Brassica napus BnPABP8 promoter and preparation method and use thereof

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: 2011-10-19
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 t

Method used

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  • Brassica napus BnPABP8 promoter and preparation method and use thereof
  • Brassica napus BnPABP8 promoter and preparation method and use thereof
  • Brassica napus BnPABP8 promoter and preparation method and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Rapeseed P BnPABP8 Expression pattern analysis of driven endogenous genes:

[0055] The rapeseed used in the present invention is Brassica napus L. . The test materials were sown in the field and managed normally.

[0056] Take roots, stems, leaves, flower buds, and siliques from fertile plants with the same growth conditions in the field. Take at least three replicates of each material, and at least one plant for each replicate. After sampling, wrap them in tin foil and place them in liquid nitrogen immediately. medium, stored at -80°C. RNA was extracted (method described above). Fluorescence quantitative PCR instrument is R TM -Cycler (Boao Biological Co., Ltd.), using rapeseed Actin (accession number AF111812) as an internal standard gene, Actin gene primers are forward primer 5'-CTGGAATTGCTGACCGTATGAG-3' and reverse primer 5'-ATCTGTTGGAAAGTGCTGAGGG-3'. P BnPABP8 The driven endogenous gene primers are 5'-ATCTGGATGCGAGTGTTACCG-3' and 5'-AGTTCAGTTCCTGGATGGCTC-3'....

Embodiment 2

[0061] A brassica napus promoter P BnPABP8 The preparation method of promoter, its step is:

[0062] A. Prepare rapeseed P BnPABP8 Promoter primer design:

[0063] Utilize the SDS cracking method to extract the rapeseed DNA (J. Sambrook.D.W.Russell, Huang Peitang et al. Translation Molecular Cloning Test Guide (Third Edition) Science Press), according to the genome walking kit (purchased from Clontech company, product Codenamed Cat.No.638904) operating procedures, genome walking, walking for two rounds of PCR amplification, cloned to obtain rapeseed P BnPABP8 (942bp). The primers used for walking are listed in Table 2.

[0064] Table 2 Genome walking clone P BnPABP8 Primers used

[0065]

[0066] B. Brassica napus promoter P BnPABP8 The preparation is:

[0067] According to the instructions of the genome walking kit (purchased from Clontech, product code Cat. For genomic DNA, four "libraries" of restriction-enzyme-treated DNA ligated with specific adapters were est...

Embodiment 3

[0071] pBI-P BnPABP8 Arabidopsis transformation and PCR detection:

[0072]Arabidopsis thaliana was transformed according to the transformation method in the above literature (Zhang X R. et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature, 2006, 1:1-6). According to the unique kanamycin resistance of the transgenic plants, the green shoots obtained were preliminarily considered to be positive shoots when they were grown on MS (described above) medium containing 50 mg / L kanamycin. After the green seedling grows two true leaves, it is transplanted into vermiculite, and after the inflorescence appears on the plant, a true leaf is taken to extract genomic DNA (described above) by SDS method for PCR identification. 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 2 1.2ul, 2mM ...

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Abstract

The invention discloses a brassica napus BnPABP8 promoter and a preparation method and use thereof. The preparation method comprises the following: A, a step of primer design, which is to design specific walking primers according to known sequences; B, a step of brassica napus promoter preparation, which is to extract brassica napus DNA by using a sodium dodecyl sulfonate (SDS) lysis method, perform genome walking according to genome walking kits, digest 5 micro liters of DNA by using DraI enzyme, detect the purity of the DNA, perform enzyme digestion of the DNA by using the incision enzymes provided by four kits, and purify the product of the enzyme digestion; C, a step of purification, which is to add ethanol into enzyme digestion solution, precipitate, centrifuge, dry in the air, dissolve in sterile water and add specific connectors after purification is accomplished, wherein GSP1 and AP1 are used as primers in primary polymerase chain reaction (PCR) reaction; and D, a step, which is to perform amplification by using the diluted solution of the product of the primary PCR reaction as a template and GSP2 and AP2 as primers in a secondary PCR reaction, recover purified product of the secondary PCR product, connect with a T vector, and obtain the PBnPABP8. The invention also discloses the use of the PBnPABP8 in roots, stems, leaves, buds and anthers of plants. When the promoter is used, the safety of the edible brassica napus oil can be improved, and the disease resistance, stress resistance and lodging resistance in crops can be improved by transgenosis.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and biotechnology. Specifically related to a Brassica napus BnPABP8 promoter (named P BnPABP8 , the same below), and also relates to a preparation method of Brassica napus BnPABP8 promoter. The present invention also relates to the carrier containing the promoter or its homologous nucleotide sequence and the application of the promoter in genetic engineering of rapeseed and other plants. Background technique [0002] Plant promoters play a key role in the regulation of gene expression. The regulation of gene expression is the result of the comprehensive action of many factors. Generally, according to the sequence of events, gene regulation is divided into transcription level regulation, translation level regulation, and protein processing level regulation. The proteins and RNAs encoded by genes and their secondary metabolites are extremely important for maintaining the whole life acti...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/63A01H5/00A01H5/02A01H5/04A01H5/06A01H5/12
Inventor 刘胜毅石磊董彩华
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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