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Plant expression vector of alfalfa malic acid channel protein gene MsALMT1, and applications thereof

A plant expression vector and channel protein technology, which is applied to the plant expression vector of alfalfa malate channel protein gene MsALMT1, and the application field of transgenic plants, can solve the problems of limited and single plant sources, and achieve the goal of enhancing malic acid secretion Effect

Inactive Publication Date: 2013-03-06
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it is worth our attention that currently used for transgenic ALMT1 The plant sources of the genes are still not extensive, and the genes are all derived from a single aluminum-resistant or model plant

Method used

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  • Plant expression vector of alfalfa malic acid channel protein gene MsALMT1, and applications thereof
  • Plant expression vector of alfalfa malic acid channel protein gene MsALMT1, and applications thereof
  • Plant expression vector of alfalfa malic acid channel protein gene MsALMT1, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: MsALMT1 Gene cDNA amplification, TA cloning and sequence analysis

[0035] Since the model plant Alfalfa truncation and Medicago truncatula have high similarity, according to the gene sequence of the aluminum-induced malate channel protein of Alfalfa truncation published on NCBI ( MtALMT1 , ABD32183), the primers were designed as follows:

[0036] Upstream primer: 5′- GGATCC ATGGTGTCTGAACCAAATTCAAG-3'( Bam H I)

[0037] Downstream primer: 5'- AAGCTT TAGTTAATTATAATAACATGTTG-3'( Hind III)

[0038] Add the GGATCC characteristic sequence to the 5' end of the upstream primer, and thus form Bam H I restriction site; downstream primer 3' plus AAGCTT characteristic sequence, forming Hind III restriction site.

[0039] After aluminum-sensitive alfalfa YM1 was treated with aluminum for 24 hours, cut 0.1g root, grind it with liquid nitrogen, add 1ml TRIzoL extract, let it stand at room temperature for 5min, then transfer it to a centrifuge tube, then a...

Embodiment 2

[0041] Example 2: Construction of intermediate vector pENTR tm 2B- MsLAMT1

[0042] use Hind III and Bam H I Digest pMD18- MsALMT1 and pENTR*-PrbcS-*T- GFP carrier ( figure 2 ), separated the excised vector and insert by agarose gel electrophoresis, and recovered pMD18- MsALMT1 produced after being cut MsALMT1 Gene fragment (about 1.4kb) and pENTR*-PrbcS-*T- GFP The vector fragment pENTRTM2B generated after being cleaved, and then pENTRTM2B and MsALMT1 The DNA fragment of the gene produces the intermediate vector pENTRTM2B- MsALMT1 ( figure 2 ). Conversion of high efficiencies (10 8 ) Escherichia coli competent cells (DH5α, purchased from Tiangen Biochemical Technology Co., Ltd.), spread the transformed Escherichia coli on a plate added with kanamycin (Km, 50 μg / ml), and cultivate overnight at 37°C. Screen the Km-resistant recombinant colony, extract the plasmid from the Km-resistant recombinant colony, and select the successfully connected plasmid vector...

Embodiment 3

[0043] Example 3: Plant expression vector pK2-35S- MsALMT1 build

[0044] Through the LR response of Gateway technology, the MsALMT1 Subcloned into the plant expression vector pK2GW7 (Gateway's destination vector, Belgium VIB / Gent company). The specific method is: use the plasmid extraction kit to purify Gateway's destination vector pK2GW7 ( Figure 6 A), add pENTRTM2B- to Gateway's LR reaction system MsALMT1 and pK2GW7 each 150ng, 1μl LR Clonase II Enzyme Mix (Invitrogen), mix well and react overnight at 25°C, through the action of integrase MsALMT1 Integrate into pK2GW7 to obtain MsALMT1 The plant expression vector pK2-35S- MsALMT1 ( Figure 5 ). Conversion of high efficiency (10 8) Escherichia coli competent cells (DH5α, purchased from Tiangen Biochemical Technology Co., Ltd.), spread the transformed Escherichia coli on a plate added with spectinomycin (Spe, 50 μg / ml), cultivate overnight at 37°C, and screen Spe-resistant recombinant colonies. Plasmids were extr...

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Abstract

The present invention discloses an alfalfa aluminum induced malic acid channel protein gene plant expression vector pK2-35S-MsALMT1, which is a plant expression vector containing a constitutive promoter (35S) of cauliflower mosaic virus and alfalfa aluminum induced malic acid channel protein gene MsALMT1. According to the present invention, MsALMT1 gene is amplified from alfalfa, and a constitutive promoter is adopted to control overexpression of the MsALMT1 gene in tobacco so as to improve aluminum toxicity resistance of the tobacco; and experiment results show that: after the trans-MsALMT1 gene tobacco is subjected to stress growth for 24 h in a solution containing 30 muM of AlCl3, malic acid secreted by root tip and aluminum resistance of the trans-MsALMT1 gene tobacco are significantly higher than malic acid secreted by root tip and aluminum resistance of the wild-type control plant, and aluminum content in root tip of the trans-MsALMT1 gene tobacco is significantly lower than aluminum content in root tip of the wild-type plant.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to an alfalfa malate channel protein gene for improving the ability of plants to resist aluminum toxicity stress MsALMT1 The plant expression vector pK2-35S- MsALMT1 The invention and its application in the preparation of transgenic plants with reduced aluminum absorption and enhanced aluminum toxicity resistance. Background technique [0002] Aluminum toxicity is a major limiting factor for plant growth and crop yield in acidic soils. Acidic soils account for 30% of the world's arable soils, and are mainly distributed in South Africa, Central Asia, and Southeast Asia, which have the greatest food demand in developing countries. The acidic soil in my country is mainly distributed in the tropical, subtropical and Yunnan-Guizhou-Sichuan areas south of the Yangtze River, covering an area of ​​2.04 million hectares. The pH value of most of the soil is less than 5.5, and a larg...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29A01H5/00
Inventor 陈丽梅陈奇武孔焕李昆志玉永雄
Owner KUNMING UNIV OF SCI & TECH
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