阅读说明：本技术 SgPAP7在提高植物利用内源有机磷能力的应用 (Application of SgPAP7 in improving capability of plants in utilizing endogenous organophosphorus ) 是由 罗佳佳 刘春� 董荣书 刘攀道 蔡泽坪 于 2021-08-13 设计创作，主要内容包括：本发明涉及柱花草紫色酸性磷酸酶SgPAP7在促进/提高植物利用内源有机磷能力的应用。柱花草紫色酸性磷酸酶SgPAP7不仅能有效水解内源有机磷底物,而且还能提高APase活性。在植物体内表达柱花草紫色酸性磷酸SgPAP7基因,促进植物利用内源性ADP克服低磷胁迫。本发明克服了SgPAP7不能利用内源有机磷促进植物生长的技术偏见,对解决植物磷利用问题,尤其是对无磷/低磷环境植物种植问题,具有重要价值。(The invention relates to application of stylosanthes guianensis purple acid phosphatase SgPAP7 in promoting/improving the capability of plants to utilize endogenous organophosphorus. The stylosanthes guianensis purple acid phosphatase SgPAP7 not only can effectively hydrolyze endogenous organophosphorus substrates, but also can improve the APase activity. The stylosanthes guianensis purple acid phosphate SgPAP7 gene is expressed in a plant body, and the plant is promoted to overcome low phosphorus stress by utilizing endogenous ADP. The invention overcomes the technical prejudice that SgPAP7 can not utilize endogenous organic phosphorus to promote plant growth, and has important value for solving the problem of plant phosphorus utilization, especially for the problem of plant planting in a phosphorus-free/low-phosphorus environment.)

1. The application of the stylosanthes guianensis purple acid phosphatase SgPAP7 in promoting/improving the capability of plants to utilize endogenous organophosphorus.

2. The application of the coding gene of the stylosanthes guianensis purple acid phosphatase SgPAP7 in promoting/improving the capability of plants to utilize endogenous organophosphorus.

3. The use according to any one of claims 1 to 2, wherein the organophosphorus is Adenosine Diphosphate (ADP).

4. Application of the coding gene of the stylosanthes guianensis purple acid phosphatase SgPAP7 in constructing transgenic plants resistant to low-phosphorus environments.

5. The application of the coding gene of the stylosanthes guianensis purple acid phosphatase SgPAP7 in constructing transgenic plants resistant to low-phosphorus environment, wherein only Adenosine Diphosphate (ADP) exists in the environment.

6. A method for improving the utilization capacity of endogenous organic phosphorus of plants is characterized in that the gene SgPAP7 of the purple acid phosphatase of stylosanthes guianensis is expressed in the plants.

7. The method as claimed in claim 6, wherein the method for expressing the Strychnos japonicus purple acid phosphatase SgPAP7 gene in the plant is to transfer the Strychnos japonicus purple acid phosphatase SgPAP7 gene into the plant for endogenous expression.

8. The method of any one of claims 6 to 7, wherein the organophosphorus is Adenosine Diphosphate (ADP).

9. The application of the purple acid phosphatase SgPAP7 in hydrolyzing organophosphorus substrates, wherein the organophosphorus substrates are adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine monophosphate, disodium 4-nitrophenylphosphate, pyrophosphoric acid, glucose-6-phosphate, phosphoserine, phosphothreonine or phosphotyrosine.

10. A method for hydrolyzing an organophosphorus substrate is characterized in that the organophosphorus substrate is dispersed in a dispersion medium, and then, a purple acid phosphatase SgPAP7 is added, wherein the organophosphorus substrate is adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine monophosphate, disodium 4-nitrophenylphosphate, pyrophosphoric acid, glucose-6-phosphate, phosphoserine, phosphothreonine or phosphotyrosine.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to application of stylosanthes guianensis purple acid phosphatase SgPAP7 in promoting plants to utilize endogenous organophosphorus to resist low-phosphorus stress and application in hydrolyzing organophosphorus substrates.

