Method for identifying induction characteristics of promoter by utilizing phenotype of transgenic plant

A technology for transgenic plants and promoters, which is applied in the field of identifying the characteristics of plant promoters, can solve the problems of fusion, restricting the process of plant genetic engineering and industrialization development, and quantitative difficulties, and achieves the effect of having identification results and intuitive identification results.

Inactive Publication Date: 2011-09-28
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these genes have some shortcomings of one kind or another, such as the need for special chemical reagents and observation equipment for experimental observation, cumbersome analysis steps, difficult quantification, or the inability to carry out amino-terminal fusion, which greatly limits the application of plant genetic engineering. Process and Industrialization Development

Method used

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  • Method for identifying induction characteristics of promoter by utilizing phenotype of transgenic plant
  • Method for identifying induction characteristics of promoter by utilizing phenotype of transgenic plant
  • Method for identifying induction characteristics of promoter by utilizing phenotype of transgenic plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1. Construction of plant expression vectors:

[0021] 1. Experimental materials:

[0022] 1.1 Plant material:

[0023] Wild-type Columbia ecotype (Col-O) Arabidopsis thaliana, "Innovation No. 1" tobacco

[0024] 1.2 Strains and plasmids:

[0025] Vector plasmid: pTA7002 was kindly provided by Professor Chua Nam-Hai, Rockefeller University, USA.

[0026] Strains: Escherichia coli DH5α, provided by the Laboratory of Plant Molecular Biology, Inner Mongolia Agricultural University; Agrobacterium GV3101, provided by the Laboratory of Plant Molecular Biology, Inner Mongolia Agricultural University.

[0027] 1.3 Reagents:

[0028] rTaq enzyme and DNA marker were purchased from TaKaRa Company, various restriction enzymes and T4 DNA ligase were purchased from NEB Company, RNaseA, pCF-T, DNA recovery kit and plasmid mini-prep kit were purchased from From Beijing Tiangen Company.

[0029] Ampicillin (Amp), Kanamycin (Kan), Gentamicin (Gent), X-gal, Isopropylthio-β-D-galactos...

Embodiment 2

[0143] Example 2: Preparation and transient expression of Agrobacterium-mediated tobacco:

[0144] 1. Reagents

[0145] 1.1 Configuration of reagents

[0146] 1M MgSO4 (Shanghai Sangong): Dissolve 3.009 grams of MgSO4 in 25ml of distilled water and autoclave;

[0147] 0.2M MES (BBI): Dissolve 1.0655g of MES in 25ml of distilled water, adjust the pH to 5.5, and sterilize by autoclaving;

[0148] 150mM AS (Proteco): Dissolve 0.0294g of AS in 1ml DMSO (Shanghai Sangong), dispense in 200μl, and store at -4°C;

[0149] 30mM DEX (Sigma): Dissolve 0.0058g of DEX in 500μl of absolute ethanol and dilute 1000 times when used;

[0150] 1.2 Configuration of the suspension

[0151] 10mM MgSO4: need 1M MgSO4 100μl

[0152] 10mM MES: 500μl of 0.2M MES is required

[0153] 150 μM AS: 100 μl of 150 mM AS is required

[0154] Dilute to 10ml with sterile water.

[0155] 2. Transient expression of tobacco:

[0156] ①. Put the Agrobacterium containing the target plasmid (pTA-Kinase1) stor...

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Abstract

The invention discloses a method for identifying the induction characteristics of a promoter by utilizing a phenotype of a transgenic plant. The method mainly comprises the following steps of: cloning a plant promoter; fusing the cloned plant promoter with an arabidopsis calcium-dependent protein kinase AtCDPK1 gene, and constructing a plant expression vector; mediating the plant expression vector by using Agrobacterium tumefaciens, and performing transient expression on tobacco leaves by adopting a transient expression system; and observing the character of necrosis spots generated on transgenic tobacco leaves. The method has the advantages that: the complicated operation process and a large number of experimental apparatuses are not required, and identification results can be observed by naked eyes and are intuitive and visible.

Description

technical field [0001] The invention relates to a method for identifying by using a biotechnology method, in particular to a method for identifying the characteristics of a plant promoter. Background technique [0002] Plant promoter is one of the important elements necessary for plant genetic engineering. According to the inducible expression characteristics and expression intensity of the promoter, different types of promoters can be selected to create transgenic plants, new plant materials and new germplasm with directional expression functions, so as to meet the needs of different genetic engineering, especially transgenic plants and bioreactors needs in agriculture, industry, health care and the environment. [0003] PCR technology has been developed and matured, and the required gene sequence can be quickly obtained. After the primer sequence of the plant promoter is determined, the entire plant promoter can be easily obtained according to the operation steps in the P...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/62C12N15/82
Inventor 李国婧王瑞刚王琪刘贞万永青祈婧
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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