CN111978384B

CN111978384B - Application of protein PNR1 in cultivating phosphorus nutrition efficient plant variety

Info

Publication number: CN111978384B
Application number: CN201910422464.4A
Authority: CN
Inventors: 陈益芳; 武维华; 王雪
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2022-11-01
Anticipated expiration: 2039-05-21
Also published as: CN111978384A

Abstract

The invention discloses application of protein PNR1 in cultivating phosphorus nutrition high-efficiency plant varieties. The invention also discloses an application of the PNR1 protein in at least one of the following (1) to (9): (1) regulating and controlling the phosphorus content of plants; (2) increasing the phosphorus content of plants; (3) regulating and controlling the phosphorus absorption rate of plants; (4) improving the phosphorus absorption rate of plants; (5) promoting plant phosphorus accumulation and/or phosphorus absorption; (6) regulating and controlling the growth and development of plants; (7) promoting the growth and development of plants; (8) regulating plant yield and/or quality; (9) improving the yield and/or quality of the plant. Experiments prove that: the PNR1 protein and the coding gene thereof have the function of regulating and controlling plant phosphorus absorption, and have 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 the technical means of genetic engineering.

Description

Application of protein PNR1 in cultivating phosphorus nutrition efficient plant variety

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a protein PNR1 in cultivation of phosphorus nutrition efficient plant varieties.

Background

Phosphorus is one of major elements required for plant growth and development, accounts for about 0.05-0.5% of the dry weight of the plant, is an important component of various substances such as nucleic acid, nucleoprotein, phospholipid and the like, and participates in the physiological processes of energy metabolism, substance metabolism and the like of the plant. The available phosphorus concentration in the soil solution is extremely low, and about 70% of agricultural planting areas worldwide are in a phosphorus-deficient state. When the plant is subjected to low phosphorus stress, the division and differentiation of plant cells are inhibited, the plant is short and small, the anthocyanin is excessively accumulated, the leaves of the plant are dark green or purple red, the flowering and seed formation of the plant are inhibited, and the growth and yield quality of the plant/crop are seriously influenced. In order to ensure the yield, phosphate fertilizers are applied in large quantities. After the phosphate fertilizer is applied to soil, the phosphate fertilizer has poor mobility in the soil, is easy to form insoluble salt with metal ions in the soil, or is converted into organic phosphorus which cannot be absorbed by plants by microorganisms, so that the utilization efficiency of the phosphate fertilizer is low, and the current utilization rate of the phosphate fertilizer is lower than 30%. Therefore, the method improves the phosphorus absorption and utilization efficiency of plants through molecular genetic breeding, is beneficial to improving the yield and quality of crops, can relieve the environmental and energy pressure, and has important significance in the aspects of production and environmental protection.

Disclosure of Invention

The invention aims to provide application of PNR1 protein and related biological materials thereof in cultivating plant varieties with high phosphorus nutrition and high efficiency.

In one aspect, the invention protects a new use of the PNR1 protein.

The invention provides an application of PNR1 protein in at least one of the following (1) to (9):

(1) Regulating and controlling the phosphorus content of plants;

(2) The phosphorus content of the plant is improved;

(3) Regulating and controlling the plant phosphorus absorption rate;

(4) Improving the phosphorus absorption rate of plants;

(5) Promoting plant phosphorus accumulation and/or phosphorus absorption;

(6) Regulating and controlling the growth and development of plants;

(7) Promoting the growth and development of plants;

(8) Regulating and controlling plant yield and/or quality;

(9) Increasing plant yield and/or quality;

the PNR1 protein is a protein shown as a) or b) or c) or d) as follows:

a) The amino acid sequence is a protein shown in a sequence 2;

b) A fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2;

c) The protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2;

d) A protein having homology of 75% or more than 75% with the amino acid sequence shown in the sequence 2 and having the same function;

in order to facilitate the purification of the protein in a), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 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
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of c) above, 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 in the c) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene encoding the protein of c) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 1, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 to the 5 'end and/or 3' end thereof.

In the above d), "homology" includes an amino acid sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology with the amino acid sequence represented by the sequence 2 of the present invention.

In another aspect, the invention protects a new use of a biological material associated with a PNR1 protein.

