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Brassica napus Bna.A08IDD7 gene promoter and application thereof

A technology of bna.a08idd7, Brassica napus, applied in the direction of application, genetic engineering, recombinant DNA technology, etc., to achieve the effect of good application potential

Active Publication Date: 2020-12-01
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But after all, it is not derived from plants, and the application of this promoter in plant genetic engineering will have potential biosafety risks

Method used

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  • Brassica napus Bna.A08IDD7 gene promoter and application thereof
  • Brassica napus Bna.A08IDD7 gene promoter and application thereof
  • Brassica napus Bna.A08IDD7 gene promoter and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, Brassica napus Bna.A08IDD7 gene promoter cloning

[0029] Search the promoter sequence of Brassica napus Bna.A08IDD7 translation initiation site (ATG) upstream 2000bp on NCBI, and use Primer Premier 5.0 to design and clone the PCR amplification primers Pro-Bna.A08IDD7-F / R and For the vector universal primer pM13F / R, a BamHI restriction site is added to the 5' end of the primer before the promoter amplification, and a NcoI restriction site is added to the 5' end of the rear primer. Table 1 is the designed primer sequence.

[0030] Table 1, Bna.A08IDD7 gene promoter amplification primers and vector universal primer sequences

[0031]

[0032]

[0033] Using the genomic DNA of Brassica napus ZS11 as a template, high-fidelity enzymes provided by TAKARA were used Amplify. Amplification system (50μL): PCR reaction program: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 56°C for 15s, extension at 72°C for 120s, 35 cycle...

Embodiment 2

[0035] Embodiment 2, plant promoter vector construction

[0036] Connect the PCR recovered product to the blunt-ended T vector pEASY-Blunt simplecloning vector provided by Quanshijin Company, and transform Escherichia coli DH5α competent; use the colony PCR to select the bacterial solution with the correct sequencing result and multiply, and extract the gene containing Bna.A08IDD7 The plasmid T-BnaIDD7-pro of the promoter sequence, the detection result is as follows image 3 . The T-BnaIDD7-pro vector and the destination vector pCAMIBIA1305.1 were double digested with restriction enzymes BamHI and NcoI from Thermo Scientific; then the promoter fragment of the Bna.A08IDD7 gene was combined with the T4 DNA Ligase provided by Promega. The fully digested pCAMIBIA1305.1 vector backbone was ligated, and the Bna.A08IDD7 gene promoter was used to replace pCAMIBIA1305.1 (Ping Li, Yan Pei, Xianchun Sang, Yinghua Ling, Zhenglin Yang, Guanghua He, Transgenic indica rice expressing a bitt...

Embodiment 3

[0043] Example 3, Transformation of plant recombinant expression vector into Agrobacterium tumefaciens GV3101

[0044] Competent Agrobacterium was purchased from the laboratory, and the constructed pCAMBIA1305.1-Bna.A08IDD7::GUS expression vector plasmid was transformed into competent cells of Agrobacterium GV3101 by liquid nitrogen cold shock method.

[0045] Specific operation: Take 1 tube of 100 μL competent Agrobacterium GV3101 stored at -80°C, add pCAMBIA1305.1-Bna.A08IDD7::GUS expression vector plasmid after thawing in an ice-water bath, mix gently with a pipette tip, and bathe in ice-water for 5 minutes After being cold-shocked with liquid nitrogen for 8 minutes, quickly heat-shocked in a water bath at 37°C for 5 minutes, added 800 μL of blank YEB liquid medium to the ultra-clean workbench, revived at 28°C and 180r for 3 hours on a shaker, and centrifuged at room temperature for 5 minutes at 5000rpm after the revival. Aspirate 600 μL of supernatant, resuspend the remain...

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Abstract

The invention discloses a brassica napus Bna.A08IDD7 gene promoter and application thereof, the nucleotide sequence of the promoter is shown as SEQ ID NO.5, and the promoter can be strongly expressedin roots, leaves, stems, flowers, siliques, seeds and other tissues of arabidopsis thaliana; therefore, it is indicated that the sequence has very strong expression activity and characteristics of a constitutive promoter, and can be used as a strong constitutive promoter to be applied to plant genetic engineering research. Therefore, the promoter creates excellent germplasm resources and other genetic engineering research aspects; the promoter has good application potential, can replace a cauliflower mosaic virus (CaMV) 35S promoter commonly used in current plant genetic engineering research,constructs a plant transgenic expression vector, is applied to plant genetic engineering, and can widely cultivate transgenic plants with high biosafety.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the Bna.A08IDD7 gene promoter of Brassica napus, and also relates to the application of the promoter. Background technique [0002] my country is the world's largest producer of rapeseed (Brassica napus) and consumer of rapeseed oil. It can provide about 5.2 million tons of high-quality edible oil every year, accounting for 47% of domestic vegetable oil. However, at present, the supply situation of edible vegetable oil in my country is still relatively severe, and the self-production is insufficient, which seriously threatens the safety of edible vegetable oil in my country. Rapeseed production mainly utilizes fallow fields in winter, and does not compete with food crops for land. Currently, there are still 66hm of fallow fields in the Yangtze River Basin 2 All of the above have great potential for developing the rapeseed industry. [0003] The plant gene promoter plays a key role ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8222
Inventor 卢坤刘绪梅段回春曲存民李加纳
Owner SOUTHWEST UNIVERSITY
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