Background

Phosphorus is one of the macronutrients necessary for plant growth and development, and is involved in various metabolic processes of plants, including synthesis of membrane phospholipids and nucleotides, photosynthesis, energy transfer, signal transduction, and the like. The main forms of soil phosphorus exist in three forms, including Inorganic soluble phosphate (Pi), Inorganic insoluble phosphorus and organic phosphorus, but the Inorganic insoluble phosphorus and the organic phosphorus with abundant contents cannot be directly absorbed and utilized by plants, and the Pi content which can be directly absorbed and utilized by the plants is less than 1.0%. In order to relieve low phosphorus stress, people excessively apply phosphate fertilizers to ensure the stable yield and high yield of crops, but the utilization efficiency of the phosphate fertilizers by the crops is low, only about 20% of the applied phosphate fertilizers can be absorbed and utilized by the plants, and meanwhile, the excessive phosphate fertilizers enable water bodies to be eutrophicated and seriously pollute the environment.

During the long-term evolution process, plants form various strategies for enhancing the absorption and utilization efficiency of phosphorus, such as changing the root system morphology and configuration, symbiosis with arbuscular mycorrhizal fungi, enhancing the expression and activity of high-affinity phosphorus transporters, secreting organic acids and Purple Acid Phosphatases (PAPs) and the like to the rhizosphere, replacing membrane phospholipids with galactolipids and thioesters, and the like. Purple Acid Phosphatases (PAPs) are a large family of polygenes, a group of metallophosphatases that are widely present in plants, and are characterized by the presence of 5 conserved domains: DXG, GDXXY, GNH (D/E), VXH, GHXH. The PAPs can effectively catalyze the hydrolysis of phosphate or anhydride in an acid environment to release phosphate groups which can be utilized by plants, and promote the utilization of phosphorus in the environment by the plants, namely the development of research on the function of purple acid phosphatase gene families of the plants, Wenming and the like, plant academy, 2019. A large number of members of the PAPs gene family are up-regulated in response to low phosphorus stress. As shown in functional studies of rice purple acid phosphatases OsPAP10a and OsPAP10c, Sanlinghong, Zhejiang university, 2016, plant can synthesize and secrete PAPs outside roots under the condition of phosphorus deficiency, so that organophosphorus compounds around plant roots are hydrolyzed, and inorganic phosphorus is released for plant growth and utilization. Studies have shown that the PAP Ia subfamily plays an important role in the utilization of organophosphorus in plants and the adaptation to phosphorus deficiency. The rice PAP Ia subfamily includes OsPAP10a, OsPAP10b, OsPAP10c, OsPAP10d and OsPAP 26. For example, the identification and low-phosphorus response characteristics of the Purple Acid Phosphatase (PAPS) gene family of maize, the report of agricultural biotechnology, Yishuang et al, 2015, the cloning and expression pattern analysis of the purple acid phosphatase gene PmPAP1 of Pinus massoniana, and the analysis of Yangting and 2016. However, none of the above studies relate to the involvement of PAPs in the utilization of organophosphorus endogenous to plants.

In addition, Stylosanthes spp is an important legume grass distributed in tropical and subtropical acid soils that is widely used for maintaining water and soil and green manure for orchard intercropping (Marques et al, 2018). Styrax floribunda has strong capability of adapting to abiotic adversity stress (including low phosphorus and aluminum toxicity) of acid soil, and Liu and other researches (2018) show that under the low phosphorus stress, Styrax floribunda strengthens the activity of SgPAP23 related to a root system and enhances the utilization of exogenous phytate phosphorus so as to adapt to the low phosphorus stress. Research shows that when dNTP is taken as a unique phosphorus source, the Stropharia ciliata purple acid phosphatase SgPAP7 gene is overexpressed in the Royal phaseoloides root, which is favorable for enhancing the utilization capacity of exogenous dNTP of the Royal phaseoloides root, namely, the molecular mechanism of activating organophosphorus by Stropharia ciliata purple acid phosphatase [ D ]. Hainan university, 2016 ]. However, it is not clear whether the Stylosanthes guianensis SgPAP7 is involved in the activation and utilization of endogenous organophosphorus.

In conclusion, a great deal of research is focused on analyzing the mechanism of the PAPs for improving the utilization of exogenous (soil) phosphorus by plants (phosphorus absorption efficiency), and less research is carried out on the participation of members of the PAPs in the high-efficiency utilization of endogenous phosphorus by plants (phosphorus utilization efficiency).

