Method for screening rice tissue specific expression type cis-acting element and flanking sequence thereof

A cis-acting element and tissue-specific technology, which is applied in the field of screening for the specific expression of cis-acting elements and their flanking sequences in rice tissues, can solve the problems of no relevant reports and small quantities

Active Publication Date: 2017-04-19
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The important reason is that the number of tissue-specific expression-related cis-elements that have been identified and have clear funct

Method used

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  • Method for screening rice tissue specific expression type cis-acting element and flanking sequence thereof
  • Method for screening rice tissue specific expression type cis-acting element and flanking sequence thereof
  • Method for screening rice tissue specific expression type cis-acting element and flanking sequence thereof

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Embodiment 1

[0036]Example 1: Screening of candidate elements related to tissue-specific expression

[0037] The information on genes specifically expressed in different tissues of rice was obtained from CREP (Collection of Rice Expression Profiles, http: / / crep.ncpgr.cn), a microarray database of rice expression profiles during the whole growth period. According to the expression pattern, all tissue-specific expression genes were divided into 3 categories: green tissue-specific expression genes (genes specifically expressed in one or more green tissues, such as: seedlings, leaves, leaf sheaths, glumes, and stems), endosperm tissue Specifically expressed genes and remaining tissue-specific expressed genes. The 2000bp sequence upstream of the gene was set as the gene promoter, and the promoters of these three types of genes were named: green tissue-specific expression promoter (GSP), endosperm-specific expression promoter (ESP) and remaining tissue-specific expression promoter (RSP). Based...

Embodiment 2

[0040] Example 2: Prediction of flanking sequences of cis-acting elements

[0041] According to the scanning results of cis-acting elements in the green tissue-specific expression promoter (GSP) and the endosperm-specific expression promoter (ESP), the flanking sequences required for its function were determined. The flanking sequence of the cis-acting element includes 3 bases respectively upstream and downstream. In the present invention, the selection principle of flanking sequence bases is based on: the frequency of occurrence in GSP is higher than that of the same site in ESP, and the frequency of occurrence in GSP is higher than 25% of the bases. The flanking sequence prediction results are as follows figure 2 shown.

Embodiment 3

[0042] Embodiment 3: Construction of plant expression vector

[0043] Unless otherwise specified, the reference method of the present invention and the corresponding routine operation of molecular biology refer to: J. Sambrook et al., "Molecular Cloning Experiment Guide (Second Edition)" (Chinese translation), Science Press, 1996 edition.

[0044] In the present invention, bioinformatics prediction is first performed on the flanking sequence of GEAT. The combination of GEAT and flanking sequence (GEATFLK, sequence shown in SEQ ID NO: 2) was repeated four times and connected downstream -46Minimal 35S promoter (named GEATFLK_MINI, structure as shown in image 3 shown), and sent to Nanjing GenScript Biotechnology Co., Ltd. for synthesis. Then GEATFLK_MINI was connected into the promoter function analysis vector pDX2181 through the Hind III and BamH I endonuclease sites respectively (for information on the vector map and multiple cloning sites, see Figure 4 ), to obtain the rec...

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Abstract

The invention belongs to the technical field of plant genetic engineering and specifically relates to a method for screening a rice tissue specific expression type cis-acting element and a flanking sequence thereof. According to the information in a rice whole-growth-period expression profile chip database, all tissue specific expression genes are classified according to expression patterns. According to the information in a plant cis-acting element database, cis-acting elements in the promoter regions of different rice tissue specific expression genes are scanned and counted. After that, cis-acting elements in the promoter regions of green tissue specific expression genes, which occur at a high frequency, are selected as candidate elements related to green tissue specific expression. Meanwhile, the flanking sequence of one candidate element GEAT is subjected to bioinformatic prediction. The combination of the above element and the flanking sequence thereof is integrated with -46 Minimal 35S promoter, so that the gene expression of a GUS gene is driven for function identification. The GUS analysis shows that, the GUS gene is associated with the green tissue specific expression. Through the site-directed mutagenesis experiment of the flanking sequence, a basic group having a critical role in maintaining the function of GEAT is found.

Description

technical field [0001] The invention belongs to the technical field of rice genetic engineering. It specifically relates to a method for screening rice tissue specifically expressing cis-acting elements and their flanking sequences. Background technique [0002] Promoter is a DNA sequence that regulates gene transcription. It is the most critical factor in the regulation of gene expression and can precisely regulate the expression pattern and abundance of target genes (Cai, M., Wei, J., Li, X. , Xu, C. and Wang, S.A rice promoter containing both novel positive and negative cis-elements for regulation of greenissue-specific gene expression in transgenic plants. Plant Biotechnol. J. 2007, 5, 664-674). With the deepening of research, people have found different types of natural promoters in the genome, such as: constitutive promoters, space-time specific promoters, inducible promoters, etc. The expression pattern and expression intensity of the promoter are mainly determined ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/82C12N15/113C12Q1/68
Inventor 林拥军王睿
Owner HUAZHONG AGRI UNIV
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