The invention provides application of biological materials related to PNR1 protein in at least one of the following (1) to (9):

(1) Regulating and controlling the phosphorus content of the plant;

(2) The phosphorus content of the plant is improved;

(3) Regulating and controlling the plant phosphorus absorption rate;

(4) Improving the phosphorus absorption rate of plants;

(5) Promoting plant phosphorus accumulation and/or phosphorus absorption;

(6) Regulating and controlling the growth and development of plants;

(7) Promoting the growth and development of plants;

(8) Regulating and controlling plant yield and/or quality;

(9) Increasing plant yield and/or quality;

(10) Cultivating a transgenic plant variety with improved phosphorus content or phosphorus absorption rate or high phosphorus nutrition efficiency;

(11) And (5) plant breeding.

In the above application, the biomaterial is any one of the following A1) to a 12):

a1 A nucleic acid molecule encoding a PNR1 protein;

a2 An expression cassette comprising the nucleic acid molecule according to A1);

a3 A recombinant vector containing the nucleic acid molecule according to A1);

a4 A recombinant vector containing the expression cassette of A2);

a5 A recombinant microorganism containing the nucleic acid molecule according to A1);

a6 A recombinant microorganism containing the expression cassette of A2);

a7 A recombinant microorganism containing the recombinant vector of A3);

a8 A recombinant microorganism containing the recombinant vector of A4);

a9 A transgenic plant cell line containing the nucleic acid molecule according to A1);

a10 A transgenic plant cell line containing the expression cassette of A2);

a11 A transgenic plant cell line containing the recombinant vector of A3);

a12 A transgenic plant cell line containing the recombinant vector of A4).

A1 The nucleic acid molecule is a gene shown in the following 1) or 2) or 3):

1) The coding sequence is cDNA molecule or genome DNA molecule shown in sequence 1;

2) A cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes PNR1 protein;

3) A cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and encodes PNR1 protein.

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.

The nucleotide sequence encoding the PNR1 protein of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence encoding the PNR1 protein are derived from and identical to the nucleotide sequence of the present invention as long as they encode the PNR1 protein and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above application, the stringent conditions are hybridization and washing of the membrane at 68 ℃ 2 times, 5min each, in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of the membrane at 68 ℃ 2 times, 15min each, in a solution of 0.5 XSSC, 0.1% SDS; alternatively, 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS in a medium, and the membrane is washed at 65 ℃.

In the above application, the expression cassette containing a nucleic acid molecule encoding a PNR1 protein according to A2) refers to a DNA capable of expressing the PNR1 protein in a host cell, and the DNA may include not only a promoter for promoting transcription of PNR1 but also a terminator for terminating transcription of PNR1. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; tissue, organ and development specific promoters and inducible promoters.

The recombinant vector containing the PNR1 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co., ltd.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translational control signals and initiation codons are wide ranging from natural to synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transformed plants can be screened directly in stress without adding any selective marker gene.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid may be pCXSN.

In the above application, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium. The agrobacterium may be GV3101.

In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

In the application, the breeding aim is to cultivate high-efficiency plant varieties.

The invention also protects a cultivation method of the transgenic plant with high phosphorus nutrition efficiency.

The cultivation method of the transgenic plant with high phosphorus nutrition and high efficiency provided by the invention comprises the steps of improving the expression quantity and/or activity of PNR1 protein in a receptor plant to obtain the transgenic plant; the transgenic plant has a higher phosphorus content and/or a higher phosphorus uptake rate than the recipient plant.

In the above method, the method for increasing the expression level and/or activity of the PNR1 protein in the recipient plant comprises overexpressing the PNR1 protein in the recipient plant;

the overexpression method may be to introduce a gene encoding the PNR1 protein into a recipient plant.

The nucleotide sequence of the coding gene of the PNR1 protein is a DNA molecule shown in a sequence 1.

In the above method, the coding gene of the PNR1 protein is introduced into a recipient plant through a recombinant expression vector, specifically, a plant cell or tissue can be transformed by using a conventional biological method such as Ti plasmid, ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissue is cultured into a plant.

In a specific embodiment of the invention, the coding gene of the PNR1 protein is introduced into a recipient plant through a recombinant expression vector 35s, wherein the recombinant expression vector 35s is obtained by inserting the PNR1 gene shown in sequence 1 into an enzyme cutting site (such as an XcmI enzyme cutting site) of a vector pCXSN.

In the above method, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the PNR1 gene into a recipient plant, but also its progeny. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In any of the above applications or methods, the phosphorus nutrient efficiency is high phosphorus absorption rate and/or high phosphorus transfer rate and/or high phosphorus content.