Disclosure of Invention

The technical problem to be solved by the invention is how to enhance the utilization of endogenous phosphorus in cells (improve the utilization efficiency of phosphorus) by plants under the condition of insufficient exogenous (environmental) phosphorus (mainly Pi) in the background technology so as to adapt to low-phosphorus stress and maintain the self growth of the plants, and the invention provides a method for improving the utilization capacity of endogenous phosphorus in the plants. Based on the research result of the invention, the excessive expression of the Strychnos prunus willd acid phosphate SgPAP7 gene in plants can promote the utilization of endogenous organophosphorus, especially endogenous ADP. The method overcomes the technical prejudice that the expression of SgPAP7 gene in the plant body can not promote the plant to utilize endogenous phosphorus.

The invention aims to provide application of the stylosanthes guianensis purple acid phosphatase SgPAP7 in promoting/improving the capability of plants to utilize endogenous organophosphorus.

Another object of the present invention is to provide the use of the gene encoding the purple acid phosphatase SgPAP7 in plants for promoting/improving the ability of plants to utilize endogenous organophosphorus.

Another objective of the invention is to provide application of the coding gene of the purple acid phosphatase SgPAP7 in constructing transgenic plants with low-phosphorus environment, wherein only Adenosine Diphosphate (ADP) exists in the environment.

Another object of the present invention is to provide a method for increasing the ability of plants to utilize endogenous organophosphorus.

Another object of the present invention is to provide the use of the purple acid phosphatase SgPAP7 for hydrolyzing organophosphorus substrates.

It is another object of the present invention to provide a method for hydrolyzing organophosphorus substrates.

In order to achieve the purpose, the invention provides the following technical scheme:

experiments prove that wild Arabidopsis thaliana and transgenic Arabidopsis thaliana (OE-1/2/3) which excessively expresses SgPAP7 are cultivated in non-nutrient soil with phosphorus content of 160mg P/kg. Compared with WT, the acid phosphatase activity (APase, taking ADP as a substrate) of transgenic Arabidopsis (OE-1/2/3) is improved by 151.0-192.6%; but the ADP concentration in vivo is reduced by 11.1 to 15.7 percent. The above results indicate that overexpression of the stylosanthes guianensis SgPAP7 gene increases the APase activity of Arabidopsis thaliana and enhances the utilization of endogenous ADP. Therefore, the following technical scheme is protected:

the application of the stylosanthes guianensis purple acid phosphatase SgPAP7 in promoting/improving the capability of plants to utilize endogenous organophosphorus.

The application of the coding gene of the stylosanthes guianensis purple acid phosphatase SgPAP7 in promoting/improving the capability of plants to utilize endogenous organophosphorus.

Wherein, preferably, the organophosphorus refers to Adenosine Diphosphate (ADP).

The application of the coding gene of the stylosanthes guianensis purple acid phosphatase SgPAP7 in constructing transgenic plants resistant to low-phosphorus environment, wherein only Adenosine Diphosphate (ADP) exists in the environment.

A method for improving the utilization capacity of endogenous organic phosphorus of plants enables the plants to express the Strychnos paniculatus purple acid phosphatase SgPAP7 gene.

Preferably, the method for expressing the stylosanthes guianensis purple acid phosphatase SgPAP7 gene in the plant body is to transfer the stylosanthes guianensis purple acid phosphatase SgPAP7 gene into the plant body to express the gene endogenously.

Wherein, preferably, the organophosphorus is Adenosine Diphosphate (ADP).

Preferably, the method for expressing the Strychnos crispus purple acid phosphatase SgPAP7 gene in the plant body is to transfer a eukaryotic expression vector connected with an open reading frame of the Strychnos crispus acid phosphatase SgPAP7 gene, and more particularly to adopt an agrobacterium strain EHA105 of the eukaryotic expression vector connected with an open reading frame of the Strychnos crispus acid phosphatase SgPAP7 gene to dip and transform the plant.

Wherein, preferably, the plant is Arabidopsis thaliana.

Wherein, the eukaryotic expression vector is pTF101.1.

