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Shepherd's-purse cold-acclimation COR15a gene promoter and application thereof in improving cold resistance of plants

A promoter and shepherd's purse technology, which is applied in the field of plant genetic engineering and molecular biology, can solve the problems that have not yet been discovered, and there are few cold-inducible specific promoters, and achieve the effect of improving cold resistance

Inactive Publication Date: 2013-10-16
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 applied to actual production, and cold-inducible specific promoters less
So far, no related reports have been found on the promoter sequence of shepherd's purse CbCOR15a gene and the use of shepherd's purse cold-regulated gene CbCOR15a gene promoter for breeding cold-resistant plants

Method used

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  • Shepherd's-purse cold-acclimation COR15a gene promoter and application thereof in improving cold resistance of plants
  • Shepherd's-purse cold-acclimation COR15a gene promoter and application thereof in improving cold resistance of plants
  • Shepherd's-purse cold-acclimation COR15a gene promoter and application thereof in improving cold resistance of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Isolation and identification of shepherd's purse COR15aP

[0024] 1. Cultivation of shepherd's purse seedlings

[0025] 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 placed at 25° C. for 4 weeks, and the photoperiod was 16 hours of light and 8 hours of darkness.

[0026] 2. Cloning of the promoter sequence

[0027]The promoter sequence of CbCOR15a gene of shepherd's purse was amplified by Genome walking technology. Experimental operation according to Universal GenomeWalker TM Kit (CLONTECH) manual. After the Genome walking library of shepherd's purse was built, two primers were designed according to the open reading frame (ORF) of the cDNA sequence of the COR15a gene: CbCOR15aF1: 5'-AGCCAACGCCTCCTTTGTCTTATCC-3' (denoted as SEQ ID NO. 2) and CbCOR15aF2: 5' -ATCAC CTTTAGCGGCGTAGACCAAC-3' (denoted as SEQ ID NO. 3), cooperates ...

Embodiment 2

[0029] Example 2 Expression characteristics of shepherd's purse COR15a gene under cold induction and cold acclimation treatment

[0030] 1. Planting and processing of shepherd's purse

[0031] Take appropriate amount of fresh shepherd's purse seeds, after aseptic treatment, place them in a sterile culture tank pre-configured with MS medium (MS powder 4.41g / L, sucrose 30g / L, agar powder 8.5g / L), and cultivate under light at 26°C. Cold acclimatization treatment Select the seedlings 4w after seed germination, and treat the shepherd's purse seedlings according to the temperature gradient of 26°C (4d), 12°C (4d), 4°C (4d), 0°C (2h), -4°C (2h). The seedlings 4 weeks after seed germination were selected for treatment. Under the condition of 4°C, the shepherd's purse seedlings were treated in a gradient of time 0h, 1h, 3h, 6h, 12h, 24h, and 48h, and total RNA was extracted from leaves, stems, and roots.

[0032] 2. RNA extraction

[0033] RNA extraction was performed according to ...

Embodiment 3

[0038] Example 3 Analysis of low temperature induction activity of shepherd's purse COR15aP promoter

[0039] In this example, the cloned shepherd's purse COR15aP sequence plus restriction sites Bgl II and Pst Ⅰ. Connect to the pCAMBA1301 vector (gifted 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. Transformed tobacco experiments showed that shepherd's purse COR15aP can enhance the expression of GUS gene under low temperature induction. Therefore, shepherd's purse COR15aP has a good application prospect in crop cold resistance genetic engineering.

[0040] Construction of expression vector

[0041] In this embodiment, construct the expression vector pCAMBA1301-COR15aP-GUS

[0042] The CaMV35S promoter of the commercialized plant expression vector pCAMBA1301 was excised and connected into shepherd's purse COR15aP to regulate the e...

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Abstract

The invention belongs to the technical field of plant genetic engineering, in particular relating to a promoter CbCOR15aP of a Shepherd's-purse cold-resistance gene CbCOR15a and application thereof in improving the cold resistance of plants. The nucleotide sequence of the promoter is shown in the SEQINDO:1, wherein the CbCOR15a gene and the promoter thereof comprise the nucleotide sequence of a 1706-locus basic group as indicated in the sequence of the SEQINDO:1 sequence table. The nucleotide sequence encodes a gene which is prone to low-temperature inducible expression and can promote cells to have cold-resistance capacity. Induced at 4 DEG C, the promoter does not only ensures expression level increase in lamina but also in root. The inducible expression vector constructed by segments of the promoter can launch expression of target genes when plants are confronted with low temperature stress. The invention also provides an application of the promoter in culturing cold resistant plants to improve the cold resistance of crops.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and plant genetic engineering, and specifically relates to the cloning and application of a plant adversity-induced promoter. 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 role in plant growth and development (Viswanathan C, Zhu JK. Molecular genetic analysis of cold-regulated gene transcription. Philos Trans R Soc Lond B Biol Sci. 2002, 357(1423):877-886). With the growing maturity of transgenic technology, a large number of genes related to cold resistance have been isolated from plants (Lin ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 林娟吴丽华沈忱周明琦
Owner FUDAN UNIV