In any of the above applications or methods, the plant is a monocot or a dicot.

Further, the dicotyledonous plant is preferably arabidopsis thaliana; the monocot is preferably maize.

Further, the Arabidopsis thaliana is Columbia type Arabidopsis thaliana (Col-0); the corn is a corn inbred line B73.

Experiments prove that: the PNR1 protein and the coding gene thereof have the function of regulating and controlling plant phosphorus absorption, and have 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 the technical means of genetic engineering.

Drawings

FIG. 1 is the identification of PNR1 Arabidopsis thaliana.

FIG. 2 shows the phosphorus content measurement of PNR1 Arabidopsis thaliana.

FIG. 3 is a graph showing the determination of the phosphorus uptake rate of PNR1 Arabidopsis thaliana.

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.

The vector pCXSN in the following examples is described in the document "Chen et a., A versatile zero background T-vector system for gene cloning and functional genes, plant physiology,2009, 1111-1121", publicly available from the Applicant, this biomaterial being only used for the repetition of the experiments related to the present invention and not for other uses.

Agrobacterium GV3101 in the following examples is described in the document "Lee et al," Agrobacterium tumefaciens proteins structures modulation by modulation method in Arabidopsis thaliana. Plant Cell,2009,21, 2948-2962 ", which is publicly available from the Applicant and is only used for repetition of experiments relating to the present invention and not for other uses.

Agrobacterium EHA105 in the following examples is described in the literature "Nyabogaetal, agrobacterium-mediated genetic transformation of yam (Dicocorarotound data)" an antigenic tool for functional study of genes and crop improvement. Fronditiors in Plant science.2014,5 463 ", which is publicly available from the applicant and is only used for repeating experiments relevant to the present invention and not for other uses.

Method for preparing MS liquid medium (Pi concentration 1.25 mM) in the following examples: mixing 1650mg NH₄NO₃、1900mg KNO₃、370mg MgSO₄·7H₂O、170mg KH₂PO₄、440mg CaCl₂·2H₂O、22.3mg MnSO₄·4H₂O、0.83mg KI、0.025mg CuSO₄·5H₂O、6.25mg H₃BO₅、0.025mg CoCl·6H₂O、8.65mg ZnSO₄·7H₂O、0.25mg Na₂MoO₄·2H₂O、27.8mg FeSO₄·7H₂O and 37.3mg Na₂EDTA dissolved in water and made up to 1L. 8g of agar powder per liter was added to the MS solid medium.

The wild type Arabidopsis thaliana (WT) in the following examples is Columbia type Arabidopsis thaliana (Col-0).

Example 1 acquisition of PNR1 protein and Gene encoding the same

Total RNA (100-200 mg) from wild type Arabidopsis seedlings was extracted by the TRizol (Invitrogen) method and checked for RNA integrity by formaldehyde-denatured RNA agarose gel electrophoresis. According to SUPERSCRIPT^IIThe instructions of (4) synthesize single-stranded cDNA. The synthesized single-stranded cDNA was diluted 5-fold and used as a template DNA, using Primer1And Primer2 were used to perform PCR reactions.

Primer1：5'-ATGATGGTGGAGATGGATTACGC-3'；

Primer2：5'-TTATGACACAGGAGTAGAAGTATTTG-3'。

Reaction system for PCR amplification (50 μ L): 10 μ L of 5 XPisuion HF Buffer,4 μ L of 2.5mM dNTP mix,2.5 μ L of Primer1 (10 μ M), 2.5 μ L of Primer2 (10 μ M), 1 μ L of template DNA,1.5 μ L of DMSO,0.5 μ L of Phusion DNA Polymerase (2U/. Mu.L), and the balance water.

Reaction procedure for PCR amplification: pre-denaturation at 98 ℃ for 3min; 15s at 98 ℃, 30s at 60 ℃, 40s at 72 ℃ and 35 cycles; extension at 72 ℃ for 10min.

And recovering a PCR product of about 1065bp, 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 1 of a sequence table and encodes a protein shown in a sequence 2 of the sequence table.

The protein shown in the sequence 2 of the sequence table is named as PNR1 protein. The coding gene of the PNR1 protein is named as PNR1 gene, and the open reading frame is shown as sequence 1 in a sequence table.

