Low-phosphor stress response regulatory factor ZmPHR1, gene for coding the protein and application

A technology of response regulator and gene, applied in the field of genetic engineering, can solve the problem of low phosphorus utilization rate of crops

Inactive Publication Date: 2013-04-03
CROP SCI RES INST SHANXI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to use molecular breeding methods to improve the problem of low phosphorus utilization in crops, the present invention provides a low phosphorus stress response regulator ZmPHR1, the gene encoding the protein and its application

Method used

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  • Low-phosphor stress response regulatory factor ZmPHR1, gene for coding the protein and application
  • Low-phosphor stress response regulatory factor ZmPHR1, gene for coding the protein and application
  • Low-phosphor stress response regulatory factor ZmPHR1, gene for coding the protein and application

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Experimental program
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Effect test

Embodiment 1

[0046] Implementation 1 Discovery and cloning of ZmPHR1 protein and its coding gene

[0047] According to the amino acid sequence of AtPHR1 (At4g28610) of Arabidopsis, the TIGR (The Institute of Genomic Research Database-TDB) corn database (http: / / maize.jcvi.org) was searched, and the length of the predicted amino acid sequence and domain, and obtained the merged sequence TC346249 that may encode the PHR1 gene, a total of 13 maize ESTs.

[0048] Using TC346249 as a template, the PCR primer sequences were designed as follows:

[0049] Forward primer: 5′-AAGCAAGGTAATGAAATG -3′;

[0050] Reverse primer: 5'-TGAATAGTGCAACCGATA-3'.

[0051] The total RNA of maize inbred line 478 was extracted with Trizol reagent from Invitrogen Company. After DNaseI (RNase-free) treatment, 4 μg was reverse-transcribed with M-MLV reverse transcriptase from Promega Company to synthesize the first strand of cDNA. used as a template for PCR amplification,

[0052] PCR reaction system: 39.5 μL of u...

Embodiment 2

[0056] Implementation 2 Construction of plant expression vectors

[0057] 1. Construction of pJIT163-ZmPHR1::GFP expression vector

[0058] Specific steps are as follows:

[0059] (1) Introduce SalI and BclI sites at both ends of the gene of the recombinant plasmid pMD18-ZmPHR1 (BclI is the homologous enzyme of BamH I)

[0060] PCR primers:

[0061] Forward primer: 5’- GC gtcgac ATGAGGAAGTTTAATC-3’

[0062] Reverse primer: 5’- GCG tgatca ACTATCTTGCAGTTT-3’

[0063] PCR reaction system: 39.5 μL of ultrapure water, 5 μL of 10×PCR buffer, 2 μL of template, 1 μL of forward and reverse primers with a concentration of 10 μM, 0.5 μL of EX-Taq DNase (5u / μL), and 1 μL of dNTPs (10 mM).

[0064] PCR reaction conditions: 94°C for 4min; 94°C for 30sec, 60°C for 30sec, 72°C for 1min30sec, 35 cycles; 72°C for 10min; 4°C for heat preservation.

[0065] (2) The recombinant plasmid pMD18-ZmPHR1 was double-digested with restriction endonucleases SalI and BclI, and the gene fragment wi...

Embodiment 3

[0073] Implementation 3 Acquisition of transgenic strains

[0074] 1. Obtaining of recombinant Agrobacterium strains

[0075] The recombinant plasmid pCAMBIA1300-ZmPHR1::GFP was introduced into Agrobacterium strain GV3101 by freeze-thaw method to obtain recombinant Agrobacterium containing ZmPHR1 gene.

[0076] 2. Obtaining of transgenic plants

[0077] (1) The recombinant Agrobacterium strain was used to transform Arabidopsis thaliana ecotype Columbia by drop method. Get T0 generation seeds;

[0078] (2) Screen the 2nd generation with hygromycin (50mg / L);

[0079] (3) Germinate positive T2 generation plants on MS plates containing antibiotics (50mg / L hygromycin), and observe the resistance reaction after 2-3 weeks. The lines without positive and negative segregation are ZmPHR1 homozygous plants Department, that is, the T3 generation (see image 3 ).

[0080] 3. Identification of transgenic homozygous lines

[0081] The T3 generation plants were detected by PCR ...

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Abstract

The invention discloses a low-phosphor stress response regulatory factor ZmPHR1, a gene for coding the protein and an application thereof, belonging to the technical field of genetic engineering and aiming at solve a problem that the phosphor utilization rate of the crops is low by a molecular breeding method. The amino acid sequence of the low-phosphor stress response regulatory factor ZmPHR1 is shown as SEQ ID NO: 2; or the sequence is replaced, deleted or added with one or more amino acid sequences, with the same function, of the amino acid. The invention further provides a gene for coding the regulatory factor ZmPHR1, wherein the sequence of the gene contains the nucleotide sequence shown as the SEQ ID NO: 1; and the invention discloses the application of the gene in plant breeding. The gene and the coding protein of the low-phosphor stress response regulatory factor ZmPHR1 provided by the invention have an important action in genetic improvement of the grains such as corns, wheat, rice and the like, and the industrial crops, and have an extensive application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a low phosphorus stress response regulator ZmPHR1, a gene encoding the protein and an application thereof. Background technique [0002] The lack of phosphorus in soil is a worldwide problem. The concentration of phosphorus in 30-40% of the arable land is only 1-10μM. Such a low phosphorus content cannot meet the needs of plant growth and development. Usually people use phosphorus fertilizers to ensure normal growth of plants and obtain higher crop yields. However, even if there is sufficient supply of phosphorus fertilizers, most phosphorus fertilizers will interact with calcium ions in alkaline soils or aluminum ions and iron in acidic soils. Ions form insoluble compounds, or interact with microorganisms to form organic substances, and only about 20% of the phosphorus is absorbed and utilized by plants; in addition, some inorganic phosphorus in the soil enters the gro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/11C12N15/84C12N1/21A01H5/00
Inventor 白建荣王秀红刘惠民孙毅史向远任志强
Owner CROP SCI RES INST SHANXI ACADEMY OF AGRI SCI
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