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Rice MIR528 gene promoter and application thereof

A promoter and gene technology, applied in the field of genetic engineering and molecular biology, can solve the problem of unclear molecular mechanism of MIR528 gene

Inactive Publication Date: 2017-02-22
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the molecular mechanism of the transcriptional regulation of the MIR528 gene itself is still unclear

Method used

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  • Rice MIR528 gene promoter and application thereof
  • Rice MIR528 gene promoter and application thereof
  • Rice MIR528 gene promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Cloning of rice MIR528 gene promoter

[0025] 1.1 Experimental method

[0026] 1.1.1 Prediction of MIR528 gene promoter

[0027] According to the rice genome annotation information in the MSU v7 database, a PERL program was written to extract the 2Kb nucleotide sequence upstream of the MIR528 precursor sequence. Use Promoter v2.0, TSSP and other online promoter prediction tools to analyze the corresponding nucleotide fragments to determine whether they are reliable promoter sequences.

[0028] 1.1.2 Cloning and bioinformatics analysis of MIR528 gene promoter

[0029] Rice genomic DNA was extracted by CTAB method. Design specific primers according to the predicted MIR528 promoter sequence (Forward: 5'-TCA TCT CAA ACT GTC ATA GAC C-3', Reverse: 5'-GTC GCT GCT ACA GCCAAA AAG-3'), and use genomic DNA as a template PCR amplification and sequencing verification of the amplified products. The cis-acting elements of the promoters were predicted and analyzed u...

Embodiment 2

[0037] Example 2: Construction and genetic transformation of rice MIR528 gene promoter and segmental deletion promoter recombinant vector

[0038] 2.1 Experimental method

[0039] 2.1.1 Construction of MIR528 full-length promoter and segmental deletion promoter recombinant vector

[0040] According to the type, quantity and distribution characteristics of the cis-acting elements of the promoter sequence predicted by PLACE, it was divided into four promoter fragments of different lengths ( figure 2 ): (1) Q528Q: full-length promoter sequence (1751bp); (2) Q528B: from the 1st to the 900th base at the 5' end of the promoter (900bp); (3) Q528C: from the 5' end of the promoter (4) Q528D: from the 901st to 1751th base (851bp) at the 5' end of the promoter. Design sequence-specific primers for Q528B(Forward:5'-TCA TCT CAA ACT GTC ATA GAC C-3', Reverse:5'-ATA CAA TAA ATGTGA GCA TTA CAG-3'), Q528C(Forward:5'-CTT TGA TGACAT TTT CAA CAT TAC-3', Reverse:5'-GTC GCT GCT ACAGCC AAAAAG-3'...

Embodiment 3

[0045] Embodiment 3: The rice MIR528 gene promoter drives the expression of the downstream GUS gene

[0046] 3.1 Experimental method

[0047] 3.1.1 GUS histochemical staining of transgenic plants

[0048] GUS histochemical staining was performed on the roots, stems and leaves of wild-type control (WT) and transgenic plants: put the corresponding test materials in X-GLUC staining solution, and place them overnight at 37°C. The stained chlorophyll-containing tissue was decolorized with 75% alcohol, and when the negative control was white, the stained tissue was photographed using a stereomicroscope.

[0049] 3.1.2 Quantitative expression analysis of GUS gene

[0050] Total RNA was extracted from 21-day-old WT and transgenic seedling roots, stems, and leaves by the Trizol method; the extracted total RNA was reverse-transcribed into cDNA using the reverse transcription kit ReverTra Ace qPCR RT Kit, and then diluted 10 times later used as a template. Quantitative RT-PCR amplifi...

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Abstract

The invention provides a rice MIR528 gene promoter and application thereof. The promoter is separated from japonica rice nipponbare, and has a sequence containing multiple adversity stress response and plant growth and development related controlling elements. Transgene proves that the promoter is a broad spectrum expression promoter; the expression activity of the promoter in rice stems is superior to that of a 35S promoter under normal conditions; in addition, the promoter can be remarkably induction expressed by arsenite in a rice root system and leaves. The invention utilizes a transgene technology to prove a biological function of the rice MIR528 gene promoter participating in response adversity stress, provides a new efficient, broad-spectrum and environmental induction type promoter element for plant stress resistance gene engineering research, and lays a foundation for further analysis of a plant response adversity stress molecular mechanism and stress resistance breeding application.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering, in particular to a rice MIR528 gene promoter and application thereof. Background technique [0002] In nature, due to the immobility of plants, they are more vulnerable to external stress factors than animals. However, plants can trigger or initiate their own defense response mechanisms to cope with various stresses by regulating the ordered spatiotemporal expression of different types of genes in their genomes. A large number of studies have proved that the promoter plays a central role in the regulation of plant gene expression, which determines the direction and efficiency of gene transcription. [0003] miRNA is a kind of non-coding small RNA with a length of about 20-24bp produced by organisms endogenously, which regulates the expression of target genes at the post-transcriptional level through complementary sequence pairing. Studies have found that one m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N1/21A01H5/00
CPCC07K14/415C12N15/8273
Inventor 刘庆坡胡海超张恒木
Owner ZHEJIANG FORESTRY UNIVERSITY