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Isolation and expression pattern identification of brown planthopper infestation inducible rice promoter zone

A promoter and brown planthopper technology, applied in the field of plant genetic engineering, can solve the problem of reduced transcription efficiency

Inactive Publication Date: 2016-08-17
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Once the base of this box is deleted or mutated, the transcription efficiency will be drastically reduced, and the CAAT box may control the frequency of transcription initiation

Method used

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  • Isolation and expression pattern identification of brown planthopper infestation inducible rice promoter zone
  • Isolation and expression pattern identification of brown planthopper infestation inducible rice promoter zone
  • Isolation and expression pattern identification of brown planthopper infestation inducible rice promoter zone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: P TG3 Structural analysis of promoters

[0018] Using promoter analysis software TSSP (http: / / www.softberry.com), PROSCAN (http: / / bimas.dcrt.nih.gov / molbio / proscan), PLACE (A Database of Plant Cis-acting Regulatory DNA Elements) The Signal Scan analysis tool in (http: / / www.dna.affrc.go.jp / PLACE / signalscan.html), TESS ( http: / / searchlauncher.bcm.tmc.edu / seq -search / gene-search.html), Match 1.0 ( http: / / www.gene-regulation.com / cgi-bin / pub / programs / match / bin / match.cgi) and AliBata2.1 ( http: / / www.gene-regulation.com / pub / programs / alibata2 / index.html ) etc. analyzed the structure of the Os01g73940 gene promoter. It was found that at -540bp upstream of the Os01g73940 promoter was a TATA box, and -552bp was a CAAT box related to transcription frequency.

Embodiment 2

[0019] Example 2: Os01g73940 gene promoter P TG3 Analysis of Inducible Functional Regions of Nilaparvata lugens

[0020] 1. Isolation of the Os01g73940 gene promoter region

[0021] Os01g73940 gene promoter specific PCR primer P TG3 -F and P TG3 -R (the primer sequence is shown in Table 1 or SEQ ID NO: 2 and 3) amplifies the complete DNA fragment P of the Os01g73940 gene promoter TG3 , the sequence length is 1953 bases, and the nucleotide sequence is shown in SEQ ID NO: 1 in the sequence table. PCR primer P TG3 -F contains restriction endonuclease Hind III site, primer P TG3 -R contains a site for the restriction enzyme PstI. The DNA fragment obtained by PCR amplification was cloned into pEASY-T 3 vector vector (Transgene, image 3 ). The cloning of the promoter fragment was verified by sequencing using SP6 and T7 universal primers (Shanghai Sangon Bioengineering Co., Ltd.) and the sequencing kit (Big Dye Kit) from Applied Biosystems, USA.

[0022] The primers involv...

Embodiment 3

[0029] Embodiment 3: Utilize transgenic plant to detect promoter P TG3 expression pattern

[0030] 1. Positive detection of transgenic plants

[0031] The transgenic rice T 0 The seeds of generation positive plants were germinated on the 1 / 2MS medium containing 50mg / L hygromycin to screen positive T 1 Substitute plants. The specific screening process is as follows

[0032] 1) Prepare 1 / 2 MS medium and add hygromycin so that the final concentration of hygromycin is 50 mg / L.

[0033] 2) Shell the rice seeds that need to be identified, and carry out disinfection treatment on the aseptic workbench, the steps are as follows: 75% alcohol cleaning for 1 minute, 0.15% HgCl 2 Soak for 20 minutes, then wash several times with sterilized water to remove residual HgCl 2 .

[0034] 3) Move the sterilized seeds to 1 / 2MS containing hygromycin for germination, and observe after one week. If the germination is normal, it is a positive seed, and if it does not germinate, it is a negative...

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Abstract

The present invention belongs to the technical field of plant genetic engineering, and in particular to isolation and expression pattern identification of a brown planthopper infestation inducible rice promoter. The invention is as below: screening to obtain a gene with specific expression in rice green tissue and expression level in up-regulation by the brown planthopper infestation; isolating a promoter zone of the gene from rice genome by a PCR method, naming the promoter as PTG3 with a nucleotide sequence shown as SEQ ID NO: 1; constructing a fusion expression gene by using PTG3 with a GUS reporter gene, and transforming a rice Zhonghua 11 by an Agrobacterium-mediated method; and conducting GUS tissue chemical staining and quantitative RT-PCR detection on the obtained transgenic rice seedling before and after inoculation of brown planthopper. The invention determines that PTG3 promoter can significantly increase the expression level of the downstream gene under the brown planthopper infestation conditions, and the characteristic has potential application value in rice insect-resistant gene engineering breeding.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. It specifically relates to the isolation and expression pattern identification of rice brown planthopper damage-inducible promoter regions. The brown planthopper damage-inducible promoter region is a DNA fragment, which can specifically enhance the expression level of the gene under the feeding induction condition of the brown planthopper. Background technique [0002] During the growth process, plants will be attacked by various pests such as brown planthopper, stem borer and armyworm. The brown planthopper is one of the main pests of rice. It may cause two consequences by sucking the rice: (1) the brown planthopper sucks the phloem juice of the rice stem and leaf tissue, resulting in a large loss of water in the rice and causing the rice plant to fall down. At the same time, the brown planthopper can also As an intermediate host to spread virus diseases, rice yields will be ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 陈浩关丽梅
Owner HUAZHONG AGRI UNIV
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