The application of the purple acid phosphatase SgPAP7 in hydrolyzing organophosphorus substrates, wherein the organophosphorus substrates are Adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), Adenosine Monophosphate (AMP), Guanosine Monophosphate (GMP), 4-nitrophenyl phosphate disodium salt (rho-NPP), pyrophosphoric acid, glucose-6-phosphate, phosphoserine, phosphothreonine or phosphotyrosine.

A method for hydrolyzing an organophosphorus substrate comprises the steps of dispersing the organophosphorus substrate in a dispersion medium, and then adding the purple acid phosphatase SgPAP7, wherein the organophosphorus substrate is adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine monophosphate, 4-nitrophenyl phosphate disodium salt, pyrophosphoric acid, glucose-6-phosphate, phosphoserine, phosphothreonine or phosphotyrosine.

Wherein, preferably, the pH value of the dispersion medium is 5-7, and the temperature is 37-43 ℃.

Wherein, preferably, the dispersion medium contains Mg²⁺。

Wherein, preferably, said Mg²⁺The concentration of (B) is 3 to 7 mM.

The invention has the following beneficial effects:

the present invention has been studied to find that:

(1) the purple acid phosphatase SgPAP7 of Stylosanthes guianensis most hydrolyzes organophosphorus substrates.

(2) The expression of the stylosanthes guianensis purple acid phosphatase SgPAP7 gene in a plant body promotes the plant to prepare absorbable phosphorus by utilizing exogenous phosphorus, promotes the plant growth, and solves the problem of low phosphorus stress of the plant, particularly the low phosphorus stress problem of an arabidopsis plant.

(3) The stylosanthes guianensis purple acid phosphatase SgPAP7 gene is expressed in a plant body to promote the growth of plants by utilizing endogenous phosphorus, so that the problem of low phosphorus/phosphorus-free stress of the plants is solved, in particular to the low phosphorus/phosphorus-free stress of arabidopsis thaliana plants.

Based on the research result, the stylosanthes guianensis purple acid phosphatase SgPAP7 and the coding gene thereof have important application values in the aspects of promoting the plant to utilize endogenous phosphorus and constructing transgenic plants resistant to low-phosphorus/phosphorus-free environments. Overcomes the technical prejudice that the plant body can not promote the growth of the plant by utilizing endogenous organophosphorus even if the plant body expresses SgPAP7 under the low-phosphorus/non-phosphorus environment condition, and has important value for solving the problem of utilizing the plant phosphorus, especially for planting the plant in the non-phosphorus/low-phosphorus environment.

Drawings

FIG. 1 shows that low phosphorus stress inhibits growth of aerial parts of Stylosanthes guianensis, where A is the plant phenotype and B is the plant dry weight.

Figure 2 shows that SgPAP7 gene is significantly up-regulated expressed in the low phosphorus stress stylosanthes guianensis root system.

FIG. 3 shows the SDS electrophoretogram (A) of Stylosanthes guianensis SgPAP7 protein, the relative enzyme activity-pH curve (B) and the relative enzyme activity-temperature curve (C) of Stylosanthes guianensis SgPAP7 protein.

Fig. 4 shows the utilization of endogenous ADP by arabidopsis Wild Type (WT) and arabidopsis transgenic lines (OE1, OE2 and OE 3). Wherein A shows the relative expression level of SgPAP7 in the upper part of Arabidopsis; b shows the activity of the endogenous acid phosphatase in the aerial part (taking ADP as a substrate); c shows the above-ground ADP concentration.

FIG. 5 shows the tolerance of Arabidopsis Wild Type (WT) and Arabidopsis transgenic lines (OE1, OE2 and OE3) to low phosphorus stress using exogenous (environmental) ADP. Wherein A shows the appearance of Arabidopsis, B shows the dry weight and C shows the total phosphorus content.

FIG. 6 shows growth conditions of Arabidopsis thaliana under high and low phosphorus conditions, respectively, wherein A shows appearance state of Arabidopsis thaliana, B shows overground part dry weight of Arabidopsis thaliana, and C shows overground part phosphorus content.

Graph indicates significant level of comparative difference between groups: p <0.05, x: p <0.01, x: p < 0.001.

