CN111066593A

CN111066593A - Method for researching functions of acid phosphatase in symbiosis of alfalfa rhizobium and mycorrhiza

Info

Publication number: CN111066593A
Application number: CN202010054520.6A
Authority: CN
Inventors: 李友国; 林会; 陈大松; 崔智强; 谢丽萍; 黄诗宸
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2020-04-28
Anticipated expiration: 2040-01-17
Also published as: CN111066593B

Abstract

The invention discloses a method for researching functions of acid phosphatase in symbiosis of alfalfa root nodule and mycorrhiza in the field of leguminous plant research, which comprises the following steps: 1) selecting alfalfa seeds of tribulus, and carrying out surface sterilization, pregermination and germination; 2) respectively carrying out pot culture tests on alfalfa sinorhizobium meliloti inoculated to the medicago truncatula and alfalfa cladosporium branchoides inoculated to the medicago truncatula under the conditions of different phosphorus concentrations; 3) detecting the tissue-specific expression of MtPA 2 in alfalfa root nodule and the tissue-specific expression of MtPA 2 in alfalfa mycorrhiza, the influence of phosphorus on the expression of MtPA 2 gene, the influence of phosphorus on alfalfa root nodule, and the influence of phosphorus on alfalfa mycorrhiza; 4) the role of alfalfa, rhizobia and mycorrhizal fungi in the symbiotic process is researched by utilizing the methods of space-time expression, promoter tissue positioning, overexpression and silencing. By obtaining direct experimental evidence, new insights are provided for plant response to low-phosphorus stress and the molecular mechanism of coordinating the self phosphorus homeostasis of the organism.

Description

Method for researching functions of acid phosphatase in symbiosis of alfalfa rhizobium and mycorrhiza

Technical Field

The invention relates to the technical field of leguminous plant research, in particular to a method for researching functions of acid phosphatase in alfalfa root nodule and mycorrhiza symbiosis.

Background

The symbiotic system formed by the leguminous plants and rhizobia or AM fungi has important significance for agricultural sustainable development. The biological nitrogen fixation process of rhizobia and leguminous plants mainly provides a nitrogen source required for the growth of the plants, and AM fungus can improve the phosphorus homeostasis in the plants; meanwhile, nitrogen and phosphorus are two major elements necessary for the growth and development of plants, and the deficient phosphorus content in the soil not only influences the growth of leguminous crops, but also has adverse effect on symbiotic nitrogen fixation of leguminous plants.

At present, researches on PAP family genes of leguminous plants mostly focus on assisting plants to cope with phosphorus stress and improving phosphorus nutrition of plants, but few reports exist about the functions of proteins of the family in the symbiotic process of plants and microorganisms. Purple Acid Phosphatase (PAP) is the most important type of acid phosphatase, and plays an important role in activating organic phosphorus around plant rhizosphere and promoting decomposition and recycling of organic phosphorus in plant tissues. The research selects MtPA 2 with high expression in rhizobium and mycorrhiza and MtPA 3 with low phosphorus and rhizobium induced expression as research objects, researches the function and action mechanism of the PAP protein in the symbiosis of leguminous plants and microorganisms, and expects to provide basis for clarifying the regulation mechanism of the PAP protein in the growth and development of plants; meanwhile, the difference of the expression modes of MtPA 2 not induced by phosphorus and MtPA 3 induced by low-phosphorus stress indicates that the MtPA 2 and the MtPA 3 have different functions in the plant phosphorus metabolism process, the research on the expression and the function of MtPA 2 and MtPA 3 in the alfalfa nodulation process can be more helpful to understand the interaction between PAP family members in plants, and a new opinion and a new insight are provided for the plant to respond to the low-phosphorus stress and coordinate the molecular mechanism of the body for self-phosphorus homeostasis.

MtPA 2 transcription levels in roots or nodules were significantly increased after inoculation with Rhizobium or AM fungi (Rhizophagusgurrriregularis); in addition, the transcription level of MtPA 2 in the nitrogen fixation region of the nodule is obviously higher than that of other parts of the nodule by combining the transcription group data applied to the symbiosis of plants, rhizobia and AM fungi by a laser microdissection technology; meanwhile, the transcription level of the gene is highest in cortex cells containing the branches; these transcriptome data suggest that MtPA 2 plays an important role in the symbiosis of Medicago truncatula with Rhizobium and AM fungi, i.e., MtPA 2 gene was the final subject.

Based on this, the present invention has devised a functional study method of acid phosphatase in alfalfa rhizobium and mycorrhiza symbiosis to solve the above-mentioned problems.

Disclosure of Invention

The present invention is directed to provide a method for functional study of acid phosphatase in symbiosis of alfalfa rhizobium and mycorrhiza, so as to solve the problems mentioned in the background art.

In order to achieve the purpose, the invention provides the following technical scheme: a method for researching the function of acid phosphatase in alfalfa root nodule and mycorrhiza symbiosis, wherein the research method comprises the following steps:

1) selecting alfalfa seeds of tribulus, and carrying out surface sterilization, pregermination and germination;

2) respectively carrying out pot culture tests on alfalfa sinorhizobium meliloti inoculated to the medicago truncatula and alfalfa cladosporium branchoides inoculated to the medicago truncatula under the conditions of different phosphorus concentrations;

3) detecting the tissue-specific expression of MtPA 2 in alfalfa root nodule and the tissue-specific expression of MtPA 2 in alfalfa mycorrhiza, the influence of phosphorus on the expression of MtPA 2 gene, the influence of phosphorus on alfalfa root nodule, and the influence of phosphorus on alfalfa mycorrhiza;

4) the role of alfalfa, rhizobia and mycorrhizal fungi in the symbiotic process is researched by utilizing the methods of space-time expression, promoter tissue positioning, overexpression and silencing.

Preferably, the specific steps of step 1) are as follows:

(1) selecting seeds of medicago truncatula A17 with uniform size;

(2) a surgical blade or a syringe needle is used for lightly scratching the surface of the seed, and the naked eye can see a slight scratch;

(3) inverting the scratched seeds into a 100mL sterile triangular flask, adding sodium hypochlorite into the triangular flask according to the proportion of 2% (V/V), and slightly shaking for 3-5min until the surface of the seed coat is brown and scratched;

(4) discarding the waste liquid, washing with sterile water for 7-10 times until the liquid in the triangular flask turns to colorless from light yellow, adding a small amount of sterile water to submerge the seeds, and standing at normal temperature for 3h until the seeds absorb water to be full;

(5) discarding the waste liquid, washing with sterile water for 5 times, spreading the seeds on water agar, and finally placing in the dark at 4 ℃ for 48 h;

(6) inverting the water agar plate containing seeds, placing in a constant temperature incubator at 28 deg.C, and dark treating for 10-12 hr until the seeds sprout to give 1cm long radicle, which can be used for potted plant and alfalfa hair root transformation.

