CN111978384A

CN111978384A - Application of protein PNR1 in cultivation of phosphorus nutrition efficient plant variety

Info

Publication number: CN111978384A
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: 2020-11-24
Anticipated expiration: 2039-05-21
Also published as: CN111978384B

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 the plant; (2) improving 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 and controlling plant yield and/or quality; (9) plant yield and/or quality is improved. 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 cultivation of 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 such as 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 efficiency.

In one aspect, the invention protects a novel 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 the plant;

(2) improving 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 and controlling plant yield and/or quality;

(9) increasing plant yield and/or quality;

the PNR1 protein is a protein shown in 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 biomaterial related to 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) improving 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 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 of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) A transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

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

1) the coding sequence is a cDNA molecule or a genome DNA molecule shown in a 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 codes 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 readily mutated by one 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 membrane washing 2 times at 68 ℃ for 5min in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing 2 times at 68 ℃ for 15min in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In the above applications, the expression cassette containing a nucleic acid molecule encoding PNR1 protein according to a2) refers to DNA capable of expressing 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 PNR 1. 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 translational control signals and initiation codons are widely derived, either naturally or synthetically. 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 transgenic plants can be directly screened and transformed in a stress environment 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 GV 3101.

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 method, the method for improving the expression amount 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 can be realized by introducing 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 can be cultivated into a plant.

In the specific embodiment of the invention, the coding gene of the PNR1 protein is introduced into a receptor plant through a recombinant expression vector 35S: PNR1, and the recombinant expression vector 35S: PNR1 is a vector obtained by inserting the PNR1 gene shown in the sequence 1 into an enzyme cutting site (such as 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 transgenic Arabidopsis thaliana, PNR 1.

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

FIG. 3 is a graph showing the determination of the phosphorus uptake rate of Arabidopsis thaliana transformed with PNR 1.

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 of the following examples is described in the literature "Chen et a., A versatile zero background T-vector system for gene cloning and functional genes plant physiology,2009,150:1111 1121", publicly available from the Applicant, the 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 literature "Lee et al, Agrobacterium tumefaciens proteins structures modulation by molecular modification plant Cell,2009,21: 2948-.

Agrobacterium EHA105 in the examples described below is described in the literature "Nyabogaiet, Agrobacterium-mediated genetic transformation of yam (microbiological research data)" an antigenic reagent for functional students and crop improvement. frontiers in Plant science 2014,5:463 ", publicly available from the applicant, this biomaterial being only used for repeating the experiments relevant to the present invention and not for other uses.

Method for preparing MS liquid medium (Pi concentration 1.25mM) 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 (100-200mg) 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 for synthesizing single-stranded cDNA. The synthesized single-stranded cDNA was diluted 5-fold and used as a template DNA, and PCR reaction was performed using a Primer pair consisting of Primer1 and Primer 2.

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 3 min; 15s at 98 ℃, 30s at 60 ℃, 40s at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min.

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 of the coding gene is shown as a sequence 1 in a sequence table.

Example 2 obtaining and characterization of transgenic Arabidopsis thaliana (PNR 1)

First, construction of recombinant vector

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

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 Primer 2(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 3 min; reacting at 98 ℃ for 15s, 60 ℃ for 30s and 72 ℃ for 40s, and performing 35 cycles; extension at 72 ℃ for 10 min.

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 complementary A product in the step (2) with the vector framework recovered in the step (3) by a TA cloning method to obtain the recombinant vector 35S: PNR 1. And the recombinant vector 35S PNR1 was sequenced. The structure of the recombinant vector 35S PNR1 is described as follows according to the sequencing result: double-stranded DNA molecules shown in a sequence 1 in a sequence table are inserted between enzyme cutting sites XcmI of a vector pCXSN.

Second, obtaining transgenic Arabidopsis thaliana (PNR 1)

1. The recombinant vector 35S PNR1 is introduced into the agrobacterium strain GV3101 to obtain recombinant agrobacterium.

