Capsella bursapastoris cold regulated protein gene promoter and application thereof in plant cold resistance improvement

A cold-regulatory protein and promoter technology, which is applied in the fields of molecular biology and plant genetic engineering, can solve the problems of few cold-inducible specific promoters and no cold-tolerant plants have been found, and achieve the effect of improving cold resistance

Inactive Publication Date: 2012-01-25
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...

Method used

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  • Capsella bursapastoris cold regulated protein gene promoter and application thereof in plant cold resistance improvement
  • Capsella bursapastoris cold regulated protein gene promoter and application thereof in plant cold resistance improvement
  • Capsella bursapastoris cold regulated protein gene promoter and application thereof in plant cold resistance improvement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Isolation and identification of CbCOR15bP.

[0025] 1. Cultivation of shepherd's purse seedlings

[0026] The shepherd's purse seeds come from Shanghai Magnolia Vegetable Seed Co., Ltd. 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.

[0027] 2. Cloning of the promoter sequence

[0028] The promoter sequence of CbCOR15b 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 COR15b gene: CbCOR15bF1: 5'-GATCAGTCTGTTGTAATGA-3' (denoted as SEQ ID NO. 2) and CbCOR15bF2: 5' - ACCATCG CCATGAGAGATATG-3' (denoted as SEQ ID NO. 3), cooperates with...

Embodiment 2

[0030] Example 2 CbCOR15b Expression characteristics under cold induction and cold acclimatization treatments.

[0031] 1. Planting and handling of shepherd's purse

[0032] Take an appropriate amount of fresh shepherd's purse seeds, after aseptic treatment, place them in a pre-configured MS medium (MS powder 4.41g / L, sucrose 30g / L, agar powder 8.5g / L) sterile culture tank, and cultivate under light at 26°C. Cold acclimation treatment The seedlings 4 weeks after seed germination were selected, and the shepherd's purse seedlings were treated according to the temperature gradient of 26°C (4d), 12°C (4d), 4°C (4d), 0°C (2h), -4°C (2h). Induction treatment Seedlings 4 weeks after seed germination were selected, and shepherd's purse seedlings were treated in a gradient of 0h, 1h, 3h, 6h, 12h, 24h, and 48h at 4°C, and total RNA was extracted from leaves, stems, and roots.

[0033] 2. RNA extraction

[0034] RNA extraction was performed according to the operating procedures of th...

Embodiment 3

[0039] Example 3 Analysis of low temperature induction activity of shepherd's purse COR15bP promoter.

[0040] In this example, the cloned CbCOR15bP 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 CbCOR15bP can enhance the expression of GUS gene under low temperature induction. Therefore, CbCOR15bP has a good application prospect in crop cold-resistant genetic engineering breeding.

[0041] Construction of expression vectors.

[0042] In this embodiment, construct the expression vector pCAMBA1301-COR15bP-GUS

[0043] The CaMV35S promoter contained in the commercialized plant expression vector pCAMBA1301 was excised and connected into shepherd's purse COR15bP to regulate the expression of GUS gene. The co...

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Abstract

The invention belongs to the technical field of plant gene engineering, and particularly relates to a capsella bursapastoris cold regulated protein gene promoter and an application thereof in plant cold resistance improvement. The nucleotide sequence of the promoter is shown in SEQ ID NO:1. The capsella bursapastoris cold regulated protein gene and the promoter thereof comprise the nucleotide sequence of the 1960th basic group in the sequence shown in SEQ ID NO:1 in a sequence table. The nucleotide sequence codes a gene which is expressed by low-temperature induction and can promote cells to generate cold resistance. In the induction condition of -4 DEG C, the expression amount of the promoter is increased in leaves, and the expression amount of the promoter is also increased in stems. The inductivity expression carrier built by the gene promoter segment can start the expression of the target gene when the plant is under low-temperature stress. The invention also provides an application of the promoter in culturing cold-resistant plants, thereby improving the cold resistance of crop varieties.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and plant genetic engineering, and in particular relates to the cloning and application of a plant adversity-induced promoter. Background technique [0002] Low temperature and cold damage is a serious natural disaster in agricultural and forestry production, which causes huge losses in the world every year, reaching 200 billion U.S. dollars according to statistics. In the natural environment, temperature is the main limiting factor that determines the geographical distribution of plants. Under cultivation conditions, it also affects the yield and quality of crops and horticultural plants. 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, ...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00C12N15/113C12N15/82
Inventor 林娟吴丽华周明琦沈忱
Owner FUDAN UNIV
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