Pathogeny evoked promoter POsDR3 and its application in improved rice resistance

A promoter, rice technology, applied in the field of plant genetic engineering, can solve problems such as reduced transcription efficiency, and achieve the effect of avoiding adverse effects

Inactive Publication Date: 2007-10-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally located around -75bp, 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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  • Pathogeny evoked promoter POsDR3 and its application in improved rice resistance
  • Pathogeny evoked promoter POsDR3 and its application in improved rice resistance
  • Pathogeny evoked promoter POsDR3 and its application in improved rice resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Isolation of clone P OsDR3 Promoter

[0025] 1. Isolate clones containing P from disease-resistant rice variety Minghui 63 OsDR3 promoter cloning

[0026]The present invention uses the cDNA clone EI12I1 as a probe, and screens a positive BAC clone 7G7 from the Minghui 63 BAC library (Peng et al. 1998). The BAC clone 7G7 was digested with the restriction endonuclease HindIII, and the digested fragment was connected to the HindIII-treated pUC19 vector (purchased from Amersham Biosciences, USA), and electrotransformed (Sambrook and Russell, 2001) Escherichia coli DH10B (purchased from Invitrogen, USA) ), to prepare subclones. The subclones were amplified and detected by PCR primers EI12I1F (5'-CAGTAGCTCCAAGGGGTGTC-3') and EI12I1R (5'-TTAAAGTTGGGGGTTCCCATTC-3') designed with the sequence of cDNA clone EI12I1, and a positive subclone 7G7H14 containing the target promoter fragment was obtained ; The foreign insert of this subclone is about 3.6kb in length.

[...

Embodiment 2

[0034] Embodiment two: P OsDR3 Functional verification of promoters

[0035] 1. Construction of genetic transformation vector

[0036] The vector used was pCAMBIA1381. The pCAMBIA1381 vector is a series vector of the commonly used Agrobacterium planta-mediated genetic transformation vector pCAMBIA1301 (Sun et al., 2004). This vector carries a promoter-less reporter gene GUS (beta-glucuronidase gene) (Fig. 3). The pCAMBIA1381 vector was kindly provided by CAMBIA Laboratory (Center for the Application of Molecular Biology to International Agriculture) in Australia.

[0037] Genetic transformation vectors were constructed using conventional recombinant plasmid construction methods (Sambrook and Russell, 2001). The main steps are: P will be obtained from subclone 7G7H14 OsDR3 Use T4 DNA Polymerase to smoothen the ends of the enzyme-digested fragments of the promoter; at the same time, digest the genetic transformation vector pCAMBIA1381 with the blunt-ended restriction endonu...

Embodiment 3

[0041] Embodiment three: P OsDR3 Promoter application

[0042] In order to test P OsDR3 The application value of the promoter in rice disease resistance, the present invention uses the reporter gene GUS in the aforementioned pCAMBIA1381 carrier to use rice bacterial blight resistance gene Xa26 (Sun et al., 2004; Patent No. ZL02139212.9, rice bacterial blight resistance gene Xa26(t); Inventors: Wang Shiping, Sun Xinli, Cao Yinglong, Yang Zhifen, Zhang Qifa; Authorized Announcement Date: 2005.5.18) instead, adopt the method described in Example 2 to convert P OsDR3 The promoter was ligated with the pCAMBIA1381 vector carrying the Xa26 gene. The obtained recombinant plasmid was named 12IPMKb (Fig. 6). The 12IPMKb plasmid was electrotransformed (Sambrook and Russell, 2001) into Agrobacterium strain EHA105 (Sun et al., 2004).

[0043] The 12IPMKb plasmid carried by the EHA105 strain was introduced into the susceptible rice variety Zhonghua 11 (Oryza sativa ssp.japonica) by the ...

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Abstract

The invention discloses a DNA fragment separating colony of rice etiological agent special induced promoter POsDR3, functional checking and appliance in plant gene engineering technical domain, which is characterized by the following: generating specific answer reaction for invading rice of pathogenic bacteria; developing answer reaction for inducement of diverse hormones (abscisic acid, ethene, activol) and chemical signal molecule (jasmine acid, salicylic acid); quick-intensifying expression of POsDR3 promoter controlling gene with pathogenic bacteria invasion and chemical signal molecular treatment. The POsDR3 promoter can intensify resistance of rice, which does not make rice generate excess protein.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. It specifically relates to the isolation and cloning, functional verification and application of a rice DNA fragment. The DNA fragment contains a rice disease resistance-related gene promoter, which can specifically drive gene expression under the induction of pathogens; it can also specifically drive gene expression under the induction of plant hormones and chemical signaling molecules . After the fragment is fused with the disease-resistant gene, it is transformed into rice, and the genetically transformed plant can specifically express the disease-resistant gene under the induction of the pathogen, and its disease resistance is enhanced. Background technique [0002] During the growth of plants, they will be attacked by various pathogens such as viruses, bacteria, molds and nematodes. Pathogens invading plants lead to two results: (1) the pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82
Inventor 王石平蔡萌曹应龙
Owner HUAZHONG AGRI UNIV
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