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Plants flower pesticide specificity promoter and uses thereof

An anther-specific, promoter technology, applied in application, plant genetic improvement, botanical equipment and methods, etc., can solve problems such as unclear regulation results, and achieve the effect of prolonging shelf life

Inactive Publication Date: 2008-05-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] Although the AtA7 gene was cloned as early as 1998 (Ma H et al., 1998, Plant Mol Biol, 37 (4): 607-619), and the anther specificity of its expression was confirmed [Yang SH et al., 2003, The Plant Cell Vol.152792-2804; Colcombet J et al., 2005, The Plant Cell, Vol.17, 3350-3361; Yang SH et al., 2005, Plant Physiology, 139(1):186-191], but for its promoter The study has not seen reports or patents, and it is still unclear whether its specific expression in the anther tapetum is the result of gene interaction or the regulation of promoter-specific elements

Method used

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  • Plants flower pesticide specificity promoter and uses thereof
  • Plants flower pesticide specificity promoter and uses thereof
  • Plants flower pesticide specificity promoter and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1, Cloning and isolation of Arabidopsis anther-specific promoter AtA7

[0072] 1. Extraction of Arabidopsis total DNA

[0073] The materials were taken from wild-type Arabidopsis thaliana of Columbia ecotype, 1-2g of young leaves, and the total DNA was extracted by CTAB method (Molecular Cloning, third edition). Take 1-5ul DNA sample, measure its concentration and purity with an ultraviolet spectrophotometer, and use 0.8% agarose gel electrophoresis to detect DNA purity and integrity. Store the extracted DNA at -20°C.

[0074] 2. PCR amplification and recovery of amplified fragments

[0075] The following two primers (Primer 1 and Primer 2) were designed and synthesized to amplify the Arabidopsis anther-specific promoter. In order to facilitate future cloning and construction, the restriction endonuclease Hind III was added to the 5' end of the two primers respectively and Bam HI recognition sequence sites (underlined sequences in the following primer sequenc...

Embodiment 2

[0084] Embodiment 2, construction of anther-specific promoter-driven GUS gene (AtA7::GUS) plant expression vector pRDAtA7

[0085] The construction process of the plant expression vector pRDAtA7 driven by the anther-specific promoter of the GUS gene (AtA7::GUS) is shown in Figure 4, and the specific methods are as follows:

[0086] After pUAtA7 was double-digested with restriction endonucleases Hind III and Bam HI, an anther-specific promoter AtA7 fragment was obtained, which was recovered with a DNA fragment recovery kit (Easy-NA Gel Extraction Kit, produced by Omeg-Bio / TEK, Germany). The fragment of the anther-specific promoter AtA7 was purified, and connected with the large fragment (about 13.6 kb) of the plant expression vector pRD410 (produced by PBI, Canada) that had been double-digested with Hind III and Bam HI to obtain a recombinant plasmid. The resulting recombinant plasmid was transformed into Escherichia coli strain DH5a by "freeze-thaw method" (Molecular Cloning, ...

Embodiment 3

[0087] Embodiment 3, the acquisition and identification of trans AtA7::GUS gene (pRDAtA7) tobacco

[0088] 1. Transformation of Agrobacterium and identification of transformants

[0089] with CaCl 2 Competent cells of Agrobacterium tumefaciens strain LBA4404 (product of LifeTechnology, USA) were prepared by the method (Molecular Cloning, third edition). The recombinant plant expression vector pRDAtA7 was transferred into the prepared LBA4404 competent cells by freeze-thaw method ("Molecular Cloning" third edition). Inoculate the transformed LBA4404 cells into YEB solid medium containing streptomycin (Str) 100mg / L and kanamycin (Kan) 50mg / L, culture in the dark at 28°C for 48-72h, pick the plate A single colony was inserted into YEB liquid medium containing 100 mg / L streptomycin and 50 mg / L kanamycin, and cultured overnight at 28°C with shaking. When the concentration of the culture reaches the OD 600 When the value is 0.4-0.6, get a small amount of bacterial liquid (1.5-2m...

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Abstract

The invention discloses a plant anther-specific promoter and application thereof. The promoter is 179 to 1174 bp in length at least containing the 996-1174th nucleotide sequence at the 5' end of sequence 1 in the sequence listing at the 3' end and extending to the 5' end according to the nucleotide arrangement of sequence 1 deoxynucleotide fragments. The anther-specific promoter of the present invention has great application prospects in the functional analysis and identification of plant anther growth and development genes, the establishment of artificial male sterile lines, the prevention of plant transgene drift or escape, and the extension of shelf life of flowers.

Description

technical field [0001] The invention relates to a plant anther-specific promoter and its application. Background technique [0002] Promoters (Promoters) are an integral part of a gene, controlling the initiation time and degree of gene expression (transcription). The promoter is like a "switch" that determines the activity of a gene. It basically determines whether, when and where a gene is expressed at the transcriptional level. According to the mode of action and function, promoters can be roughly divided into three categories: constitutive promoters, specific promoters and inducible promoters [Wang Guanlin, Fang Hongyun, 2002, Principles and Technology of Plant Genetic Engineering (Second Edition), Beijing, Science and Technology Press]. This classification basically reflects the respective functional characteristics of different types of promoters, but in some cases, one type of promoter often has the characteristics of other types of promoters. [0003] An inducible...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82
Inventor 肖兴国聂绚丽朱建伟耿安奇
Owner CHINA AGRI UNIV
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