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Constitutive promoter and application thereof

A constitutive promoter and constitutive technology, applied in recombinant DNA technology, using vectors to introduce foreign genetic material, DNA/RNA fragments, etc.

Active Publication Date: 2014-04-16
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the ubiquitin gene promoters from plants are mainly isolated from maize, sunflower, tobacco, potato, sugarcane, Arabidopsis and soybean, but the ubiquitin gene promoters in rice are rarely reported

Method used

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  • Constitutive promoter and application thereof
  • Constitutive promoter and application thereof
  • Constitutive promoter and application thereof

Examples

Experimental program
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Effect test

no. 1 example

[0060] The first embodiment, the synthesis of prOsRUBQ2 promoter sequence

[0061] Obtain the constitutive promoter prOsRUBQ2 of the present invention, as shown in SEQ ID NO: 1 in the sequence listing; the prOsRUBQ2 promoter sequence (shown in SEQ ID NO: 1 in the sequence listing) was synthesized by Nanjing KingScript Biotechnology Co., Ltd. ; The 5' end of the synthetic prOsRUBQ2 promoter sequence (SEQ ID NO: 1) is also connected with a HindIII restriction site, and the 3' end of the prOsRUBQ2 promoter sequence (SEQ ID NO: 1) is also connected with BamHI Restriction sites.

no. 2 example

[0062] The second embodiment, construction of recombinant expression vector p100017 containing prOsRUBQ2 promoter sequence and transformation of recombinant expression vector into Agrobacterium

[0063] 1. Construction of the recombinant cloning vector pT-prOsRUBQ2 containing the prOsRUBQ2 promoter sequence

[0064] The prOsRUBQ2 promoter 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-prOsRUBQ2. The construction process is as follows: 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; prOsRUBQ2 is the rice variety Nipponbare Ubiqutin (ubiquitin) 2 gene promoter (SEQ ID NO: 1); MCS is a multiple clon...

no. 3 example

[0075] The third embodiment, the acquisition of maize plants transferred to the prOsRUBQ2 promoter sequence

[0076] According to the routinely used Agrobacterium infection method, the immature embryos of the aseptically cultivated corn variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 4 in the second example, so as to infect 2 and 3 in the second example. The T-DNA (including the promoter sequence of maize Ubiquitin gene, prOsRUBQ2 promoter sequence, control sequence, CTP-EPSPS gene, PMI gene and Nos terminator sequence) in the constructed recombinant expression vectors p100017 and p100078 (control sequence) was transferred into In the maize genome, maize plants transfected with the prOsRUBQ2 promoter sequence and maize plants transfected with the control sequence (positive control) were obtained; at the same time, wild-type maize plants were used as a negative control.

[0077] For Agrobacterium-mediated maize transformation, briefly, immature immatu...

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Abstract

The invention relates to a constitutive promoter and application thereof. The nucleotide sequence of the constitutive promoter comprises the following sequence (a) or (b): (a) nucleotide sequence disclosed as SEQ ID NO:1; or (b) nucleotide sequence crossbred with the nucleotide sequence defined in (a) under strict conditions. The constitutive promoter generally has activity in most tissues and many types of cells of plants, and obviously enhances the expression level of the target heterologous nucleotide sequence in plant tissues especially in leaves and stems.

Description

technical field [0001] The invention relates to a constitutive promoter and its use, in particular to a constitutive rice ubiquitin gene promoter and its use. Background technique [0002] Major advances in cell biology and transgenic technology have allowed the incorporation of foreign genes into many crops. Foreign genes are transferred into plants mainly to express proteins, which are called "transgenic plants", and the proteins will impart beneficial traits, such as resistance to pathogenic microorganisms or insects, resistance to herbicides, tolerance to drought or other unfavorable environment. Usually, the DNA coding region of the foreign gene is linked to a strong and constitutive DNA promoter region to ensure efficient expression of the foreign gene in transgenic cells. [0003] A large number of common promoters are used to drive the expression of foreign genes in transgenic plants. The cauliflower mosaic virus (CaMV) 35S promoter is widely used in both dicots a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/62C12N15/63
Inventor 庞洁张成伟鲍晓明刘海利王登元
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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