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osago18 promoter and its application

A DNA molecule and molecular technology, applied in the field of OsAGO18 promoter, can solve problems such as affecting affinity and affecting gene expression level.

Active Publication Date: 2017-05-17
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It is now known that the initiation of transcription is a key stage of gene expression, and an important issue at this stage is the interaction between RNA polymerase and the promoter: the structure of the promoter affects its affinity with RNA polymerase, thereby affecting level of gene expression

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. It is found that the OsAGO18 promoter can be induced to express by rice stripe virus

[0053] 1. Obtaining the OsAGO18 promoter

[0054] 1. Obtaining rice cDNA template

[0055] According to the instructions, the total RNA of Nipponbare rice (Oryzasativa L.japonica.cv.Nipponbare) was extracted with Invitrogen's TRIzol Reagent, and reverse transcription was performed with the company's SuperScript II reverse transcriptase. The primer used for reverse transcription is a 16-nucleotide Oligod (T) primer. For specific methods, please refer to invitrogen M-MLVReverse Transcriptase (Cat. No. 28025-021). Finally, the rice cDNA template was obtained.

[0056] 2. Obtaining rice OsAGO18 promoter

[0057] The rice cDNA obtained by reverse transcription was used as a template, and the following primers were used for PCR amplification.

[0058] AGO18promoter-HindIII-F: 5’-ATA aagctt TGCAAAACTCTAAGCCACAGAGCT-3' (the underlined part is the recognition sequence of HindIII, and the fol...

Embodiment 2

[0114] Example 2, OsAGO18p: GUS transgenic rice was subjected to GUS staining analysis after RSV infection

[0115] 1. Material acquisition

[0116] Laodelphax striatellus with RSV virus infects the non-transgenic wild-type rice varieties Nipponbare, T 1 Substitution of OsAGO18promoter-GUS rice seedlings and the empty carrier rice obtained in step 2 (experimental group). The non-toxic Laodelphax striatellus infested the corresponding rice as the control group. Two weeks after the infection, the whole rice plant was collected as the material for GUS dyeing.

[0117] 2. GUS staining experiment

[0118] GUS histochemical staining was performed according to the method described by Wang Guanlin and others ("Principles and Techniques of Plant Genetic Engineering", Beijing Science Press, 1998). Combined with the characteristics of the rice itself, a simple optimization was carried out. The specific operations are as follows:

[0119] 1. Use a double-sided blade to cut the whole rice plant i...

Embodiment 3

[0128] Example 3. The OsAGO18 promoter can well start the expression of the target gene in plants

[0129] 1. The construction of a vector for OsAGO18 gene expression initiated by OsAGO18 promoter

[0130] 1. Obtaining rice cDNA template

[0131] According to the instructions, the total RNA of Nipponbare rice (Oryzasativa L.japonica.cv.Nipponbare) was extracted with Invitrogen's TRIzol Reagent, and reverse transcription was performed with the company's SuperScript II reverse transcriptase. The primer used for reverse transcription is a 16-nucleotide Oligod (T) primer. For the specific method, please refer to invitrogen M-MLVReverse Transcriptase (Cat. No. 28025-021). Finally, the rice cDNA template was obtained.

[0132] 2. Obtaining the expression vector of OsAGO18 gene from rice OsAGO18 promoter

[0133] Using the rice cDNA obtained in step 1 as a template, PCR amplification was performed with the following primers.

[0134] AGO18cds-F: 5’-ATAATGGCGAGCCGAGGAGGAGGC-3’ (positions 4-24 ...

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Abstract

The invention discloses an OsAGO18 promoter and an application thereof. The OsAGO18 promoter is any one of the following DNA molecules: 1) a DNA molecule of nucleotide at the 795-3000 site as shown in sequence 1; 2) a DNA molecule as shown in the sequence 1; 3) a DNA molecule which is hybridized with a DNA molecule which is strictly defined in 1) or 2) and has a promoter function; and ) a DNA molecule which has more than 90% of homology with a DNA molecule defined in 1), 2) and 3) and has the promoter function. The experiment shows that the resistance to a plant RSV virus can be recovered after an AGO18 gene recombinant promoted by the OsAGO18 promoter is transferred into an ago18 rice mutant, and the promoter can be normally adjusted and controlled inside rice without changing the endogenous adjusting and control system of rice. The invention provides a novel tool for studying the resistance of rice and other plants in future.

Description

Technical field [0001] The invention belongs to the field of biotechnology and relates to OsAGO18 promoter and its application. Background technique [0002] Rice is one of the main food crops in my country, and its production safety is directly related to the country’s economic lifeline and people’s livelihood. Plant virus diseases are known as “plant cancers”. There is currently no effective control method. It is devastating, especially for rice plants that will face no harvest. At present, rice virus diseases have become one of the important diseases that threaten the safety of my country’s food production in important rice producing areas in my country. According to data from the National Agricultural Technology Center, the areas of major rice virus diseases in the country from 2008 to 2012 were 1,492.3 and 1,746.6 respectively. , 2,604, 1,601.9 and 13.836 million acres, the prevention and control of rice yield 965,512.2, 996,212.5, 1,337,007, 929,870.8 and 714,494.9 tons, but...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63A01H5/00
Inventor 李毅戚益军吴建国杨志蕊郑立佳魏春红
Owner PEKING UNIV
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