CN110845588A

CN110845588A - Application of protein ZmPT3 in regulation and control of phosphorus content in plants

Info

Publication number: CN110845588A
Application number: CN201810816721.8A
Authority: CN
Inventors: 陈益芳; 武维华; 王芳
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-07-24
Filing date: 2018-07-24
Publication date: 2020-02-28
Anticipated expiration: 2038-07-24
Also published as: CN110845588B

Abstract

The invention discloses application of protein ZmPT3 in regulation and control of phosphorus content in plants. The coding gene of protein ZmPT3 is introduced into a maize inbred line B73 to obtain transgenic maize OE-1 and OE-2. At the seedling stage, compared with the maize inbred line B73, the phosphorus content and biomass of the young leaves and stems of OE-1 and OE-2 are both significantly increased; in the pollen-dispersing period, the phosphorus content of ear position leaves and female ears of OE-1 and OE-2 is obviously increased compared with that of the maize inbred line B73. Therefore, the protein ZmPT has important theoretical significance and practical significance for further clarifying the molecular mechanism of plant phosphorus nutrition and cultivating new crop varieties with high phosphorus nutrition efficiency by a technical means of genetic engineering. The invention has great application value.

Description

Application of protein ZmPT3 in regulation and control of phosphorus content in plants

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of protein ZmPT3 in regulation and control of phosphorus content in plants.

Background

Phosphorus is one of essential macro-elements of plants and is involved in the cellular constitution, growth and development, substance metabolism, energy metabolism and the like of the plants. Plants absorb inorganic phosphorus from soil mainly through root systems, and then are transferred to the overground part to participate in the growth and development of the plants. The concentration of available phosphorus in the soil solution is very low, typically below 10 μ M. Plant cells are typically maintained at millimolar (mM) levels of phosphorus, and plants are therefore often subject to low phosphorus stress. In order to ensure the normal growth of plants and improve the yield, people apply a large amount of phosphate fertilizer. After the phosphate fertilizer is applied to soil, the phosphate fertilizer is easily fixed by heavy metals and the like in the soil and becomes phosphorus which is not easily absorbed by plants, the utilization efficiency of the phosphate fertilizer in season is generally lower than 30%, a large amount of the phosphate fertilizer is wasted, and the environment is also polluted. Therefore, the low efficiency of phosphorus uptake by plants becomes an important limiting factor in agricultural production.

Corn is a grass of the family gramineae, known as maize. The plants are planted in all parts of China, especially in northeast, northwest and southwest provinces. Corn is a good health product in coarse grain, and the edible corn is very beneficial to the health of human bodies. Corn plays an extremely important role in food safety in China, is an important feed crop, and is an important raw material in industries such as food, chemical engineering, fuel, medicine and the like. The phosphorus plays an important role in the growth and development of the corn, the yield and the quality of the corn kernels. When the phosphorus is sufficient, the corn is early-maturing, the color and the quality of the grains are good, and the yield is high. Phosphorus deficiency in the seedling stage of the corn can cause slow growth of the corn, nitrate nitrogen accumulation, protein synthesis obstruction and purple red leaves. When the male and female spikes are differentiated and lack phosphorus, the development of the spikes is hindered, the tops of the spikes shrink, and empty stalks are easily formed. If the pollination period is lack of phosphorus, the pollination is poor, the fruit cluster curls, the fruit cluster is lack of rows, grains or bald tip, and the quality is reduced.

The efficiency of phosphorus absorption and redistribution of the corn is improved, the utilization efficiency of the corn on phosphate fertilizer is improved, the application of the phosphate fertilizer is reduced, and the environmental pollution is reduced.

Disclosure of Invention

The invention aims to promote the absorption and redistribution of phosphorus by plants under low-phosphorus conditions.

The invention firstly protects the application of the protein ZmPT3, which can be at least one of the following X1) to X6);

x1) regulating the phosphorus content of the plant;

x2) regulating the growth of a plant;

x3) regulating the biomass of the plant;

x4) regulating phosphorus element transfer in plants;

x5) regulating phosphorus uptake of plants under low phosphorus conditions;

x6) cultivating plants with increased phosphorus content and/or improved growth and/or increased biomass and/or improved phosphorus transfer to the growth center and/or low phosphorus tolerance.

In the above application, the protein ZmPT3 may be a1) or a2) or a 3):

a1) the amino acid sequence is protein shown as a sequence 1 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 1 in the sequence table;

a3) the protein with the same function correlation is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 1 in the sequence table.

Wherein, the sequence 2 in the sequence table is composed of 535 amino acid residues.

