Brassica napus p76247 promoter and its preparation method and application

A technology of Brassica napus, pbi-p76247, applied in the field of plant genetic engineering and biology, can solve problems such as unsuitable production practice, unfavorable normal plant growth, and harmful ecological environment

Active Publication Date: 2015-11-18
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.
In addition, methyl dehydrocortisol (dex, dexamethasone), estradiol (estradiol) and tetracycline (tetracycline) used as inducers in the chemical regulation system are harmful to the ecological environment and should not be used in production practice

Method used

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  • Brassica napus p76247 promoter and its preparation method and application
  • Brassica napus p76247 promoter and its preparation method and application
  • Brassica napus p76247 promoter and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Prediction of anther-specific expression gene Bn76247 and promoter P76247:

[0078] The used rapeseed of the present invention is Brassica napus Zhongshuang No. 11 (mentioned above). The test materials were sown in the field and managed normally. Eleven tissues including roots, stems, leaves, flower buds, ovules, siliques, silique peels, petals, pistils, sepals and petals were obtained from rapeseed plants grown under field growth conditions. Take at least three replicates of each material, and each replicate has at least one plant. After sampling, wrap it in tin foil, quickly place it in liquid nitrogen quick-freezing, and store it at -80°C. RNA was extracted using the Trirol extraction kit (described above) according to the requirements of the kit. The extracted RNA was sent to Shenzhen BGI Institute for reverse transcription, library construction, and transcriptome sequencing using the solexa sequencing technology (described above). The amount of sequencing data f...

Embodiment 2

[0080] Analysis of expression patterns of endogenous genes driven by rapeseed P76247:

[0081] The test materials were sown in the field and managed normally. Get roots, stems, leaves, flower buds, siliques, pistils, petals and petals from the rapeseed Zhongshuang No. 11 (as described above) plant grown under field growth conditions. Take at least three replicates of each material, and each replicate has at least one plant. After sampling, wrap it in tin foil, place it quickly in liquid nitrogen, and store it at -80°C. Extract RNA (method described above). The fluorescent quantitative PCR instrument was IQ5 (Bio-Rad Company), and rape Actin (accession number AF111812) was used as the internal standard gene. The Actin gene primers were the forward primer 5′-CTGGAATTGCTGACCGTATGAG-3′ and the reverse primer 5′-ATCTGTTGGAAAGTGCTGAGGG-3′. The primers of the endogenous gene Bn76247 driven by P76247 were 5'5'TTTGTTACCGTGCTGCTCA3' and 5'AAAGCCGTCCATCTATCAT3'. Fluorescent quantitati...

Embodiment 3

[0085] A method for preparing a Brassica napus promoter P76247 promoter, the steps are:

[0086] 1. The primer sequence of rapeseed promoter P76247:

[0087] According to the upstream 2kb sequence of the Bn76247 gene obtained from rape genome sequencing, a pair of primers were designed for PCR amplification.

[0088] 2. Preparation of rapeseed promoter P76247:

[0089] Use the SDS lysis method (described above) to extract genomic DNA from rapeseed leaves, and use this as a template for PCR amplification. The steps are: extract 25 μl of genomic DNA, and use this as a template for amplification. The reaction system is 50 μl, and 5 μl of 10×ExTaqbuffer is added respectively. 4 μl of dNTP, 1 μl of 5’ primer, 1 μl of 3’ primer, 0.5 μl of ExTaq, 1 μl of DNA template, about 100 ng, ddH 2O 37.5 μl. The primers used in PCR design were P76247S and P76247A (described above) based on the upstream 2kb sequence of the Bn76247 gene obtained from rapeseed whole genome sequencing. The size...

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Abstract

The invention discloses a Brassica napus promoter P76247 and a preparation method and application thereof. The method comprises the steps: (1) designing a pair of primers according to an upstream 2kb sequence of a gene Bn76247 which is obtained through sequencing all genomes of napus, and carrying out PCR (Polymerase Chain Reaction) amplification, so as to obtain a 5' upstream promoter sequence of the napus Bn76247; (2) preparing the napus promoter P76247: extracting genomic DNA (Deoxyribonucleic Acid) of napus leaves by using a SDS (Sodium Dodecyl Sulfate) lysis method, taking the genomic DNA as a template, carrying out PCR amplification with the primers, purifying and recovering with a gel recovery kit, and then, connecting to a pMD18-T vector, thereby obtaining an upstream 2kb flanking sequence of a target gene, wherein shown by bioinformatics analysis, the upstream flanking sequence is P76247. Shown by fluorescent quantitative PCR and GUS (glucuronidase) staining results, P76247 is efficiently expressed in the stamens and petals of napus and is not expressed in tissues, such as roots, stems, leaves, fruits, seeds and the like. The promoter has good application potential to the safety of transgenic napus edible oil and the aspects of improving crop quality, artificially creating germplasm resources and the like.

Description

technical field [0001] The present invention relates to the fields of plant genetic engineering and biotechnology, more specifically to a Bn76247 gene promoter of Brassica napus (named P76247, the same below), and also to a preparation method of the Brassica napus P76247 promoter. It also relates to a vector containing the promoter or its substantially homologous nucleotide sequence and the application of the promoter in genetic engineering of rapeseed and Arabidopsis thaliana. Background technique [0002] A plant gene promoter is a DNA sequence located in the upstream region of the 5' end of a structural gene and contains cis-acting elements. It determines the specificity, direction and efficiency of downstream gene transcription, and is the most critical factor in gene transcription regulation mechanism and expression mode. . In addition, the expression of exogenous genes in plant cells is the key to plant genetic engineering research, and the expression of exogenous gen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/82A01H5/02
Inventor 刘胜毅董彩华黄军艳程晓辉童超波于景印刘越英
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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