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An activation tag ac/ds transposition system and its application in the construction of plant mutant library

A technology for activating tags and transposons, which is applied in the field of plant genetic engineering, can solve the problems of difficult genetic transformation technology and heavy workload, and achieve the effect of overcoming recessive mutations and avoiding artificial transgenic operations

Inactive Publication Date: 2016-02-10
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As mentioned above, T-DNA-based activation labeling technology requires a lot of heavy tissue culture transformation work to obtain tens of thousands of transgenic plants. It is even more difficult for crops

Method used

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  • An activation tag ac/ds transposition system and its application in the construction of plant mutant library
  • An activation tag ac/ds transposition system and its application in the construction of plant mutant library
  • An activation tag ac/ds transposition system and its application in the construction of plant mutant library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The construction of embodiment 1.Ac transposase plant expression vector

[0045] 1. The amplification primer sequence of the Ac transposase gene is:

[0046] Reverse primer: TCATGGAGAGGAGCCACTTGC

[0047] Forward primer: AAACCGCGGATGACGCCTCCGGTTGGAAAT

[0048] 2. PCR amplification reaction system: the reaction system with a total volume of 50ml contains 5.0ml of 10×buffer, 1.0ml of 10mMdNTPs, 0.5ml of 10mM forward and reverse primers, 1ml of plasmid template, 2ml of PfuDNA polymerase, sterile double distilled water 40ml;

[0049] 3. PCR amplification reaction conditions: melting at 94°C for 5 minutes; 30 cycles at 94°C for 40s, 58°C for 40s, and 72°C for 5 minutes; extension at 72°C for 5 minutes;

[0050] 4. Purification of PCR products: After PCR amplification, the products are directly purified with the DNA recovery kit of Dingguo Biotech Co., Ltd., and purified according to the operating instructions;

[0051] 5. Enzyme digestion of PCR products: a reaction syst...

Embodiment 2

[0069]Example 2. Construction of transposon (Ds) vector with activation tag

[0070] 1. Construct the transposon element containing the pBSK vector inside:

[0071] (1)-(8) of this implementation process connected the 5' end of the transposon element to a section of the pBSK vector, and (9)-(18) connected the 3' end of the transposon element to the pBSK vector Another segment, and finally a transposon element containing the pBSK vector inside.

[0072] (1) PCR amplification of the 5' fragment of the transposon element: the forward primer is AGCTTGATATCGAATTCCTGC, the reverse primer is AAACTCGAGCGGCGGTACCCCGCGC, the amplification template is the plasmid vector DsLaunchPadT-DNA vector, and the amplification system is the same as that of Example 1-2; the amplification reaction Conditions: melting at 94°C for 5 minutes; 30 cycles of 94°C for 40s, 58°C for 40s, and 72°C for 2 minutes; extension at 72°C for 5 minutes; after that, the PCR product was purified and recovered, and the ...

Embodiment 3

[0126] Example 3. Agrobacterium transformation of plasmid DNA

[0127] 1. Materials and reagents:

[0128] Agrobacterium LBA4404;

[0129] YEB medium (1 liter): beef extract 5g, yeast extract 1g, tryptone 5g, MgSO4 7H 2 O0.5g, add 800ml of distilled water to adjust the pH to 7.0, constant volume (solid medium plus 1.5% agar powder), and autoclave after aliquoting.

[0130] 1. Preparation of Agrobacterium Competent: Pick a single colony of LBA4404 and inoculate it into 5ml bacterial solution (containing rifampicin at a concentration of 50mg / ml), and cultivate overnight; ml); 28°C, 200rpm culture to OD600=0.6-0.8; ice bath for 20 minutes, 5000rpm, 4°C, centrifuge for 15 minutes to collect the bacteria; suspension in 10% glycerol of equal volume; collect the bacteria as above, 1 / 2 volume 10 % glycerol suspension, repeat once; collect bacteria, suspension in glycerol prepared by 50ml ultrapure water; collect bacteria, suspension in 10% glycerol prepared by 1ml ultrapure water; ...

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Abstract

The present invention provides an activation tag Ac / Ds transposition system, comprising a transposase Ac expression vector and a transposon Ds carrier, characterized in that the transposon Ds carrier has an activation tag, and the activation tag is 4 A series of 35S enhancers, 4×35S enhancers are placed inside the 3' end of Ds. The Ac / Ds transposition system of the present invention can express the Ac transposase gene at a high level in various plants, activate the expression level of genes adjacent to the transposon insertion site at a high level, to create dominant mutants, and efficiently identify and Isolation of sequences flanking transposable element insertion sites for mutant library creation in polyploid plants. The invention is an ideal plant functional genomics research tool, and has great significance for the establishment of mutants and functional genomics research of crops, especially polyploid crops.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically relates to the construction of the novel activation tag Ac / Ds transposition system and its application in the creation of plant mutant libraries. Background technique [0002] With the completion of genome-wide sequencing of various plants, the era of plant post-genomics, which aims to seek gene functions, has arrived. Facing the massive data and resources generated in these genome sequencing work, how to discover new genes, understand gene functions and understand how all genes coordinate and function during plant growth and development has become the most urgent task for functional genomics research. To accurately understand the function of each gene and the interaction between genes, it is necessary to analyze the mutant phenotype of a single gene and multiple genes, and analyze and identify the gene function through the mutant phenotype. The most direct and effective...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66A01H5/00A01H1/02
Inventor 张洪博王文静刘贯山马浩然
Owner SOUTHWEST UNIV
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