In order to facilitate the purification of the protein in a1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 1 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein according to a3), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 2 of the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The invention also protects the application of a nucleic acid molecule for coding the protein ZmPT3, which can be at least one of the following X1) to X6);

x1) regulating the phosphorus content of the plant;

x2) regulating the growth of a plant;

x3) regulating the biomass of the plant;

x4) regulating phosphorus element transfer in plants;

x5) regulating phosphorus uptake of plants under low phosphorus conditions;

In the above application, the nucleic acid molecule encoding the protein ZmPT3 can be a DNA molecule shown in b1) or b2) or b3) or b4) as follows:

b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

b2) the nucleotide sequence is a DNA molecule shown in a sequence 2 in a sequence table;

b3) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b1) or b2), is derived from corn and encodes the ZmPT3 protein;

b4) DNA molecules which are derived from maize and code for the protein ZmPT3 and which hybridize under stringent conditions with the nucleotide sequences defined under b1) or b 2).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 2 in the sequence table consists of 1608 nucleotides, and encodes the amino acid sequence shown in the sequence 1 in the sequence table.

In any of the above applications, the regulating the phosphorus content of the plant may be increasing the phosphorus content of the plant. The regulating the growth of the plant may be promoting the growth of the plant. The modulating biomass of the plant may be increasing biomass of the plant. The regulating phosphorus transfer of the plant can be promoting phosphorus transfer of the plant. The regulating phosphorus uptake of the plant under low phosphorus conditions can be promoting phosphorus uptake of the plant under low phosphorus conditions.

The invention also protects a method for cultivating transgenic plants, which can comprise the steps of introducing a substance for improving the expression quantity and/or activity of the protein ZmPT3 into a receptor plant to obtain a transgenic plant;

the transgenic plant satisfies at least one phenotype of (d1) to (d5) as follows:

(d1) (ii) a phosphorus content higher than that of the recipient plant;

(d2) a biomass higher than the recipient plant;

(d3) (ii) a low phosphorus tolerance greater than that of the recipient plant;

(d4) growing faster than the recipient plant;

(d5) the phosphorus element is transferred to the growth center to a higher extent than the recipient plant.

In the above method, the "introducing a substance that increases the expression level and/or activity of the protein ZmPT3 into a recipient plant" may be carried out by introducing a nucleic acid molecule encoding the protein ZmPT3 into a recipient plant.

In the above method, the nucleic acid molecule encoding the protein ZmPT3 may be a DNA molecule represented by b1) or b2) or b3) or b4) as follows:

In the above method, said "introducing into a recipient plant a nucleic acid molecule encoding the protein ZmPT 3" may be effected by introducing into a recipient plant a recombinant vector; the recombinant vector may be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the protein ZmPT3 into an expression vector.

The recombinant vector can be specifically a recombinant plasmid UBI ZmPT 3. The recombinant plasmid UBI ZmPT3 can be specifically a recombinant plasmid obtained by inserting a double-stranded DNA molecule shown in a sequence 2 in a sequence table into a recognition site of a restriction enzyme XcmI of a vector pCXUN.

The invention also provides a plant breeding method, which comprises the following steps: increasing the expression level and/or activity of the protein ZmPT3 in the plant, thereby improving the phosphorus content and/or biomass and/or low phosphorus tolerance and/or growth speed of the plant and/or the transfer degree of phosphorus element to the growth center.

In the plant breeding method, the expression level and/or activity of the protein ZmPT3 in the plant can be increased by using methods known in the art such as multiple copies, changing promoters, regulatory factors, transgenes and the like, so as to achieve the effect of increasing the content and/or activity of the protein ZmPT in the plant.

Any of the above phosphorus contents may be an inorganic phosphorus content and/or a total phosphorus content.

Any of the above described biomass can be root dry weight and/or crown dry weight.

Any of the above phosphorus element transfers may be a phosphorus element transfer to a growth center.

Any of the plants described above may be c1) or c2) or c3) or c4) or c5) or c 6): c1) a dicotyledonous plant; c2) a monocot plant; c3) corn; c4) maize inbred line B73; c5) a cruciferous plant; c6) arabidopsis thaliana.

The application of any of the methods described above in plant breeding also falls within the scope of the present invention.

The phosphorus content of any of the above-described plants can be increased by increasing the phosphorus content of young leaves or stems of corn at the seedling stage.

Any of the above plants can be used to increase the phosphorus content of the ear leaves or the ears at the corn pollen stage.

Any of the low phosphorus described above may have a phosphorus content of less than 10 μ M.

ZmPT3 gene is introduced into a maize inbred line B73 to obtain transgenic maize OE-1 and OE-2. At the seedling stage, compared with the maize inbred line B73, the phosphorus content and biomass of the young leaves and stems of OE-1 and OE-2 are both significantly increased; in the pollen-dispersing period, the phosphorus content of ear position leaves and female ears of OE-1 and OE-2 is obviously increased compared with that of the maize inbred line B73. Therefore, the protein ZmPT has important theoretical significance and practical significance for further clarifying the molecular mechanism of plant phosphorus nutrition and cultivating new crop varieties with high phosphorus nutrition efficiency by a technical means of genetic engineering.

Drawings

FIG. 1 is an identification of transgenic maize.

