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Rice histone deacetylases gene HDT701 promoter and application thereof

A deacetylase and histone technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as restricting the promotion and development of transgenic crops, achieve good application prospects, increase local expression, and avoid unnecessary waste. Effect

Inactive Publication Date: 2012-02-29
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In recent years, with the development of modern genetic engineering technology, many genetically modified plants including many food crops have been obtained, but food safety issues have restricted the promotion and development of genetically modified crops

Method used

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  • Rice histone deacetylases gene HDT701 promoter and application thereof
  • Rice histone deacetylases gene HDT701 promoter and application thereof
  • Rice histone deacetylases gene HDT701 promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Promoter of OSHDT701 Gene: Cloning of HDT701 Promoter

[0033] 1. The japonica rice variety Nipponbare was used as the experimental material, and the genomic DNA was extracted by the CTAB method as a template.

[0034] 2. According to the sequence of the known rice gene OSHDT701 (Genebank accession number is AK072845) on NCBI, primers were designed 2 KB before the start codon, and PCR amplification was performed using the genomic DNA of the japonica rice variety Nipponbare as a template.

[0035] Upstream primer: F: 5'-TGAAGCTTTAAGGCGAATAAGCGAAAC-3'

[0036] Downstream primer: R: 5'-CTCTGCAGGAAGAACCCTAGAAAAGAAA-3'

[0037] The PCR reaction was carried out in a 50ul system, and the reaction parameters were: pre-denaturation at 94°C for 5min; denaturation at 94°C for 30s, annealing at 58.9°C for 45s, extension at 72°C for 2min, and 35 cycles; extension at 72°C for 5min. PCR amplification products were separated by 1.0% agarose gel electrophoresis ( figure 1 ...

Embodiment 2

[0039] Embodiment 2: Construction of HDT701-GUS fusion expression vector

[0040] A large number of plasmids were extracted from positive clones with correct sequencing in Example 1. The extracted plasmid and vector PCAMBIA1391Z (its structure is as follows figure 2 shown) were digested with HindIII and PstI at the same time, the target fragment was recovered and ligated to obtain the expression vector PCAMBIA1391Z-HDT701P with HDT701 as the promoter and its downstream gene as GUS. There is a SpeI restriction site at 700bp of the HDT701P fragment, and there is no SpeI restriction site on PCAMBIA1391Z, so a small fragment of 0.7 KB ( image 3). The positive plasmid was transferred into Agrobacterium EHA105 by freeze-thaw method, and the positive clone transferred into the expression vector PCAMBIA1391Z-HDT701P was identified by colony PCR and used for rice transformation.

Embodiment 3

[0041] Embodiment 3: Agrobacterium tumefaciens transformation rice mature embryo callus

[0042] The genetic transformation method mediated by Agrobacterium EHA105 (Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, 1994, Plant Journal 6: 271-282) will The constructed plant fusion expression vector PCAMBIA1391Z-HDT701P was introduced into the callus of mature embryos of the japonica rice variety Zhonghua 11 to obtain transgenic plants.

[0043] The steps of Agrobacterium-mediated genetic transformation are as follows:

[0044] 1. Callus induction

[0045] Ripe rice seeds are dehulled, treated with ethanol with a volume fraction of 70% for 1 min, and sterilized with a mercuric chloride solution with a mass fraction of 1% for 15 min; the seeds are washed with sterilized water for 3 to 5 times, the first few times for 3-5 min, and the last time for 10 min . The sterilized seeds were in...

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Abstract

The invention discloses a rice histone deacetylases gene HDT701 promoter and an application thereof. The sequence of the promoter is one of the following nucleotide sequences: (a) the nucleotide sequence shown in the sequence table SEQ ID NO.1; (b) the nucleotide sequence which can be hybridized with a DNA sequence of 1) under rigorous conditions and has same function; (c) the nucleotide sequencewhich has more than 90% homology with the DNA sequence of a) or b) and also has the function of the promoter. According to the invention, the promoter of the HDT701 gene is cloned from the rice, wherein the cloned promoter is the rice histone deacetylases gene HDT701 promoter which can promote the expression of downstream gene in some organs of the rice, and the rice histone deacetylases gene HDT701 promoter is a tissue specificity promoter, so that the promoter can be used in the transgenic engineering, the expression products of the target gene can be accumulated at certain organ or tissue so as to increase local expression quantity, and simultaneously, unnecessary waste of plant nutrient can be reduced, consequently, the promoter has good application prospect in the transgenic engineering.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering and rice breeding, and in particular relates to the cloning of the upstream promoter sequence of the rice histone deacetylase gene HDT701 coding region-the rice histone deacetylase gene HDT701 promoter, and the promoter in the Application in transgenic rice. Background technique: [0002] Promoter refers to the region on a DNA molecule that can bind to RNA polymerase and form a transcription initiation complex, usually located upstream of the coding gene. There are a large number of cis-acting elements in the promoter fragment, and transcription factors can regulate the transcription of genes by combining with cis-acting elements, so that the gene has temporal and spatial expression specificity. Therefore, the isolation and functional analysis of promoters is an important part of genetic engineering research. [0003] Promoters commonly used in plant genetic engineering are divided ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N1/21C12N15/84A01H5/00C12R1/01
CPCC12N15/8222
Inventor 段俊张伟赵金会刘夏
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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