Detailed Description

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

Example 1: response of Stylosanthes guianensis SgPAP7 expression levels to Low phosphorus stress

1.1 plant cultivation

The gumbola plant TF0277 is used as a material, and gumbola plant seeds are provided by the grass research institute of tropical crop variety resources of the institute of tropical agriculture academy of sciences in China. Taking a proper amount of plump stylosanthes guianensis seeds, sterilizing the surfaces of the seeds, and putting the seeds without KH₂PO₄1/2MS (Murashige)&Skoog) solid medium, selecting well-growing and uniform stylosanthes guianensis seedlings, transferring the seedlings to a normal phosphorus supply (containing 250 mu M KH) after 3d germination₂PO₄) 1/2Hoagland nutrient solution is pre-cultured for 10 days, and then transferred to a new 1/2Hoagland nutrient solution to be treated with different phosphorus concentrations, including normal phosphorus supply treatment (HP, added with 250. mu.M KH)₂PO₄) And low phosphorous treatment (LP, 5. mu.M KH added₂PO₄) After the treatment culture for 18d, the biomass was measured. After the roots of the harvested stylosanthes guianensis are quickly frozen in liquid nitrogen, the stylosanthes guianensis are stored in a refrigerator at the temperature of-80 ℃ for subsequent analysis.

As a result: the low phosphorus stress severely inhibited the growth of the aerial parts of stylosanthes guianensis, significantly reduced its biomass, which was reduced by 30.39% under low phosphorus treatment (LP) compared to normal phosphorus supply (HP) (fig. 1A, 1B).

1.2 SgPAP7 Gene expression quantification

First, RNA of Stylosanthes guianensis TF0277 was extracted by the TRIzol (Invitrogen Inc, USA) method, and double-stranded cDNA was synthesized by reverse transcription using the HiScript II 1st Strand cDNA Synthesis Kit (+ gDNA wiper) Kit from Vazyme. Then, qRT-PCR primer pairs (SgPAP7-RT-F/R) of SgPAP7(NCBI accession number: KU315544) were designed using Oligo7 software, and specific sequence information is shown in Table 1. Meanwhile, SgEF-1 alpha (NCBI accession number: JX164254) is used as an internal reference gene, wherein the quantitative primer pair of the SgEF-1 alpha is SgEF-1 alpha-F and SgEF-1 alpha-R (table 1). Finally, real-time fluorescent quantitative analysis of SgPAP7 Gene was performed using the SYBR qPCR Master Mix (Vazyme, China) quantitative kit reference protocol using Rotor-Gene Q (Hilden, Germany) instrument platform.

TABLE 1 primer sequences

As a result: the SgPAP7 gene was significantly up-regulated in the low phosphorus stress stylosanthes guianensis root system (fig. 2).

Example 2: enzymological characteristics of Stylosanthes guianensis SgPAP7

The Open Reading Frame (ORF) sequence of SgPAP7 gene was amplified using a primer pair (SgPAP7-GST-F/R, Table 1), and the obtained sequence was cloned into pGEX6P-3 vector (GE Healthcare, USA), followed by transformation of E.coli strain BL21 for heterologous expression. The SgPAP7 protein expressed in Escherichia coli BL21 has a fusion protein GST (glutaminone S-transferase) tag at the N-terminus. Then, the protein SgPAP7 is purified by GST tag and glutathione agarose gel electrophoresis.

A100. mu.l reaction system was prepared, containing 45mM buffers of different pH, 5mM MgCl₂5mM of the different substrates, and then adding about 100ng of the fusion protein, incubating at 37 ℃ for 30min for reaction, using a malachite green molybdate reagent, detecting inorganic phosphate Pi released from the reaction system at 650nm by spectrophotometry, and expressing the activity of the GST: SgPAP7 protein as micromoles Pi released per minute per mg of protein, each reaction comprising 3 repeats.

(1) The optimum substrate of SgPAP7 protein is screened, 10 substrates of 4-nitrophenyl disodium phosphate (rho-NPP), Adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), Adenosine Monophosphate (AMP), Guanosine Monophosphate (GMP), pyrophosphate, phosphoserine, phosphothreonine and glucose-6-phosphate are respectively utilized for reaction, Na-acetic acid (pH 5.0) is used as a buffer solution, the reaction temperature is 37 ℃, the hydrolytic activity of the GST: SgPAP7 protein on different substrates is respectively detected, and the relative enzyme activity of each substrate is calculated by taking the enzyme activity of the hydrolytic substrate rho-NPP as a reference (100%).