FIG. 2 shows the phenotype and biomass measurements of transgenic maize.

FIG. 3 shows the measurement of inorganic phosphorus content and total phosphorus content in transgenic corn.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Maize inbred line B73 is described in the following documents: wei et al, the medical and genetic frame of the mail B73 genome, PLoS Genetics 2009, 5: e1000715. maize inbred line B73 is known in the literature as Maize B73.

Agrobacterium EHA105 is described in: agrobacterium-mediated transformation of yam (Dioscoreatorunnata): frontiers in plantascice 2014, 5: 463.

hogaland nutrient solutions (described in Liang and Li, Differencens in cluster-root formation and carboxylate evaluation in Lupinusalburses L. plant and Soil 2003, 248: 221-: the solvent is water; solutes and their concentrations were as follows: 0.75mMK₂SO₄，0.25mM KH₂PO₄，0.1mM KCl，0.65mM MgSO₄，2mM Ca(NO₃)₂，0.1mMFeNaEDTA，1μM H₃BO₃，1μM MnSO₄，1μM ZnSO₄，4μM CuSO₄，5μM(NH₄)₆Mo₂O₄。

5 XPPhusion HF Buffer, Phusion DNA Polymerase and restriction enzyme XcmI are all products of NEB corporation. SUPERSCRIPT^IIAnd Trizol are both products of Invitrogen corporation. The 10 XPCR buffer is a product of the company TaKaRa.

Example 1 discovery of ZmPT3 protein and Gene encoding the same

Trizol was used to extract total RNA (about 100-200mg) from maize inbred line B73 seedlings, and the integrity of the RNA was checked by formaldehyde-denatured RNA agarose gel electrophoresis. According to SUPERSCRIPT^IIThe instructions for synthesizing single-stranded cDNA. The synthesized single-stranded cDNA was diluted 10-fold and used as a template DNA, and PCR reaction was performed using a Primer pair consisting of ZmPT3-F and Primer 2.

ZmPT3-F：5'-tataagcttATGGCACACGATCACAAGGT-3'；

Primer 2：5'-gctctagaTTATTCACTCAAATCTGTCGGCAC-3'。

PCR System (50. mu.L): 10 μ L of 5 XPisusion HF Buffer, 4 μ L of dNTP mix (concentrations of dATP, dTTP, dGTP and dCTP were all 2.5mM), 2.5 μ L of ZmPT3-F in water (concentration of 10 μ M), 2.5 μ L of LPrimer 2 in water (concentration of 10 μ M), 1 μ L of template DNA, 1.5 μ L of DMSO, 0.5 μ L of LPHUsion DNA Polymerase (concentration of 2U/. mu.L), and the balance water.

PCR procedure: pre-denaturation at 98 ℃ for 3 min; 15s at 98 ℃, 30s at 63 ℃, 1min at 72 ℃ and 20s for 35 cycles; extension at 72 ℃ for 10 min.

And recovering a PCR product of about 1620bp, connecting the PCR product to a pMD18-T vector, and performing enzyme digestion and sequencing identification in sequence. Sequencing results show that the PCR product has an open reading frame shown in a sequence 2 of a sequence table and encodes a protein shown in a sequence 1 of the sequence table.

The protein shown in the sequence 1 of the sequence table is named ZmPT3 protein. The encoding gene of the ZmPT3 protein is named as ZmPT3 gene, and the open reading frame is shown as a sequence 2 in a sequence table. The recombinant plasmid constructed by connecting ZmPT3 to pMD18-T vector was named pMD18-ZmPT 3.

Example 2 cultivation of transgenic plants with increased phosphorus content Using the ZmPT3 Gene

Construction of recombinant expression vector

1. And (3) carrying out PCR amplification by using the pMD18-ZmPT3 recombinant plasmid as a template DNA and adopting a primer pair consisting of ZmPT3-F and ZmPT3-R to obtain a PCR amplification product.

ZmPT3-F：5'-tataagcttATGGCACACGATCACAAGGT-3'；

ZmPT3-R：5-gactagtTTATTCACTCAAATCTGTCGGCAC-3’。

2. The blunt end of the PCR amplification product obtained in step 1 was subjected to A addition using Taq enzyme to have an A sticky end.

3. Vector pCXUN (GenBank: FJ905215.1) was digested with restriction enzyme XcmI, linearized and possessing a T-sticky end.

4. The product of step 2 was ligated to the product of step 3 by TA cloning to give recombinant plasmid UBI: ZmPT 3.

According to the sequencing results, the structure of the recombinant plasmid UBI: ZmPT3 is described as follows: the double-stranded DNA molecule shown in sequence 2 of the sequence table is inserted into the XcmI enzyme cutting site of the vector pCXUN.

