Promoter sequence based on BnaCnng52950D gene, recombinant vector and application

A promoter and gene technology, applied in the fields of genetic engineering and molecular biology, can solve the problems of wasting energy, easily affecting the physiological metabolism of plants, adverse pleiotropic reactions, etc., and achieve the effect of increasing diversity

Active Publication Date: 2019-04-26
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since constitutive promoters can drive the expression of downstream genes at different times and in different tissues, waste energy and easily affect the normal physiological metabolism of plants, resulting in some adverse pleiotropic reactions, so effective use of specific expression promoters is a must. The key to addressing the need for GM crops

Method used

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  • Promoter sequence based on BnaCnng52950D gene, recombinant vector and application
  • Promoter sequence based on BnaCnng52950D gene, recombinant vector and application
  • Promoter sequence based on BnaCnng52950D gene, recombinant vector and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Promoter sequence cloning

[0042] 1. In the early stage, the Brassica napus transcriptome data was used to screen the gene BnaCnng52950D with tissue-specific expression, and the upstream 1500bp promoter amplification primers PR and PF were designed according to the Brassica napus genome sequence.

[0043] 2. Using the genomic DNA of Brassica napus ZS11 as a template, use the Q5 high-fidelity enzyme of NEB Company in the United States to amplify. The PCR program is: 98°C pre-denaturation for 30s; 98°C denaturation for 10s; 56°C annealing for 20s; 35 cycles; the final incubation at 72°C for 2 minutes. The amplified products were detected by electrophoresis, and the electrophoresis results were observed in a gel imaging system. The fragments with the correct band size were recovered by using the gel recovery kit method to recover the target fragments.

[0044] 3. Mix 4 μl of the amplified PCR product with 1 μl of Salt Solution, take 2.5 μl into a new centrifuge tube, add...

Embodiment 2

[0046] Promoter Homeopathic Element Analysis

[0047] Using the PlantCARE database to analyze the cis-acting elements in the amplified sequence of the BnaCnng52950D gene promoter, the results showed that the Brassica napus BnaCnng52950D gene promoter contained a large number of core elements TATA-box and CAAT-box and many other important cis-acting elements, Including many light response-related regulatory elements, such as Box 4, GT1-motif and MRE, etc., there are also circadian cis-regulatory elements related to circadian rhythm control and cis-regulatory elements GCN4_motif involved in endosperm expression, as well as anaerobic-induced cis-regulatory factor etc.

[0048] Table 1 Cis-acting elements in the BnaCnng52950D promoter

[0049]

Embodiment 3

[0051] Construction of recombinant expression vectors

[0052] 1. Digest the plasmid pEntry-52950 with a combination of restriction endonucleases MluI to linearize the circular plasmid, which is beneficial to improve the efficiency of the LR reaction. Enzyme digestion system (50 μl): ddH2O, 14 μl; 10×NEB buffer 3.1, 5 μl; pEntry-52950 plasmid, 30 μl; MluI enzyme, 1 μl.

[0053] 2. After digestion for 30 minutes, perform LR recombination reaction between the linearized pEntry-52950 plasmid and the plant expression vector pEarly502, and recombine the BnaCnng52950D promoter sequence into the pEarly502 vector. LR recombination reaction system (6 μl): digested pEntry-52950, ​​3 μl; plant expression vector pEarly502, 2 μl; 5×LR Clonase Enzyme, 1 μl.

[0054] 3. After mixing the connection system, place it at room temperature for 4 minutes, add 0.5 μl proteinase K, and place it at 37°C for 10 minutes, then transfer it to Escherichia coli TOP10 competent, screen it on an LB plate con...

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Abstract

The invention belongs to the field of gene engineering and molecular biology, and particularly relates to a promoter sequence of a cabbage type rape BnaCnng52950D gene, a recombinant vector built according to the promoter sequence and application. A promoter element included in the sequence is predicted through bioinformatic software, and a plant expression vector is built for performing qualitative detection on the promotor activity. The result shows that a cloned sequence can drive the expression of a GUS reporter gene in the bases of pod stalks and leaves. The acquisition of the promoter provides a powerful tool for the transgenic research of rapeseed, and lays a certain foundation for the research of the rapeseed and genetically close species thereof.

Description

technical field [0001] The invention belongs to the field of genetic engineering and molecular biology, and in particular relates to a promoter sequence of BnaCnng52950D gene of Brassica napus, a recombinant vector and a transgenic plant constructed therefrom. Background technique [0002] As one of the four major oil crops in the world, Brassica napus is the only winter oil crop in the world, and it is also the main oil, feed and energy crop in my country. Its perennial planting area and total output rank first in the world. But rape is still facing problems such as low yield and oil content, weak resistance and unsuitable for mechanized operation. [0003] The promoter is located at the 5' end of the upstream of the gene and plays a vital role in the regulation of gene expression. It can be recognized by RNA polymerase and bind to it, and accurately initiate the transcription of a specific DNA sequence. The regulation of transcription level is the most cost-effective and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/70C12N15/10C12N15/82A01H5/00A01H6/20C12N15/11
CPCC12N15/10C12N15/70C12N15/8223C12N15/8261C07K14/415
Inventor 卢坤李加纳刘淼张凯曲存民王瑞
Owner SOUTHWEST UNIVERSITY
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