Brassica napus embryo-specific promoter pbnaa09g21960d and its application

A pbnaa09g21960d, promoter technology, 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: 2019-12-13
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, methylcortisol, estradiol, and 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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  • Brassica napus embryo-specific promoter pbnaa09g21960d and its application
  • Brassica napus embryo-specific promoter pbnaa09g21960d and its application
  • Brassica napus embryo-specific promoter pbnaa09g21960d and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: The primer sequence design of rapeseed promoter pBnaA09g21960D:

[0035] According to the BnaA09g21960D gene (Sequence ID: XM_009109941.1) upstream gene spacer sequence 990bp obtained by rapeseed genome sequencing, a pair of primers were designed for PCR amplification, and the 5' upstream promoter sequence of rapeseed BnaA09g21960D was amplified, and the primers used were pBnaA09g21960D- F: 5′-GAGATCTACAGCGCTAAGCTTaaccgtctttgctcgaatttc-3′ and pBnaA09g21960D-R: 5′-GGACTGACCACCCGGGGATCCtgaacacgtagacagcttttaa-3′. In the primer pBnaA09g21960D-F, the sequence GAGATCTACAGCGCT is the upstream sequence of the fusion site of the vector DX2181, and the sequence AAGCTT is the restriction site of Hind III; in the primer pBnaA09g21960D-R, the sequence GGACTGACCACCCGG is the downstream sequence of the fusion site of the vector DX2181, and the sequence is GGATCC Restriction site for BamH I.

Embodiment 2

[0036] Example 2: Preparation of rapeseed promoter pBnaA09g21960D:

[0037] Rapeseed used in the present invention is Brassica napus L. Darmor (provided by researcher Liu Shengyi, Institute of Oil Crops, Chinese Academy of Agricultural Sciences, hereinafter the same), and rapeseed is sown in the field and managed in the normal field. Genomic DNA of rapeseed leaves was extracted by CTAB method, and polymerase chain reaction PCR (Polymerase chain reaction) was carried out using the extracted whole genome DNA of rapeseed as a template. PCR system: 2×Mix buffer 25 μL, pBnaA09g21960D-F: 1 μL, pBnaA09g21960D-R: 1 μL, DNA 1 μL, ddH 2 O 22 μL. The PCR program was: 94°C for 5 min; 35 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min; 72°C for 10 min; 4°C∞. The PCR product size is 990bp (see figure 1 ), identified by 1.0% agarose gel electrophoresis, purified and recovered, and tested for concentration according to the instructions of the kit.

[0038] The DX2181 vector pla...

Embodiment 3

[0039] Example 3: Sequence Analysis and Function Prediction of Rapeseed Promoter pBnaA09g21960D:

[0040]The pBnaA09g21960D sequence obtained by cloning and sequencing was used in PlantCARE (Lescot M, Déhais P, Thijs G, et al. PlantCARE, a database of plantcis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences [J]. Nucleic acids research, 2002, 30 (1): 325-327. http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) online predictive analysis of functional components. The results show that if figure 2 As shown, pBnaA09g21960D contains core elements TATA box and CAAT box necessary for eukaryotic promoters. Further analysis of the promoter sequence found that in addition to the necessary core elements, there are also a variety of promoter functional elements in the pBnaA09g21960D sequence: ABRE (TACGTG): cis-acting element involved in the abscisic acid responsiveness, abscisic acid responsive cis-acting element ; G-Box(CACGTA / CA...

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Abstract

The invention discloses a cabbage-type rape embryo specific promoter pBnaA09g21960D and an application thereof. According to the invention, the promoter of a gene BnaA09g21960D is cloned from the cabbage-type rape, a plant expression vector DX2181-pBnaA09g21960D of a report gene GUS regulated and controlled by the promoter is constructed, an agrobacterium-mediated inflorescence dipping method is employed for conveying arabidopis thaliana, GUS histochemical method staining is carried out by the screened positive transgene lines, the result shows that pBnaA09g21960D has function for driving a downstream gene for specific expression in the embryo, and does not expression in other tissue organs. The promoter has good application potential for enhancement of the crop quality by the rape transgene and artificial creation of germplasm resource.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and biotechnology, in particular to a promoter of Brassica napus BnaA09g21960D gene, which is named pBnaA09g21960D (hereinafter the same), and the promoter can be used for specific expression of Arabidopsis embryos. Background technique [0002] The plant gene promoter plays a key role in the regulation of gene expression. It is located in the upstream region of the 5' end of the structural gene and contains a DNA sequence of cis-acting elements, which determines the specificity, direction and efficiency of downstream gene transcription. In addition, the promoter plays a key role in the process of constructing a heterologous expression vector capable of high-level expression, which determines the temporal and spatial sequence, expression intensity, transcription efficiency, and gene expression level of exogenous gene expression. Therefore, the study of the function of the promoter has very...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/82A01H5/10A01H6/20
CPCC12N15/8234C12N15/8241
Inventor 华玮李俊范世航胡志勇
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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