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Morus alba dihydroflavonol reductase promoter and its recombinant expression vector and application

A technology of dihydroflavonol and expression vector, which is applied in the field of genetic engineering, can solve problems such as inefficiency, and achieve high-efficiency expression

Active Publication Date: 2016-05-04
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Sequence analysis shows that the N-terminus of MnDFR has a predicted NADP-binding domain, and the key amino acid residue that determines substrate specificity is aspartic acid, indicating that MnDFR belongs to the aspartic acid type DFR, which cannot effectively catalyze two Reduction of hydrokaempferol to geranoid anthocyanins
The expression pattern and regulatory activity of MnDFR are determined by the MnDFR promoter sequence, but there is no report on the MnDFR promoter in the prior art

Method used

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  • Morus alba dihydroflavonol reductase promoter and its recombinant expression vector and application
  • Morus alba dihydroflavonol reductase promoter and its recombinant expression vector and application
  • Morus alba dihydroflavonol reductase promoter and its recombinant expression vector and application

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Experimental program
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Effect test

Embodiment 1

[0017] Embodiment 1, the acquisition of mulberry MnDFR gene promoter

[0018] The primers for amplifying the MnDFR promoter were designed, the upstream primer was 5'-cgtctgaacccggtcctaa-3' (SEQ ID NO.1), and the downstream primer was: 5'-cttcgatcccatattgctgc-3' (SEQ ID NO.2). Then use the Chuanmulus genomic DNA as a template, and the sequences shown in SEQIDNO.1 and SEQIDNO.2 as primers for PCR amplification. The PCR amplification system is: 10×Ex-taqbuffer2.5μL, 25mMMgCl 2 2μL, 2.5mMdNTP 2μL, Chuanmulberry genomic DNA 30ng, 10μM upstream and downstream primers 1μL each, 5U / μl Ex-taq polymerase 0.2μL, sterilized water to make up to 25μL; PCR amplification conditions are 94°C pre-denaturation for 4 minutes; 94°C denaturation 40 seconds, annealing at 55°C for 40 seconds, extension at 72°C for 2 minutes, and 30 cycles; finally, extension at 72°C for 10 minutes, and storage at 4°C. The PCR amplification product was subjected to agarose gel electrophoresis, and the electrophoresis...

Embodiment 2

[0019] The construction of embodiment 2 plant expression vector pCAMBIA1303-MnDFR

[0020] According to the mulberry MnDFR gene promoter sequence and pCAMBIA1303 carrier sequence obtained in Example 1, select a suitable enzyme cutting site, design and construct the primer of the plant expression vector, specifically: upstream primer: 5 '-cg ggatcc cgtctgaacccggtcctaa-3' (SEQ ID NO.4), the underline indicates the BamHI restriction site, downstream primer: 5'-g Actagt cttcgatcccatattgctgc-3' (SEQ ID NO.5), the underline indicates the SpeI restriction site. Then use the pMD19-Tsimple-MnDFR carrier obtained in Example 1 as a template, and the sequences shown in SEQIDNO.4 and SEQIDNO.5 as primers for PCR amplification. The PCR amplification system is: 10×Ex-taqbuffer2.5μL, 25mMMgCl 2 2μL, 2.5mMdNTP2μL, pMD19-Tsimple-MnDFR plasmid 50pg, 10μM upstream and downstream primers 1μL each, 5U / μLEx-taq polymerase 0.2μL, sterilized water to make up to 25μL; PCR amplification conditions ar...

Embodiment 3

[0022] Embodiment 3, Morus alba MnDFR gene promoter expresses exogenous protein in Arabidopsis

[0023] The plant expression vector pCAMBIA1303-MnDFR was transformed into competent cells of Agrobacterium strain LBA4404, and cultured in YEB medium to OD 600 0.6-0.8, collected the cells by centrifugation, transformed the permeate with Arabidopsis (1 / 2MS medium 2.17g, sucrose 50g, MES 0.5g, constant volume to 1L, KOH adjusted pH value to 5.7, added 1mg / mL6 -BA 10 μL and Silwet 200 μL) were resuspended to make a transformed bacterial solution. The plant expression vector pCAMBIA1303-MnDFR was transformed into the Arabidopsis genome by the Agrobacterium-mediated flower dipping method, and then the Arabidopsis seeds were cultivated and harvested.

[0024] The specific method for screening transgenic plants is as follows: place the harvested Arabidopsis seeds at 4°C for vernalization for two days, dry them in an oven at 30°C for one day, and then place the seeds in a disinfectant so...

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Abstract

The invention discloses a mulberry dihydroflavonol reductase promoter, a recombinant expression vector of the mulberry dihydroflavonol reductase promoter, and application of the mulberry dihydroflavonol reductase promoter. A nucleotide sequence of the mulberry dihydroflavonol reductase promoter is shown in SEQ ID NO. 3, and the length of the nucleotide sequence is 1812bp; the nucleotide sequence comprises a 12bp coding sequence starting from a dihydroflavonol reductase gene initiation codon ATG and an upstream 1800bp sequence. The promoter can mediate foreign protein to perform specific expression on a root, and the promoter with important values is provided for the research and application of plant genetic engineering.

Description

technical field [0001] The invention belongs to the field of genetic engineering, specifically relates to a mulberry dihydroflavonol reductase promoter, and also relates to a recombinant expression vector containing the mulberry dihydroflavonol reductase promoter and its application. Background technique [0002] Mulberry is a perennial woody plant. For a long time, mulberry, as the main food source of silkworm, has played a vital role in the development of silk industry. Mulberry tree is rich in flavonoid compounds. The dihydroflavonol4-reductase (DFR) gene is a key gene in the plant flavonoid biosynthesis pathway. The encoded product of this gene can catalyze the reduction reaction of dihydroflavonol to Produces colorless anthocyanins, which belong to the reduced coenzyme II-dependent reductase family. At present, the gene has been cloned in various plants. Sequence analysis shows that its sequence in the functional region is highly conserved, and the gene structure is co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N9/24
Inventor 何宁佳亓希武向仲怀
Owner SOUTHWEST UNIV
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