Preferably, in the step 2), the alfalfa inoculated sinorhizobium meliloti pot culture test specifically comprises the following steps:

(1) taking a proper amount of river sand, washing the river sand clean by tap water, filling the river sand into a can bottle, pouring a proper amount of nitrogen-free nutrient solution (NFS) into one half of the bottle, pouring a proper amount of low-phosphorus (5 mu mol/LKH2PO4) nitrogen-free nutrient solution (LP-NFS) into the other half of the bottle, sealing and tightly wrapping kraft paper, and sterilizing for 1h at 121 ℃;

(2) transplanting strong and consistent alfalfa seedlings into can bottles, planting 4 seedlings in each bottle, covering a layer of preservative film after the seedlings are planted in each bottle, reducing water evaporation, and placing the bottles in an illumination room;

(3) after the first true leaf of the seedling grows out (about 6-7 days), inoculating 1mLOD 600-0.5-1.0 Sinorhizobium meliloti1021 bacterial liquid to each seedling;

(4) replacing the nitrogen-free nutrient solution and the low-phosphorus nitrogen-free nutrient solution in the bottle every 3 days respectively;

(5) after 102121 days of inoculation with Sinorhizobium meliloti, root sample and nodule sample are collected, marked, and stored at-80 deg.c for gene expression analysis.

Preferably, in the step 2), the alfalfa inoculation arbuscular mycorrhizal fungi pot culture test of the caltrop comprises the following specific steps:

(1) cleaning appropriate amount of river sand, packaging into cloth bag, and oven drying; simultaneously, hammering soil collected from a test field into pieces, sieving, uniformly mixing with river sand according to the proportion of 1:3(V/V) to form a sand-soil mixture matrix, filling the sand-soil mixture matrix into a cloth bag, sterilizing for 1h at 121 ℃, and intermittently sterilizing for 3 times; washing a pot with the diameter of 10cm with tap water, sterilizing at 121 deg.C for 1h, and intermittently sterilizing for 3 times;

(2) transplanting the strong and consistent alfalfa seedlings into a pot filled with sterile sand, simultaneously pouring 1/2Hoagland nutrient solution containing 0.2mmol/LKH2PO4, covering a layer of preservative film after finishing the pouring, reducing water evaporation, and placing the container in an illumination chamber;

(3) after the first true leaf of the seedling grows out, weighing a proper amount of the AM fungus Rhizopharmarsiregularis inoculant according to the inoculation amount of 10% (V/V) and uniformly mixing the inoculant with the sandy soil mixture matrix according to the corresponding proportion; as a control, the other group was not inoculated with AM fungal inoculant; respectively placing the two groups of matrixes into pots with equal quantity, transferring seedlings into the pots, planting 5 seedlings in each pot, simultaneously pouring 1/2Hoagland nutrient solution containing 0.2mmol/LKH2PO4, covering a preservative film after finishing the planting, reducing water evaporation, and placing the pots in an illumination chamber;

4) watering 1/2Hoagland low-phosphorus nutrient solution containing 20 mu mol/LKH2PO4 in the first two weeks, equally dividing three groups from the third week, watering 1/2Hoagland low-phosphorus nutrient solution containing 20 mu mol/LKH2PO4 in one group, watering 1/2Hoagland medium-phosphorus nutrient solution containing 0.2mmol/LKH2PO4 in the second group, watering 1/2Hoagland high-phosphorus nutrient solution containing 1mmol/LKH2PO4 in the third group, and watering sterile deionized water in the rest time to ensure the water required by the normal growth of the plants;

5) after two weeks of treatment at different phosphorus concentrations, root samples were harvested, labeled, stored at-80 ℃ and used for gene expression analysis.

Preferably, the method for researching the influence of phosphorus on the expression of the MtPA 2 gene comprises the following steps: collecting plant root samples treated by nutrient solutions of Hoagland (1mmol/LPi) and LP-Hoagland (5 mu mol/LPi) for 21 days, extracting RNA, carrying out transcription level analysis, and taking a phosphorus transport gene MtPT1 induced and expressed in low-phosphorus stress roots as a control; the method for researching the influence of phosphorus on alfalfa root nodules comprises the following steps: collecting roots and nodules 21 days after NFS (containing 1mmol/LPi) and LP-NFS (containing 5 mu mol/LPi), extracting RNA, and performing reverse transcription to cDNA for performing transcript level analysis; the method for researching the influence of phosphorus on the alfalfa mycorrhiza comprises the following steps: after inoculation with the AM fungus, plants were irrigated with 1/2LP (20. mu. mol/LPi) Hoagland nutrient solution for 2 weeks, then with 1/2HP (1mmol/LPi), 1/2MP (0.2mmol/LPi) and 1/2LP (20. mu. mol/LPi) Hoagland nutrient solution for 2 weeks, and finally the collected root samples were divided into two portions, one for TB staining, the other for RNA extraction and reverse transcription into cDNA for transcript level analysis.

Preferably, the spatiotemporal expression comprises the spatiotemporal expression of MtPA 2 in alfalfa rhizobium, the spatiotemporal expression of MtPA 2 in alfalfa mycorrhiza and the spatiotemporal expression of MtPA 2 in mycorrhiza symbiosis,

the specific implementation steps of the spatio-temporal expression of the MtPA 2 in the alfalfa nodule are as follows: collecting plant materials of 9 days, 15 days, 21 days and 28 days after the inoculation of the sinorhizobium meliloti s.meliloti1021, extracting RNA, carrying out reverse transcription to cDNA for RT-qPCR analysis, and taking a nodulin gene MtN5 stably expressed in the rhizobium as a positive control;

the specific implementation steps of the spatiotemporal expression of the MtPA 2 in the alfalfa mycorrhiza are as follows: collecting plant materials of 9 days, 15 days, 21 days and 28 days after the inoculation of the AM fungus R.irregularis, dividing the collected root sample into two parts, using one part for TB staining, statistically analyzing symbiotic level data of the AM fungus, extracting RNA from the other part, performing transcription level analysis, and using a phosphorus transport gene MtPT4 participating in the formation of the arbuscular branches as a control;

the specific implementation steps of the spatio-temporal expression of the MtPA 2 in mycorrhizal symbiosis are as follows: transferring the obtained positive plants into sterile sandy soil, inoculating AM fungi after 3-5 days of phosphorus starvation treatment, and collecting root samples after 14 days and 28 days of inoculation respectively.

