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Detection method for promoter activity

A detection method and promoter technology, which are applied in the direction of material analysis by observing the effect on chemical indicators, fluorescence/phosphorescence, analysis by chemical reaction of materials, etc., can solve the problem of heavy workload, time-consuming, induction of differentiation It can reduce production costs, improve screening efficiency, and avoid transformation specificity.

Active Publication Date: 2013-09-04
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a method for detecting promoter activity, which can effectively overcome technical defects such as time-consuming, heavy workload, difficulty in inducing differentiation and rooting, and specificity of genotype transformation in the prior art.

Method used

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  • Detection method for promoter activity
  • Detection method for promoter activity
  • Detection method for promoter activity

Examples

Experimental program
Comparison scheme
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no. 1 example

[0038] The first embodiment, the construction of the recombinant expression vector containing the promoter sequence

[0039] 1. Construct a recombinant cloning vector containing the promoter sequence to be tested

[0040] The prZmUbi nucleotide sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector pT-prZmUbi, and its construction process like figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; prZmUbi is the maize variety B73 Ubiqutin (ubiquitin) gene promoter (SEQ ID NO: 1); MCS is a multiple cloning site).

[0041]Then, the recombinant cloning vector pT-prZmUbi was transformed into Escherichia coli T1 comp...

no. 2 example

[0055] The second embodiment, the acquisition of maize embryos transferred to the promoter sequence to be tested

[0056] According to the commonly used Agrobacterium infection method, the immature embryos of the aseptically cultured maize variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 3 in the first example, so that the T-DNA (including prZmUbi promoter sequence, prOsAPX promoter sequence, prOsAct1 promoter sequence, prOsPGD1 promoter sequence and GUS gene) in recombinant plant expression vectors p90001, p90030, p90031, p90039 and p80000 (empty vector control) were transferred into In the maize genome, the maize embryos transferred to the prZmUbi promoter sequence, the maize embryos transferred to the prOsAPX promoter sequence, the maize embryos transferred to the prOsAct1 promoter sequence, the maize embryos transferred to the prOsPGD1 promoter sequence and the Empty vector was used as control corn germ, and wild-type corn germ was used as negativ...

no. 4 example

[0058] The fourth embodiment, activity determination of the promoter sequence to be tested

[0059] Experiment 1. GUS staining

[0060]Take 50-100 corn embryos transformed with prZmUbi promoter sequence, corn embryos transformed with prOsAPX promoter sequence, corn embryos transformed with prOsAct1 promoter sequence, corn embryos transformed with prOsPGD1 promoter sequence and positive control corn embryos as samples , refer to the method of Jefferson et al. (Jefferson R.A., Burgess S.M., Hirsh D.Beta-glucuronidase from Escherichia coliasa gene fusion marker.Proc.Natl.Acad.Sci., 1986,83:8447-8454) and make appropriate improvements, through Seal staining in staining solution for two days at 37°C, and examine the expression of GUS histochemically. The main components of the staining solution are: 0.1M NaPO 4 , 0.01M EDTA (pH8.0), 0.5mM potassium ferricyanide, 0.5mM potassium ferrocyanide, 0.5mg / ml 5-bromo-4-chloro-3-indole-β-D-glucuronide (X -gluc). The GUS enzyme produced i...

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Abstract

The invention relates to a detection method for promoter activity. The detection method comprises the steps of infecting a plant embryo by agrobacterium, co-culturing for at least two days, and carrying out GUS identification. The detection method for the promoter activity can identify the promoter activity through GUS staining analysis and / or GUS protein content determination only by co-culturing the plant embryo (particularly corn embryo) for three days, without a tissue culture process lasting for a plurality of weeks or even a plurality of months, and omits links such as corn genotype transformed specificity, induced differentiation, difficulty to take roots, etc. Therefore, time is saved; save time, screening efficiency is increased; production cost is reduced; and experimental results are intuitive and accurate.

Description

technical field [0001] The invention relates to a method for detecting promoter activity, in particular to a method for rapidly detecting constitutive promoters. Background technique [0002] Expression of a heterologous DNA sequence in a plant host is dependent on having an operably linked promoter that is functional in the plant host. The choice of promoter sequence will determine when and where the heterologous DNA sequence is expressed in the plant host. Thus, when expression in plant-preferred tissues is desired, tissue-specific promoters are used. Conversely, when expression in whole plant cells is desired, constitutive promoters are used. Transformed plants can be obtained by directly treating the meristems of plant embryos, and the transformed plants can be analyzed to determine whether the promoter sequence drives expression of the heterologous DNA sequence of interest in the target tissue. [0003] Maize transgenic technology has been greatly developed in recent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/82G01N21/64
Inventor 贾志伟王娜
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD