Extract the RNA of the above samples, reverse transcribe it into cDNA using a reverse transcription kit (Vazyme, Nanjing), and then use the cDNA as a template for real-time fluorescence quantitative PCR and gene fragment cloning to obtain the target gene. The cloning reaction system is shown in Table 1.

[0043] Table 1 Cloning reaction system

[0044]

[0045] Example 2: Construction of vector

[0046] Step 1: Ligate the target gene to the pLB zero-background expression vector

[0047] S1, Refer to the instruction manual of the Tiangen zero-background rapid ligation kit to ligate the pLB vector and the target gene to obtain ligation product 1 (pLB-GmPP2-10);

[0048] S2, Refer to the instruction manual of the Vazyme DH5α Chemically Competent Cell product, and transfer ligation product 1 into Escherichia coli DH5α competent cells by PCR amplification to obtain the expression vector;

[0049] S3, Spread the Escherichia coli into which the ligation product has been transferred in S2 on a solid medium plate and culture it. After the Escherichia coli grows into single colonies on the plate, under sterile conditions, inoculate the single colonies into a centrifuge tube, and then place the centrifuge tube on a shaker for shaking culture to allow the bacteria to proliferate in large numbers to obtain the expression vector bacterial liquid;

[0050] S4, Perform PCR detection of the expression vector bacterial liquid, and identify the target band by SDS polyacrylamide gel electrophoresis to determine whether the target gene has been ligated into the expression vector. Take the bacterial liquid with correct identification and culture it overnight with shaking, extract the plasmid and send it for sequencing, and use the DNAMAN software to compare the sequencing results. The plasmid extraction refers to the instruction manual of the Novoprotein DC20. The bacterial liquid PCR amplification reaction system is shown in Table 2.

[0051] Table 2 Bacterial liquid PCR system

[0052] Reagent Name Reagent Dosage 2×Accurate Taq Master Mix 25 μl Bacterial Solution 2 μl Forward Primer F 1.5 μl Reverse Primer R 1.5 μl <![CDATA[ddH2O]]> 20 μl

[0053] Step 2: Construction of the overexpression vector

[0054] The expression vector obtained in Step 1 was identified by single and double enzyme digestion, and the recombinant fragment containing the CDS sequence of the GmPP2-10 gene was cloned. The vector pTF101 was linearized with XbaI and SacI and ligated with the recombinant fragment, followed by transformation to obtain the ligation product II (pTF101-GmPP2-10). After successful transformation, the ligation product II bacterial solution was identified, the bacterial solution with the correct band was selected, and the ligation product II bacterial solution was sequenced. The bacterial solution with successful sequence alignment was amplified and cultured, and the plasmid was extracted.

[0055] The plasmid with correct sequencing was transformed into Agrobacterium rhizogenes K599 and Agrobacterium tumefaciens GV3101. After successful transformation, the bacterial solution was identified, the bacterial solution with the correct band was selected, the plasmid was extracted, and the overexpression vector was obtained.

[0056] Step 3: Construction of transient expression vector

[0057] The vector pCAMBIA1300 was linearized with the vector-specific restriction enzymes NcoI and SpeI, and then ligated with the recombinant fragment, followed by transformation to obtain the ligation product III. After successful transformation, the ligation product III bacterial solution was identified, the bacterial solution with the correct band was selected, and the ligation product III bacterial solution was sequenced. The bacterial solution with successful sequence alignment was amplified and cultured, and the plasmid was extracted to obtain the transient expression vector.

