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Preparation method and application of brassica napus BnPABP5-3 promoter

A Brassica napus and promoter technology, applied in the field of plant genetic engineering and biology, can solve problems such as unsuitable production practice, unfavorable normal growth of plants, and harmful ecological environment

Inactive Publication Date: 2011-10-26
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of inducible promoters also has certain limitations. The external condition treatment of recipient plants, such as heat shock, hormone treatment, etc., may cause a series of physiological and biochemical reactions in the organism, which is not conducive to the normal growth of plants.
Moreover, the methyl dehydrocortisol (dex, dexamethasone), estradiol (estradiol) and tetracycline (tetracycline) used as inducers in the chemical regulation system are all harmful to the ecological environment and should not be used in production practice.

Method used

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  • Preparation method and application of brassica napus BnPABP5-3 promoter
  • Preparation method and application of brassica napus BnPABP5-3 promoter
  • Preparation method and application of brassica napus BnPABP5-3 promoter

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] A kind of Brassica napus P BnPABP5-3 The preparation method of the promoter, its steps are:

[0045] A, rape P BnPABP5-3 Promoter primer design:

[0046] Use SDS lysis method to extract rapeseed DNA (by J. Sambrook. DW Russell, Huang Peitang et al. Translated Molecular Cloning Test Guide (Third Edition) Science Press), according to the Genome Walking Kit (purchased from Clontech, product Code Cat.No.638904) operation program, carry out genome walking, walking carry out two rounds of PCR amplification, clone and obtain rape P BnPABP5-3 (942bp). The primers used for walking are shown in Table 2.

[0047] Table 2 Genome walking clone P BnPABP5-3 Primer used

[0048]

[0049] B. Brassica napus P BnPABP5-3 The promoter preparation is:

[0050] According to the instructions of the Genome Walking Kit (purchased from Clontech, product code Cat.No.638904), approximately 2.5μg of DNA were digested with the restriction enzymes DraI, EcoRV, PvuII and StuI to produce blunt ends in 100μl sys...

Embodiment 2

[0054] pBI-P BnPABP5-3 Arabidopsis transformation and PCR detection:

[0055] According to the above-mentioned document (Zhang X R. et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature, 2006, 1:1-6) the transformation method of Arabidopsis thaliana was transformed. According to the unique kanamycin resistance of the transgenic plants, they were grown on MS (described above) medium containing 50 mg / L kanamycin, and the green shoots obtained were initially considered to be positive shoots. After the green seedlings grow two true leaves, they are transplanted into vermiculite. After the plants appear inflorescence, one true leaf is taken to extract genomic DNA (described above) by the SDS method, and PCR identification is performed. The primer sequences are 5'-GAATTGTGAGCGGATAAC-3' and 5'-ACATAAGGGACTGACCAC-3'; the PCR reaction system is as follows: Genomic DNA template 1μL (about 50ng), 10×Taq enzyme reaction buffer 2ul, 25mMMgCL ...

Embodiment 3

[0057] Brassica napus P BnPABP5-3 Functional analysis of the promoter:

[0058] The present invention clones P for the first time BnPABP5-3 The sequence and its function are analyzed. From Example 3, the T1 generation of the positive seedlings screened in the Arabidopsis transformation and PCR detection steps were selfed to harvest the seeds (ie, the T2 generation). The tissues of 10 strains of T2 generation at different periods were stained with GUS.

[0059] The dyeing process of T2 substitution is as follows: soak the sample in GUS dye solution (X-gluc 0.5mg / mL, phosphate buffer 50mmol / L, potassium ferricyanide and potassium ferrocyanide each 0.5mmol / L, EDTA 10mmol / L , Triton-x-100 0.001%, methanol 20% (volume ratio), vacuum for 5 minutes, overnight at 37°C. Decolorize with alcohol-acetic acid (volume ratio 1:1) the next day until the leaves turn white, then Rinse 3-5 times with distilled water, and take photos with a stereo microscope (OLYMPUS SZX16). Take the whole plant at ...

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Abstract

The invention discloses a preparation method and an application of a brassica napus BnPABP5-3 promoter (PBnPABP5-3). A genome walking method is utilized to clone a PBnPABP5-3 sequence: an SDS (sodium dodecyl sulphate) cracking process is applied to extract genome DNA; in different systems, endonucleases DraI, EcoRV, PvuII and StuI are used to digest DNA, and the digested DNA is respectively connected with joint segments, then primary PCR (Polymerase Chain Reaction) amplification is carried out by taking the DNA connected with the joint segments as templates, and secondary PCR amplification is carried out by taking 50-time diluted solution of products of the primary PCR as a template, thus the PBnPABP5-3 is obtained. The PBnPABP5-3 can be applied to the anther, pollen grain, root tip and ovule of a plant. The promoter has the function of driving expression of a foreign gene, the expression part is in the anther, pollen grain, root tip and ovule of the plant, and the promoter has application potential in the improvement of the safety of rape edible oil, the improvement of the stress resistance and lodging resistance, improvement of the quality of crops, artificial sterile line creation, germ plasma resource enrichment and the like.

Description

Technical field [0001] The invention relates to the fields of plant genetic engineering and biotechnology. Specifically related to a Brassica napus BnPABP5-3 promoter (named P BnPABP5-3 , The following is the same), and also relates to a preparation method of the BnPABP5-3 promoter of Brassica napus. The invention also relates to a vector containing the promoter or its homologous nucleotide sequence and to the application of the promoter in rape and other plant genetic engineering. Background technique [0002] Plant promoters play a key role in the regulation of gene expression. The regulation of gene expression is the result of multiple factors. Generally according to the sequence of an event, gene regulation is divided into transcription level regulation, translation level regulation, and protein processing level regulation. The protein and RNA encoded by genes and their secondary metabolites are extremely important for maintaining the entire life activities of organisms. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/84A01H5/02A01H5/06
Inventor 刘胜毅石磊董彩华刘越英
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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