Second, obtaining transgenic corn

1. The recombinant plasmid UBI ZmPT3 is introduced into agrobacterium EHA105 to obtain recombinant agrobacterium.

2. Genetic transformation of embryogenic callus of recipient material (framework and Wang, Agrobacterium tumefaciens-mediated transformation of maize tissue culture a stationary vector system. plant Physiology 2002, 129: 13-22) was performed using the recombinant Agrobacterium obtained in step 1 to obtain transgenic maize. The receptor material is a maize inbred line B73.

OE-1 and OE-2 are two homozygous transgenic maize lines selected at random.

Identification of transgenic corn

T with OE-1 plant to be detected₃Plant-substitute OE-2T₃Generation plants or plants of maize inbred line B73.

ZmPT3 insertion identification

1. Culturing the plant to be tested to the three-leaf one-heart stage, shearing leaves and extracting genome DNA.

2. And (3) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and a primer pair (the target sequence is about 1600bp) consisting of Ubip-F and ZmPT 3-R.

Ubip-F：5’-TTGATCTTGATATACTTGGATG-3’；

ZmPT3-R：5’-gactagtTTATTCACTCAAATCTGTCGGCAC-3’。

Reaction system for PCR amplification (20 μ L): 10 XPCR buffer 2 uL, 0.4 uL dNTPmix (dATP, dTTP, dGTP and dCTP concentration is 2.5mM), 0.4 uLUbip-F aqueous solution (10 uM concentration), 0.4 uL ZmPT3-R aqueous solution (10 uM concentration), 0.2 uLTaq DNA polymerase (15U/. mu.L concentration), the balance is water.

Reaction conditions for PCR amplification: pre-denaturation at 95 ℃ for 5 min; 30s at 95 ℃, 30s at 63 ℃, 1min at 72 ℃ and 40s for 35 cycles; extension at 72 ℃ for 10 min.

Ubip-F was replaced by ZmPT3-F according to the above procedure, and the other procedures were not changed.

The partial PCR amplification results are shown in FIG. 1A. Both OE-1 and OE-2 are ZmPT3 gene-transferred corn, and the recombinant plasmid UBI ZmPT3 is inserted.

(II) ZmPT3 gene expression identification

1. Culturing the plant to be tested to three-leaf one-heart stage, taking the whole plant, extracting total RNA and carrying out reverse transcription to obtain cDNA.

2. The cDNA obtained in step 1 was used as a template, and a Real-Time quantitative PCR (qRT-PCR) was performed using a Primer pair consisting of an Applied Biosystems (Applied Biosystems, Foster City, Calif., USA) model 7500, ABI POWER SYBR GREEN PCR MASTER MIX kit, Primer 4 and Primer 5. The ZmUBQ gene is an internal reference gene.

Primer 4：5’-ATGGCACACGATCACAAGGT-3’；

Primer 5：5’-GTCCGAGTAGTAGATGCGGC-3’。

Part of the test results are shown in B of FIG. 1. The qRT-PCR result shows that the expression level of ZmPT3 genes in OE-1 and OE-2 is obviously higher than that of a maize inbred line B73.

Phenotypic detection of transgenic maize

Phosphorus rich medium: get

calyy ore (Goron, t.l., Watts, s., Shearer, c.andraizada, m.n.2015, Growth in

Adding 0.43g of urea, 0.11g of KCl, 0.38gKH g of urea and/or BMC in the form of urea chloride, potassium₂PO₄、0.25g MgSO₄·7H₂O, 1mL1000 Xmicro (containing 1mM H)₃BO₃、1mM MnSO₄、1mMZnSO₄、4mM CuSO₄And 5mM (NH)₄)₆Mo₂O₄Aqueous solution of (b) and 10mL of 100 Xiron salt (aqueous solution containing 10mM FeNaEDTA), a phosphorus-rich medium was obtained.

Low-phosphorus medium: only 0.38gKH in phosphorus rich medium₂PO₄Replacement is 0.076gKH₂PO₄The balance is completely consistent with the composition and content of the phosphorus-rich medium.

T with OE-1 seed to be detected₃Seed-generating, OE-2T₃Generation seeds or seeds of maize inbred line B73.

Phenotypic assay of transgenic maize

1. And (3) taking the seeds to be tested which germinate on the wet filter paper for 36 hours, sowing the seeds to be tested to a flowerpot filled with 5Kg of phosphorus-rich culture medium or low-phosphorus culture medium, and culturing the seeds at 25 +/-2 ℃ for 45 days to obtain the plants to be tested.

2. And (4) after the step 1 is finished, observing the crown and the root of the plant to be detected and respectively taking pictures.

The crown of the part of the plant to be tested is shown in A in figure 2. The roots of the part of the plants to be tested are shown in C in FIG. 2. The results show that under the condition of sufficient phosphorus, the phenotypes of the crowns of the maize inbred lines B73, OE-1 and OE-2 are not significantly different; under the condition of low phosphorus stress, compared with a maize inbred line B73, OE-1 and OE-2 have obviously larger crowns and have obvious growth advantages; under the condition of sufficient phosphorus and the condition of low phosphorus stress, the roots of OE-1 and OE-2 are obviously larger than those of a maize inbred line B73.

