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Soybean GmPHR1 gene and encoded protein and application thereof

A gene and protein technology, applied in the field of soybean GmPHR1 gene, can solve the problems of limited application range and unclear molecular mechanism of plant phosphorus efficiency

Inactive Publication Date: 2012-09-05
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some phosphorus efficient genes have been obtained, their application range is still limited, and the molecular mechanism of plant phosphorus efficiency has not yet been clarified.

Method used

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  • Soybean GmPHR1 gene and encoded protein and application thereof
  • Soybean GmPHR1 gene and encoded protein and application thereof
  • Soybean GmPHR1 gene and encoded protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Molecular cloning of embodiment 1GmPHR1 gene

[0023] 1. Soybean seedling cultivation and plant phosphorus deficiency treatment: After the seeds are germinated, select materials with consistent germination and sow them in flowerpots covered with vermiculite. After the opposite true leaves are unfolded, perform phosphorus deficiency (0mM) treatment, and the control phosphorus treatment is 1.0 mM.

[0024] 2. Extraction of total RNA: extraction of total RNA from soybean plants (phosphorus deficiency treatment and control treatment) was carried out according to the operating instructions of TRNzol Total RNA Reagent (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0025] 3. cDNA reverse transcription synthesis: the first strand of cDNA is obtained after the total RNA of the plant is reverse transcribed, and the synthesis process refers to PrimeScript TM 1st Strand cDNA Synthesis Kit (purchased from Treasure Bioengineering (Dalian) Co., Ltd.) operatio...

Embodiment 2

[0053] Prokaryotic expression of embodiment 2GmPHR1 gene

[0054] 1. Digest the recombinant plasmid pGM-GmPHR1 and the prokaryotic expression vector pET-32a(+) with KpnI / SacI double enzymes, and recover the target fragment. The enzyme digestion reaction system of the two plasmids is as follows:

[0055] Kpn I 1U

[0056] Sac I 1U

[0057] 10×L buffer 2μL

[0058] Plasmid 10 μL

[0059] Make up to 20μL with double distilled water

[0060] 2. Ligate the recovered pET-32a(+) expression vector with the GmPHR1 restriction fragment to construct the pET-32a(+)-GmPHR1 vector. The connection system is as follows:

[0061] GmPHR1 fragment 5 μL

[0062] pET-32a(+) vector 1 μL

[0063] T 4 DNA Ligase 1U

[0064] 10× ligation buffer 1 μL

[0065] Make up to 10 μL with double distilled water

[0066] After mixing, connect overnight at 16°C.

[0067] 3. Induced expression of fusion recombinant protein: Transform the above ligation product into Escherichia coli DH5α, detect by PCR ...

Embodiment 3

[0069] Example 3 Analysis of subcellular localization of protein encoded by GmPHR1

[0070] 1. The design of primers used in PCR: use the biological software DNAMAN to design a pair of PCR primers, amplify the open reading frame of GmPHR1 after removing the stop codon, and carry out the subcellular localization of the encoded protein. The primer sequences are as follows:

[0071] PHR3: 5'-TCTAGATGTATCACACAAAGAAATTTTCACCGGC-3',

[0072] PHR4: 5'-GGTACCCTGTCCACTCTTCATTTATCTTCATCC-3'.

[0073] 2. Amplification of the open reading frame required for subcellular localization: use the pGM-GmPHR1 plasmid as a template, use PHR3 and PHR4 primers, amplify the open reading frame sequence of the gene without a stop codon, and recover the target item after electrophoresis detection band, connected with pGM-T vector, blue-white screening, sequencing, and the positive clone pGM-PHR1 with correct open reading frame sequence was selected for subsequent tests.

[0074] 3. Construction of the...

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Abstract

The invention relates to the field of plant molecular biology, in particular to a soybean GmPHR1 gene and encoded protein and application thereof. The invention provides a soybean GmPHR1 protein as follows: (1) a protein composed by amino acid as shown in SEQ ID No.2; or (2) a protein derived from (1) with the same activity by substituting, deleting or adding one or more amino acids on the amino acid sequence as shown in SEQ ID No.2. The invention also provides the gene for encoding the protein and the application thereof in culturing new species of low phosphorus resistant plants. Compared with wild type plants, GmPHR1 transgenic plants show stronger low phosphorus stress resistant capacity.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and in particular relates to a soybean GmPHR1 gene, its encoded protein and its application. Background technique [0002] Among the 17 essential elements required by plants, phosphorus plays an extremely important role in plant metabolism and normal growth and development. At the same time, due to the unique "genetic phosphorus deficiency" phenomenon in cultivated land soil, phosphorus deficiency has become an important limiting factor affecting crop yield and quality improvement. It is an important way to solve the above problems by exploiting the potential of plants, isolating phosphorus-efficient genes through modern gene cloning technology, and transferring them into different crops through transgenic technology, so as to cultivate new varieties of phosphorus-efficient crops. At present, the cloned P-efficiency genes mainly include the following categories: (1) Phosphorus-efficiency-r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A01H5/00
Inventor 张彩英李喜焕常文锁王运杰张俊红
Owner HEBEI AGRICULTURAL UNIV.