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Inducible promoter for pathogenic bacteria of rice

A technology of promoters and pathogenic bacteria, applied in the field of plant genetic engineering, can solve problems such as quality decline and yield reduction

Inactive Publication Date: 2013-05-01
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Rice is one of the most important food crops. Fungal diseases and bacterial diseases of rice will reduce yield and quality

Method used

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  • Inducible promoter for pathogenic bacteria of rice
  • Inducible promoter for pathogenic bacteria of rice
  • Inducible promoter for pathogenic bacteria of rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Cloning and sequence analysis of rice 2H16 gene promoter p2H16

[0033] Primers were designed according to the upstream 2300bp sequence of rice Nipponbare 2H16 gene (GENBANK accession number: AK058338) (5ˊ-CAGACGGCTACTGTCCATCA-3ˊ, whose sequence is shown in SEQ ID NO.2; 5ˊ-GCGAGAGCAGGAGGAGAGAC-3ˊ, whose sequence is shown in SEQ ID NO.3 shown) was used to obtain the promoter of the gene.

[0034] Genomic DNA of rice IRBB13 was extracted and used as a template for PCR amplification. The reaction procedure was as follows: pre-deformation at 94°C for 5 min, denaturation at 94°C for 40 s, annealing at 54°C for 40 s, extension at 72°C for 2 min, reaction for 35 cycles, post-extension at 72°C for 7 min, After the end, the amplified fragment was recovered and purified with the DNA recovery kit of Kangwei Company, and then the purified DNA fragment was connected to the vector pGEM-T (Promega Company), incubated at room temperature for 1 hour, and transformed into E.col...

Embodiment 2

[0042] Example 2 Constructing the recombinant vector of 2H16 promoter sequence and GFP gene and transforming rice Zhonghua 11

[0043] Using rice IRBB13 DNA as a template, using a forward primer, its sequence is shown in SEQ ID NO.4 (5′-ATG GTC GAC CAGACGGCTACTGTCCATCA-3′, the underlined base is the restriction endonuclease SalI recognition site) and the reverse primer, its sequence is shown in SEQ ID NO.5 (5′-ATG CTGCAG .GCGAGAGCAGGAGGAGAGAC-3ˊ, the underlined base is the restriction endonuclease PstI recognition site) to amplify the promoter fragment, the PCR reaction procedure is as follows: pre-denaturation at 94°C for 5min, denaturation at 94°C for 40s, annealing at 62°C for 40s, extension at 72°C 2min, react for 35 cycles, and then extend at 72°C for 7min to obtain the -2197bp-+60bp region of the sequence SEQ ID NO.1, and add recognition sites for restriction endonucleases SalI and PstI at both ends of the sequence. After the reaction, the PCR product was detected by ...

Embodiment 3

[0045] Example 3 Detection of induced responses of p2H16 transgenic plants to pathogenic bacteria, salt and plant hormones

[0046] The transgenic seedlings of the T1 generation rice Zhonghua 11 grown in the greenhouse for about 3 weeks were subjected to various treatments. Xanthomonas PXO99, PXO61, the pathogen of bacterial blight of rice, RS105 of rice bacterial spot, YWK-196 of corn sheath blight, NaCl and three plant hormones of salicylic acid, gibberellic acid and abscisic acid were used respectively. Treat rice seedlings, and set untreated as control. The intensity of GFP activity before and after different treatments of different transgenic lines was detected. Among them, bacterial blight and thin streak bacteria were cultured on PSA medium at 28°C for 2 days, resuspended in sterile water, and inoculated by injection. One day after inoculation of pathogenic bacteria, the leaves of plants were taken for analysis; Cultivate on PDB medium at 25°C for 2 days, inoculate wi...

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Abstract

The invention relates to the technical field of plant gene engineering and provides an inducible promoter for pathogenic bacteria of rice. The promoter can be activated by rice bacterial leaf blight bacterium xanthomonas PXO99 and PXO61, a rice baeterial leaf streak bacterium RS105 and a corn banded sclerotial blight bacterium YWK-196, and can also be activated by abscisic acid, gibberellic acid, salicylic acid and sodium chloride. Truncation of the promoter verifies that two sections from -513bp to -411bp and from -411bp to -309bp are critical areas induced by the pathogenic bacteria. Various plant expression vectors can be constructed with the adoption of the promoter, and the promoter can be used for improving plant quality and other beneficial production traits through a plant gene engineering technology.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, and provides a rice pathogen-induced promoter, which can be used to improve plant disease resistance and other beneficial production traits through plant genetic engineering technology. Background technique [0002] Plant diseases caused by fungi, bacteria, viruses and nematodes have a huge impact on crop production. Plants have two different resistance mechanisms: one is the passive defense mechanism, which refers to some resistance-related morphological structures, such as epidermis and hard cell wall (Brady JD, Fry S. Formation of di-isodityrosine and loss of isodityrosine in the cell walls of tomato cell-suspension cultures treated with fungal eli citors or H 2 o 2 . Plant Physiol 1997, 115:87-92.). The other is active defense, which is caused by the recognition and interaction between resistance genes and pathogenic bacteria. The elicitor encoded by the pathogen avirule...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A01H5/00
Inventor 储昭辉丁新华李宁
Owner SHANDONG AGRICULTURAL UNIVERSITY
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