The results show that the improvement of the expression level of the ZmPT3 gene can promote the growth of the corn in the seedling stage.

(II) Biomass detection of transgenic maize roots and crowns

The experiment is repeated three times to obtain an average value, and the number of seeds to be detected in each repetition is 30.

And (3) taking the seeds to be tested which germinate on the wet filter paper for 36 hours, sowing the seeds to be tested to a flowerpot filled with 5Kg of phosphorus-rich culture medium or low-phosphorus culture medium, and culturing the seeds at 25 +/-2 ℃ for 45 days to obtain the plants to be tested. And (4) carrying out statistical analysis on crown biomass (DW) and root biomass (DW) of the plant to be tested. The crown biomass (DW) of the plant to be detected is the mass of the crown of the plant to be detected after being dried in an oven at 80 ℃ to constant weight and being placed in a dryer for cooling. The root biomass (DW) of the plant to be detected is the mass of the root of the plant to be detected after being dried in an oven at 80 ℃ to constant weight and being put into a dryer for cooling.

The crown biomass (DW) statistics of some of the plants tested are shown in B of FIG. 2. The statistical results of biomass (DW) of the roots of some of the plants to be tested are shown in FIG. 2D. The results show that under the condition of sufficient phosphorus, the crown biomass (DW) of the maize inbred lines B73, OE-1 and OE-2 has no significant difference, but the root biomass (DW) of the OE-1 and OE-2 is significantly larger than that of the maize inbred line B73; under the condition of low phosphorus stress, the biomass (DW) of the roots and the biomass (DW) of the crowns of OE-1 and OE-2 are both significantly larger than that of the maize inbred line B73.

The above results indicate that increasing the expression level of ZmPT3 gene increased biomass (DW) in the root and crown of maize.

(III) detection of inorganic phosphorus content in transgenic corn

The specification of the EP tube was 10 mL.

Color reaction stock solution: to 3.5g (NH)₄)₆Mo₇O₂₄·4H₂Adding a proper amount of deionized water into O and 23.39mL of 98% sulfuric acid, fully dissolving, and then using the deionized water to fix the volume to 1L.

Color reaction working solution: taking a color development reaction stock solution, and adding ascorbic acid to obtain a color development reaction working solution; the concentration of ascorbic acid in the working solution for color development reaction was 1.4% (m/v). The color reaction working solution needs to be prepared at present.

Inorganic phosphorus extract 5mL of 1M Tris-HCl buffer (pH8.0), 1mL of 0.5M EDTA (pH8.0), 2.92g of NaCl, 35. mu.L of β -mercaptoethanol, and 5mL of PMSF were dissolved in water, and the volume was adjusted to 500mL with water.

1. And (3) taking the seeds to be detected which germinate on the wet filter paper for 36 hours, sowing the seeds to be detected in a flowerpot filled with 5Kg of phosphorus-rich culture medium or low-phosphorus culture medium, and culturing the seeds at 25 +/-2 ℃ for 45 days to obtain the seedlings to be detected.

2. And (3) after the step (1) is finished, respectively taking each leaf (the leaf is counted from bottom to top according to the growth sequence, the 1 st leaf is the 1 st leaf from bottom to top, the 2 nd leaf is the 2 nd leaf from bottom to top, and the like), and recording the fresh weight value of each group of samples, putting the samples into liquid nitrogen for freezing storage, and crushing the samples by using a grinder.

3. An EP tube was first filled with 4.3mL of glacial acetic acid.

4. Taking a sample tube, adding 70mg of sample and 700 mu L of inorganic phosphorus extracting solution (the proportion is 10 mg: 100 mu L), and turning upside down and mixing uniformly to obtain a mixed solution.

5. And (4) taking the EP pipe completing the step (3), and adding the mixed liquid obtained in the step (4).

6. The sample tube that completed step 5 was taken, 1mL of glacial acetic acid was added, washed upside down, and then transferred to the EP tube that completed step 5.

7. The sample tube that completed step 6 was taken, 1mL of glacial acetic acid was added, washed upside down, and then transferred to the EP tube that completed step 6.

8. After completion of step 7, the EP tube was carefully inverted and mixed, water-washed at 42 ℃ for 30min, then centrifuged at 4 ℃ and 4000rpm for 15min, and the supernatant was collected.

9. Preparation of phosphorus Standard Curve

(1) Respectively taking KH₂PO₄Stock solution of standard substance (i.e. KH)₂PO₄Aqueous standard solution at a concentration of 1mM) 0. mu.L, 5. mu.L, 10. mu.L, 20. mu.L, 40. mu.L, 60. mu.L, 80. mu.L and 100. mu.L with ddH₂Supplementing O to 300 μ L, and adding 700 μ L to develop colorThe working solution was mixed by inversion to give dilutions of phosphorus at concentrations of 0. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 60. mu.M, 80. mu.M and 100. mu.M in this order.