(2) The SgPAP7 protein was screened for optimal hydrolysis conditions.

(2.1) screening the optimum reaction pH of the GST SgPAP7 protein, applying 4 different buffers including Gly/HCl (pH 3.0-4.5), Na-acetate (pH 5.0-5.5), Tris/HCl/MES (pH 6.0-7.0) or Tris/HCl (pH 7.5-9.0), setting 10 different pHs, namely 3, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 and 9, hydrolyzing a substrate to be rho-NPP, detecting the enzyme activities under the different pHs respectively at the reaction temperature of 37 ℃, and calculating the relative enzyme activities under each pH by taking the highest enzyme activity as a reference (100%).

(2.2) screening the optimum reaction temperature of GST: SgPAP7 protein, setting the system reaction temperature to be 20-70 ℃, including 7 temperature gradients, namely 20 ℃,30 ℃, 37 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃, hydrolyzing a substrate to be rho-NPP, and a buffer solution to be Na-acetic acid (pH 5.0), respectively determining the enzyme activities of GST: SgPAP7 protein at different temperatures, and calculating the relative enzyme activity at each reaction temperature by taking the highest one of the enzyme activities as a reference (100%).

As a result: as shown in the SDS protein electrophoresis (FIG. 3A), the recombinant protein GST: SgPAP7, which was successfully purified by E.coli induced expression, had a molecular weight of 63 kD. Using 9 different organophosphates as hydrolysis substrates, SgPAP7 protein was found to have a certain hydrolytic activity on 9 substrates, indicating that the hydrolysis substrate of SgPAP7 protein has a broad spectrum, with SgPAP7 protein having the highest relative activities for hydrolysis of ADP and ATP, 155.8% and 131.6%, respectively, and relatively low relative activities for hydrolysis of other substrates (below p-NPP), particularly phosphotyrosine and GMP, both of which are below 10% (table 2). Secondly, using p-NPP as the hydrolysis substrate, the optimum pH and temperature for proteolytic activity of SgPAP7 were 6.0 and 40 ℃ (fig. 3B and fig. 3C), respectively.

TABLE 2 SgPAP7 protein substrate specificity analysis

Example 3: SgPAP7 transgenic Arabidopsis construction

Arabidopsis thaliana expressing the Stylosanthes SgPAP7 gene was created. The ORF sequence of the SgPAP7 gene was amplified using a primer set (SgPAP 7-OE-F/R; Table 1), and the obtained sequence was cloned into the vector pTF101.1 using a homologous cloning method and then transformed into the Pseudomonas strain EHA 105. The method for transforming arabidopsis thaliana by adopting the flower invasion method comprises the following specific steps:

culturing the transformed agrobacterium continuously at 28 deg.c and 200rpm to culture agrobacterium OD₆₀₀To 0.8.

② subpackaging the agrobacterium into centrifuge tubes, centrifuging at 6000rpm for 15min, pouring out the upper liquid, leaving the agrobacterium precipitate, finally adding the suspension for suspension precipitation.

Thirdly, cutting off pods of the arabidopsis thaliana, only reserving inflorescences, soaking the inflorescences in the suspension for 2min, taking out the inflorescences, sealing the inflorescences with a preservative film, and finally carrying out dark treatment for 24 h.

And fourthly, after 24 hours, normally growing and culturing the arabidopsis, infecting the arabidopsis for the second time after 7 days, and after the arabidopsis is mature, harvesting seeds of T0 generations.

Screening positive plants by using herbicide Basta. qRT-PCR verification of transgenic Arabidopsis positive plants expressing the SgPAP7 gene preliminarily screened by Basta is utilized, a primer pair (SgPAP7-OE-F/R, table 1) is utilized to analyze the relative expression quantity of SgPAP7 in transgenic Arabidopsis (OE) and wild type Arabidopsis (Col-0, WT), an AtEF-1 alpha (Elongation factor 1-alpha) gene is used as an internal reference, and a real-time fluorescence quantitative primer pair (AtEF-1 alpha-F/R) is shown in table 1.

