Vector for analyzing expression specificity of plant promoter, preparation method and application thereof

A promoter-specific technology, applied in the field of genetic engineering, can solve the problems of prolonged screening time, influence on differentiation and seedling formation, slow growth of resistant callus, etc., to reduce biosafety risks, reduce non-specific interactions, improve specific effect

Pending Publication Date: 2021-09-24
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the fly in the ointment is that the activity of the tCUP1 promoter in rice is less than that of the 35S promoter, the growth of resistant callus is slow, and the screening time is significantly prolonged, which affects the later differentiation and seedling formation, and cannot meet the requirements of large-scale gene expression analysis.

Method used

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  • Vector for analyzing expression specificity of plant promoter, preparation method and application thereof
  • Vector for analyzing expression specificity of plant promoter, preparation method and application thereof
  • Vector for analyzing expression specificity of plant promoter, preparation method and application thereof

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specific Embodiment approach

[0045] As a specific implementation manner of the embodiment of the present invention, the initiation codon of the coding sequence of the β-glucuronidase gene is ATG.

[0046] As a specific implementation of the embodiment of the present invention, the nucleotide sequence of the vector is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0047] The embodiments of the present invention also relate to host bacteria or transgenic strains containing the above-mentioned vectors. Preferably, the recombinant plasmid is a recombinant plasmid containing QHB, CYCB, DR5 or PR1b promoter.

[0048] The embodiment of the present invention also relates to a method for analyzing the expression specificity of a plant gene promoter in rice using the above-mentioned vector, at least including the following steps:

[0049] S1. Connect the upstream promoter fragment of the target gene or the synthetic promoter fragment into the multiple cloning site in front of the reporter gene in the vector to obtain a re...

Embodiment 1

[0057] Vectors for analysis of expression specificity of plant promoters:

[0058] 1) The a4 vector composed of deoxyribonucleotides shown in SEQ ID NO: 1, its T-DNA region contains a hygromycin gene expression cassette driven by a callus specific promoter, and a multiple cloning site GUS gene expression cassette.

[0059] Among them, the callus-specific promoter is close to the left border LB, far away from the GUS gene, specifically as figure 1 Shown in a4.

[0060] 2) The c4 vector composed of deoxyribonucleotides shown in SEQ ID NO: 2, its T-DNA region contains a hygromycin gene expression cassette driven by a callus-specific promoter, and a multiple cloning site GUS gene expression cassette. Wherein the callus-specific promoter is reversely close to the GUS gene, specifically as figure 1 Shown in c4.

[0061] Such as figure 1 As shown, in a4, the CSP promoter in the vector is close to the left border and away from the GUS gene; in c4, the CSP promoter in the vector ...

Embodiment 2

[0072] Construction of embodiment 2 promoter expression vector

[0073] In order to construct a transformation vector capable of accurately expressing foreign gene promoters in rice for GUS histochemical staining analysis, the GUS expression vector tCUP1-HPT-GUS-Tnos(a3) was screened using the tCUP1 promoter to drive the hygromycin resistance gene and T35S-HPT-tCUP1-GUS-Tnos(c3) (Zhou et al., 2014) as the backbone to construct the GUS expression vector CSP-HPT-GUS- Tnos(a4) and T35S-HPT-CSP-GUS-Tnos(c4) (attached figure 1 ), the specific construction method and steps are as follows:

[0074] Construction method of CSP-HPT-GUS-Tnos(a4):

[0075] 1) Digest the tCUP1-HPT-GUS-Tnos(a3) vector with AscI (purchased from NEB Company), cut off the tCUP1 promoter, and recover the vector fragment.

[0076] 2) The rice callus-specific high-expression promoter (SEQ ID NO: 3) was excised from the pMD19-T vector (purchased from Takara Company) with AscI, and after recovery, it was combine...

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Abstract

The invention relates to the field of genetic engineering, and particularly relates to a vector for analyzing the expression specificity of a plant promoter, a preparation method and application thereof. A T-DNA region of the vector sequentially comprises a screening marker gene expression cassette driven by a callus specific promoter and a reporter gene expression cassette with multiple cloning sites from the 5' end to the 3' end; and the screening marker gene expression cassette driven by the callus specific promoter comprises the callus specific promoter, a screening marker gene coding sequence and a terminator. The vector utilizes the rice callus specific promoter to drive the expression of a screening marker gene, can greatly reduce the non-specific interaction of a target gene promoter, improves the specificity of target gene expression, can effectively reduce the biological safety risk caused by the screening marker gene in transgenic plants, and has important application value in basic theoretical research and molecular breeding.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a carrier for analyzing the expression specificity of a plant promoter, its preparation method and application. Background technique [0002] Using transgenic technology to study gene function in plants and cultivate biosafe transgenic crops requires accurate expression of the target gene, but in fact, independent transgenic lines often show different gene expression levels and genetic traits (Hansen & Wright, 1999). This inadvertently introduced influence that has nothing to do with the target gene mainly comes from two aspects: one is the interaction between the transgenic sequence and the external flanking gene sequence caused by the different T-DNA insertion position during the transgenic process, which is called the position effect The second is the influence of the selection marker gene carried by the transgenic vector itself and the regulatory elements driving the expres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00A01H6/46
CPCC12N15/8205C12N15/8209C12N15/8222
Inventor 周洁王栩鸣杨勇余初浪程晔羊健严成其陈剑平
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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