2. Infecting Arabidopsis thaliana of Columbia type with the recombinant Agrobacterium obtained in step 1 by flower bud soaking (Clough and Bent, Floral dip: a amplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. plant Journal,1998,16:735-₁And (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 transgenic Arabidopsis single copy homozygous lines were generated: 35S PNR1-8 and 35S PNR 1-23.

Identification of transgenic PNR1 Arabidopsis thaliana

Respectively combine T with₃Generation-transferred PNR1 Arabidopsis line 35S, PNR1-8, T₃The transgenic PNR1 Arabidopsis line 35S PNR1-23 and wild type Arabidopsis plants were identified as follows: the Trizol method is used for extracting total RNA of plants and reversely transcribing the total RNA into cDNA, qRT-PCR expression quantity detection is carried out by adopting a Primer pair (Primer 3: 5'-GTCGATTCCTAAACGCGCTT-3'; Primer 4: 5'-ATATGCCTCCAACCACCACA-3') consisting of Primer3 and Primer4, 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. Reverse transcription of RNA into cDNA was performed 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 to 20 μ L in sequence: 5 XFirst-Strand buffer 4. mu.L, 0.1M DTT 2. mu.L, SuperScript^TMII Reverse Transcriptase 1. mu.L. And (3) fully and uniformly mixing 20 mu L of sample, reacting at 25 ℃ for 15min, at 42 ℃ for 50min, at 70 ℃ for 15min, and freezing the reverse transcription product at-20 ℃ or performing qRT-PCR experiment.

The results are shown in FIG. 1. The results show that: t is₃Generation-transferred PNR1 Arabidopsis line 35S, PNR1-8, T₃The PNR1 gene expression level in the transgenic PNR1 Arabidopsis strain 35S, PNR1-23 is obviously higher than that of the wild Arabidopsis plant.

Example 3 physiological Properties of transgenic Arabidopsis thaliana (PNR 1)

Detecting phosphorus content index

1. Respectively combine T with₃Generation-transferred PNR1 Arabidopsis line 35S, PNR1-8, T₃Transfer PNR1 Arabidopsis line 35S PNR1-23 and wild type Arabidopsis seeds are sowed on a 1/2MS solid culture medium plate to germinate and grow for 10 days, and then the whole plant is obtained.

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 a 10mL EP tube in advance for later use. Inorganic phosphorus extract (1L inorganic phosphorus extract formulation: 10mL of 1M Tris-HCl (pH 8.0), 2mL of 0.5M EDTA (pH 8.0), 5.844g of NaCl, 700. mu.L of beta-mercaptoethanol, 10mL of 100mM PMSF (as prepared)) was added to the sample tube at a ratio of 100. mu.L of extract per 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 6.3 mL. The mixture was carefully inverted and reacted in a water bath at 42 ℃ for 30 min. 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 μ L of the supernatant was aspirated, added to 350 μ L of developing solution, and mixed by inversion. Meanwhile, a standard curve is prepared, and the reaction is carried out in water bath at 42 ℃ for 30 min. 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-transferred PNR1 Arabidopsis line 35S, PNR1-8, T₃The inorganic phosphorus content in the transgenic PNR1 Arabidopsis strain 35S, PNR1-23 is obviously higher than that in wild Arabidopsis, which shows that the improvement of the expression of PNR1 is beneficial to the phosphorus accumulation of plants (Arabidopsis).

Detection of phosphorus absorption rate index

Respectively combine T with₃Generation-transferred PNR1 Arabidopsis line 35S, PNR1-8, T₃Generation of 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 parallel) and carried out³²P-absorption rate detection, the detection procedure can be referred to in the literature "Wang et al, 2014, Arabidopsis WRKY45 transformation factors activated phosphatates trap 1; 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:

firstly, pre-treating liquid is packaged in 2mL of EP tubes in advance, and 1.5mL of pre-treating liquid is added into each tube;

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

The experimental process comprises the following steps:

Firstly, 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;

absorbing: 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.

Thirdly, 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.

Drying the materials: 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.

