Brassica napus BnGLABRA2 promoter and preparation method and application thereof
A promoter and technology of Arabidopsis thaliana, applied in the field of molecular biology 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: 2012-10-03
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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Problems solved by technology
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.
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Embodiment 1
[0059] The preparation steps of a rape BnGL2 promoter are:
[0060] 1. Cloning of rapeseed BnGL2 gene promoter GL2B:
[0061] Utilize the SDS cracking method to extract Brassica napus DNA (J. Sambrook.D.W.Russell, Huang Peitang et al. Translate Molecular Cloning Test Guide (Third Edition) Science Press), according to the genome walking kit (purchased in Clontech company ) operating procedure, two genome walks were performed, and two rounds of PCR were amplified for each walk to clone the rapeseed GL2 gene promoter GL2B (2882bp). The primers used for the two walks are listed below.
[0062] Table 1 Primers used for genome walking cloning of Brassica napus GL2 gene promoter GP2
[0063]
[0064] 2. Bioinformatics analysis of GL2B sequence of rapeseed GL2 promoter:
[0065] Using BLASTN to compare the promoter sequence of the Arabidopsis GL2 gene (about 2Kb) (GenBank accession number: NM_106633) and the promoter GL2B sequence of the rape GL2 gene, it was found that the homo...
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The invention discloses a brassica napus BnGLABRA2 promoter and a preparation method and application thereof. The method comprises the following steps of: A, cloning a promoter sequence by genomic walking, namely extracting genomic DNA (Deoxyribose Nucleic Acid) by using an SDS (Sodium Dodecyl Sulfonate) cracking method, performing genomic walking, and amplifying PCR (Polymerase Chain Reaction) cloned rape twice during each genomic walking; B, performing bioinformatics analysis on the promoter sequence, namely performing homology comparison on a cloned sequence by BLASTN, wherein a promoter sequence and an upstream promoter element are obtained from a promoter core element and an upstream cis acting element by using promoter prediction software; and C, amplifying a full-length promoter and a missing promoter and constructing a pMD18-T-GP vector, namely designing and amplifying a primer of each missing segment by taking the genomic DNA as a template according to the previously obtainedpromoter sequence for PCR amplification. The application of the brassica napus BnGL2 genomic full-length promoter SEQ ID NO:1 to leaf epidermal hairs, seeds, root parts, entire veins and stele leaf veins is disclosed. The method has the advantages of easiness in operating, stable and reliable result. The brassica napus BnGLABRA2 promoter has biological safety and development and application values.
Description
technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a BnGLABRA2 promoter of Brassica napus, a preparation method of the BnGLABRA2 promoter of Brassica napus, and the application of the BnGLABRA2 promoter of Brassica napus in genetic engineering of rapeseed. 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 important for maintaining the whole life activities of organisms. Any misregulation of gene expression can have serious consequences for life. Therefore, the researc...
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IPC IPC(8): C12N15/11C12N15/10C12N15/82A01H5/00
Inventor 白泽涛董彩华刘胜毅柴国华黄军艳刘越英
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI