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Application of peanut AhFRDL1 gene in improving aluminum toxicity stress resistance of plants

A peanut and gene technology, applied in the field of genetic engineering, achieves broad application prospects and the effect of improving yield and quality

Active Publication Date: 2015-04-29
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in peanut, the MATE gene related to aluminum stress resistance has not yet been reported.

Method used

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  • Application of peanut AhFRDL1 gene in improving aluminum toxicity stress resistance of plants
  • Application of peanut AhFRDL1 gene in improving aluminum toxicity stress resistance of plants
  • Application of peanut AhFRDL1 gene in improving aluminum toxicity stress resistance of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Cloning of peanut AhFRDL1 gene

[0049] (1) Peanut hydroponic culture

[0050] The peanut seeds Luhua 14 (purchased from the Chinese Academy of Agricultural Sciences) were disinfected with 10% hydrogen peroxide for 30 minutes, cleaned and placed in a saturated calcium sulfate solution for about 8 hours, and ventilated and protected from light. Then place it in a tray covered with absorbent paper, pour an appropriate amount of water, and place it in an artificial incubation room protected from light. The incubation room was set up at 30°C for 14 hours in light and 22°C in darkness for 10 hours. After a day or so, the peanuts will send out small buds and be cultured in quartz sand. After 2 leaves have grown, they will be cultured in hydroponic nutrient solution. The formula of hydroponic nutrient solution is as follows: KH 2 PO 4 (0.5mM), K 2 SO 4 (0.75mM), KCl (0.1mM), MgSO 4· 7H 2 O (0.65mM), CaSO 4· 2H 2 O(2mM), H 3 BO 3 (1μM), MnSO 4· 4H 2 O (1μM), ZnSO 4· ...

Embodiment 2

[0060] Example 2 Construction of recombinant expression vector

[0061] 1. Construct the vector

[0062] (1) Add restriction sites XmaI and BamHI through primers, and use the vector pMD20-T-AhFRDL1 as a template for PCR;

[0063] The primer sequence is:

[0064] 35s-AhFRD1-F: 5'-ACGCCCCGGGATGGCTGAGAAGCAG-3' (SEQ ID NO. 5);

[0065] 35s-AhFRD1-R: 5'-CGGGATCCTTTCTCCATAGGAATTCCCAAG-3' (SEQ ID NO.6);

[0066] (2) Clone the PCR product into the pGEM-T vector, transform DH5α competent cells, select positive clones, culture the bacteria and extract the plasmid to obtain the vector pGEM-T-AhFRDL1;

[0067] (3) The vector pGEM-T-AhFRDL1 and pBAR1 vector were simultaneously digested with XmaI and BamHI, and the target fragment of AhFRDL1 gene and pBAR1 vector fragment were recovered respectively, and then the target fragment of AhFRDL1 gene was ligated with the digested pBAR1 with T4 ligase Vector fragment to construct a vector with 35S strong promoter driving gene expression;

[0068] (4) Transfor...

Embodiment 3

[0080] Example 3 Functional complementarity test of peanut AhFRDL1 gene to Arabidopsis single mutant AtALMT1-Ko, AtMATE-Ko and double mutant Atdouble-Ko

[0081] 1.1 Obtainment of Arabidopsis plants transformed with AhFRDL1 gene

[0082] The recombinant expression vector pBAR1-AhFRDL1 obtained in Example 2 was transformed into three Arabidopsis mutants with lack of aluminum resistance by transgene technology: (1) The Arabidopsis aluminum-tolerant gene AtALMT1 (malate transporter) knocked out The single mutant AtALMT1-Ko that was deleted; (2) the single mutant AtMATE-Ko knocked out of the Arabidopsis aluminum-tolerant gene AtMATE (citrate transporter); (3) the Arabidopsis double knocked out of AtALMT1 and AtMATE Mutant Atdouble-Ko.

[0083] The specific operation steps are as follows: soak the Arabidopsis seeds with 1% agarose, and place the seeds in the nutrient soil mixed with vermiculite with a pipette (the ratio of vermiculite to nutrient soil is 1:1, and it is placed in a plasti...

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Abstract

The invention provides an application of peanut citric acid transport protein gene AhFRDL1 in improving aluminum toxicity stress resistance of plants. The application comprises: transferring the peanut citric acid transport protein gene AhFRDL1 or a recombinant expression vector containing the peanut citric acid transport protein gene AhFRDL1 into plant cells, screening transgenic plants, and making the transgenic plants express the peanut citric acid transport protein gene so as to improve the aluminum toxicity stress resistance of the plants. In the invention, the fact that the peanut citric acid transport protein gene AhFRDL1 is gene for regulating and controlling secretion of citric acid in vitro of the plants and participates in the biological process of the aluminum toxicity stress resistance of plants is proved at first, and the peanut citric acid transport protein gene AhFRDL1 is used for improving the aluminum toxicity stress resistance of plants, which has long-term practical significance for further breeding aluminum toxicity resistant main crops on acid soil.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and particularly relates to the application of the peanut citrate transporter gene AhFRDL1 in improving the resistance of plants to aluminum toxicity stress. Background technique [0002] Aluminum is the most abundant metal element in the earth's crust, usually in the form of insoluble silicate or alumina, which is not harmful to plants, but under acidic conditions (pH <5), soluble aluminum (mainly Al 3+ ) It is toxic to most plants. China’s acidic soil accounts for about 21% of the country’s total land area. Aluminum is not only the main source of soil acidity on acid soils, but also because the exchange of aluminum accounts for 20-80% of the soil cation exchange. As a result, cations in the soil are prone to leaching, resulting in the lack of nutrients such as P, K, Ca, Mg, B, and Mo. Therefore, aluminum toxicity has become an important limiting factor restricting the normal growth and developme...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00
Inventor 左元梅邱巍纪春巧熊宏春郭笑彤
Owner CHINA AGRI UNIV
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