[0058] Step 4: Construction of gene editing vector

[0059] S1. Four target sites for the GmPP2-10 gene were designed using the Huazhong Agricultural University sgRNA website: CRISPR-P 2.0 Chttp: / orispr.hzau.edu Cn / CRISPR2A, and four target sites (guide2, guide4, guide11, and guide16) were obtained. The primers for the four target sites are as follows:

[0060] guide2

[0061] F (SEQ ID NO.9): GGATTGTAGAGTTAGAGGAAGAAAG

[0062] R (SEQ ID NO.10): AAACTAGAGTTAGAGGAAGAAAGCA

[0063] guide4

[0064] F (SEQ ID NO.11): GGATTGAGTCCCAAGACTTGCCGGA

[0065] R (SEQ ID NO.12): AAACAGTCCCAAGACTTGCCGGACA

[0066] guide11

[0067] F (SEQ ID NO.13): GGATTGGTAATAGAAATATCCCTAG

[0068] R (SEQ ID NO.14): AAACGTAATAGAAATATCCCTAGCA

[0069] guide16

[0070] F (SEQ ID NO.15): GGATTGGTGCTACATGCTTTCCGCT

[0071] R (SEQ ID NO.16): AAACGTGCTACATGCTTTCCGCTCA

[0072] The lengths of the said primers are all 25 bp.

[0073] S2. Anneal the four synthesized target primers, ligate the annealed primers with the pUC19 vector to construct pUC19 vector 1, 3, 5, and 6 ligation vectors. Subsequently, transfer the pUC19 vector 1, 3, 5, and 6 ligation vectors into Escherichia coli DH5α respectively, then spread the Escherichia coli DH5α on the LB medium supplemented with ampicillin. After the bacteria grow to form single colonies, pick the single colonies and use the M13-26 universal primer for single-direction sequencing, and then conduct sequence alignment. After successful alignment, culture the Escherichia coli containing the correct recombinant plasmid by shaking, and then extract the plasmid using a plasmid extraction kit and measure the plasmid concentration.

[0074] S3. Ligate the plasmid extracted in step S2 to the pCas9 vector to obtain ligation body four. Transfer ligation body four into Escherichia coli DH5α, then spread the Escherichia coli DH5α on the LB medium supplemented with ampicillin. After the bacteria grow to form single colonies, pick bacterial samples from the culture medium plate, and then conduct electrophoresis analysis on the bacterial samples. A band at about 1000 bp indicates successful vector construction, and a gene editing vector is obtained. The ligation system of pUC19 is shown in Table 3.

[0075] Table 3 pUC19 ligation system

[0076]

[0077]

[0078] Example 3: Soybean genetic transformation experiment

[0079] Take out Huachun 6 that has germinated for about 3 days (d), wash the vermiculite, cut off the parts below the root base, and inject the K599 bacterial liquid containing the empty vector (pTF101), overexpression vector (pTF101-GmPP2-10), and gene editing vector (GmPP2-10) with an OD of about 0.6 - 0.8 into the stem about 2 cm below the cotyledons of the seedlings with a syringe. Place the seedlings injected with the bacterial liquid on moist vermiculite covered with two layers of filter paper on the surface and seal them with plastic wrap. First, culture them in the dark for 1 d, then carry out 16 h of light, perform 8-hour dark culture at a temperature of 26 °C, and then carry out light culture at a temperature of 24 °C. After successful rooting and the root length reaching 3 - 5 cm, transfer the seedlings to nutrient solutions containing different phosphorus concentrations for cultivation. Observe the phenotypic differences of soybean plants with different vectors under low phosphate (LP) and normal phosphate (NP) concentration treatments, count the relevant data and take pictures.

[0080] Figure 1 This is the expression pattern analysis diagram of soybean GmPP2-10 in this example, and the expression patterns of soybean GmPP2-10 under normal phosphate treatment (NP) and low phosphate treatment (NP) are compared. It can be seen from the experimental comparison that the expression of soybean GmPP2-10 is up-regulated under low phosphate stress.

[0081] As Figure 3 shown, where part a and part b are the phenotypes of the control group (CK: pTF101 empty vector) soybean lines, overexpression (OE: pTF101-GmPP2-10) soybean lines, and GmPP2-10 gene-edited (Cas9) soybean lines under normal phosphate and low phosphate concentration treatments, and the scale bar in the figure is 1 cm.

