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A wheat promoter induced by rust fungus

A promoter, wheat technology, applied in the field of plant genetic engineering

Active Publication Date: 2017-10-27
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But rust-inducible promoters are rarely reported

Method used

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  • A wheat promoter induced by rust fungus
  • A wheat promoter induced by rust fungus
  • A wheat promoter induced by rust fungus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The cloning of embodiment 1 wheat GER4 gene promoter:

[0033] Using the coding sequence of the barley germin-like protein gene GER4c (GenBank: 418E01), in the Wheat Chinese Spring Genome Database ( http: / / www.cerealsdb.uk.net) to search for homologous sequences, and obtained a sequence spanning contig with a length of 1.4 kb through sequence splicing, which included two exons at the 5' end of the wheat GER homologous gene and its upstream sequence. Primers were designed according to the upstream sequence of the wheat homologous gene, and the sequences of StuI and SpeI restriction sites were added to the ends of the primers respectively. The forward primer sequence: 5'-AAAAGGCCTCTTTGCAGGCACTAACTTCATA-3', its nucleotide sequence is shown in SEQ ID NO:2 ; Reverse primer sequence: 5'-TTTACTAGTTGATTTCAGTCCTTTGGGTC-3', the nucleotide sequence of which is shown in SEQ ID NO:3.

[0034] PCR amplification uses Phusion high-fidelity enzyme (Thermo Scientific Company, Cat. No. ...

Embodiment 2

[0038] The construction of embodiment 2 plant expression vectors:

[0039] Cloning of introns:

[0040] Primers were designed according to the wheat WIR1 gene (GenBank: M95500), its intron fragment (151bp) was amplified, and Spe I and Hind III restriction sites were introduced at the ends, respectively. Forward primer sequence: 5'-TTTACTAGTGGAGCCACGGCCGTCCACG-3', its nucleotide sequence is shown in SEQ ID NO:4; reverse primer sequence: 5'-TTTAAGCTTTGCCTGGACGGGAAACCATGGA-3', its nucleotide sequence is shown in SEQ ID NO:5 shown.

[0041] Construction of intermediate vectors:

[0042] First, the wheat PTaGER4 gene promoter obtained in the previous step was double-digested with StuI and SpeI, and then cloned into the pUC-T vector (Kangwei Century Company, product number CW0802). Then, the recombinant plasmid was subjected to double digestion with SpeI and HindIII, and ligated with the restriction fragment of the WIR1 gene intron. Finally, the "promoter-intron" fragment was ob...

Embodiment 3 2

[0046] Embodiment 3 Genetic transformation and screening verification of Brachypodium distachyon

[0047] Genetic transformation of Brachypodium distachyon by Agrobacterium-mediated transformation. Take 2 μl of the carrier, and transform Agrobacterium strain AGL1 competent cells by electroporation. The Brachypodium callus was infected with the Agrobacterium solution with an OD value of 0.6, then placed on three layers of sterile filter paper, and cultured in the dark at 22°C for 3 days. Then put them on the induction medium containing hygromycin 40 mg / L for selection for 3 weeks, and subculture once in the middle, and the culture condition is dark culture at 28°C. If the callus is in a good state, it can be transferred to the regeneration medium (REM). The regeneration culture conditions are: light for 16 hours, darkness for 8 hours, and 28°C. When the young leaves grow to 1-2cm, they are transferred to a culture bottle containing rooting medium (MS) to make them take root. ...

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Abstract

The invention relates to the technical field of plant genetic engineering, and provides a wheat promoter induced by rust fungus and its application. The promoter is derived from the wheat germin-like protein gene, its nucleotide sequence is shown in SEQ ID NO: 1, and the length is 672bp. The inventor transforms it with a recombinant expression vector containing the promoter and the green fluorescent protein (GFP) gene The model plant, Brachypodium, verified that the promoter was induced by rust to enhance expression. Utilizing this characteristic, the promoter can be applied to inducible overexpression or RNAi silencing research of target genes or gene fragments in wheat crops, so as to realize functional analysis of genes or genetic engineering breeding.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, and provides a wheat promoter induced by rust fungus, which can be used for wheat genetic engineering breeding and the like. Background technique [0002] Promoter is a specific DNA sequence that directly combines with RNA polymerase and its transcription factors to determine whether gene transcription starts or not. It is located at a fixed position upstream of the gene regulated by it, close to the transcription start site, and the length is generally About one hundred to one thousand bases. According to the mode of action and function, promoters can be divided into constitutive, specific and inducible promoters. Among them, constitutive promoters can promote the expression of downstream genes in all tissues, and the expression of the promoted genes is continuous, such as the promoters of plant housekeeping genes such as actin and tubulin; specific promoters It has the funct...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82
Inventor 吴佳洁刘强陈凤娟杨超付道林
Owner SHANDONG AGRICULTURAL UNIVERSITY
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