Brassica napus constitutive promoter pbnac05g31880d and its application

A promoter and constitutive technology, applied in the field of plant genetic engineering and biology, can solve problems such as unsuitable production practice, unfavorable normal plant growth, harmful ecological environment, etc., and achieve good application potential

Active Publication Date: 2022-03-15
SHIHEZI UNIVERSITY +1
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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 constitutive promoter pbnac05g31880d and its application
  • Brassica napus constitutive promoter pbnac05g31880d and its application
  • Brassica napus constitutive promoter pbnac05g31880d and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: The primer sequence design of rapeseed promoter pBnaC05g31880D:

[0034] According to the BnaC05g31880D gene (Sequence ID: GCF_000686985.2) upstream gene spacer sequence 1525bp obtained by rapeseed genome sequencing, a pair of primers were designed for PCR amplification, and the 5' upstream promoter sequence of rapeseed BnaC05g31880D was amplified, and the primers used were pBnaC05g31880D- F: 5′-GAGATCTACAGCGCTAAGCTTtatcagacatggagaagaac-3′ and pBnaC05g31880D-R: 5′-GGACTGACCACCCGGGGATCCaaagaactgttgttttgctc-3′. In the primer pBnaC05g31880D-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 pBnaC05g31880D-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

[0035] Example 2: Preparation of rapeseed promoter pBnaC05g31880D:

[0036] The rape used in the present invention is brassica napus L. (Brassica napus L.) (provided by the Institute of Oil Crops, Chinese Academy of Agricultural Sciences, the same below), and the rape 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 chainreaction) was carried out using the extracted whole genome DNA of rapeseed as a template. PCR system: 2×Mix buffer 25 μL, pBnaC05g31880D-F: 1 μL, pBnaC05g31880D-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.

[0037] The DX2181G vector plasmid was comp...

Embodiment 3

[0038] Example 3: Sequence Analysis and Function Prediction of Rapeseed Promoter pBnaC05g31880D:

[0039] The pBnaC05g31880D 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, as shown in Table 1 and figure 2 As shown, pBnaC05g31880D contains the core elements TATAbox and CAAT box necessary for eukaryotic promoters. Further analysis of the promoter sequence found that, in addition to the necessary core elements, there are a variety of promoter functional elements in the sequence of pBnaC05g31880D: ARE: cis-acting element involved in light responsiveness, light cis-acting element involved in light responsiveness; ...

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Abstract

The invention discloses a constitutive promoter of Brassica napus and its application, and clones the gene from Brassica napus BnaC05g31880D promoter, construct the plant expression vector of the reporter gene GUS regulated by the promoter, transform Arabidopsis thaliana by the inflorescence dipping method mediated by Agrobacterium thaliana, and perform GUS histochemical staining on the screened positive transgenic lines, the results show that, The downstream genes driven by pBnaC05g31880D were strongly expressed in roots, stems, leaves, flowers, siliques, ovules, seed coats and mature seeds. The promoter has a good application potential in the fields of genetic engineering research fields such as the improvement of crop quality by transgenic rapeseed and the artificial creation of germplasm resources.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and biotechnology, and in particular relates to a brassica napus constitutive promoter pBnaC05g31880D, which can be used to constitutively express a target gene in plants. 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 important scientific significance for the r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/02A01H5/04A01H5/06A01H5/08A01H5/10A01H5/12A01H6/20
CPCC12N15/8222
Inventor 华兆晖范世航李俊
Owner SHIHEZI UNIVERSITY
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