Brassica napus L promoter pBnUnng0942890 and application thereof

A Brassica napus and promoter technology, applied in the field of plant genetic engineering and biology, can solve problems such as limited potential

Active Publication Date: 2019-03-08
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, conventional breeding techniques have limited potential to continue to improve crop yield and quality

Method used

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  • Brassica napus L promoter pBnUnng0942890 and application thereof
  • Brassica napus L promoter pBnUnng0942890 and application thereof
  • Brassica napus L promoter pBnUnng0942890 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Primer sequence design for rapeseed promoter pBnUnng0942890

[0037] According to the 1525bp intergenic sequence upstream of the rapeseed BnUnng0942890 gene sequence obtained by sequencing the entire genome of rapeseed, a pair of primers were designed for PCR amplification, and the 5'upstream promoter sequence of rapeseed BnUnng0942890 was amplified. 3' and pBnUnng0942890-R: 5'-GGACTGACCACCCGGGGATCCcttccttaactttctacacata-3'. In the primer pBnUnng0942890-F, the sequence GAGATCTA CAGCGCT is the upstream sequence of the fusion site of the vector DX2181G, and the sequence AAGCTT is the restriction site of Hind III; in the primer pBnUnng0942890-R, the sequence GGACTGACCACCCGG is the downstream sequence of the fusion site of the vector DX2181G, the sequence GGATCC is the restriction site of BamH I.

Embodiment 2

[0038] Example 2: Preparation of Rapeseed Promoter pBnUnng0942890

[0039] The rapeseed used in the present invention is Brassica napus L. Darmor, and the rapeseed is sown in the field and managed in the field normally. 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, pBnUnng0942890-F: 1 μL, pBnUnng0942890-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 1525bp (see figure 1 ), identified by 1.0% agarose gel electrophoresis, purified and recovered, and tested for concentration according to the instructions of the kit.

[0040] The DX2181G vector plasmid was completely digested by Hind III and BamH I, and recovered by 1% agarose gel electrophoresis. The recover...

Embodiment 3

[0041] Example 3: Sequence Analysis and Function Prediction of Rapeseed Promoter pBnUnng0942890

[0042] The pBnUnng0942890 sequence obtained by cloning and sequencing was used in the 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, pBnUnng0942890 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 pBnUnng0942890 sequence: G-Box: cis-acting regulatory element involved in light responsiveness light signal regulation cis-acting element; GT1...

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Abstract

The invention discloses brassica napus L promoter pBnUnng0942890 and application thereof. By cloning a promoter of a gene pBnUnng0942890 in the brassica napus L, a plant expression vector of a reporter gene GUS regulated by the promoter is constructed; by using an agrobacterium-mediated inflorescence impregnating method to transform arabidopsis, GUS histochemical staining is carried out on screened positive transgenic strains; the result shows that the pBnUnng0942890 has the functions of driving preferential expression of downstream genes in roots, flowers and seeds, and not expressing or realizing low expression in other tissues and organs. The promoter has good application potential in the aspects of improving crop quality by rapeseed transgene, artificially creating germplasm resourcesand the like.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and biotechnology, and in particular relates to a promoter of Brassica napus BnUnng0942890 gene, named as pBnUnng0942890 (the same below), which can be used to predominately express the target gene in the roots, flowers and seeds of 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 functi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8227C12N15/823C12N15/8234
Inventor 范世航李俊匡琛胡志勇
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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