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Plant expression vector for expressing dicarboxylate-tricarboxylate carrier (DTC) genes and application thereof in improving aluminum-resistant performance of Medicago sativa L

A transporter and transgenic technology, which is applied in the field of plant genetic engineering to achieve the effects of increased root elongation, huge economic benefits, and improved aluminum tolerance traits of alfalfa

Inactive Publication Date: 2012-12-26
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no report on the creation of Al-tolerant alfalfa varieties by utilizing the gene encoding the carboxylate transporter

Method used

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  • Plant expression vector for expressing dicarboxylate-tricarboxylate carrier (DTC) genes and application thereof in improving aluminum-resistant performance of Medicago sativa L
  • Plant expression vector for expressing dicarboxylate-tricarboxylate carrier (DTC) genes and application thereof in improving aluminum-resistant performance of Medicago sativa L
  • Plant expression vector for expressing dicarboxylate-tricarboxylate carrier (DTC) genes and application thereof in improving aluminum-resistant performance of Medicago sativa L

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The preparation of embodiment one alfalfa genome

[0045] 1. Extraction of DNA

[0046] Select 0.5-1 g of young alfalfa leaves, quickly grind them into powder in liquid nitrogen, add 3 mL of 65°C preheated CTAB extract (100 mmol / L Tris-HCl (pH8.0), 20 mmol / L EDTA (pH8.0), 1.5mol / L NaCl, 2% CTAB (W / V), 4% PVP40 (W / V) and 2% mercaptoethanol (V / V), add PVP and mercaptoethanol before use), shake and mix quickly. Bath at 65°C for 30min, then add 1mL of 5mol / L KAc, ice-bath for 20min, extract once with an equal volume of chloroform:isoamyl alcohol (24:1) (10,000r / min, centrifuge at 4°C for 5min), and take the To clean it, add 2 / 3 times the volume of -20°C pre-cooled isopropanol, mix well, and let it stand for about 30 minutes. Rinse once, blow dry, and resuspend in 500 μL TE. Add 1 μL RNaseA (10mg / ml), and treat at 37°C for 1h. Then phenol (pH8.0): chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1) were extracted once each (10,000r / min, centrifug...

Embodiment 2

[0059] Embodiment two expresses the nucleotide of carboxylic acid transporter gene and the preparation of plant expression vector

[0060] Bacterial strains containing the pUC-CjDTC cloning vector containing the CjDTC coding gene sequence and the plant expression vector p5 (~500ng) were incubated in 5mL LB liquid medium (containing corresponding antibiotics) at 37°C for 12-16 hours with shaking. Vector construction process see figure 1 . The constructed plant expression vector structure is as follows figure 2 As shown, it includes the nucleotide (SEQ ID NO. 1) for expressing the carboxylic acid transporter gene and each element required for expression screening. pUC-CjDTC is a common cloning vector containing the target gene, and p5 is a backbone vector for constructing plant expression vectors. The p5 vector is modified on the basis of pBI121, and has the GUS gene under the control of the CaMV 35S promoter, which facilitates the process of genetic transformation in plants ...

Embodiment 3

[0068] Preparation of embodiment three transformants and transgenic plants

[0069] 1. Introduce the constructed plant expression vector plasmid into Agrobacterium LBA4404 by electric shock method

[0070] Referring to the Bio-RAD MicroPulser user manual, the above-mentioned vectors were introduced into Agrobacterium LBA4404 by electric shock transformation.

[0071] 2. The vector for overexpressing the CjDTC gene was integrated into the alfalfa genome

[0072] Genetic transformation of alfalfa by an Agrobacterium tumefaciens mediated approach.

[0073] Table 1 Agrobacterium tumefaciens-mediated alfalfa genetic transformation medium formula

[0074]

[0075] MS: Murashige & Skoog, 1962; Gelrite: Sigma, Item No.: G1910; SH: Schenk & Hildebrandt, 1972

[0076] 3. The expression vector is introduced into alfalfa through the method of Agrobacterium-mediated embryogenic healing, and the specific method is as follows:

[0077] (1) Generation of sterile seedlings

[0078] The...

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Abstract

The invention discloses a method of improving the aluminum-resistant performance of Medicago sativa L. The method comprises the following steps: dicarboxylate-tricarboxylate carrier (DTC) genes under regulation of overexpression promoters are integrated into Medicago sativa L genomes, so that the overexpression of the DTC genes in the Medicago sativa L is realized, thus obtaining transgenic Medicago sativa L. By utilizing the method, the content of organic acid in the Medicago sativa L is obviously increased, the relative elongation of the root of the transgenic Medicago sativa L is larger than that of the root of a wild reference under aluminum stress, the content of aluminum accumulated in plants is low, and the aluminum-resistant performance of the Medicago sativa L is obviously improved.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a plant expression vector and its application, in particular to a plant expression vector containing a carboxylic acid transporter gene and its application in improving the aluminum tolerance of alfalfa. The invention also relates to a method for preparing transgenic plants based on the plant expression vector. Background technique [0002] Among the 8 billion hectares of arable land in the world, acidic soil accounts for about 30%, and more than 50% of the arable land is potential acidifying soil (von Uexküll and Mutert, 1995). Acidic soils are mostly distributed in tropical and subtropical developing countries, which account for about 60% of the world's acidic soils. As far as my country's current land resources are concerned, southern my country is located in tropical and subtropical regions, and the area of ​​acidic soil is about 2.035 million km 2 , acc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00
Inventor 李德谋裴炎罗小英侯磊张觅白文钦陈佐友邓伟
Owner SOUTHWEST UNIV
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