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Application of recombinant protein harpinzδ89-124

A recombinant protein and anti-bacterial blight technology, applied in the field of genetic engineering, can solve the problems of lack of research on plant growth promotion and disease resistance, and achieve the effects of easy in vitro expression, cost saving, and good biological effects

Active Publication Date: 2016-03-16
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In Pseudomonas syringae, the functional domain of the bean pathogenic variety P. The research is limited to the study of physical and chemical properties, such as peptide binding sites, plasma membrane lipid binding sites and the formation of holes, etc., lack of research on plant growth promotion and disease resistance

Method used

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  • Application of recombinant protein harpinzδ89-124
  • Application of recombinant protein harpinzδ89-124
  • Application of recombinant protein harpinzδ89-124

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Implementation Example 1: hrpZ PsgS1 Design, cloning and sequencing of gene recombination fragments

[0041] According to the hrpZ sequence with the accession number L41862 in Genbank, a complete open reading frame (ORF) primer was designed, and BamH1 and EcoR1 restriction sites were introduced at the 5' end and 3' end, respectively;

[0042] wxya PsgS1 -P1: 5'-GG GGATCC ATGCAGAGTCTCAGTCTTAAC-3' (SEQ ID No. 1);

[0043] wxya PsgS1 -P2: 5'-GG GAATTC TCAGGCAGCAGCCTGGTTG-3' (SEQ ID No. 2).

[0044] Using the genomic DNA of the S1 strain (CGMCCNo.6699, the same below) as a template, use the above primers to amplify hrpZ by PCR PsgS1 Full-length gene sequence, reaction conditions: pre-denaturation at 94°C for 4 minutes, denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 90 seconds, 35 cycles, and extension at 72°C for 10 minutes. The PCR product was connected to the pMD18-T vector, transformed into Escherichia coli DH5α, and pos...

Embodiment 2

[0049] Example 2: HarpinZ Δ89-124 Expression and purification of recombinant proteins

[0050] 2.1hrpZ Δ89-124 Construction of Genetic Engineering Expression Strains

[0051] Correctly sequenced positive recombinant strain DH5α / pMD18-T-hrpZ Δ89-124 Plasmid DNA was extracted, digested by BamH1 and EcoR1, and connected to the expression vector pET30a(+), and then transformed into Escherichia coli BL21 by plasmid PCR ( figure 2 A) and digestion ( figure 2 B) Positive strains were identified and sequenced correctly. Simultaneously DH5α / pMD18-T-hrpZ PsgS1 Connect pET30a(+) in the same way, transform Escherichia coli BL21, as a full-length control, and name the two strains BL21 / pET30a(+)-hrpZ respectively Δ89-124 , BL21 / pET30a(+)-hrpZ PsgS1 .

[0052] 2.2 HarpinZ Δ89-124 Expression of recombinant protein

[0053] (1) IPTG induced recombinant protein expression: recombinant strain BL21 / pET30a(+)-hrpZ Δ89-124 , BL21 / pET30a(+)-hrpZ PsgS1 Inoculate in LB liquid medium (km5...

Embodiment 3

[0057] Example 3: HarpinZ Δ89-124 Application of Recombinant Proteins in Plants

[0058] 3.1 HarpinZ Δ89-124 Recombinant protein enhances tobacco-induced HR response

[0059] Detection of Recombinant Protein HarpinZ in Tobacco Δ89-124 The ability to induce HR while HarpinZ PsgS1 Compared with water treatment, inject 200μl into the intercellular space on the back of tobacco leaves with a headless syringe, and the concentration of pure protein used is 10nmol / ml, 7nmol / ml, 2.5nmol / ml, 1.5nmol / ml, 1.2nmol / ml, 0.5nmol / ml, observe the development of leaf HR after 24h.

[0060] The results showed that the recombinant protein HarpinZ Δ89-124 with HarpinZ PsgS1 In contrast, the concentration that induces HR response is lower, as low as 0.5nm / ml, indicating that HarpinZ Δ89-124 The ability to induce HR is higher than that of full-length HarpinZ PsgS1 ( Figure 4 ).

[0061] 3.2 HarpinZ Δ89-124 Recombinant protein effectively improves tobacco resistance to mosaic virus diseas...

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Abstract

The invention discloses an application of recombinant protein HarpinZ[delta]89-124 and provides the functional fragment HarpinZ[delta]89-124 of HarpinZPsgS1 protein. The protein functional fragment can be stably expressed in bodies of escherichia coli and is better in biological effect on plants than the HarpinZPsgS1 protein in overall length. After treatments on tobacco and rice by the two proteins in the same concentration, a tobacco anti-mosaic virus effect, a growth-promoting effect on roots of the rice, and a rice anti-bacterial blight effect of the HarpinZ[delta]89-124 are all significantly higher than those of the HarpinZPsgS1 protein in overall length. The recombinant protein HarpinZ[delta]89-124 can be applied in enhancements of the anti-mosaic virus effect of the tobacco and the anti-bacterial blight effect of the rice and in promotion of growth of the rice.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to recombinant protein HarpinZ Δ89-124 Applications. Background technique [0002] Harpin protein is a kind of protein elicitor produced by plant pathogenic bacteria, which can stimulate non-host plants to produce allergic cell death (hypersensitive cell death, HCD), induce plant defense response, and promote plant growth. These beneficial effects of Harpin protein may It is realized through different functional domains of the protein, so it is very important to isolate and identify the functional regions in the Harpin protein that are related to disease resistance and growth promotion. [0003] In Pseudomonas syringae, the functional domain of the bean pathogenic variety P. The research is limited to the physical and chemical properties, such as peptide binding sites, plasma membrane lipid binding sites and the formation of holes, etc., lack of research on plant growth promotion a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N47/44A01P21/00A01P1/00A01P3/00C07K14/21C12N15/70
Inventor 高学文张阳李响伍辉军
Owner NANJING AGRICULTURAL UNIVERSITY