Preferably, the construction of the promoter tissue localization vector is used for researching the spatial and temporal expression localization of the MtPA 2 promoter in the symbiosis of the alfalfa root nodule and mycorrhiza, and comprises the following steps: the promoter sequence of MtPA 2 is fused to the N end of GUS reporter gene, and the correct alignment of colony PCR, enzyme digestion and sequencing shows that the MtPA 2 promoter tissue expression positioning vector is successfully constructed.

Preferably, the alfalfa nodule research method of the overexpression pair of the MtPA 2 gene comprises the steps of construction of an MtPA 2 gene overexpression vector, detection of the transcription level of MtPA 2 overexpression in alfalfa nodule and influence of MtPA 2 gene overexpression on nodule symbiosis, and the research method of MtAP 2 gene silencing comprises the steps of construction of an MtPA 2 gene silencing vector, detection of the transcription level of MtAP 2 silencing in nodule and influence of MtAP 2 gene silencing on nodule symbiosis.

Preferably, the alfalfa mycorrhiza research method of the MtPA 2 gene overexpression pair comprises the steps of construction of an MtPA 2 gene overexpression vector, detection of the transcription level of MtPA 2 overexpression in alfalfa mycorrhiza and influence of MtAP 2 gene overexpression on alfalfa mycorrhiza symbiosis, and the MtAP 2 gene silencing research method comprises the steps of construction of an MtAP 2 gene silencing vector, detection of the transcription level in alfalfa mycorrhiza after MtAP 2 silencing and influence of MtAP 2 gene silencing on alfalfa mycorrhiza symbiosis.

Compared with the prior art, the invention has the beneficial effects that:

1. MtPA 2 has expression in the rhizome and leaf of alfalfa, and the expression level in leaf is the highest; after inoculation of rhizobium or AM fungi, the expression level of the gene in rhizobium or mycorrhiza is obviously improved. The phosphorus induction results show that the expression of MtPA 2 in roots is not induced by phosphorus stress, indicating that the protein is not involved in the response of plants to low phosphorus stress.

2. Spatio-temporal gene expression and promoter tissue expression localization showed that mtpp 2 is expressed under induction by rhizobia and AM fungi and is expressed predominantly in meristematic regions, invasive regions, vascular bundle tissue and mycorrhiza containing arbuscular branches and adjacent cortical cells of the nodule.

3. The overexpression of the MtPA 2 gene and the symbiotic phenotype of silent plants show that the overexpression of the MtPA 2 can obviously improve the root nodule number and the azotase activity, and increase the infection rate of mycorrhiza and the arbuscular abundance; silencing of the MtPA 2 gene can obviously inhibit the development of the root nodule and the activity of azotase, the formation and development of arbuscular branches in mycorrhiza are also influenced, and the abundance of the arbuscular branches is obviously reduced, so that the protein has an important role in the symbiosis of alfalfa, the root nodule and mycorrhizal fungi, and the protein can regulate and control the symbiosis of plants and microorganisms by regulating the metabolism of phosphorus in root tissues and the balanced distribution in the tissues.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph of the transcript levels of MtN5 and MtPA 2 in the roots and nodules of a non-inoculated and inoculated Rhizobium S.meliloti1021 plant of the present invention;

FIG. 2 is a structural diagram of the AM fungus R.irregularis according to the invention in different stages of symbiosis;

FIG. 3 is a graph of symbiotic levels of the AM fungus of the invention 9, 15, 21 and 28 days after inoculation;

FIG. 4 is a graph of the transcriptional levels of MtPT4 and MtPA 2 of the present invention in the roots of inoculated and uninoculated AM fungus R.irregularis plants;

FIG. 5 is a graph of the transcriptional levels of MtPT1 and MtPA 2 in roots under low phosphorus treatment in accordance with the present invention;

FIG. 6 is a graph of the transcription levels of MtPA 2 in roots and nodules under low phosphorus treatment in accordance with the present invention;

FIG. 7 is a graph showing the effect of different phosphorus concentration treatments of the present invention on the growth of Medicago truncatula;

FIG. 8 is a micrograph of alfalfa root treated with different phosphorus concentrations according to the present invention after TB dyeing;

FIG. 9 is a graph of the transcript levels of MtPA 2 of the present invention in roots of both inoculated and uninoculated AM fungus R.irregularis plants under conditions of varying phosphorus concentrations;

FIG. 10 is a schematic diagram of the MtPA 2-Pro: GUS vector of the present invention;

FIG. 11 is a diagram showing the construction of the tissue expression localization vector of the MtPA 2 promoter according to the present invention;

FIG. 12 is a diagram of spatiotemporal expression of MtPA 2 in mycorrhizal symbiosis according to the present invention;

FIG. 13 is a schematic diagram of the MMtPA 2-Overexpression vector of the present invention;

FIG. 14 is a diagram of the construction of the overexpression vector of MtPA 2 according to the invention;

FIG. 15 is a graph of the transcription levels of MtPA 2 of the present invention in the hairy roots and nodules of over-expressed plants;

FIG. 16 is a representative of the nodulation of the overexpression plant of MtPA 2 of the present invention;

FIG. 17 is a graph depicting the determination of the symbiotic phenotype of MtPA 2 overexpressing and control plants according to the invention;

FIG. 18 is a schematic diagram of the MtPA 2-RNAi vector of the present invention;

FIG. 19 is a diagram of the construction of the MtPA 2-RNAi interference vector of the present invention;

fig. 20 is a graph of transcriptional levels in nodules of a silenced plant of mtpp 2 of the present invention after rooting and inoculation with s.meliloti 1021;

FIG. 21 is a representative of the nodulation of the MtPA 2-RNAi plant of the present invention;

FIG. 22 is a graph depicting determination of symbiotic phenotype of MtPA 2 silenced and control plants according to the present invention;

FIG. 23 is a paraffin section view of nodules of plants overexpressing and silencing MtPA 2 according to the invention;

FIG. 24 is a graph of transcription levels in rooting and mycorrhizal colonizing root samples of the overexpressing plant MtPA 2 of the invention;

FIG. 25 is a symbiotic phenotype diagram of the overexpression plant of MtPA 2 of the present invention and AM fungus;