Example 2 acquisition and characterization of PNR1 transgenic Arabidopsis

1. Construction of recombinant vectors

1. Synthesizing a double-stranded DNA molecule shown in a sequence 1 in the sequence table, taking the double-stranded DNA molecule as a template DNA, and performing PCR amplification by adopting a Primer pair consisting of a Primer1 and a Primer2 to obtain a PCR amplification product.

Primer1：5'-ATGATGGTGGAGATGGATTACGC-3'；

Primer2：5'-TTATGACACAGGAGTAGAAGTATTTG-3’。

2. The blunt end of the PCR amplification product obtained in step 1 was A-supplemented with Taq enzyme to have an A-sticky end.

3. The vector pCXSN was digested with the restriction enzyme XcmI, linearized and provided with T-sticky ends, and the vector backbone was recovered.

4. And (3) connecting the supplement A product obtained in the step (2) with the vector framework recovered in the step (3) by a TA cloning method to obtain a recombinant vector 35S. And sequencing recombinant vector 35s. The recombinant vector 35s was structurally described according to the sequencing results as follows: double-stranded DNA molecules shown in a sequence 1 in a sequence table are inserted between enzyme cutting sites XcmI of a vector pCXSN.

2. Obtaining of transgenic PNR1 Arabidopsis

1. The recombinant vector 35S.

2. The recombinant Agrobacterium obtained in step 1 was used to infect Columbia type Arabidopsis thaliana by the Floral bud soaking method (Clough and Bent, floral dip: a amplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal,1998,16, 735-743.₁And (5) seed generation. T is₂Generation represents T₁Seeds produced by generation selfing and plants grown from them, T₃Generation represents T₂Seeds produced by generation selfing and plants grown from the seeds. Screening for T on MS solid Medium plates containing 50. Mu.g/L hygromycin₁Plant generation and T₂Generation and T₃Segregation ratio statistics of generations at T₃Two PNR1 Arabidopsis thaliana single copy homozygous lines are obtained by generation: 35S.

3. Identification of transgenic PNR1 Arabidopsis

Respectively combine T with₃Generation-transfer PNR1 Arabidopsis strain 35S₃The transgenic PNR1 Arabidopsis line 35S and wild type Arabidopsis plants were identified as follows: the Trizol method is used for extracting total RNA of plants and carrying out reverse transcription to obtain cDNA, a Primer pair consisting of Primer3 and Primer4 (Primer 3:5'-GTCGATTCCTAAACGCGCTT-3'; primer4: 5'-ATATGCCTCCAACCACCACA-3') is adopted for qRT-PCR expression quantity detection, and ACTIN2/8 is used as an internal reference gene. The method comprises the following specific steps:

1. total RNA was first treated by dnase digestion. Reaction system (10 μ L): contains total RNA 8 μ g,10 XDaseI buffer 1 μ L, RNase-free DNaseI 1 μ L, DEPC-H₂And O is supplemented to 10 mu L. The reaction was carried out at 37 ℃ for 20 minutes and at 65 ℃ for 10 minutes, and the reaction mixture was placed on ice.

2. The RNA was reverse transcribed into cDNA using a reverse transcription kit. Reaction system (20 μ L): containing 4. Mu.g total RNA, 1. Mu.L Random primers, 1. Mu.L 2.5mM dNTPs, DEPC-H₂O is supplemented to 13 mu L. Denaturation at 65 ℃ for 5min, cooling immediately on ice for 5min, and then adding the following components in sequence to 20 μ L:5 XFirst-Strand buffer 4. Mu.L, 0.1M DTT 2. Mu.L, superScript^TMII Reverse Transcriptase 1. Mu.L. 20 mu L of sample is fully and evenly mixed, reaction is carried out for 15min at 25 ℃, reaction is carried out for 50min at 42 ℃, reaction is carried out for 15min at 70 ℃, and reverse transcription products are frozen at-20 ℃ or qRT-PCR experiments are carried out.

The results are shown in FIG. 1. The results show that: t is₃Generation-transfer PNR1 Arabidopsis strain 35S₃The PNR1 gene expression level in the transgenic PNR1 Arabidopsis strain 35S is obviously higher than that of a wild Arabidopsis plant.

Example 3 physiological Properties of PNR1 transgenic Arabidopsis

1. Phosphorus content index detection

1. Respectively combine T with₃Generation-transfer PNR1 Arabidopsis strain 35S₃The transgenic PNR1 Arabidopsis line 35S and wild type Arabidopsis seeds are sown on a 1/2MS solid medium plate for germination and growth for 10 days, and then the whole plant is taken.

