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Acidic phosphatase, its encoding gene and application

A coding gene and coding technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as improving crop varieties, and achieve the effects of reducing dependence, improving efficiency, and reducing environmental pollution

Inactive Publication Date: 2011-10-05
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of understanding of genes encoding root surface acid phosphatase, it is currently impossible to use genetic engineering to genetically manipulate this type of gene to achieve the purpose of improving crop varieties

Method used

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  • Acidic phosphatase, its encoding gene and application
  • Acidic phosphatase, its encoding gene and application
  • Acidic phosphatase, its encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Discovery of genes

[0043] In order to identify and clone the secretory acid phosphatase gene induced by low phosphorus stress in plants, the model plant Arabidopsis thaliana was used as material, and the mutants lacking acid phosphatase activity on the root surface were screened on a large scale under low phosphorus conditions. . The screening method is to germinate the Arabidopsis seeds directly on the phosphorus-deficient MS medium. After 7-8 days of growth, the roots of the plant seedlings are covered with acid phosphatase catalytic substrate BCIP (5-bromo-4-chloro-3 -indolyl-phosphate, 5-bromo-4chloro-3 indolyl-phosphate) agar. After incubating for 12 hours at 25°C, the acid phosphatase on the root surface can catalyze BCIP to form a blue precipitate, while the roots of plants that cannot produce acid phosphatase activity are white. A total of 22 mutants that could not produce acid phosphatase were obtained by this method. Genetic analysis showed that t...

Embodiment 2

[0044] Example 2. Determination of purple acid phosphatase activity on the surface of plant roots

[0045] It is further proved that the acid phosphatase encoded by AtPAP10 is mainly attached to the surface of plant roots after being induced by low phosphorus conditions. The evidence comes from three aspects:

[0046] 1) In the AtPAP10 mutant nop1, the acid phosphatase spectrum in the root cells and secreted into the surrounding medium did not change significantly. Extraction of total protein inside root cells: Arabidopsis wild-type (WT) and mutants grown vertically on solid MS medium (1.2% agar) with normal phosphorus (P+) or phosphorus-deficient (P-) for 14 days The roots of nop1 plants were separated; then ground with liquid nitrogen, and then according to 1g tissue / 2ml protein extract (0.1M NaAc, 20mM CaCl 2 , 2mM EDTA, 0.1mM phenylmethylsulfonyl fluoride, glycerol 20% (v / v), pH 5.4) and the corresponding amount of protein extract; ice bath for 15min; at 4℃, centrifuge at 1300...

Embodiment 3

[0051] Example 3. Application of genes-overexpression

[0052] 1. Gene preparation

[0053] Using the total DNA extracted from wild-type Arabidopsis as a template, primers 5'-GGGGGATCCCCCTTTGTTTATCGG-3' (SEQ ID NO: 3) (containing BamHI restriction site) and 5'-GGGGAGCTCGGCACAAGAACTCTC-3' (SEQ ID NO :4) (Contains SacI restriction site) for PCR amplification, ligate the PCR amplified product to the pGM-T vector, transform the ligated product into E. coli, pick the positive single clone, culture the positive single clone in liquid, and extract The plasmid was sequenced, and the sequencing result showed that the nucleotide sequence of the inserted AtPAP10 gene is shown in SEQ ID NO: 2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 1. The positive recombinant vector was designated as pGM-T-AtPAP10.

[0054] 2. Preparation and verification of recombinant expression vector

[0055] The pBI121 vector has been disclosed in the document "Jefferson RA, Kavanagh TA, ...

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Abstract

The invention discloses an acidic phosphatase, its encoding gene and an application. The protein provided in the invention is a following protein as a) or b): a) a protein composed by an amino acid sequence which is described as SEQ ID NO:1; b) a protein with acidic phosphatase activity derived from a) that the amino acid sequence is conducted a substitution and / or deficiency and / or addition through one or a plurality of amino acid residue which is described as SEQ ID NO:1. According to the invention, a gene resource is provided to the novel crops varieties capable of efficiently using soil nutrition by employing a gene engineering approach, which is applied to cultivate the novel crops varieties capable of efficiently using soil nutrition.

Description

Technical field [0001] The invention relates to an acid phosphatase and its coding gene and application. Background technique [0002] Phosphorus is one of the most important nutrients needed for plant growth and development. Phosphorus is not only a structural component of various biological macromolecules in plant cells, but also participates in various physiological and biochemical processes in plant life activities. Therefore, whether crops can efficiently absorb and rationally use phosphorus in the soil has an extremely important impact on their final yield. However, most arable land in the world today suffers from phosphorus deficiency. In many farmlands, although the absolute content of phosphorus is not low, the concentration of inorganic phosphate that can be absorbed by plants is far below the requirements for optimal crop growth. 50%-70% of the phosphorus in the soil is in the form of organic phosphorus, including organic phosphorus contained in phytic acid and vari...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/11C12N15/63C12N1/00C12N5/10C12N7/01C12N15/82A01H5/00C12R1/91
Inventor 刘栋王良省李征
Owner TSINGHUA UNIV
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