[0082] Figure 4 This is a comparison diagram of the relative expression levels, primary root lengths, root fresh weights, and root phosphorus contents of the control group (CK: pTF101 empty vector) soybean lines, overexpression (OE: pTF101-GmPP2-10) soybean lines, and GmPP2-10 gene-edited (Cas9) soybean lines under high and low phosphate treatments. It can be seen from Figure 4 that under normal phosphate and low phosphate concentrations, the relative expression levels, primary root lengths, fresh weights, and root phosphorus contents of the transgenic lines are significantly higher than those of the control lines, showing significant differences.

[0083] Example 4: Arabidopsis thaliana genetic transformation

[0084] Step 1: Inflorescence infiltration of Arabidopsis thaliana

[0085] Plant wild-type Arabidopsis thaliana in nutrient soil. Cut off the main bolt when it first bolts, and prepare for infection when the side bolts are about to flower; transfer the vector pTF101-GmPP2-10 into Agrobacterium tumefaciens GV3101. Screen the Agrobacterium tumefaciens that has successfully transferred the vector, put the positive bacteria into a liquid medium for culture until the OD600 value reaches 0.6, then centrifuge the positive bacteria in a centrifuge, resuspend the bacterial cells with a resuspension solution, then immerse the Arabidopsis flowers in the resuspension solution for about 1 min, seal and moisturize, and culture in the dark for 1 d, and then culture normally.

[0086] Step 2: Screening and identification of positive seedlings

[0087] Harvest the Arabidopsis thaliana seeds after infection in nutrient soil. Regularly spray herbicide multiple times after two true leaves have fully grown. Screen out some false positive seedlings. When the plants grow strong, select fresh leaves, quickly extract DNA with TPS extraction solution, conduct DNA molecular level identification, and obtain 9 independent lines (L1-L9) with overexpression of the GmPP2-10 gene. Identify and backcross to the T3 generation by this method.

[0088] The results are as Figure 2 shown. Among them, the substance in lane 1 of part a is the 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 plasmid of the gene overexpression vector, and the substances in lanes 5-14 are the DNA samples of positive seedling plants.

[0089] Extract the RNA of Arabidopsis thaliana of different lines, and through fluorescence quantitative identification, select the lines with high expression levels for large-scale seed multiplication. The results of fluorescence quantitative identification are as Figure 2 shown in part b. In the figure, WT is the wild-type line; L1-L9 are transgenic Arabidopsis thaliana lines with overexpression of GmPP2-10; the comparison results show that the expression levels of L3, L6, and L7 in Arabidopsis thaliana plants are the highest.

[0090] Among them, the quantitative primers for Arabidopsis thaliana GmPP2-10 are: GmPP2-10-RT-F and GmPP2-10-RT-R. Use the Arabidopsis thaliana housekeeping gene as the internal reference gene, and the housekeeping gene of Arabidopsis thaliana uses Tubulin quantitative primers. The specific primer sequences are as follows:

[0091] GmPP2-10-RT-F (SEQ ID NO.17): RTCTCCTCTACCATTTCTCCACT

[0092] GmPP2-10-RT-R (SEQ ID NO.18: CTGGTCGATGATGGTGGGAC

[0093] Tubulin-F(SEQ ID NO.19): ATCGATTCCGTTCTCGATGT;

[0094] Tubulin-R(SEQ ID NO.20): ATCCAGTTCCTCCTCCCAAC.

[0095] Example 5: Identification of Arabidopsis thaliana low-phosphorus phenotype

[0096] Step 1: Germination culture

[0097] Disinfect wild-type and transgenic Arabidopsis thaliana seeds (OE-3, OE-6, OE-7) with 10% sodium hypochlorite for about 10 min, then wash the seeds 4-5 times with sterile water, and sow them on a square plate of 1 / 2MS solid medium (pH 5.8). Seal the plate with breathable tape and place it at 4°C for 3-4 d, and then transfer it to normal culture conditions for culture.