2. Taking the materials obtained in the step 1, recording the fresh weight value of each group of materials, freezing and storing in liquid nitrogen, crushing the samples by using a grinder, and adding 1% glacial acetic acid into a10 mL EP tube in advance for later use. Inorganic phosphorus extract (1L formulation of inorganic phosphorus extract: 1M Tris-HCl (pH 8.0) 10mL,0.5M EDTA (pH 8.0) 2mL, naCl 5.844g, beta-mercaptoethanol 700. Mu.L, 100mM PMSF (as-prepared) 10 mL) was added to the sample tube at a ratio of 100. Mu.L of extract to 10mg of sample. For example, a 70mg sample was added with 700. Mu.L of the extract solution, and the mixture was inverted and mixed. The sample was drawn into an EP tube containing 4.3mL of glacial acetic acid using a gun. Since the original sample tube is stained with the sample, 1mL of glacial acetic acid is sucked into the sample tube by the gun, the EP tube is cleaned by reversing the upper part and the lower part, the sample is sucked into the 10mL of EP tube, and the cleaning is repeated twice, wherein the volume of the glacial acetic acid in the 10mL of EP tube is 6.3mL. The mixture was carefully mixed by inversion and reacted in a water bath at 42 ℃ for 30min. Centrifuge at 4000rpm for 15min at 4 ℃.

3. And (3) taking the leaching liquor obtained in the step (2), and carrying out phosphorus content determination by using a vanadium molybdenum blue method. Color development: 150. Mu.L of the supernatant was aspirated, added to 350. Mu.L of a developing solution, and mixed by inversion. Meanwhile, a standard curve is prepared, and the reaction is carried out in water bath at 42 ℃ for 30min. And (3) light absorption value determination: and (3) sucking 200 mu L of the reacted solution into an enzyme label plate, and measuring at the wavelength of 820nm by a continuous enzyme label instrument.

The results are shown in FIG. 2. The measurement results showed that T₃Generation-transfer PNR1 Arabidopsis strain 35S₃The content of inorganic phosphorus in the transgenic PNR1 Arabidopsis strain 35S is obviously higher than that of wild Arabidopsis, which shows that the improvement of PNR 1expression is beneficial to phosphorus accumulation of plants (Arabidopsis).

2. Phosphorus absorption Rate index detection

Respectively combine T₃Generation-transfer PNR1 Arabidopsis strain 35S₃Transgenic PNR1 Arabidopsis line 35S PNR1-23 and wild type Arabidopsis seeds were sown on MS solid medium plates and cultured until germination, and seedlings that grew for 7 days were divided into 3 groups (15 plants each, one in parallel) and subjected to germination³²P absorption Rate detection, the detection procedure can be referred to in the literature "Wang et al, 2014, arabidopsis WRKY45transformation factors activated PHOSPHATE TRANSPORTER1;1expression in response to phosphate actuation. Plant Physiology 164 "2020-2029". The method comprises the following specific steps:

preparation in the early stage of the experiment:

(1) pre-treatment liquid is packaged in 2mL of EP tubes in advance, and 1.5mL of the pre-treatment liquid is added into each tube;

(2) preparing absorption liquid, adding H into the absorption liquid₃PHO₄The final concentration of the labeled isotope was 0.2Ci/mL. 1.5mL of absorbent solution was added to each sample.

The experimental process comprises the following steps:

(1) material taking: putting the pre-treatment liquid prepared in advance on a balance, returning to zero, putting 15 seedlings into the pre-treatment liquid, weighing on the balance, recording the weight, and pre-treating for 20-30min, wherein 3 seedlings are parallel to each other;

(2) absorption: according to the sequence of putting the materials into the pretreatment liquid, the pretreatment liquid is sucked out by a gun at a constant speed in sequence, and then 1.5mL of absorption liquid is added in sequence for 4 hours and 6 hours.

(3) Rinsing: the absorption liquid is sequentially sucked out by a gun according to the sequence of adding the absorption liquid, then 1.5mL of precooled desorbent is added, the mixture is placed for 30-45min, and the washing is repeated twice.

(4) Drying the material: and (3) taking out the washed seedlings by using tweezers, sucking surface liquid by using absorbent paper, putting the seedlings into a new 1.5mL EP tube, sucking 20 mu L of absorption liquid serving as an internal reference, opening the cover, putting the box into a radiation-proof box, and drying the box in a 65 ℃ oven.