As a result: the constructed expression vector PTF101s-SgPAP7 is used for transforming Arabidopsis thaliana to obtain a positive transgenic Arabidopsis thaliana plant. qRT-PCR analysis showed (FIG. 4A) that the relative expression level of SgPAP7 gene in Arabidopsis transgenic line (OE-1/2/3) was significantly higher than that of wild type Arabidopsis (WT).

Example 4: analysis of endogenous ADP utilization by SgPAP7 transgenic Arabidopsis

Taking appropriate amount of wild type Arabidopsis thaliana (WT) and three lines of homozygous T3 generation transgenic Arabidopsis thaliana (OE-1, OE-2, OE-3) seeds, sterilizing the surface, and spreading on a medium containing 500 μ M KH₂PO₄And performing germination acceleration on 1/2MS solid medium. After the arabidopsis thaliana sprouts and grows for 7 days, uniform seedlings are selected and transplanted into a non-nutrient peat soil matrix (Jiffy, the Netherlands), and KH is added in a soil-mixing manner₂PO₄The phosphorus content was 160mg P/kg of non-nutritive soil, and the Arabidopsis seedlings were watered with MS liquid culture medium containing no phosphorus every three days, and the experiment contained 4 biological replicates. After the seedlings were cultured for 21d (3 weeks), the overground parts were harvested, respectively, and analyzed for the overground part APase activity (using ADP as a hydrolysis substrate), and the overground part ADP concentration was absolutely quantified by High Performance Liquid Chromatography (HPLC).

Wherein, the determination of the APase activity of the overground part of Arabidopsis thaliana: taking 0.1-0.2 g of fresh plant sample, adding 1.2mL of Tris-HCl buffer (0.1M, pH 6.8), fully homogenizing, centrifuging (4 ℃,14,000rpm,30min), and collecting supernatant as sample solution to be detected. Taking a proper amount of sample solution to be tested, adding 0.3mL of 45mM acetic acid-sodium acetate buffer solution with pH of 5.0, adding 1.5mL of 1mM ADP substrate reaction solution (prepared by 45mM acetic acid-sodium acetate buffer solution with pH of 5.0), mixing well, and incubating at 37 deg.C15min, adding 0.2mL of 2M NaOH solution to stop the reaction, mixing uniformly, centrifuging at 12,000rpm for 2min, collecting supernatant as sample solution to be tested, and determining OD₄₀₅An absorbance value.

Detecting yellow p-nitrophenol (characteristic absorption OD) in the sample solution to be detected₄₀₅) To evaluate the APase activity when ADP is used as a substrate. Sampling the sample to be tested, and measuring OD by using ultraviolet spectrophotometer₄₀₅The absorbance value of (a). Preparation of a standard curve: 6 pieces of 2mL centrifuge tubes were charged with 45mM of 1mM ADP standard solution 0, 0.1, 0.2, 0.3, 0.4, and 0.5mL in acetic acid-sodium acetate buffer solution (pH 5.0), and then the volume was adjusted to 1.8mL with acetic acid-sodium acetate buffer solution (45mM, pH 5.0), 0.2mL of 2M NaOH solution was added thereto, the mixture was mixed, and the mixture was left to stand for 2min to measure OD₄₀₅An absorbance value. Finally, the standard curve is used to determine the absolute OD₄₀₅The APase activity was calculated as absorbance values and expressed as the hydrolysis of ADP (. mu.mol) per unit protein (mg protein) per unit time (min), while 1. mu. mol of ADP hydrolyzed per unit time (min) was defined as 1U.

Protein content determination: taking appropriate amount of the above sample solution to be tested, adding double distilled water to make up 0.2mL, adding 1.8mL Coomassie brilliant blue G250 solution, mixing, standing for 5min, and measuring OD with spectrophotometer₅₉₅The absorbance value of (a). Preparation of a standard curve: firstly, 0.25 mug/muL of bovine serum albumin standard solution is prepared, 0, 40, 80, 120, 160 and 200 muL of bovine serum albumin standard solution are respectively added into 6 centrifuge tubes, 0.2mL of double distilled water is used for supplementing, 1.8mL of Coomassie brilliant blue G250 solution is added, the mixture is mixed evenly and stands for 5min, and OD is measured₅₉₅An absorbance value. Finally, the absorbance value OD is determined by using the standard curve₅₉₅And calculating the protein content.