FIG. 26 is a micrograph of a TB-stained root segment of an overexpressed plant of the invention from MtPA 2;

FIG. 27 is a statistical plot of the root segment symbiosis levels of the MtPA 2-OE plants of the present invention;

FIG. 28 is a graph of transcription levels in roots of a silencing plant of MtPA 2 of the present invention and in roots after inoculation with AM fungi;

FIG. 29 is a phenotype of the symbiosis of the MtPA 2-RNAi interfering plant and AM fungus;

FIG. 30 is a micrograph of a TB-stained root section of an MtPA 2RNAi interfering plant of the present invention;

FIG. 31 is a statistical chart of the symbiotic level of the root segments of the plants interfered by MtPA 2-RNAi.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Examples

The invention provides a technical scheme that: a method for researching the function of acid phosphatase in alfalfa root nodule and mycorrhiza symbiosis, wherein the research method comprises the following steps:

4) the role of alfalfa, rhizobia and mycorrhizal fungi in the symbiotic process is researched by utilizing the methods of space-time expression, chemical tissue localization, overexpression and silencing.

In this embodiment, the specific steps of step 1) are as follows:

(1) selecting seeds of medicago truncatula A17 with uniform size;

(6) inverting the water agar plate containing seeds, placing in a constant temperature incubator at 28 deg.C, and dark treating for 10-12 hr until the seeds sprout to give 1cm long radicle, which can be used for potted plant and alfalfa hair root transformation. In the embodiment, in the step 2), the alfalfa inoculated sinorhizobium meliloti pot culture test specifically comprises the following steps:

Under the condition of different phosphorus concentrations, the alfalfa inoculation arbuscular mycorrhizal fungi pot culture test of the caltrop specifically comprises the following steps:

4) the first two weeks are irrigated with 1/2Hoagland low-phosphorus nutrient solution containing 20 mu mol/LKH2PO4, the plants are potted and divided into three groups from the third week, one group is irrigated with 1/2Hoagland low-phosphorus nutrient solution containing 20 mu mol/LKH2PO4, the second group is irrigated with 1/2Hoagland low-phosphorus nutrient solution containing 0.2mmol/LKH2PO4, the third group is irrigated with 1/2Hoagland high-phosphorus nutrient solution containing 1mmol/LKH2PO4, and the rest time is irrigated with sterile deionized water to ensure the water required by the normal growth of the plants.

In this embodiment, the method for studying the influence of phosphorus on mtpp 2 gene expression includes the following steps: collecting plant root samples treated by Hoagland (1mmol/LPi) and LP-Hoagland (5 mu mol/LPi) nutrient solution for 21 days, extracting RNA, carrying out transcription level analysis, and taking a phosphorus transport gene MtPT1 induced to be expressed in roots with low phosphorus stress as a control, as shown in a figure 5(A, C), wherein the result shows that the relative expression amount of MtPA 2 in the roots after the HP and LP treatment is not significantly different, as shown in a figure 5(B, C), which shows that the expression of the gene in the roots is not influenced by the phosphorus concentration in the environment, and the protein is not involved in the response reaction of the plants to the low phosphorus stress, and is mainly related to the metabolism and distribution of phosphorus in plant tissues;

the method for researching the influence of phosphorus on alfalfa root nodules comprises the following steps: collecting roots and nodules 21 days after NFS (containing 1mmol/LPi) and LP-NFS (containing 5 mu mol/LPi), extracting RNA, and performing reverse transcription to cDNA for performing transcript level analysis; the results show that the relative expression level of MtPA 2 in the nodules is higher than that in the roots, and the relative expression level in the nodules after LP-NFS treatment is highest, and that MtPA 2 in the nodules after LP-NFS treatment is higher than that in the nodules after NFS treatment, but the expression of the gene in the roots is not changed by LP treatment as shown in FIG. 6; the expression level of MtPA 2 in the root after inoculation is not affected by the phosphorus concentration in the external environment, but the expression level of the gene in the root nodule is increased due to the reduction of the phosphorus concentration in the rhizosphere environment; it is presumed that under the condition of LP stress, the plant organism excessively expresses MtPA 2 to decompose organophosphorus stored in each tissue and provide phosphorus elements for the formation of the root nodule and nitrogen fixation in order to maintain the phosphorus homeostasis in the root nodule.

The method for researching the influence of phosphorus on the alfalfa mycorrhiza comprises the following steps: after inoculation of the AM fungus, plants were irrigated with 1/2LP (20. mu. mol/LPi) Hoagland nutrient solution for 2 weeks, then with 1/2HP (1mmol/LPi), 1/2MP (0.2mmol/LPi) and 1/2LP (20. mu. mol/LPi) Hoagland nutrient solution for 2 weeks, respectively, as shown in FIG. 7, and finally collected root samples were divided into two portions, one for TB staining as shown in FIG. 8, and the other for RNA extraction and reverse transcription into cDNA for transcript level analysis; the results show that the expression level of mtpp 2 was not significantly different between roots and mycorrhiza during low-phosphorus and high-phosphorus treatments, and further indicate that the expression level of mtpp 2 in roots and mycorrhiza was not affected by changes in the external phosphorus concentration. Comparing the expression of MtPA 2 in roots and mycorrhiza, the expression level of MtPA 2 in mycorrhiza was significantly higher than that in roots, which is consistent with the previous test results in mycorrhiza, as shown in FIG. 9,

in the embodiment, the spatiotemporal expression comprises the spatiotemporal expression of MtPAP2 in alfalfa rhizobia and the spatiotemporal expression of MtPAP2 in alfalfa mycorrhiza,

the specific implementation steps of the spatio-temporal expression of MtPA 2 in alfalfa root nodules are as follows: collecting plant materials of 9 days, 15 days, 21 days and 28 days after the inoculation of the sinorhizobium meliloti s.meliloti1021, extracting RNA, carrying out reverse transcription to cDNA for RT-qPCR analysis, and taking a nodulin gene MtN5 stably expressed in the rhizobium as a positive control; as shown in FIG. 1(A), the results show that the relative expression level of MtPA 2 in the root nodules in each period of symbiotic nitrogen fixation is higher than that in the roots, and the relative expression level in the root nodules at 15 days and 28 days after inoculation is highest and is 29.37 times of that in the roots of the inoculation experimental group; in addition, the relative expression level of MtPA 2 in roots at each period under symbiotic conditions is not significantly different from that under non-symbiotic conditions as shown in FIG. 1(B), which indicates that MtPA 2 is expressed in roots and stems in a small amount under non-symbiotic conditions, but after inoculation of rhizobia, the expression level of the gene in nodules at the symbiotic metaphase (15dpi) and symbiotic anaphase (28dpi) of nodule nitrogen fixation is obviously increased and is significantly higher than that in other tissues. Its abundant expression in nodules at various stages of symbiosis indicates that the protein may be involved in phosphorus metabolism during the symbiosis of nodules.