Measuring: 1mL of scintillation fluid was added to the baked material to ensure that all of the seedlings were immersed in the scintillation fluid, and the measurement was performed on a scintillation counter model HIDEX300SL, and the measured DPM was used to calculate the phosphorus absorption rate.

The results are shown in FIG. 3. The measurement results showed that T₃Generation-transferred PNR1 Arabidopsis line 35S, PNR1-8, T₃The phosphorus absorption rate of the transgenic PNR1 Arabidopsis strain 35S, PNR1-23 is obviously higher than that of wild Arabidopsis, which indicates that the improvement of PNR1 expression is beneficial to the 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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<213> Artificial Sequence (Artificial Sequence)

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cttgtcactc aagcgatcga agcttgtcgg aaggagttat ctggtacgac gacaactaca 180

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attcctatca agaaaattag ttccttgtgt gaagaagtac aagaagaaga agaagaagat 300

ggtgaacatg aatcttctcc agaacttgtg aataataaga aatcagattg gcttagatct 360

gttcagctat ggaatcattc accggatcta aatccaaaag aggagcgtgt agctaagaaa 420

gcgaaagtgg tggaggtgaa accaaaaagc ggtgcgtttc agccgtttca aaagcgcgtt 480

ttggagactg atttgcaacc ggcggtgaaa gtagctagtt cgatgccagc gacgacgacg 540

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tcgccggaat tacaccgtcg attcctaaac gcgcttcagc agcttggagg atctcatgtt 720

gctacaccaa agcaaatcag ggatcacatg aaggttgatg gattaacaaa cgacgaagtt 780

aaaagccatt tacagaaata tagacttcac acaagaaggc cagcagcaac atccgtggcg 840

gcacaaagta ccgggaatca gcaacaacca caatttgtgg tggttggagg catatgggta 900

ccatcgtcac aagattttcc accaccgtcc gatgtagcca acaagggtgg tgtatatgct 960

ccggttgcgg tggcgcaatc tccaaaacgt tcgttggaga gaagttgcaa ctcgccggcg 1020

gcatcttcct ctacaaatac aaatacttct actcctgtgt cataa 1065

<210>2

<211>354

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Met Met Val Glu Met Asp Tyr Ala Lys Lys Met Gln Lys Cys His Glu

1 5 10 15

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

Use of the PNR1 protein in at least one of the following (1) to (9):

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

(2) improving 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 and controlling plant yield and/or quality;

(9) increasing plant yield and/or quality;

the PNR1 protein is a protein shown in 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) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 2 and having the same function.
2. Use of a biomaterial 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) improving 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 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.
3. Use according to claim 2, characterized in that: the biomaterial is any one of the following A1) to A12):

A1) a nucleic acid molecule encoding a PNR1 protein;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).
4. Use according to claim 3, characterized in that: A1) the nucleic acid molecule is a gene shown in the following 1) or 2) or 3):

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

2) a cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding the PNR1 protein of claim 1;

3) A cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) and encodes a PNR1 protein as claimed in claim 1.
5. Use according to any one of claims 1 to 4, characterized in that: the breeding aims at cultivating high-efficiency plant varieties.
6. Use according to any one of claims 1 to 5, characterized in that: the plant is a monocotyledon or a dicotyledon;

or, the dicotyledonous plant is preferably arabidopsis;

alternatively, the monocot is preferably maize.
7. A method for producing a transgenic plant having a high phosphorus nutrition efficiency, comprising the steps of increasing the expression level and/or activity of PNR1 protein in a recipient plant to obtain a transgenic plant; the transgenic plant has a higher phosphorus content and/or a higher phosphorus uptake rate than the recipient plant.
8. The method of claim 7, wherein: 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;

alternatively, the overexpression method is to introduce a gene encoding the PNR1 protein into a recipient plant.
9. The method of claim 8, wherein: the nucleotide sequence of the coding gene of the PNR1 protein is a DNA molecule shown in a sequence 1.
10. The method according to any one of claims 7-9, wherein: the plant is a monocotyledon or a dicotyledon;

or, the dicotyledonous plant is preferably arabidopsis;

alternatively, the monocot is preferably maize.