(2) Taking the diluent obtained in the step (1), and carrying out water bath at 42 ℃ for 30min to obtain a reaction solution.

(3) After completion of

step

2, 200. mu.L of the reaction solution was placed on an microplate and the absorbance at 820nm was measured using a microplate reader.

And (4) drawing a standard curve by taking the concentration of the diluent as an abscissa and the corresponding light absorption value as an ordinate.

10. Determination of inorganic phosphorus content by vanadium molybdenum blue method

(1) And (3) adding 350 mu L of the color reaction working solution into 150 mu L of the supernatant collected in the step (8), reversing and uniformly mixing, and carrying out water bath at 42 ℃ for 30min to obtain a reaction solution 1.

(2) 200 mu L of the reaction solution 1 is placed on an ELISA plate, and the light absorption value at 820nm is detected by using an ELISA reader.

And (4) calculating the inorganic phosphorus content in the supernatant collected in the step (8) according to the standard curve, and further obtaining the inorganic phosphorus content in the sample.

The results of some of the experiments are shown in FIG. 3A (6L for the 6 th leaf, 7L for the 7 th leaf, 8L for the 8 th leaf, 9L for the 9 th leaf, 10L for the 10 th leaf). The results show that the inorganic phosphorus content of the young leaves and stems of OE-1 and OE-2 is obviously higher than that of the maize inbred line B73 in the maize seedling stage. Thus, the inorganic phosphorus content of the young leaves and stems of OE-1 and OE-2 is significantly increased compared with that of the inbred corn line B73 at the seedling stage of corn.

(IV) detection of Total phosphorus content in transgenic maize

(1) Sowing seeds to be detected to an experimental base of a farm institute of Jilin province, Guillain-public-Main Ling, Jilin province, conventionally culturing in a field, and taking lower leaves, upper leaves, ear leaves, tassels and female ears as samples after the seeds grow to a pollen scattering period.

(2) And (3) after the step (1) is finished, taking samples (lower leaves, upper leaves, ear leaves, tassels or female ears), drying in an oven at 80 ℃ to constant weight, putting into a dryer for cooling, weighing and recording the dry weight value.

(3) After the step (2) is finished, taking a sample, carefully putting the sample into a crucible, carbonizing the sample in a muffle furnace at 300 ℃ for 10h, adjusting the temperature to 575 ℃ for ashing for 14h, cooling, and adding a proper amount of 0.1N hydrochloric acid to dissolve the sample overnight.

(4) And (4) taking the overnight dissolved sample obtained in the step (3), and detecting according to the method in the step (III) 3 to the step (10) to obtain the total phosphorus content of the sample.

Part of the experimental results are shown in fig. 3B. The results show that the total phosphorus content of ear position leaves and the female ears of OE-1 and OE-2 is obviously higher than that of the maize inbred line B73 in the pollen scattering period. Therefore, in the powder scattering period, compared with the maize inbred line B73, the total phosphorus content of ear position leaves and female ears of OE-1 and OE-2 is obviously increased; the increase of the expression level of the ZmPT3 gene can promote the transfer of phosphorus to the corn growth center.

<110> university of agriculture in China

Application of <120> protein ZmPT3 in regulation and control of phosphorus content in plants

<160>2

<170>PatentIn version 3.5

<210>1

<211>535

<212>PRT

<213> corn (Zea mays L.)

