Carrier for enhancing aluminum-tolerance of plant, and method for establishing the same

A plant expression vector and vector technology, applied in the field of plant genetic engineering, can solve the problem of carbon source consumption

Inactive Publication Date: 2011-09-28
KUNMING UNIV OF SCI & TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these studies are all overexpression of one gene in plants, and there is no report that overexpression of two or more genes in plants significantly improves the ability of plants to tolerate aluminum
In addition, existing studies have used constitutive promoters to overexpress genes related to org

Method used

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  • Carrier for enhancing aluminum-tolerance of plant, and method for establishing the same
  • Carrier for enhancing aluminum-tolerance of plant, and method for establishing the same
  • Carrier for enhancing aluminum-tolerance of plant, and method for establishing the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: PEPC Gene amplification and TA cloning:

[0060] PEPC Amplification of gene DNA coding region and TA cloning strategy such as figure 2 As shown, first a pair of primers are designed according to the PEPC gene sequence, the sequence is as follows:

[0061] pepc5:5'-CACC GCATGC CATCAGTCCTCGATGTGACC-3'

[0062] pepc3: 5'- GATATC TTAGCCTGTATTGCGCATCCCCGC-3'

[0063] Add the CACC characteristic sequence to the end of the 5' end primer pepc5, and thus form Nco I restriction site; 3' end primer pepc3 end plus xho I restriction site.

[0064] A thermophilic cyanobacteria mutant PEPC Gene entry vector pENTR TM 2B-KsPEPC (Chen LM. Genetic Engineering of photosynthesis in C3 plant. Yunnan: Yunnan Science and Technology press, 2008) was used as a template, and the mutant was amplified with PEPC-specific primers pepc5 and pepc3 PEPC The DNA fragment of the coding region of the full-length gene (3.0Kb), recovered and purified the PEPC full-length gene f...

Embodiment 2

[0065] Example 2: The construction strategy of the entry cloning vector pENTR*-PrbcS-PEPC:

[0066] PEPC The construction strategy of the gene entry vector pENTR*-PrbcS-PEPC is as follows: Figure 4 As shown, first cut the purified plasmid vectors pENTR*-PrbcS-*T-GFP and pUCm-PEPC with NcoI and XhoI, separate the cut vector and insert fragments by agarose gel electrophoresis, and recover pENTR from the gel The vector fragment pENTR*-PrbcS (4.0kb) and pUCm-PEPC produced by cutting *-PrbcS-*T-GFP PEPC The DNA fragment (3.0kb) of the gene, and then use the ligase kit of TaKaRa to connect pENTR*-PrbcS and PEPC A DNA fragment of the gene generates the entry vector pENTR*-PrbcS-PEPC. Conversion of high efficiencies (10 8 ) Escherichia coli competent cell DH5α, spread the transformed Escherichia coli on the plate added with kanamycin (Km, 50 mg / ml), at 37 o C culture overnight, screen the Km-resistant recombinant colony, extract the plasmid from the Km-resistant recombinant c...

Embodiment 3

[0067] Embodiment 3: Construction strategy of PEPC gene plant expression vector pH2-35S-PrbcS-PEPC:

[0068] The PrbcS-PEPC was subcloned into the plant expression vector pH2GW7 (Gateway's destination vector) by the LR reaction of Gateway technology ( Figure 6 ). The specific method is: use the plasmid extraction kit to purify Gateway’s target vector pH2GW7, add pENTR*-PrbcS-PEPC and pH2GW7 each 150 ng, 1 μl LR Clonase II Enzyme Mix (Invitrogen) to the LR reaction system of Gateway, mix both at 25 o C reacted overnight, and integrated PrbcS-PEPC into pH2GW7 through the action of integrase to obtain the plant expression vector plasmid pH2-35S-PrbcS-PEPC of PEPC ( Figure 6 ). Conversion of high efficiency (10 8 ) Escherichia coli competent cell DH5α, spread the transformed Escherichia coli on the plate added with spectinomycin (Spe, 50 mg / ml), at 37 o C cultured overnight, screened the Spe-resistant recombinant colonies, extracted plasmids from the Spe-resistant recombina...

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Abstract

The invention provides a carrier for enhancing aluminum-tolerance of a plant, and a method for establishing the same. The carrier is a plant expression vector having photoinduction promoters and phosphoenolpyruvate carboxylase (PEPC) genes. The method for establishing the carrier comprises the following steps: searching for the sequence of the full length gene of Synechococcus vulcanus PEPC in GenBank and designing a pair of primers with sequences as described in the specification; recovering and purifying PEPC full length gene segments and connecting the segments to a pUCm-T vector; establishing an entry vector pENTER*-PrbcS-PEPC; establishing a plant expression vector pH2-35S-PrbcS-PEPC. In the invention, the activity of citrate synthase of tabacoo with transgenic PEPC and CS genes is 2.4 to 2.6 times that of wild tobacco, and the activity of phosphoenolpyruvate carboxylase of such tabacco is 2.2 to 2.4 times that of wild tobacco. The special-purpose carrier provided in the invention can exert great influence on the improvement of aluminum-tolerance of a plant, and particularly, can significantly promote aluminum-tolerance of plants grown in acid red soil in southern China, thereby providing a novel approach for variety improvement of plants.

Description

technical field [0001] The invention specifically relates to a carrier and a construction method for constructing a new citric acid synthesis pathway in a plant to improve the plant's aluminum tolerance, and belongs to the field of plant genetic engineering. Background technique [0002] Aluminum is the third most abundant element on the earth's surface and is highly toxic to plants. When the pH value of the soil solution drops to a certain value after acidification, aluminum ions will be released from the silicate or oxide and dissolved into the soil solution (Ma JF, Furukawa J. 2003. Recent progress in the research of external Al detoxification in higher plants: a minireview. J Inorg Biochem . 97:46-51). Soluble aluminum can be divided into the following categories: free aluminum or Al(H 2 O) 6 3+ , polymerized aluminum Al 13 and low molecular weight aluminum compounds. With the increase of soil pH, Al(H 2 O) 6 3+ Convert to Al(OH) 2+ , Al(OH) 2 + . In neutra...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66
Inventor 陈丽梅王奇峰玉永雄易琼胡清泉赵玥李昆志
Owner KUNMING UNIV OF SCI & TECH
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