Preparation method and applications of rape BnRabGDI3 promoter

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

Active Publication Date: 2014-06-25
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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  • Preparation method and applications of rape BnRabGDI3 promoter
  • Preparation method and applications of rape BnRabGDI3 promoter
  • Preparation method and applications of rape BnRabGDI3 promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] A brassica napus promoter P BnRabGDI3 The preparation method of promoter, its step is:

[0061] 1. Rapeseed promoter P BnRabGDI3 Primer sequences:

[0062] According to the upstream 2kb sequence of the BnRabGDI3 gene obtained by sequencing the rapeseed whole genome, a pair of primers were designed for PCR amplification. 3'. The forward primer PBnRabGDI3S used contained a Kpn I restriction site, and the reverse primer PBnRabGDI3A contained an Xba I restriction site.

[0063] 2. Rapeseed promoter P BnRabGDI3 Preparation of:

[0064] The rapeseed used in the present invention is Brassica napus L. Zhongshuangjiu (mentioned above). Zhongshuang No. 9 was sown in Daejeon and managed in the field normally. 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 10×Ex ...

Embodiment 2

[0068] pBI-P BnRabGDI3 Arabidopsis transformation and PCR detection:

[0069] Transform Arabidopsis thaliana by the inflorescence infection method (described above), and grow on MS (described above) medium containing 50 mg / L kanamycin according to the unique kanamycin resistance of the transgenic plants , the obtained green shoots were preliminarily considered to be transformed plants. After the transformed plant grew two true leaves, it was transplanted into vermiculite. After the plant appeared inflorescences, a true leaf was taken to extract genomic DNA (as described above) by SDS method for PCR identification. The primer sequences are NPTIIF and NPTIIR (mentioned above), and the PCR reaction system is as follows: template DNA 1μL (about 50ng), 10×Taq buffer (containing MgCl 2 ) 1μl, 1.5mmol / L dNTP (10mmol / L) 1μl, 5’ primer (10μmol / L) 0.5μl, 3’ primer (10μmol / L) 0.5μl, Taq (5U / μl) 0.5μl, ddH 2 O5.5 μl. The reaction program was: denaturation at 94°C for 5min, 32 cycles a...

Embodiment 3

[0071] Brassica napus P BnRabGDI3 Functional analysis of promoters:

[0072] The present invention clones for the first time to obtain P BnRabGDI3 sequence and its functional analysis. From Example 2, T1 generation of transgenic positive seedlings were screened through the transformation of the model plant Arabidopsis thaliana (described above) and PCR detection (described above), and the seeds (ie T2 generation) were harvested by selfing. At different stages, different tissues of transformed plants from 10 lines of T2 generation were used for GUS staining.

[0073] The GUS staining process of T2 generation transgenic plants and tissues is as follows: soak the samples in GUS staining solution (described above) and vacuum for 5 minutes, overnight at 37°C. The next day, the leaves were decolorized with alcohol-acetic acid (volume ratio 1:1) until the leaves turned white, then rinsed with distilled water for 3-5 times, and photographed under a stereomicroscope (OLYMPUS SZX16)....

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Abstract

The invention discloses a preparation method and applications of a cabbage type rape BnRabGDI3 promoter. The preparation method comprises the steps of: firstly, obtaining a primer sequence amplified by the PBnRabGDI3 promoter: designing a primer according to an upstream 2kb sequence of a PBnRabGDI3 gene obtained from rape whole genome sequencing for PCR (Polymerase Chain Reaction) amplification; and secondly, extracting rape leaf genome DNA (Deoxyribose Nucleic Acid) by use of an SDS (Sodium Dodecyl Sulfate) cracking method, carrying out PCR amplification, connecting to a pMD18-T vector after carrying out purification and recycling of a gel extraction kit, converting the competent cell of a gold strain by a heat shock method, picking positive clones to obtain the upstream 2kb flanking sequence of the target gene, namely PBnRabGDI3. A GUS (glucosiduronide) staining result shows that PBnRabGDI3 is efficiently expressed in rape anthers and pollen grains and not expressed in the tissues of root, stem, leaf, silique, seed and the like. The promoter has good application potentiality in the aspects of improving crop quality, manually establishing germplasm resources, and the like.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and biotechnology, in particular to a BnRabGDI3 gene promoter (named P BnRabGDI3 , the same below), and also relates to a method for preparing a Brassica napus BnRabGDI3 promoter. The present invention also relates to the vector containing the promoter or its substantially homologous nucleotide sequence and the application of the promoter in genetic engineering of rapeseed and other plants. Background technique [0002] Plant gene 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, the regulation of genes is divided into regulation at the transcriptional level, regulation at the translational level, and regulation at the protein processing level. The proteins and RNAs encoded by genes and their secondary metabolites are extremely impor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/84A01H5/02
Inventor 刘胜毅董彩华黄军艳李振波刘越英程晓辉
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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