As a result: qRT-PCR analysis showed that the relative expression level of SgPAP7 gene in Arabidopsis transgenic line (OE-1/2/3) was significantly higher than that of wild type Arabidopsis (WT) (FIG. 4A). Furthermore, overexpression of SgPAP7(OE-1/2/3) increased the acid phosphatase activity (APase, using ADP as a substrate) of transgenic Arabidopsis thaliana by 151.0% to 192.6% as compared with WT (FIG. 4B); but the ADP concentration decreased by 11.1% to 15.7% (fig. 4C). The above results indicate that overexpression of the stylosanthes guianensis SgPAP7 gene increases the APase activity of Arabidopsis thaliana and enhances the utilization of endogenous ADP.

Example 5: analysis of exogenous ADP utilization by SgPAP7 transgenic Arabidopsis

Taking a proper amount of filled seeds of wild arabidopsis (WT) and three lines of homozygous T3 generation transgenic arabidopsis (OE-1, OE-2 and OE-3), carrying out surface disinfection by using 75% ethanol and 10% NaClO, flatly paving the seeds on a 1/2MS solid culture medium, germinating at 22 ℃, selecting healthy and uniformly grown seedlings to be transferred to a 1/2MS solid culture medium after the radicles grow to about 1cm, and adding 150 mu M ADP (+ ADP) as a unique phosphorus source. The experiment contained 4 biological replicates. After Arabidopsis thaliana was cultured for 18 days, phenotype was recorded and plant dry weight and total phosphorus content were measured.

As a result: as shown in FIG. 5A, overexpression of SgPAP7 resulted in significantly better growth of transgenic Arabidopsis (OE1/2/3) than wild-type (WT) when ADP was the sole source of phosphorus; meanwhile, the dry weight ratio of the transgenic arabidopsis thaliana is increased by 38.3-45.1% compared with WT (figure 5B); the total phosphorus content is improved by 73.1.3-81.3 percent compared with WT (figure 5C). The results show that the overexpression of the stylosanthes guianensis SgPAP7 gene improves the utilization capacity of arabidopsis thaliana to exogenous ADP.

Example 6 adaptability of SgPAP7 transgenic Arabidopsis to Low phosphorus stress

To evaluate the involvement of the SgPAP7 gene of Stylosanthes guianensis in the adaptation of Arabidopsis thaliana to low phosphorus stress, the transgenic Arabidopsis thaliana (OE-1/2/3) overexpressing SgPAP7 and the wild type Arabidopsis thaliana (WT) were subjected to High phosphorus (High Pi: KH) respectively, with reference to the soil culture method of "example 4"₂PO₄Soil mix, 160mg P/kg non-nutrient soil) and Low phosphorus (Low Pi: KH (Perkin Elmer)₂PO₄Soil mixing, 20mg P/kg non-nutrient soil) and culturing at 22 deg.C in a greenhouse. The Arabidopsis seedlings were watered with MS liquid culture medium containing no phosphorus every three days. Different treatments comprised 4 biological replicates and after 21d (3 weeks) of treatment culture, samples of different treated Arabidopsis were harvested and the dry weight of the aerial parts and the total phosphorus content were determined separately.

As a result, it was found that: no significant difference in overground growth phenotype, overground dry weight and overground phosphorus content between WT and transgenic Arabidopsis (OE1/2/3) under High Pi treatment; however, overexpression of SgPAP7 promoted overground growth of transgenic Arabidopsis (OE-1/2/3) under Low Pi treatment (FIG. 6A); meanwhile, the dry weight of the overground part of the transgenic arabidopsis (OE-1/2/3) is improved by 36.3-61.1 percent compared with that of the WT (figure 6B), and the phosphorus content of the overground part is improved by 40.8-62.6 percent compared with that of the WT (figure 6C). The above results suggest that overexpression of the SgPAP7 gene contributes to improvement of the adaptability of Arabidopsis to low phosphorus stress.

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