The specific implementation steps of the spatiotemporal expression of MtPA 2 in alfalfa mycorrhiza are as follows: collecting plant materials of 9 days, 15 days, 21 days and 28 days after the inoculation of the AM fungus R.irregularis, dividing the collected root sample into two parts, using one part for TB staining, statistically analyzing the symbiotic level data of the AM fungus, another part of the RNA was extracted, and transcript level analysis was performed, and the phosphorus transporter MtPT4 involved in the formation of the clump was used as a control, as shown in FIG. 4(A), the symbiotic level data show that as the mycorrhizal symbiosis relationship is established, the infection rate (F), the infection intensity (M) and the arbuscular abundance (A) are sequentially increased, the infection rate (F) of the middle and later symbiosis period (21-28dpi) is up to 80.4 percent, the infection intensity (M) is up to 61.3 percent, the arbuscular abundance (A) is up to 40.6 percent, as shown in FIG. 3, it was shown that the AM fungus had invaded most of the root segments of the host plant at this time, as shown in FIG. 2, and formed a number of arbuscular structures. At this time, the mycorrhizal fungi can transport phosphorus absorbed by the hyphae outside the root to the host plant through the arbuscular branches, so that phosphorus is accumulated in cells containing the arbuscular branches and cells of adjacent cortex in a large amount.

The RT-qPCR results show that the expression level of MtPA 2 in roots at each period has no significant difference in the control group which is not inoculated with AM fungus. At 9d after inoculation of AM fungus, the expression level in roots was not significantly different from that in uninoculated roots, while in the 15-28d root segment, the gene expression was significantly increased, reaching the highest at 28d, 28 times that in uninoculated AM fungus roots, as shown in FIG. 4 (B).

In combination with the mycorrhizal symbiosis level results, no arbuscular formation occurred in the 9d roots after inoculation, at which time no phosphorus transport occurred between the mycorrhizal fungi and the plant. Formation of the arbuscular branches and increase in arbuscular abundance at 15d-21d after inoculation indicate that the transport efficiency of phosphorus between the mycorrhizal fungi and the plant cell is enhanced and phosphorus begins to accumulate gradually in the cells containing the arbuscular branches and adjacent cells. Therefore, after inoculation of 28d, the arbuscular abundance (A) in the whole root segment reaches 28.7%, and although the arbuscular abundance in the root is slightly reduced, the expression level is still about 5.4 times higher than that of 15-21d, which indicates that the protein is related to the balance and distribution of phosphorus in root tissues during the mycorrhizal fungi colonization symbiosis process.

The specific implementation steps of the spatio-temporal expression of MtPA 2 in mycorrhizal symbiosis are as follows: transferring the obtained positive plants into sterile sandy soil, inoculating AM fungi after 3-5 days of phosphorus starvation treatment, and collecting root samples after 14 days and 28 days of inoculation respectively. The results show that under non-symbiotic conditions, mtpp 2 is predominantly expressed in the vascular bundles in the columella in roots (see fig. 12A, D, M, P); after inoculation with AM fungus, mtpp 2 was expressed not only in the pericycle tissue in roots at pre-symbiotic (14dpi) and post-symbiotic (28dpi) stages, but also strongly in cortical cells adjacent to the pericycle in roots (fig. 12G, J, S, V), and these cortical cells contained or were adjacent to the pericycle-containing cortical cells (fig. 12I, L, U, X); the results of the space-time expression positioning further verify that the MtPA 2 is subjected to AM fungus induced expression, and meanwhile, the expression part of the gene also shows that the gene is highly expressed mainly in cells containing arbuscular branches and adjacent cortex, which are accumulated by phosphorus, suggesting that the protein can be transported to other tissues through the action of phosphorus transport protein by releasing a large amount of phosphorus accumulated in the cells containing arbuscular branches and adjacent cortex, and further, the balance of phosphorus in root tissues in the mycorrhizal symbiosis process is regulated.

In FIG. 12, A-L are observed for 14dpi positive transformed plants; M-X is the observation of positive transformation plants of 28 dpi; A-F, M-R is observation of a missed plant; G-L and S-X are observed for inoculated plants; the diagrams D, E, J, K, P, Q, V and W are respectively partial enlargements of the diagrams A, B, G, H, M, N, S and T; panels A, D, G, J, M, P, S and V are brightfield shots, and panels B, E, H, K, N, Q, T and W are WGA 488-stained GFP fluorescence field shots; a, B, C, G, H, I, M, N, O, S, T, and U, wherein bar is 100 μ M, D, E, F, J, K, L, P, Q, R, V, W, and X, wherein bar is 50 μ M.

In this example, the construction of a promoter tissue localization vector for studying spatiotemporal expression localization of the mtpp 2 promoter in alfalfa nodule and mycorrhizal symbiosis includes: the promoter sequence of MtPA 2 is fused to the N end of GUS reporter gene, and the correct alignment of colony PCR, enzyme digestion and sequencing shows that the MtPA 2 promoter tissue expression positioning vector is successfully constructed. As shown in fig. 10 and 11. Wherein, A is MtPA 2 promoter cloning; b, carrying out PCR verification on a transformant; c, enzyme digestion verification of a promoter tissue expression positioning vector;

lane 1 in M:1kbladderA is the MtPA 2 promoter amplified from genomic DNA;

lanes

2 and 3 in B are target bands obtained by PCR amplification of positive transformants respectively; lane 4 in C is a band obtained by double digestion of the promoter tissue expression localization vector with HindIII and BamHI, and the excised band is 2157 bp.

In this example, the construction of the overexpression vector of the mtpp 2 gene:

in order to further explore the functions of MtPA 2 in the interaction of biological nitrogen fixation and bacterial planting, an overexpression vector is constructed, as shown in FIG. 13, the colony PCR, enzyme digestion and sequencing comparison are correct, which indicates that the construction of the MtPA 2 overexpression vector is successful, as shown in FIG. 14, wherein A is the MtPA 2 coding region cloning; b, carrying out PCR verification on a transformant; c, enzyme digestion verification of a overexpression vector pUB-PAP2-OE, wherein M1 is DL2000 Plus; m2, Marker 12; m3:1 kblader

Lane 1 is the MtPA 2 coding region amplified from cDNA;

lanes

2 and 3 in B are target bands obtained by PCR amplification of positive transformants respectively; lane 4 in C is the band of the overexpression vector pUB-PAP2-OE after XbaI and AscI double digestion, the band cut out is 1204bp, which is in line with the expectation, and the overexpression vector is successfully constructed.

