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miR399 fusion gene, construction method thereof and application thereof in plant breeding

A technology of 1. mir399 and gene fusion, which is applied in the field of plant genetic engineering to achieve the effect of reducing production costs

Active Publication Date: 2011-10-05
INST OF SOIL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there have been no literature and patent reports on successfully inducing the expression of phosphorus-related microRNAs and obtaining transgenic crops that absorb phosphorus fertilizers efficiently in the soil at home and abroad.

Method used

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  • miR399 fusion gene, construction method thereof and application thereof in plant breeding
  • miR399 fusion gene, construction method thereof and application thereof in plant breeding
  • miR399 fusion gene, construction method thereof and application thereof in plant breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Arabidopsis thaliana environmental stress-specific expression promoter RD29A promoter clone

[0020] Using Arabidopsis genomic DNA as a template, the 652bp promoter of RD29A gene was amplified by PCR method, and the amplified product was recovered and cloned by TA.

[0021] (1) PCR amplification of the target fragment

[0022] Design specific primers according to the promoter region of Arabidopsis thaliana environmental stress-specific expression RD29A gene (GenBank accession number D13044) published by GenBank, add a HindIII restriction site at the 5′ end of the upstream primer, and add a BamHI restriction enzyme at the 5′ end of the downstream primer location.

[0023] Upstream primer: 5′-TCG AAGCTT GGTGAATTAAGAGGAGAGAG-3′(add HindIII restriction site)

[0024] Downstream primer: 5′-GAC GGATCC TGAGTAAAACAGAGGAGGGTC-3′(add BamHI restriction site)

[0025] Genomic DNA of Arabidopsis thaliana was extracted according to a conventional method, and the gen...

Embodiment 2

[0043] Embodiment 2: Arabidopsis microRNA miR399 gene cloning

[0044] Specific primers were designed according to the published DNA information of the Arabidopsis thaliana ath-miR399 gene (DBE#2109) by ASRP. A BamHI restriction site was added to the 5' end of the upstream primer, and a SacI restriction site was added to the 5' end of the downstream primer. The PCR product is 306bp

[0045] Upstream primer: 5′-TCG GGATCC TTGCTAGTCCAACTTCCAAT-3′ (Add BamHI restriction site)

[0046] Downstream primer: 5′-GC GAGCTC AGGATTCGATCTATAAAACCT-3′ (Add SacI restriction site)

[0047] Genomic DNA of Arabidopsis thaliana was extracted according to conventional methods, and the precursor of ath-miR399d gene was amplified by PCR method with genomic DNA template, and then TA cloned with pMD18simple vector. The PCR reaction system, reaction conditions, target fragment recovery, cloning and sequencing are the same as in Example 1.

Embodiment 3

[0048] Example 3: Construction of RD29A-miR399 fusion gene using pBI121 vector

[0049] (1) Extract vector plasmid pBI121 from Escherichia coli (this strain can be purchased from a reagent company), and use BamHI / SacI double enzyme digestion to recover a large vector fragment.

[0050] (2) Extract the plasmid from the TA clone prepared in Example 1, and cut back the ath-miR399 gene fragment with BamHI / SacI double enzymes.

[0051] (3) The above two fragments were ligated overnight at 16° C. under the catalysis of ligase to complete the construction of the ath-miR399 gene on the pBI vector.

[0052] Reagent

Amount added

10×T4DNA ligase Buffer

1.0 μL

T4DNA ligase

0.5μL

pBI121 vector recovery fragment

2.0 μL

ath-miR399 gene recovery fragment

6.5μL

[0053] (4) Transform E.coli DH5α competent cells with the ligation mixture, and select white colonies on the transformation plate, the method is the same as in Exa...

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Abstract

The invention discloses a miR399 fusion gene, a construction method thereof and application thereof in plant breeding. The construction method comprises the following steps of: respectively cloning a promoter of a plant environmental stress specific expression gene RD29A and a low-phosphorous specific response microRNA gene miR399, and constructing an RD29A-ath-miR399 fusion gene capable of being specifically induced to be expressed under the environmental stress condition. The fusion gene transforms plants, and the miR399 which is specifically induced to be expressed under environmental stress can be obtained, so that a new transgenic crop variety capable of efficiently utilizing soil phosphorin resources is obtained and has important significance for reducing production cost of agricultural planting and water and soil pollution caused by application of a large quantity of phosphate fertilizer.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, specifically, it uses the promoter of Arabidopsis thaliana environmental stress specific expression gene RD29A and low phosphorus specific response microRNA gene miR399 to construct RD29A-ath-miR399 fusion gene and its breeding in transgenic plants in the application. Background technique [0002] In agricultural production, in order to ensure the economic output of crops, high amounts of phosphate fertilizers are often applied, which increases production costs and lowers the utilization rate of phosphate fertilizers. Moreover, long-term excessive phosphorus fertilizers have caused enormous pressure on the soil environment and surrounding water bodies. This phenomenon is especially evident in facility agricultural production. Falling into a dilemma where crop yields will decrease if no fertilization is applied, and soil quality degradation such as secondary salinization and acidificatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/82C12N15/10A01H5/00A01H4/00
Inventor 施卫明高南闵炬
Owner INST OF SOIL SCI CHINESE ACAD OF SCI