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Recombinant barley stripe mosaic virus-mediated wheat target miRNA overexpression method

A striped mosaic and overexpression technology, which is applied in the directions of botanical equipment and methods, biochemical equipment and methods, and applications, can solve problems such as the lack of non-coding miRNA, and achieve the effect of simple operation and shortening of the cycle.

Inactive Publication Date: 2017-06-13
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there is no report on the overexpression technology of wheat endogenous small non-coding miRNA

Method used

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  • Recombinant barley stripe mosaic virus-mediated wheat target miRNA overexpression method
  • Recombinant barley stripe mosaic virus-mediated wheat target miRNA overexpression method
  • Recombinant barley stripe mosaic virus-mediated wheat target miRNA overexpression method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The applicant adopts the recombinant virus BSMV carrying the artificial miRNA precursor sequence (Pre-amiR-PDS) fragment to verify the method of the present invention, and the specific method is:

[0040] First, the applicant constructed the gamma of recombinant BSMV (BSMV: amiR-PDS) using PCR methods well known in the art pre-amiR-PDS cDNA;

[0041] Secondly, α, β, γ of BSMV are transcribed with the in vitro transcription method known in the art pre-amiR-PDS cDNA obtained α, β, γ PDS RNA;

[0042] Then, take α, β, γ pre-amiR-PDS Each 300ng of RNA was mixed with 25 μl of FES Buffer (containing 1% by weight of glycine, 0.75% by weight of K 2 HPO 4 , 1% by weight of diatomite, 1% by weight of bentonite, 1% by weight of Na 3 PO 4 .10H 2 O) Mix well between thumb and index finger tip;

[0043] Finally, rub the wheat ears up and down gently for 3-5 times when the seedlings at the 3-5 leaf stage fully unfold the new leaves or at the flowering stage.

[0044] Afte...

Embodiment 2

[0049] The applicant constructed a recombinant γ that carries the sequence fragment of the wheat endogenous miR56 precursor pre-miR156 pre- miR156 cDNA. The build method is as follows:

[0050] The applicant designed upstream primers and downstream primers based on the wheat miR56 primary transcript pri-miR156 (Genbank accession number CL902915) and pre-miR56 sequence ((Scoles et al., 2008) known in the art, wherein:

[0051] Upstream primer: 5'-CGCTTCGGCTCCTCTCTCTCT-3';

[0052] Downstream primer: 5'-CGGACAAGATAGGGCTGAGGT-3';

[0053] Genomic DNA of wheat was extracted by the CTAB method known in the art, and the sequence fragment of the miR56 precursor (pre-miR156) was obtained by the PCR amplification method known in the art using the genomic DNA as a template.

[0054] The fragment was connected to the linearized BSMV gamma cDNA plasmid (Ma et al., 2012) to obtain gamma pre-miR156 cDNA. α, β, γ of BSMV are transcribed in vitro by methods well known in the art pre-mi...

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Abstract

The invention discloses a recombinant barley stripe mosaic virus (BSMV)-mediated wheat target miRNA overexpression method. The overexpression of wheat endogenous miRNA can be realized mainly by performing balanced mixing the alpha, beta, gama cDNA in-vivo transcripts of target miRNA precursor segment-carried recombinant BSMV and then inoculating fully developed new leaves of wheat seedlings in three-leaf to five-leaf stage or wheatear parts in blooming stage. Compared with the existing transgene-based wheat miRNA overexpression technical method, the recombinant barley stripe mosaic virus-mediated wheat target miRNA overexpression method has the characteristics of being simple, rapid, accurate, and good in repeatability.

Description

technical field [0001] The invention relates to a method for overexpressing wheat endogenous target miRNA mediated by recombinant barley streak mosaic virus (BSMV), which can be used to study the biological role of wheat endogenous miRNA in wheat growth and development or in response to adversity . Background technique [0002] In recent years, a large number of studies have found that miRNA in organisms is a type of endogenous small non-coding RNA with a length of 18-24 nucleotides. It is transcribed and processed by endogenous genes with a stem-loop structure. produced from long single-stranded RNA precursors (pre-miRNA) (Chen 2009; Bartel 2004). Plant endogenous miRNA can bind to mRNA complementary to its sequence, and inhibit the expression of target mRNA by cutting the sequence or inhibiting the translation of the sequence. Response to adversity (Chen 2004; Sunkar et al. 2006; Xue et al. 2009; Xin et al. 2010; Kantar et al. 2011; Wang et al. 2011). The first miRNA in...

Claims

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Application Information

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IPC IPC(8): C12N15/83A01H5/00
Inventor 赵惠贤简超韩冉
Owner NORTHWEST A & F UNIV
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