Overexpression of mtpp 2 was detected at the transcript level in alfalfa root tumors:

in order to determine whether the constructed overexpression vector obtains overexpression in alfalfa hair roots and root nodules, a transgenic plant is obtained through alfalfa hair root transformation, after the plant hair roots grow for 10 days, root samples are harvested, RNA is extracted, reverse transcription is carried out to obtain cDNA, and overexpression efficiency detection is carried out; the result shows that the relative expression amount of MtPA 2 in the hairy roots of the over-expressed plants is 8.6 times (n is 9, p is less than 0.001) of that of an empty vector control group (control) (as shown in FIG. 14, A), which indicates that the MtPA 2 gene obtains gene over-expression; inoculating sinorhizobium meliloti S.meliloti1021 to the obtained positive plants, harvesting a rhizobium sample after 28 days, extracting RNA, performing reverse transcription to obtain cDNA, and performing transcription level detection. The results showed that the relative expression of mtpp 2 in the root nodules of the over-expressed plants was 17.5 times (n 9, p <0.001) that of the empty vector control plants (control) (as shown in fig. 14, B), indicating that this batch of plants can be used for subsequent symbiotic phenotype identification.

Overexpression of the MtPA 2 gene affects nodule symbiosis:

the symbiotic phenotype identification is carried out on the obtained over-expression plants, and the result shows that compared with the empty vector control (control) plants, the MtPA 2 over-expression plants have better overground growth vigor, longer and dense underground roots and large and more nodules, and as shown in figure 16, in order to quantify the influence degree of MtPA 2 over-expression on symbiotic nitrogen fixation, the statistical analysis is respectively carried out on symbiotic indexes such as overground fresh weight, nitrogen-fixing enzyme activity, nodule number, fresh weight of nodules and the like of the two groups of plants; the results show that the overground part fresh weight of the control group plant (control) is 0.09 g/piece on average, and the overground part fresh weight of the over-expression plant is 0.12 g/piece on average (as shown in figure 17, A), compared with the control group, the overground part fresh weight of the over-expression plant is obviously increased (p is less than 0.01, and n is 18); compared with the control group, the nitrogen-fixing enzyme activity of the over-expression plant is remarkably increased (p is less than 0.01, and n is 9); the average number of nodules per plant in the control group was 17.5, while the average number of nodules per plant in the overexpression group was 30.72 (fig. 17, C), and the number of nodules in the overexpression plant was significantly increased compared to the control group (p <0.001, n ═ 9); the fresh root nodule weight of the control plant (control) is 0.004 g/grain on average, while the fresh root nodule weight of the over-expressed plant is 0.009 g/grain on average (as shown in fig. 17, D), and compared with the control group, the fresh root nodule weight of the over-expressed plant is significantly increased (p <0.001, and n is 9).

In conclusion, the mass expression of the MtPA 2 gene promotes the formation and development of the nodule, improves the nitrogen-fixing enzyme activity, and further promotes the growth of the overground part.

Construction of MtPA 2 gene silencing vector:

since overexpression of the mtpp 2 gene promotes root nodule development and plant growth, is silencing the expression of this gene also detrimental to root nodule development or inhibits plant growth? In order to further determine the function of the gene in nodule symbiosis, an RNAi interference vector is constructed (as shown in FIG. 18), and the colony PCR, enzyme digestion and sequencing alignment are correct, which indicates that the MtPA 2RNAi interference vector is successfully constructed (as shown in FIG. 19). Wherein, A is MtPA 2 interference fragment cloning; b, carrying out PCR verification on a transformant; c, enzyme digestion verification of an interference vector p1301-PAP2-Ri, wherein M1 is DL2000 Plus; m2, Marker 1; m3:1 kblader

Lanes

1 and 2 are the sense and antisense strands, respectively, of the interfering fragment amplified from the cDNA;

lanes

3 and 4 and

lanes

5 and 6 in B are target bands obtained by PCR amplification of positive transformants of the sense strand and the antisense strand of the interference fragment, respectively; lane 7 in C is the band of interference vector p1301-PAP2-Ri after SacI and PstI double digestion, the band cut is 742bp, it accords with expectation, the interference vector construction is successful.

Transcriptional level measurements in nodules following silencing of mtpp 2:

in order to determine whether the constructed RNAi interference vector plays a role in alfalfa hair roots and root nodules, a transgenic plant is obtained through alfalfa hair root transformation, after the plant hair roots grow for 10 days, root samples are harvested, RNA is extracted, the RNA is reversely transcribed into cDNA, and silencing efficiency detection is carried out; the results showed that the relative expression of mtpp 2 in the roots of the silenced plants was 0.35 times (n-9, p <0.001) that of the empty vector control plants (control) (fig. 20, a); the MtPA 2 gene expression is inhibited; inoculating sinorhizobium meliloti S.meliloti1021 to the obtained positive plants, harvesting a rhizobium sample after 21 days, extracting RNA, performing reverse transcription to obtain cDNA, and performing transcription level detection; the results showed that the relative expression of mtpp 2 in silenced plant nodules was 0.07 times (n-9, p <0.001) that of empty vector control plants (control) (fig. 20, B); indicating that the batch of plants can be used for the subsequent symbiotic phenotype identification.

Mtpp 2 gene silencing affects nodule symbiosis:

carrying out symbiotic phenotype identification on the obtained silent plants; the results showed that the mtpp 2 silenced plants grew less overground, had shorter underground roots, had small nodules and mostly necrotic nodules with faint color, compared to the empty vector control (control) plants (fig. 21, a-D).