<400>1

Met Ala His Asp His Lys Val Leu Asp Ala Leu Asp Ala Ala Lys Thr

1 5 10 15

Gln Trp Tyr His Phe Thr Ala Val Val Val Ala Gly Met Gly Phe Phe

20 25 30

Thr Asp Ala Tyr Asp Leu Phe Ser Ile Ser Leu Val Thr Lys Leu Leu

35 40 45

Gly Arg Ile Tyr Tyr Ser Asp Pro Ser Ser Lys Thr Pro Gly Ser Leu

50 55 60

Pro Pro Asn Val Ser Ala Ala Val Asn Gly Val Ala Phe Cys Gly Thr

65 70 75 80

Leu Ala Gly Gln Leu Phe Phe Gly Trp Leu Gly Asp Lys Met Gly Arg

85 90 95

Lys Lys Val Tyr Gly Met Thr Leu Met Leu Met Ala Ile Cys Cys Leu

100 105 110

Ala Ser Gly Leu Ser Phe Gly Ser Thr Pro Lys Asp Val Met Val Thr

115 120 125

Leu Cys Phe Phe Arg Phe Trp Leu Gly Val Gly Ile Gly Gly Asp Tyr

130 135 140

Pro Leu Ser Ala Thr Ile Met Ser Glu Tyr Ala Asn Lys Arg Thr Arg

145 150 155 160

Gly Ala Phe Ile Ala Ala Val Phe Ala Met Gln Gly Phe Gly Asn Leu

165 170 175

Ala Gly Gly Ile Val Ala Ile Ala Val Ser Ala Ala Phe Lys Ser Arg

180 185 190

Phe Asp Ala Pro Ala Tyr Lys Asp Asp Pro Ala Gly Ser Thr Val Pro

195 200 205

Gln Ala Asp Tyr Val Trp Arg Ile Val Leu Met Phe Gly Ala Val Pro

210 215 220

Ala Leu Leu Thr Tyr Tyr Trp Arg Met Lys Met Pro Glu Thr Ala Arg

225 230 235 240

Tyr Thr Ala Leu Val Ala Lys Asn Ala Lys Gln Ala Thr Ser Asp Met

245 250 255

Ala Arg Val Leu Asp Val Asp Leu Ala Glu Glu Arg Gln Lys Pro Val

260 265 270

Glu Glu Leu Glu Arg Arg Arg Glu Glu Phe Gly Leu Phe Ser Arg Gln

275 280 285

Phe Ala Lys Arg His Gly Leu His Leu Leu Gly Thr Thr Val Cys Trp

290 295 300

Phe Thr Leu Asp Ile Ala Phe Tyr Ser Gln Asn Leu Phe Gln Lys Asp

305 310 315 320

Met Tyr Ala Ala Val Asn Trp Leu Pro Arg Ala Asp Thr Met Asn Ala

325 330 335

Leu Glu Glu Met Phe Arg Ile Ser Arg Ala Gln Thr Leu Val Ala Leu

340 345 350

Cys Gly Thr Ile Pro Gly Tyr Trp Phe Thr Val Phe Phe Ile Asp Ile

355 360 365

Val Gly Arg Phe Ala Ile Gln Leu Gly Gly Phe Phe Phe Met Thr Ala

370 375 380

Phe Met Leu Gly Leu Ala Ile Pro Tyr His His Trp Thr Thr Pro Gly

385 390 395 400

His His Val Gly Phe Val Val Met Tyr Ala Leu Thr Phe Phe Phe Ala

405 410 415

Asn Phe Gly Pro Asn Ser Thr Thr Phe Ile Val Pro Ala Glu Ile Phe

420 425 430

Pro Ala Arg Leu Arg Ser Thr Cys His Gly Ile Ser Ser Ala Ala Gly

435 440 445

Lys Cys Gly Ala Ile Val Gly Ser Phe Gly Phe Leu Tyr Ala Ala Gln

450 455 460

Ser Thr Asp Pro Thr Lys Thr Asp Ser Gly Tyr Pro Pro Gly Ile Gly

465 470 475 480

Val Arg Asn Ser Leu Phe Met Leu Thr Gly Cys Asn Val Ile Gly Phe

485 490 495

Leu Phe Thr Phe Leu Val Pro Glu Ser Lys Gly Lys Ser Leu Glu Glu

500 505 510

Leu Ser Gly Glu Asn Asp Glu Glu Ala Ala Pro Gln Gln Gln Gln Thr

515 520 525

Val Pro Thr Asp Leu Ser Glu

530 535

<210>2

<211>1608

<212>DNA

<213> corn (Zea mays L.)

