Arabidopis thaliana flower and embryo specific promoters EL5 and EL6, and application thereof

A specific promoter, Arabidopsis flower technology, applied in Arabidopsis flower and embryo-specific promoters EL5 and EL6 and their application fields

Inactive Publication Date: 2012-06-06
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few reports of promoters that c...

Method used

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  • Arabidopis thaliana flower and embryo specific promoters EL5 and EL6, and application thereof
  • Arabidopis thaliana flower and embryo specific promoters EL5 and EL6, and application thereof
  • Arabidopis thaliana flower and embryo specific promoters EL5 and EL6, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Cloning of embodiment 1 Arabidopsis EL5 and EL6 promoter

[0036] Use forward primer 5'-CTCCCTTATCGTGTTGACA-3' and reverse primer 5'-CGCTAGGGAAATCTGAGGAA-3' to amplify and sequence the EL5 promoter with a length of 2500bp from the Arabidopsis genome, and its nucleotide sequence is as SEQ ID Shown in NO.1.

[0037] Utilize forward primer 5'-CTGCTGATTATCTCGTTC-3' and reverse primer 5'-CCAGTATTATTCTGGCGCCG-3' from the Arabidopsis genome to PCR amplify and clone and sequence to obtain the EL6 promoter with a length of 2501bp, its nucleotide sequence is as SEQ ID NO.2 shown.

[0038] The above PCR amplification system is: (20μl system)

[0039] LA Taq enzyme 0.2μl

[0040] 10x buffer 2.0μl

[0041] dNTP (2.5mM each) 1.0μl

[0042] Primer 1 (10uM) 1.0μl

[0043] Primer 2 (10uM) 1.0μl

[0044] DNA template 1.0μl

[0045] wxya 2 O 13.8 μl

[0046]

[0047] Total volume 20.0μl

[0048] PCR program:

[0049]

Embodiment 2

[0050] Example 2 Arabidopsis thaliana EL5 and EL6 promoters regulate the expression of the downstream gene GUS

[0051]The EL5 and EL6 promoters were cloned and fused with the GUS gene respectively, and then the obtained fusion gene was constructed into the expression vector pGreen-GW-GUS (the construction process of the plasmid pGreen-GW-GUS is: adopt the method of GateWay recombination cloning (Invitrogen), The PCR product was first recombined into pDONR207 by BP reaction, and then recombined into the target vector pGreen-GW-GUS containing the GUS reporter gene by LR reaction. The structure of pGreen-GW-GUS is as follows figure 1 shown), to obtain binary expression vectors pEL5-GUS and pEL6-GUS; the plant expression vectors pEL5-GUS and pEL6-GUS were transformed into Escherichia coli DH5α for propagation. Transformation of Escherichia coli competent cells, including: (1) Preparation of LB solid medium containing ampicillin; (2) Take out the competent cells stored at -80°C, m...

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Abstract

The invention provides Arabidopis thaliana flower and embryo specific promoters EL5 and EL6. The nucleotide sequences of the promoters are respectively represented by SEQ ID No.1 and SEQ ID No.2. According to results of overexpression of Arabidopis thaliana and soybean GUS genes by using the promoters EL5 and EL6, the promoters EL5 and EL6 can obviously promote the expression level of the GUS genes in plant floral organs and embryos; therefore, the promoters EL5 and EL6 have specificity in plant floral organs and embryos and are applicable to specific expression of a target gene in flowers and embryos of the plants like a variety of crops, forest trees, vegetable, flowers, pasture, etc.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and in particular relates to Arabidopsis flower and embryo-specific promoters EL5 and EL6 and applications thereof. Background technique [0002] The promoter is an important cis-acting element in the gene, generally located at dozens of bases upstream of the transcription start site, its core structure includes TATA-box and CAAT-box, in addition to the core structure, it also contains the transcription start site Points, tissue-specific expression elements and other regulatory elements, such as biotic stress and abiotic stress-induced elements, which play an important role in the study of gene expression and function, while the inducible promoter can control the gene in a specific Expressed in time and specific space, and perform specific functions. [0003] Promoters can be divided into three types according to different transcription modes: constitutive prom...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21
Inventor 傅永福江颖柳霖坡张晓玫
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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