In order to quantify the degree of influence of the silenced MtPA 2 on symbiotic nitrogen fixation, the overground fresh weight, the azotobacter activity, the number of nodules and the fresh weight of nodules of the two groups of plants are respectively subjected to statistical analysis; the results showed that the fresh weight of aerial parts of the control plants (control) averaged 0.061 g/grain, while the silenced plants averaged 0.048 g/grain, with a significant decrease in the fresh weight of the silenced plants above ground compared to the control (p <0.05, n-9) (fig. 22, a); the average of the azotase activity of the plants in the control group is 3.93 mu molg-1NDWh-1, the azotase activity of the silenced plants is 2.48 mu molg-1NDWh-1, and compared with the control group, the azotase activity of the silenced plants is remarkably reduced (p is less than 0.001, and n is 9) (figure 22, B); the average number of nodules per plant in the control group was 17.8, with an average number of nodules per plant of 16.5 for the roses, an average number of nodules per 1.3 for the xanthomas, and an average number of nodules per plant of 16.8 for the silenced plants, with an average number of nodules per 10.6 for the roses and an average number of nodules per 6.1 for the xanthomas, there was no significant difference in the total number of nodules of the silenced plants compared to the control group, but the number of nodules was significantly reduced and the number of necrotic yellow nodules was significantly increased (p >0.05, n-9) (fig. 22, C); the fresh root nodule weight of the control plant (control) was 0.003 g/piece on average, while that of the silenced plant was 0.002 g/piece on average, and the fresh root nodule weight of the silenced plant was significantly reduced compared to the control (p <0.05, n-9) (fig. 22, D).

In conclusion, due to silencing of the MtPA 2 gene, phosphorus elements required by symbiotic nitrogen fixation cannot be timely transported to the root nodule, so that the activity of the root nodule nitrogen fixation enzyme is reduced, and the overground growth of the plant is influenced.

To further examine the role of mtpp 2 in nodule formation and development, 9-12 positive nodules were randomly picked from the empty vector control, overexpression and silencing groups, respectively, and fixed by FAA, paraffin-embedded, slit, stained with toluidine blue, and observed under a stereomicroscope. The result shows that compared with the plant of a control group, the root nodule morphology of the MtPA 2 overexpression plant is well developed, and the bacterial cells in the root nodule meristematic region and the infection region are obviously increased; while MtPA 2 silenced the abnormal development of plant nodules, the number of germ-containing cells in the nitrogen fixation region was significantly reduced, the symbiont membrane was disrupted and the periplasmic space was increased (FIG. 23, A-F). The method comprises the following steps of A, longitudinally cutting an empty vector control plant 21dpi root nodule, B, longitudinally cutting an overexpression plant 21dpi root nodule, C, longitudinally cutting a silencing plant 21dpi root nodule, D, E and F are partial enlargements of a nitrogen fixing area in pictures A, B and C respectively, in the pictures, mer represents a meristem area, infz represents an infection area, and fixz represents a nitrogen fixing area. In the figure, bar is 100 μm.

In this example, mtpp 2 overexpression was detected at the transcriptional level in alfalfa mycorrhiza:

obtaining a transgenic plant by an alfalfa hairy root transformation method, harvesting a root sample after the plant hairy root grows for 10 days, extracting RNA, performing reverse transcription to obtain cDNA, and performing overexpression efficiency detection; the results show that the relative expression amount of mtpp 2 in the hairy roots of the over-expressed plants is 8.6 times (n is 12, p is less than 0.001) of that of the empty vector control group (control) (fig. 24, a), indicating that the mtpp 2 gene is over-expressed; inoculating AM fungi to the obtained positive plants, harvesting root samples 28 days later, randomly selecting a part of the root samples for TB staining, extracting RNA from the rest root samples, performing reverse transcription to obtain cDNA, and performing transcription level detection; the results showed that the relative expression of mtpp 2 in the root-like of the over-expressed plants was 11.6 times (n 12, p <0.001) that of the empty vector control plants (control) (fig. 24, B), indicating that this batch of plants could be used for subsequent symbiosis level statistics.

Carrying out symbiosis level index statistics on the obtained root segments of the over-expression plants; the results showed that the overground growth of the mtpp 2 overexpressing plants was better compared to the empty vector control (control) plants, as shown in figure 25,

in order to quantify the influence degree of the overexpressed mtpp 2 on mycorrhizal symbiosis, the infection rate of root-like plants, well-developed branches, degraded branches, intraradicular hyphae and vesicle abundance after TB staining of two groups of plants are respectively counted (fig. 26), and the result shows that the infection rate of plants in a control group is 49.7%, while the infection rate of overexpressed plants is 68.4%, compared with the control group, the infection rate of overexpressed plants is increased by 18.7% (p is less than 0.05, and n is 12); the abundance of well-developed branches of the plants in the control group is 27.6%, while that of well-developed branches of the over-expression plants is 42.7%, and compared with the control group, the abundance of well-developed branches of the over-expression plants is increased by 15.1% (p <0.05, n is 12); the abundances of the degraded branches of the plants in the control group are 1.4%, the abundances of the degraded branches of the over-expression plants are 1.9%, and compared with the control group, the abundances of the degraded branches of the over-expression plants are not significantly different (p is greater than 0.05, and n is 12); the abundance of hyphae in the roots of the plants in the control group is 18.5%, the abundance of hyphae in the roots of the over-expression plants is 20.8%, and compared with the control group, the abundance of hyphae in the roots of the over-expression plants is not significantly different (p is more than 0.05, and n is 12); the vesicle abundance of the control plant was 2.3%, while that of the silenced plant was 3.1%, and there was no significant difference in the vesicle abundance of the overexpressed plants compared to the control (p >0.05, n ═ 12) (fig. 27).

In conclusion, the overexpression of the MtPA 2 gene improves the infection rate and branch abundance of AM fungi, and shows that the protein plays an important role in the symbiosis of mycorrhizal fungi and the formation and development of arbuscular branches.

Transcript levels in mycorrhiza were measured following silencing of mtpp 2:

obtaining a transgenic plant by an alfalfa hairy root transformation method, harvesting a root sample after the plant hairy root grows for 10 days, extracting RNA, performing reverse transcription to obtain cDNA, and performing silencing efficiency detection; the results show that the relative expression amount of mtpp 2 in the hairy roots of the silenced plants is 0.35 times (n is 12, p is less than 0.001) of that of the empty vector control group (control) (fig. 28, a), indicating that the expression of mtpp 2 gene is inhibited; inoculating AM fungi to the obtained positive plants, harvesting root samples 28 days later, randomly selecting a part of the root samples for TB staining, extracting RNA from the rest root samples, performing reverse transcription to obtain cDNA, and performing transcription level detection; the results show that the relative expression of mtpp 2 in the roots of the silenced plants was 0.21 times (n is 12, p is <0.001) that of the empty vector control plants (control) (fig. 28, B), indicating that this batch of plants can be used for subsequent symbiosis level statistics.