[0098] Step 2: Phosphorus treatment experiment

[0099] S1. Prepare the long-term treatment medium for Arabidopsis thaliana, adjust the pH of the medium to 4.5, and then sterilize it. Different amounts of NH4H2PO4 need to be added to the medium during preparation so that the Pi concentrations in the medium are 1000 μΜ, 100 μΜ, and 10 μΜ respectively.

[0100] S2. When the germinated Arabidopsis thaliana seedlings grow to about 1 cm, select uniform seedlings and transfer them to media with different phosphorus concentrations for continued culture for about 2 weeks. Observe the phenotypic differences between wild-type and transgenic Arabidopsis thaliana under each phosphorus concentration treatment, count the relevant data and take phenotypic photos.

[0101] The experimental results are as Figure 5 shown: There is no obvious difference between wild-type plants and transgenic lines under normal phosphorus and phosphorus-free concentrations. Under low phosphorus concentration, the main root length and fresh weight of transgenic lines are significantly higher than those of wild-type lines, showing a significant difference.

[0102] Example 6: Tobacco subcellular localization analysis

[0103] The Agrobacterium tumefaciens GV3101-superp19 strains carrying super-1300 and super-1300-GmPP2-10 respectively were inoculated into YEP liquid medium supplemented with kan and cultured with shaking at 28 °C for 12 h. Take 50 ml of the bacterial liquid and centrifuge it in a centrifuge. The rotation speed of the centrifuge is set at 12000 rpm, and the centrifugation time is 10 min. Subsequently, the collected bacterial cell precipitate is resuspended with a tobacco epidermal cell transformation resuspension to OD600 = 0.45 - 0.55. It should be noted that before transforming the tobacco leaves, the bacterial suspension needs to be kept in the dark at 22 °C - 24 °C for 2 - 3 h.

[0104] Then the resuspension was injected into the leaves of 3 - 4-week-old tobacco. Three days after transformation, a laser confocal scanning microscope was used to observe the distribution of fluorescence in tobacco epidermal cells under the excitation light of 488 nm (GFP) / 587 nm (mChe) wavelengths.

[0105] The results are as Figure 6 shown. In the figure, 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 image. The GmPP2-10–GFP fusion protein was transiently expressed only in the nucleus and had a strong fluorescent signal, while the control GFP protein was distributed throughout the cell, and the GmPP2-10 protein was localized in the nucleus.

[0106] The data in this example are the mean and standard error of 3 replicates. Asterisks indicate significant differences (Student’s t-test) between the control (CK: pTF101 empty vector) and overexpression (OE: pTF101-GmPP2-10) and gene editing (GmPP2-10), *: P < 0.05, **: P < 0.01, ***: P < 0.001), ****: P < 0.0001).

Claims

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

2.

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

2.

3. Use of the soybean low phosphorus tolerance gene GmPP2-10 in cultivating low phosphorus tolerant plants, characterized in that, The nucleotide sequence of the soybean low phosphorus tolerance gene GmPP2-10 is shown in SEQ ID NO.1, or the soybean low phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown in SEQ ID NO.

2.

4. A preparation for improving the low phosphorus stress tolerance of plants, characterized in that, The active ingredient of the preparation comprises the soybean low phosphorus tolerance gene GmPP2-10 or the protein encoded by the soybean low phosphorus tolerance gene GmPP2-10. The nucleotide sequence of the soybean low phosphorus tolerance gene GmPP2-10 is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.

2.

5. A method for promoting plant root growth, characterized in that, Overexpress the soybean low phosphorus tolerance gene GmPP2-10 in plants. The nucleotide sequence of the soybean low phosphorus tolerance gene GmPP2-10 is shown in SEQ ID NO.1, or the soybean low phosphorus tolerance gene GmPP2-10 encodes an amino acid sequence shown in SEQ ID NO.

2.

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

2.

7. Use of a genetically engineered bacterium in increasing the root length of plants under low phosphorus conditions and improving the low phosphorus tolerance of transgenic plants, characterized in that, The genetically engineered bacterium contains the recombinant expression vector described in claim 6.

Images