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Plant epidermal hair specific expression promoter

A plant epidermis and promoter technology, applied in the field of plant bioengineering and plant improvement genetic engineering, can solve the problem of lack of plant epidermal hair cis-regulatory elements and the like

Inactive Publication Date: 2006-05-31
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no cis-regulatory elements specifically expressed in plant cuticular hairs (cotton fibers) have been reported so far

Method used

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  • Plant epidermal hair specific expression promoter
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  • Plant epidermal hair specific expression promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Isolation of GaRDL1 promoter

[0057] Cotton DNA was extracted by cold phenol method. 2 g of material (cotton fiber of Asian cotton (Gossypium arboreum)), ground into powder in liquid nitrogen, transferred to a 50 ml centrifuge tube, added 8 ml of extraction buffer (1M Tris HCl, 50 mM EDTA, 1% SDS, pH 9.0 ) and an equal volume of water-saturated phenol: chloroform: isoamyl alcohol (25:24:1) were shaken and mixed, placed on ice for 1 hour, and mixed every 10 minutes. Centrifuge at 13000g for 20 minutes at 4°C. Repeat phenol: chloroform: isoamyl alcohol extraction 2 to 4 times, and finally extract once with chloroform: isoamyl alcohol (24:1). Take the supernatant, add 1 / 2 volume of high salt solution (0.8M sodium citrate, 1.2M NaCl) and 1 / 2 volume of isopropanol, mix well, and place at -70°C for 1 hour. Centrifuge at 13000g for 20 minutes at 4°C. The following operations are performed according to the extraction of DNA and RNA. Remove the supernatant, wash ...

Embodiment 2

[0061] Example 2 Vector Construction and Agrobacterium Transformation

[0062] According to the distribution of the multi-cloning region of the vector, the GaRDL1 promoter and the restriction site of the target gene, determine the restriction site for the fusion fragment to be inserted into the vector, and then introduce the restriction site into the GaRDL1 promoter and the target gene by PCR . After digestion and ligation, heat shock method was used to transform Escherichia coli, positive clones were identified by PCR, and sequenced for verification.

[0063] According to the full-length coding sequence of the exogenous gene GUS gene, primers (corresponding to the first 20 bp and the last 20 bp) were designed to amplify the complete coding reading frame, and the GUS sequence was obtained after PCR amplification, and then directly connected to the GaRDL1 promoter (located at downstream of the promoter). Then the DNA sequence (GaRDL1::GUS sequence) was cloned into the interme...

Embodiment 3

[0065] Example 3 Plant Transformation and Screening of Transgenic Progeny

[0066] In this example, the transgenes of cotton and Arabidopsis are taken as examples, which can be used as a reference for the transformation of other plants.

[0067] a. Transgenic cotton

[0068]Cotton was transformed using an Agrobacterium-mediated method.

[0069] The upper part (0.5 cm) of the hypocotyl of the 5-day-old aseptic seedling was pre-cultured for 36 hours. Medium: SH+2,4-D 0.1mg / L+KT 0.1mg / L.

[0070] The hypocotyl sections were soaked in the Agrobacterium bacterial solution (SH+2,4-D 0.1 mg / L+KT 0.1 mg / L+acetosyringone 100 μmol / L) for 20 minutes.

[0071] Co-cultivate for 3 days. Medium: SH+2,4-D 0.1mg / L+KT 0.1mg / L+acetosyringone 100μmol / L.

[0072] The callus was initially induced for 25-30 days. Medium: SH+2,4-D 0.1mg / L+KT 0.1mg / L+Km50mg / L+cephalosporin (cef) 300mg / L or MSB+2,4-D 0.1mg / L+IAA 0.1mg / L L+ZT0.1mg / L+Km 50mg / L+cef 300mg / L.

[0073] Embryogenic callus was induced ...

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Abstract

The invention provided promoter elements of plant cuticular trichomes attribute expression®ñ®ñGaRDL1 promoter of the cotton, it includes the construction, vector and its use of GaRDL1 promoter element. The GaRDL1 promoter of this invention can be used in the gene engineering of the cotton fiber and glands secondary metabolite.

Description

technical field [0001] The invention belongs to the fields of plant bioengineering and plant improvement genetic engineering. Specifically, the present invention relates to the sequence of a novel plant hair-cut specific expression promoter GaRDL1 and its cis-regulatory elements related to the specific expression of cuticle hair. The invention also discloses the use of the GaRDL1 promoter, especially the application in the genetic engineering of the secondary metabolism of cotton fiber and glandular hairs. Background technique [0002] Plant epidermal trichomes are a model system for studying cell differentiation, polar growth and morphogenesis at the single-cell level (Hulskamp, ​​2004, Nat. Rev. Mol. Cell Biol. 5, 471-480). Plant epidermal hairs are divided into glandular hairs and non-glandular hairs, the main function of which is to protect plants, such as resisting insect damage, reducing evaporation, improving frost resistance and UV protection (Marks et al., 1997, An...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/63C12N15/82
Inventor 陈晓亚王水李春红王凌健
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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