Carrying out symbiotic level index statistics on the obtained silent plant root segments; the results showed that mtpp 2 silenced plants grew less extensively compared to empty vector control (control) plants (fig. 29).

In order to quantify the influence degree of MtPA 2 on mycorrhizal symbiosis after silencing, respectively counting the infection rate of root samples, well-developed branches, degraded branches, root hyphae and vesicle abundance of two groups of plants after TB staining (figure 30, A-D), wherein A is an empty carrier plant 28dpi staining root segment C, and RNAi interferes with the plant 28dpi staining root segment; the C and D are partially enlarged views A and B

In fa denotes well-developed shoots, da denotes shoots that have degraded, ih denotes intraradicular hyphae, and v denotes vesicles. Bar 100 μm

The results show that the infection rate of plants in the control group is 70.6%, while the infection rate of silent plants is 52.1%, and compared with the control group, the infection rate of silent plants is reduced by 18.5% (p <0.05, n is 12); the abundance of well-developed branches of the plants in the control group is 44.7 percent, while that of well-developed branches of the silenced plants is 4.6 percent, and compared with the control group, the abundance of well-developed branches of the silenced plants is reduced by 40.1 percent (p is less than 0.05, and n is 12); the abundances of the degraded branches of the plants in the control group are 4.8%, the abundances of the degraded branches of the silenced plants are 20.5%, and the abundances of the degraded branches of the silenced plants are increased by 15.7% compared with the abundances of the degraded branches of the control group (p <0.05, n is 12); the abundance of hyphae in the roots of the plants in the control group is 15.4%, the abundance of hyphae in the roots of the silenced plants is 16.2%, and compared with the control group, the abundance of hyphae in the roots of the silenced plants is not significantly different (p is more than 0.05, and n is 12); the vesicle abundance of the control plant was 5.6% and that of the silenced plant was 10.7%, which increased the vesicle abundance of the silenced plant by 5.1% (p <0.05, n-12) compared to the control (fig. 31).

In conclusion, the silencing of MtPA 2 gene expression inhibits the infection of mycorrhizal fungi, especially the formation and development of arbuscular structures, and further proves that MtPA 2 plays an important role in the symbiosis of mycorrhizal fungi and the formation and development of arbuscular structures.

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. The method for researching the function of acid phosphatase in alfalfa root nodule and mycorrhiza symbiosis is characterized in that: the research method comprises the following steps:

2. The method of claim 1, wherein the acid phosphatase function is selected from the group consisting of: the specific steps of the step 1) are as follows:

(1) selecting seeds of medicago truncatula A17 with uniform size;

3. The method of claim 1, wherein the acid phosphatase function is selected from the group consisting of: in the step 2), under the condition of different phosphorus concentrations, the alfalfa inoculation sinorhizobium meliloti pot culture test specifically comprises the following steps:

4. The method of claim 1, wherein the acid phosphatase function is selected from the group consisting of: in the step 2), under the condition of different phosphorus concentrations, the experiment of inoculating the medicago truncatula into the arbuscular mycorrhizal fungi pot culture specifically comprises the following steps:

5. The method of claim 1, wherein the acid phosphatase function is selected from the group consisting of: the method for researching the influence of phosphorus on the expression of the MtPA 2 gene comprises the following steps: collecting plant root samples treated by nutrient solutions of Hoagland (1mmol/LPi) and LP-Hoagland (5 mu mol/LPi) for 21 days, extracting RNA, carrying out transcription level analysis, and taking a phosphorus transport gene MtPT1 induced and expressed in low-phosphorus stress roots as a control; the method for researching the influence of phosphorus on alfalfa root nodules comprises the following steps: collecting roots and nodules 21 days after NFS (containing 1mmol/LPi) and LP-NFS (containing 5 mu mol/LPi), extracting RNA, and performing reverse transcription to cDNA for performing transcript level analysis; the method for researching the influence of phosphorus on the alfalfa mycorrhiza comprises the following steps: after inoculation with the AM fungus, plants were irrigated with 1/2LP (20. mu. mol/LPi) Hoagland nutrient solution for 2 weeks, then with 1/2HP (1mmol/LPi), 1/2MP (0.2mmol/LPi) and 1/2LP (20. mu. mol/LPi) Hoagland nutrient solution for 2 weeks, and finally the collected root samples were divided into two portions, one for TB staining, the other for RNA extraction and reverse transcription into cDNA for transcript level analysis.

6. The method of claim 1, wherein the acid phosphatase function is selected from the group consisting of: the spatiotemporal expression comprises the spatiotemporal expression of MtPAP2 in alfalfa rhizobium, the spatiotemporal expression of MtPAP2 in alfalfa mycorrhiza and the spatiotemporal expression of MtPAP2 in mycorrhiza symbiosis,

7. The method of claim 1, wherein the acid phosphatase function is selected from the group consisting of: the construction of the promoter tissue localization vector is used for researching the space-time expression localization of the MtPA 2 promoter in the symbiosis of the alfalfa root nodule and mycorrhiza, and comprises the following steps: the promoter sequence of MtPA 2 is fused to the N end of GUS reporter gene, and the correct alignment of colony PCR, enzyme digestion and sequencing shows that the MtPA 2 promoter tissue expression positioning vector is successfully constructed.

8. The method of claim 1, wherein the acid phosphatase function is selected from the group consisting of: the alfalfa nodule research method of the MtPA 2 gene overexpression pair comprises the construction of an MtPA 2 gene overexpression vector, the detection of the transcription level of MtPA 2 overexpression in alfalfa root tumors and the influence of MtPA 2 gene overexpression on nodule symbiosis, and the research method of MtPA 2 gene silencing comprises the construction of an MtPA 2 gene silencing vector, the detection of the transcription level in nodules after MtPA 2 silencing and the influence of MtPA 2 gene silencing on nodule symbiosis.

9. The method of claim 1, wherein the acid phosphatase function is selected from the group consisting of: the alfalfa mycorrhiza research method of the MtPA 2 gene overexpression pair comprises the construction of an MtPA 2 gene overexpression vector, the detection of the transcription level of MtPA 2 overexpression in alfalfa mycorrhiza and the influence of MtPA 2 gene overexpression on alfalfa mycorrhiza symbiosis, and the research method of MtPA 2 gene silencing comprises the construction of an MtPA 2 gene silencing vector, the detection of the transcription level in alfalfa mycorrhiza after MtAP 2 silencing and the influence of MtAP 2 gene silencing on alfalfa mycorrhiza symbiosis.