<400>2

atggcacacg atcacaaggt gctggacgcg ctggacgcag ccaagacaca gtggtaccac 60

ttcacggcgg tggtggtcgc cggcatgggc ttcttcaccg atgcgtacga cctcttctcc 120

atctccctcg tcaccaagct cctcggccgc atctactact cggaccccag ctccaagacc 180

cccggctccc tcccgcccaa cgtctccgca gccgtcaacg gcgtcgcctt ctgcggcacg 240

ctcgccgggc agctcttctt cggctggctc ggcgacaaga tgggccgcaa gaaggtgtac 300

gggatgacgc tcatgctcat ggccatctgc tgcctcgcct ccggcctctc gttcgggtcc 360

acgcccaagg acgtcatggt cacgctctgc ttcttccgct tctggctcgg cgtcggcatc 420

ggcggcgact acccgctgtc cgcgaccatc atgtccgagt acgccaacaa acggacgcgc 480

ggcgcattca tcgcggccgt cttcgccatg cagggcttcg gcaacctcgc cggcggcatc 540

gtcgccattg ccgtctcggc ggcgtttaag tcccgcttcg acgcgccggc gtacaaggac 600

gaccccgccg gctccacggt gccacaggcc gactacgtgt ggcgcatcgt cctcatgttc 660

ggcgccgtcc cggctctgct cacctactac tggcgcatga agatgcccga gacggcgcgg 720

tacaccgcgc tggtcgccaa gaacgccaag caggccacgt ccgacatggc gcgtgtgctc 780

gacgtcgacc tggccgagga gcggcagaag ccggtagagg agttggagcg ccgccgcgag 840

gagttcggtc tcttctcccg ccagttcgcg aagcggcatg gcctccacct gctgggcacg 900

acggtgtgct ggttcacgct ggacatcgcc ttctactcgc agaacctgtt ccagaaggac 960

atgtacgccg ccgtgaactg gctgccgagg gcggacacca tgaacgccct ggaggagatg 1020

ttcaggatct cccgcgcgca gacgctcgtc gcgctgtgcg gcaccatccc gggctactgg 1080

ttcaccgtct tcttcatcga catcgtcggc cgcttcgcca tccagctcgg cggcttcttc 1140

ttcatgacgg cgttcatgct gggcctcgcc atcccgtacc accactggac gaccccaggg 1200

caccacgtcg gcttcgtcgt catgtacgcc ctcactttct tcttcgccaa cttcgggccc 1260

aactccacca ccttcatcgt gccggcggag atcttcccag caaggctgcg gtccacctgc 1320

cacggtattt cttcagctgc aggaaagtgt ggcgccattg tcgggtcatt tgggttcctg 1380

tacgcggcgc agagcacgga tcccaccaag acggactccg ggtacccgcc gggcatcgga 1440

gtgcgcaact cgctgttcat gctcaccgga tgcaatgtta tcggtttcct gttcacgttc 1500

ctggtgccgg agtccaaggg aaaatcgctg gaagagctct ccggcgagaa cgacgaggag 1560

gcagcaccgc agcaacagca gaccgtgccg acagatttga gtgaataa 1608

Claims

1. The application of the protein ZmPT3 is at least one of the following X1) to X6);

x1) regulating the phosphorus content of the plant;

x2) regulating the growth of a plant;

x3) regulating the biomass of the plant;

x4) regulating phosphorus element transfer in plants;

x5) regulating phosphorus uptake of plants under low phosphorus conditions;

x6) cultivating plants with increased phosphorus content and/or improved growth and/or increased biomass and/or improved phosphorus element transfer to the growth center and/or low phosphorus tolerance;

the protein ZmPT3 is a1) or a2) or a 3):

2. Use of a nucleic acid molecule encoding the protein ZmPT3 of claim 1, which is at least one of X1) to X6);

x1) regulating the phosphorus content of the plant;

x2) regulating the growth of a plant;

x3) regulating the biomass of the plant;

x4) regulating phosphorus element transfer in plants;

x5) regulating phosphorus uptake of plants under low phosphorus conditions;

3. Use according to claim 2, characterized in that: the nucleic acid molecule encoding the protein ZmPT3 of claim 1 is a DNA molecule represented by b1) or b2) or b3) or b4) as follows:

b3) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined in b1) or b2), derived from maize and encoding the protein ZmPT3 according to claim 1;

b4) a DNA molecule which is derived from maize and which codes for the protein ZmPT3 as claimed in claim 1, which hybridizes under stringent conditions with the nucleotide sequence defined under b1) or b 2).

4. Use according to any one of claims 1 to 3, wherein: regulating the phosphorus content of the plant to increase the phosphorus content of the plant; regulating the growth of the plant to promote the growth of the plant; the biomass of the plant is regulated and controlled to increase the biomass of the plant; regulating and controlling the phosphorus transfer of the plant to promote the phosphorus transfer of the plant; the regulation and control of phosphorus uptake of plants under low phosphorus conditions is to promote phosphorus uptake of plants under low phosphorus conditions.

5. Use according to any one of claims 1 to 4, wherein: the phosphorus content is inorganic phosphorus content and/or total phosphorus content; the biomass is root dry weight and/or crown dry weight; the phosphorus element transfer is the transfer of phosphorus element to the growth center.

6. A method for producing a transgenic plant, comprising the step of introducing a substance which increases the expression level and/or activity of the protein ZmPT3 according to claim 1 into a recipient plant to obtain a transgenic plant;

(d1) (ii) a phosphorus content higher than that of the recipient plant;

(d2) a biomass higher than the recipient plant;

(d3) (ii) a low phosphorus tolerance greater than that of the recipient plant;

(d4) growing faster than the recipient plant;

7. The method of claim 6, wherein: the "introduction of a substance that increases the expression level and/or activity of the protein ZmPT3 into a recipient plant" is achieved by introducing a nucleic acid molecule encoding the protein ZmPT3 into a recipient plant.

8. A method of plant breeding comprising the steps of: increasing the expression level and/or activity of the protein ZmPT3 as defined in claim 1 in a plant, thereby increasing the phosphorus content and/or biomass and/or low phosphorus tolerance and/or growth rate and/or the extent of phosphorus transfer to the growth center of the plant.

9. The method of any of claims 6 to 8, wherein: the phosphorus content is inorganic phosphorus content and/or total phosphorus content; the biomass is root dry weight and/or crown dry weight.

10. The use according to any one of claims 1 to 5, or, the method according to any one of claims 6 to 9, wherein: the plant is c1) or c2) or c3) or c4) or c5) or c 6): c1) a dicotyledonous plant; c2) a monocot plant; c3) corn; c4) maize inbred line B73; c5) a cruciferous plant; c6) arabidopsis thaliana.