(5) And (3) determination: adding 1mL of scintillation fluid into the baked material to ensure that all seedlings are immersed in the scintillation fluid, measuring on a scintillator with the model of HIDEX300SL, and calculating the phosphorus absorption rate by using the measured DPM.

The results are shown in FIG. 3. The measurement results showed that T₃Generation-transfer PNR1 Arabidopsis strain 35S₃The phosphorus absorption rate of the transgenic PNR1 Arabidopsis strain 35S is obviously higher than that of wild Arabidopsis, which shows that the improvement of PNR 1expression is beneficial to phosphorus absorption of plants (Arabidopsis).

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> university of agriculture in China

Application of <120> protein PNR1 in cultivation of phosphorus nutrition efficient plant variety

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<170>PatentIn version 3.5

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Tyr Val Glu Ala Leu Glu Glu Glu Gln Lys Lys Ile Gln Val Phe Gln

20 25 30

Arg Glu Leu Pro Leu Cys Leu Glu Leu Val Thr Gln Ala Ile Glu Ala

35 40 45

Cys Arg Lys Glu Leu Ser Gly Thr Thr Thr Thr Thr Ser Glu Gln Cys

50 55 60

Ser Glu Gln Thr Thr Ser Val Cys Gly Gly Pro Val Phe Glu Glu Phe

65 70 75 80

Ile Pro Ile Lys Lys Ile Ser Ser Leu Cys Glu Glu Val Gln Glu Glu

85 90 95

Glu Glu Glu Asp Gly Glu His Glu Ser Ser Pro Glu Leu Val Asn Asn

100 105 110

Lys Lys Ser Asp Trp Leu Arg Ser Val Gln Leu Trp Asn His Ser Pro

115 120 125

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

130 135 140

Glu Val Lys Pro Lys Ser Gly Ala Phe Gln Pro Phe Gln Lys Arg Val

145 150 155 160

Leu Glu Thr Asp Leu Gln Pro Ala Val Lys Val Ala Ser Ser Met Pro

165 170 175

Ala Thr Thr Thr Ser Ser Thr Thr Glu Thr Cys Gly Gly Lys Ser Asp

180 185 190

Leu Ile Lys Ala Gly Asp Glu Glu Arg Arg Ile Glu Gln Gln Gln Ser

195 200 205

Gln Ser His Thr His Arg Lys Gln Arg Arg Cys Trp Ser Pro Glu Leu

210 215 220

His Arg Arg Phe Leu Asn Ala Leu Gln Gln Leu Gly Gly Ser His Val

225 230 235 240

Ala Thr Pro Lys Gln Ile Arg Asp His Met Lys Val Asp Gly Leu Thr

245 250 255

Asn Asp Glu Val Lys Ser His Leu Gln Lys Tyr Arg Leu His Thr Arg

260 265 270

Arg Pro Ala Ala Thr Ser Val Ala Ala Gln Ser Thr Gly Asn Gln Gln

275 280 285

Gln Pro Gln Phe Val Val Val Gly Gly Ile Trp Val Pro Ser Ser Gln

290 295 300

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

305 310 315 320

Pro Val Ala Val Ala Gln Ser Pro Lys Arg Ser Leu Glu Arg Ser Cys

325 330 335

Asn Ser Pro Ala Ala Ser Ser Ser Thr Asn Thr Asn Thr Ser Thr Pro

340 345 350

Val Ser

Claims

1. A method for cultivating transgenic plant with high phosphorus nutrition and high efficiency comprises the steps of increasing expression quantity and/or activity of PNR1 protein in receptor plant to obtain transgenic plant; the transgenic plant has a higher phosphorus content and/or higher phosphorus uptake rate than the recipient plant;

the method for improving the expression quantity and/or activity of the PNR1 protein in the receptor plant comprises the steps of over-expressing the PNR1 protein in the receptor plant;

the overexpression method is to introduce a coding gene of PNR1 protein into a receptor plant;

the coding gene of the PNR1 protein is expressed by a recombinant expression vector35S:PNR1Introduced into a recipient plant, said recombinant expression vector35S:PNR1To be shown in sequence 1PNR1The gene is inserted into a vector obtained by the enzyme cutting site of the vector pCXSN;

the recipient plant is Arabidopsis thaliana.