Promoter of shepherd spurse CBF path key gene CbCBF and applications thereof

A promoter and shepherd's purse technology, applied in the fields of plant genetic engineering and molecular biology, can solve the problems of few cold-inducible specific promoters, and have not yet discovered the cultivation of cold-resistant plants, etc., and achieve the effect of improving cold resistance

Inactive Publication Date: 2010-04-14
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of specific inducible promoters to avoid non-specific sustained and high-efficiency expression of foreign genes in host plants has been widely recognized, but there are not many specific promoters that can be app

Method used

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  • Promoter of shepherd spurse CBF path key gene CbCBF and applications thereof
  • Promoter of shepherd spurse CBF path key gene CbCBF and applications thereof
  • Promoter of shepherd spurse CBF path key gene CbCBF and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Isolation and Identification of Shepherd's Purse CBFP Promoter

[0023] 1. Cultivation of shepherd's purse seedlings

[0024] The shepherd's purse seeds come from Shanghai Seed Company. After the shepherd's purse seeds are sterilized, they are sown in the 0 The culture tank of the culture medium was cultured at 25°C for 4 weeks, and the photoperiod was 16 hours of light and 8 hours of darkness.

[0025] 2. Cloning of the promoter sequence

[0026] The promoter sequence of CbCBF gene of shepherd's purse was amplified by Genome walking technology. Experimental operation according to UniversalGenomeWalker TMKit (CLONTECH) manual. After the Genome walking library of shepherd's purse was built, 2 primers were designed according to the open reading frame (ORF) of the cDNA sequence of the CBF gene (GenBank Accession No.: AY391121): 5'-TCCGACGAACTCCTC TGTAAAC TGG-3' (denoted as SEQ ID NO .2) and 5′-CGGGTCTCACGAAACTTCTTCCTAC-3′ (denoted as SEQ ID NO.3), cooperate ...

Embodiment 2

[0028] Example 2 Induced expression of shepherd's purse CBF pathway key gene CbCBF gene

[0029] The expression of CbcbfmRNA was verified to be induced by low temperature by semi-quantitative RT-PCR. The wild-type shepherd's purse was used as the material, and the material was treated according to three different low-temperature induction conditions: 28°C for 4d; 4°C for 8h; 28°C for 4d. Sequentially extract the total RNA of three kinds of shepherd's purse leaves in three different stages, and detect and measure the OD of the extracted RNA by electrophoresis 260 value. Semi-quantitative RT-PCR was performed using One Step PCR Kit (Takara). The RT-PCR reaction conditions were 50°C for 30 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 2 min. Uninduced RNA was extracted for the amplification of the ubiquitin gene (about 250nt) (AY189972), which was used as a control for better comparative analysis.

[0030] The results showed that only under low tem...

Embodiment 3

[0031] Example 3 Analysis of Low Temperature Inducible Activity of Shepherd's Purse CBFP Promoter

[0032] In this example, the cloned shepherd's purse CBFP promoter sequence was connected to the pCAMBA1301 vector (provided by CAMBIA [the Center of the Application of Molecular Biology to International Agriculture, Australia] in Australia) to construct a plant expression vector that drives the GUS gene . Transient expression experiments showed that in both monocotyledonous and dicotyledonous plants, the CBFP promoter of shepherd's purse can enhance the expression of GUS gene under low temperature induction. Therefore, the shepherd's purse CBFP promoter has a good application prospect in crop cold resistance genetic engineering.

[0033] Construction of transient expression vector

[0034] In this embodiment, the transient expression vector pCAMBA1301-CBFP-GUS is constructed

[0035] The CaMV35S promoter of the commercialized plant expression vector pCAMBA1301 was excised a...

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Abstract

The invention pertains to the technical field of plant genetic engineering, and particularly relates to a promoter CBFP of a key gene CbCBF of a shepherd spurse CBF path and applications thereof. The nucleotide sequence of the promoter is showed as SEQ ID NO:1, wherein CbCBF gene and the promoter thereof contain the nucleotide sequence of the 2283rd basic group in the sequence showed in the sequence table of SEQ ID NO:1, and the nucleotide sequence encodes a transcription factor which is induced by low temperature and can regulate and control a cold-inductive gene. The promoter area contains two ABRE cis-acting elements which are serially arranged, thus being capable of specifically playing a response reaction to low temperature and ABA; and the promoter area contains three MYCRE cis-acting elements CANNTG, thus being capable of being combined with an upstream control element (ICE) (inducer of CBF expression). An inductive expression carrier structured by using the genetic promoter can powerfully induces the expression of a target gene when the plant is stressed by low temperature. The invention also provides the applications of the promoter in cultivating cold-resistant plants, which realizes the breed improvement of crops.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and plant genetic engineering. It specifically relates to the cloning and application of plant stress-induced promoters. By identifying and cloning a promoter of a shepherd's purse that is induced by low temperature and can regulate the transcription factor gene of cold-induced genes, it is applied to the genetic transformation of plant stress genes, especially for plant cold resistance genes, so as to improve the cold resistance of plants Purpose. Background technique [0002] Plants have sufficient adaptability and resistance to environmental changes and adverse environments. This stress resistance is not only controlled by the genetic type of phylogenetic evolution, but also restricted by physiological and ecological factors in individual development. As one of the important environmental factors, temperature limits the distribution, growth and yield of plants, and plays a decisive...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/82
Inventor 林娟周明琦吴丽华
Owner FUDAN UNIV
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