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Plant anther specific promoter and its application

An anther-specific and promoter-based technology, applied in the fields of application, plant genetic improvement, botany equipment and methods, etc., can solve problems such as poor effect and achieve the effect of extending shelf life

Inactive Publication Date: 2007-02-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] Although the use of anther-specific promoters to create genetically engineered male sterile lines and restorer lines has achieved great success, most rely on the anther-specific promoter TA29 from tobacco, other specific promoters, such as A9 (Worrall D et al., 1992, Plant Cell, 4:759-771; Paul et al., 1992), Anther-box (van der Meer et al., 1992), PS1 (Zuo JT et al., 1994, Amer J Bot, 81:552- 561), Osg6B [Tsuchiy T et al., 1995, Plant and Cell Physiol, 36 (3): 487-494; Matsuda N, 1996, Plant and Cell Physiol, 37 (2): 215-222; Kitashiba H et al., 1999, Mol Breeding, 5(2):209-218; Kawanab T et al., 2006, Plant and Cell Physiol, 47(6):784-787] and LAT52 [Muchietti J et al., 1994, Plant J, 6(3):321 -328], etc. have also been reported to cause male sterility, but most of them are not effective.

Method used

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  • Plant anther specific promoter and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1, Cloning and Isolation of Petunia Anther-specific Promoter PhFD

[0050] 1. Extraction of petunia total DNA

[0051] Total DNA was extracted from 1-2 g of tender leaves of Petunia x hybrida variety "Big Bleu" (available in various flower companies) grown in pots in the greenhouse, and extracted with the 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.

[0052] 2. PCR amplification and recovery of amplified fragments

[0053] The following two primers (primer 1 and primer 2) were designed and synthesized to amplify the petunia anther-specific promoter. In order to facilitate subsequent cloning and construction, restriction enzymes HindIII and Recognition sequence site of Bam HI (underlined sequence in the following primer sequences)

[0054] Primer 1: ...

Embodiment 2

[0070] Example 2, Construction of anther-specific promoter-driven GUS gene (PhFD::GUS) plant expression vector pRDFDG

[0071] The construction process of the anther-specific promoter-driven GUS gene (PhFD::GUS) plant expression vector pRDFDG is as follows: image 3 As shown, the specific method is as follows:

[0072] After pUFD was double-digested with restriction endonucleases Hind III and Bam HI, the anther-specific promoter PhFD fragment was obtained, and the fragment was freeze-thawed ("Molecular Cloning" third edition) or DNA fragment recovery kit (Easy-NA GelExtraction Kit, Germany Omeg-Bio / TEK product) recovers and purifies the anther-specific promoter PhFD fragment, and connects with the large fragment (about 13.6kb) of the plant expression vector pRD410 (Canada PBI product) that has been digested with Hind III and Bam HI , to obtain the recombinant plasmid. The resulting recombinant plasmid was transformed into Escherichia coli strain DH5a by "freeze-thaw method" ...

Embodiment 3

[0073] Embodiment 3, identification of transgenic (PhFD) tobacco

[0074] 1. Transformation of Agrobacterium and identification of transformants

[0075] 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 pRDFDG 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 0.4-0.5, take a small amount of bacterial liquid (1.5-2ml), extract the plasmid according...

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Abstract

The invention discloses plant anther specificity promoter and its application. The promoter nucleotide sequence can be the one as (1) sequence table number one in question; or the one as (2), (1) in question nucleotide sequence complementary; or the one as (3), (1), or (2) in question which has 60% or above homology; or the one as (4), (1), (2), or (3) in question which can hybridized at strict hybridization condition. The inverse gene, small RNA, or expression vector can be used to transform host cell or tissue and its progeny cell to culture male sterile line plant, prevent transgene from polluting its wild species, landscaping plant, or crop species, etc.

Description

technical field [0001] The invention relates to a plant anther-specific promoter and its application. Background technique [0002] Promoter is the most important factor among gene expression regulators, which basically determines whether, when and where a gene is expressed. 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 promoter (inducible promoter) means that the gene controlled by it can greatly increase the transcription level under the stimulation of certain specific physi...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/63C12N15/82C12N5/10
Inventor 肖兴国耿安奇赵占军聂绚丽
Owner CHINA AGRI UNIV
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