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Isolation of rice brown planthopper damage-inducible promoter region and identification of its expression pattern

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

Inactive Publication Date: 2018-05-25
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 of rice brown planthopper damage-inducible promoter region and identification of its expression pattern
  • Isolation of rice brown planthopper damage-inducible promoter region and identification of its expression pattern
  • Isolation of rice brown planthopper damage-inducible promoter region and identification of its expression pattern

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) Signal Scan analysis tool (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 -540 bp upstream of the Os01g73940 promoter was a TATA box, and -552 bp 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 Os01g73940 gene promoter region

[0021] Os01g73940 gene promoter specific PCR primer P TG3 -F and P TG3 ‐R (primer sequence is shown in Table 1 or SEQID NO: 2 and 3) to amplify 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 a 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 involved in ...

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 invention belongs to the technical field of plant genetic engineering. In particular, it relates to the isolation and expression pattern identification of a brown planthopper damage-inducible rice promoter. A gene specifically expressed in rice green tissue and up-regulated by brown planthopper damage was obtained through screening. The promoter region of the gene was isolated from the rice genome by PCR, and the promoter was named PTG3, and its nucleotide sequence is shown in SEQ ID NO:1. The fusion expression gene was constructed by using PTG3 and GUS reporter gene, and the rice Zhonghua 11 was transformed by Agrobacterium-mediated method. GUS histochemical staining and quantitative RT‑PCR detection of the obtained transgenic rice seedlings before and after inoculation with BPH nymphs confirmed that the PTG3 promoter can significantly increase the expression level of downstream genes under the condition of BPH infestation. It has potential application value in 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 Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/46
Inventor 陈浩关丽梅
Owner HUAZHONG AGRI UNIV