Escherichia coli-yeast-agrobacterium shuttle expression vector and application thereof

A shuttle expression vector, Escherichia coli technology, applied in the field of genetic engineering, can solve the problems of long time, low efficiency, low efficiency, etc., and achieve the effects of shortening the process and time, the preparation process is simple, and the work efficiency is high

Active Publication Date: 2014-01-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The disadvantages of the above vector construction methods are that the efficiency is very low, time-consuming, and not suitable for high-throughput DNA transformation and gene knockout operations
This makes vector construction inefficient and unsuitable for high-throughput ATMT operations

Method used

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  • Escherichia coli-yeast-agrobacterium shuttle expression vector and application thereof
  • Escherichia coli-yeast-agrobacterium shuttle expression vector and application thereof
  • Escherichia coli-yeast-agrobacterium shuttle expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The construction method of Escherichia coli-yeast-Agrobacterium shuttle expression vector pKO1B or pKO1B-RED, including:

[0046] 1 Construction of Escherichia coli-Agrobacterium shuttle expression vector carrying H3 promoter and fluorescent protein gene

[0047] (1) Acquire the H3 promoter of Magnaporthe grisea

[0048] Using the pKD5 plasmid as a template, high-fidelity PCR amplification was performed using primers a1 / a2 to obtain a partial sequence (1.2kb) of the H3 promoter sequence (1.5kb in length). The base sequences of the primers a1 / a2 are as follows:

[0049] Upstream primer a1: 5'-TT CTCGAG GTGTATTTTCTTTCTGCTCG-3' (Xho I restriction site is underlined);

[0050] Downstream primer a2: 5'-TT GAATTC GGCCATTGTGATTGATTTGT-3' (the EcoRI restriction site is underlined).

[0051] The PCR reaction system is: pKD5DNA 10ng, high-fidelity DNA polymerase 0.5μL, dNTP (50mM) 0.4μL, upstream and downstream primers 0.5μL, 10x PCR buffer 5μL, add water to 50μL.

[0052]...

Embodiment 2

[0082] The construction method of Escherichia coli-yeast-Agrobacterium shuttle expression vector pKO1B-HIS3, including:

[0083] 1 Construct the pKO1A vector according to the method of step 1-(1)(2) of Example 1;

[0084] 2 Construction of pKO1B-HIS3

[0085] (1) Obtain 2micro2_origin DNA fragment

[0086] Using the pYES2 plasmid as a template, use primers e1 / e2 for PCR amplification to obtain a 1.1kb 2micro2_originDNA fragment. The base sequence of primers e1 / e2 is:

[0087]Upstream primer e1: 5'-GTATCGACGGAGCCGATTTTGAAA CCGCGG

[0088] GCTAGCTTTTCAATTCAATTCATC-3' (the underline is the Sac II restriction site);

[0089] Downstream primer e2: 5'-TTCGATGTAACCCACTCGTGCA-3';

[0090] The PCR reaction system is: pYES2DNA 10ng, high-fidelity DNA polymerase 0.5μL, dNTP (50mM) 0.4μL, upstream and downstream primers 0.5μL, 10x PCR buffer 5μL, add water to 50μL.

[0091] The PCR reaction conditions were: 94°C for 3 minutes, 35 cycles (94°C for 30 seconds, 57°C fo...

Embodiment 3

[0104] The construction method of the Escherichia coli-yeast-Agrobacterium shuttle expression vector pKO1B-LEU2, including:

[0105] 1 Construct the pKO1A vector according to the method of step 1-(1)(2) of Example 1;

[0106] 2 Construction of pKO1B-LEU2

[0107] (1) Obtain the 2micro2_origin DNA fragment according to the method of step 2-(1) of Example 2;

[0108] (2) Obtain LEU2 gene fragment

[0109] Using the pTAL4_Leu plasmid as a template, use primers g1 / g2 for PCR amplification to obtain a 2.2kb LEU2 gene fragment. The base sequence of primers g1 / g2 is:

[0110] Upstream primer g1: 5'-ATCAGTTGGGTGCACGAGTGGGTTACAT

[0111] CGAATCATATAATTTCTGTTGAATTAC-3';

[0112] Downstream primer g2: 5'-ACAGAGCGTTGCTGCCTGTGATCA CCGCGG

[0113] CGTCTACCCTATGAACATATTCCA-3' (the underline is the Sac II restriction site);

[0114] The PCR reaction system is: pTAL4_Leu DNA 10ng, high-fidelity DNA polymerase 0.5μL, dNTP (50mM) 0.4μL, upstream and downst...

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Abstract

The invention discloses an escherichia coli-yeast-agrobacterium shuttle expression vector and an application thereof. The escherichia coli-yeast-agrobacterium shuttle expression vector is a dual-element vector which contains a yeast DNA (Deoxyribose Nucleic Acid) replicon and a yeast selection marker gene. The escherichia coli-yeast-agrobacterium shuttle expression vector is applied as a DNA transformed into a fungus or plant cell. The escherichia coli-yeast-agrobacterium shuttle expression vector disclosed by the invention is reported first in the world, is a plasmid which can be copied, is proliferated and stably exists in yeast, escherichia coli and agrobacterium at the same time, can be directly transformed into the fungus or plant cell through an agrobacterium tumefaciens mediated transformation way and used for shortening the gene operation flow and time, and is convenient to use, high in work efficiency as well as simple, convenient, rapid and efficient in preparation process.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an Escherichia coli-yeast-Agrobacterium shuttle expression vector and its application. Background technique [0002] Transgenic technology is a method of introducing artificially isolated or modified DNA fragments into the genome of an organism, causing the expression or non-expression of a certain gene, and causing heritable changes in the traits of the organism. In production and scientific research, plants and fungi are two types of genetically modified objects that are widely carried out. Transformed DNA fragments are generally used for gene expression, gene knockout or gene silencing, etc., with the purpose of studying gene functions, improving plant disease resistance, increasing production, improving quality, and increasing production efficiency of industrial fungi. There are many methods for DNA transformation of plants and fungi, among which Agrobacterium tu...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12N1/21C12N15/82C12R1/19
Inventor卢建平曹惠娟张莉林林福呈
OwnerZHEJIANG UNIV