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A method for rapid diagnosis and visual dynamic monitoring of plant phosphorus nutrition and the application of its recombinant expression vector

A rapid diagnosis and dynamic monitoring technology, applied in the field of genetic engineering, can solve problems such as application research gaps, and achieve the effect of sensitive dynamic monitoring

Active Publication Date: 2016-02-24
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the research and application of anthocyanins are mainly concentrated in the fields of cultivating new varieties of flowers and developing anti-oxidative health foods [13], and the application research of anthocyanins as a visual reporter gene in the field of biotechnology engineering is still blank

Method used

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  • A method for rapid diagnosis and visual dynamic monitoring of plant phosphorus nutrition and the application of its recombinant expression vector
  • A method for rapid diagnosis and visual dynamic monitoring of plant phosphorus nutrition and the application of its recombinant expression vector
  • A method for rapid diagnosis and visual dynamic monitoring of plant phosphorus nutrition and the application of its recombinant expression vector

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Experimental program
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Effect test

Embodiment 1

[0067] Cloning of embodiment 1.OsPT6 promoter and Pr gene:

[0068] 1) Genomic DNA extraction Take rice and cauliflower leaves that grow normally under natural conditions, and quickly put them in liquid nitrogen for cryopreservation. Weigh about 0.1g of the sample, grind it fully with liquid nitrogen and transfer it to a 2ml centrifuge tube, add 500μl of DNA for extraction After buffer, vortex thoroughly to mix. 65°C constant temperature water bath for 15-20min, vortex the sample every 5min. Add 500 μl of chloroform-isoamyl alcohol (ratio 24:1), and mix slowly on a shaker for 10-15 minutes. Centrifuge at 7500rpm for 15min at 4°C. Transfer the supernatant to a new 1.5ml centrifuge tube, extract once with 500μl phenol / chloroform (1:1), and then extract once with an equal volume of chloroform. Carefully transfer the supernatant to a new 1.5ml centrifuge tube, add 1ml of absolute ethanol, turn it upside down several times, and place it at -20°C for 10min. Centrifuge at 7500rpm...

Embodiment 2

[0081] Embodiment 2. Construction of plant expression vector pS1aG-3-OsPT6-Pr:

[0082] 1) Construction of the intermediate vector of OsPT6 promoter and Pr gene. The PCR products of OsPT6 promoter and Pr gene were separated by agarose electrophoresis and recovered by gel cutting. The recovered fragments were respectively connected with pMD19-T vector. The total volume of the enzyme-linked system was 10 μl , containing 5 μl of ligation solution, 1 μl of pMD19-T vector, 3-4 μl of PCR purified product, supplemented with 10 μl of water, and then ligated overnight at 16°C; then transferred to Escherichia coli DH5α competent cells and coated with 100 μg / mL ambenzyl After growing on LB solid medium for 12h-14h, pick positive colonies for DNA sequencing. The bacteria solution with correct sequencing was added to an equal volume of 30% glycerol and stored at -70°C for later use. Recombinant plasmids containing the OsPT6 promoter sequence and the full-length sequence of the Pr gene were...

Embodiment 3

[0098] Example 3. Verification of Phosphorus Deficiency Indicating Effect of Transgenic Tobacco

[0099] 1) Cultivation of wild-type and T1-generation transgenic tobacco Put the wild-type and T1-generation transgenic tobacco seeds into a 1.5ml sterile centrifuge tube, soak in 30% sodium hypochlorite solution for 10 minutes; pour off the sodium hypochlorite solution, and wash with sterile water 4-5 times , soak for 30min for the last time. Carefully transfer the seeds to sterile filter paper to blot dry, carefully place the seeds on sterile 1 / 2 MS solid medium with sterile tweezers, and culture them in a tissue culture room at 28°C for 4-6 weeks.

[0100] 2) Phosphorus deficiency treatment of transgenic tobacco. The wild-type and transgenic tobacco that had grown to two true leaves were aseptically cultured and moved into sterilized sand. The phosphorus nutrient solution was cultivated for 14 days, and the formula and concentration of the nutrient solution were: 2mM KNO 3 , 1...

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Abstract

The invention discloses a rapid plant phosphorus nutrition diagnosis and visual dynamic monitoring method and application of a recombinant expression vector. A promoter which specifically responds to a phosphorus deficiency signal is used for regulating and controlling a recombinant expression vector for plant anthocyanin synthetic route gene expression to be converted into a plant so as to obtain a transgenic plant; when sufficient phosphorus is supplied, the leaves of the transgenic plant are maintained to be original green; when the plant is lack of the phosphorus, the promoter specifically drives overexpression of the anthocyanin synthesis gene, the anthocyanin is greatly accumulated on the leaves of the transgenic plant, and then the leaves of the plant are turned into dark pulp; when sufficient phosphorus is supplied again, the transgenic plant can be recovered to be green within a short time, so that the purpose of visual dynamic monitoring of the phosphorus of the plant is achieved. Due to the adoption of the method, the dynamic change of the phosphorus inside the plant can be sensitively and specifically monitored, and meanwhile with the application of the remote sensing technique, the supply state of the plant phosphorus supply condition can be rapidly monitored in large area, so that instruction of rational application of fertilizers in the field can be realized.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a rapid diagnosis and visualization dynamic monitoring method of plant phosphorus nutrition and the application of the recombinant expression vector. Background technique [0002] Phosphorus is one of the most important mineral nutrients in the process of plant growth and development, and is widely involved in metabolic processes such as energy transfer, signal transduction, and biochemical synthesis in plants[1,2]. However, because phosphorus is easily fixed and precipitated in the soil, and plants absorb mainly inorganic orthophosphate from the soil, compared with other nutrients, the mobility and availability of phosphorus in the soil are very low, which also It often becomes one of the main limiting factors of plant growth in farmland and natural ecosystems[3‐5]. [0003] Applying phosphorus fertilizer is an important means to solve crop phosphorus deficiency and i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84C12N15/66A01H5/00G01N21/25
Inventor 徐国华李依婷顾冕
Owner NANJING AGRICULTURAL UNIVERSITY
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