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Non-inducing expressing gene engineering strain and structural process and application thereof

A genetically engineered strain and genetic engineering technology, applied in the field of plant disease prevention and control, can solve the problems of lack of homology, unclear effect, and low homology.

Inactive Publication Date: 2006-06-28
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these two proteins have similar properties and functions, there is a lack of homology between the hrpN and hrpZ genes, that is to say, the homology of the harpin coding gene between genera is very low, but the homology within the same genus is very low. High source
[0007] Harpin plays a role in stimulating non-host HR and inducing disease resistance in plant pathogenic bacteria, but its role in the interaction between pathogens and compatible hosts is still unclear

Method used

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  • Non-inducing expressing gene engineering strain and structural process and application thereof
  • Non-inducing expressing gene engineering strain and structural process and application thereof
  • Non-inducing expressing gene engineering strain and structural process and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Embodiment 1PCR amplification Harpin Z gene

[0122] Synthesis of amplification primers: primers were designed according to the ORF of the Harpin Z gene and the vector, and detected by oligo6.0 software. Upstream primer: G GAATTC G ATG CAG AGC TCT AGT CTT AAC (underlined is EcoR I restriction site): downstream primer: G CTCGAG TCA GGC CAC AGC CTGGTT AGT C (the Xho I restriction enzyme site is underlined). The above primers were synthesized by Shanghai Sangong. Using the plasmid containing the hrpZ gene stored in our laboratory as a template, the 1.1kb hrpZ gene fragment was amplified using a PCR instrument with upstream primers and downstream primers.

[0123] PCR system components: 5μl 10×PCR buffer, 4μl 2.5mM dNTPs, 3μl 25mMMgCl 2 , 1 μl upstream primer (20 μM), 1 μl downstream primer (20 μM), 2 μl hrpZ plasmidDNA plate, 0.5 μl Taq DNA polymerase, supplemented with sterile ddH 2 0 to 50 μl.

[0124] Suction and mix with a pipette gun, and finally add 50 μl o...

Embodiment 2

[0127] The recovery and purification of embodiment 2PCR product

[0128](1) Electrophoresis the remaining PCR product on 1.0% agarose gel (1×TAE), observe the electrophoresis situation with ultraviolet light, stop the electrophoresis when the DNA band to be recovered is completely separated from other bands, and perform electrophoresis under ultraviolet light Cut off the band to be recovered with a razor blade, and purify it with a PCR product purification kit. (2) Crush the glue in an Eppendorf tube, add an equal volume of sol solution Binding Buffer, bathe in water at 65°C for 7 minutes, and shake the Eppendorf tube every 2 minutes until the glue is completely melted. (3) Add the melted sample to the chromatographic column, centrifuge at 12000 rpm for 1 min, and discard the liquid. (4) Add 300μl Binding Buffer, centrifuge and discard the liquid. (5) Add 750μl Washing Buffer, centrifuge and discard the liquid. (6) Repeat step 5). (7) Centrifuge the empty column at 12000rp...

Embodiment 3

[0129] Example 3 The PCR product is cloned in pGEM-T-Vector

[0130] The above purified PCR product was cloned into pGEM-T-Vector. The ligation system is: 5.0 μl 2×ligation buffer; 0.5 μl pGEM-T-Vector; 4.0 μl PCR products; 0.5 μl T4 DNA ligase, supplemented with sterile ddH 2 0 to 10 μl. Mix the above liquids on ice, put 10 μl of the mixed solution in a 250 μl sterile centrifuge tube, use a pipette gun to gently suck a few times to mix, centrifuge briefly at 5000 rpm, concentrate the mixed solution at the bottom of the tube, and then connect at 4 °C overnight.

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Abstract

The invention discloses a non-induced expression genetic engineering strain and the constructing method and the application. The host cell of the strain is enteric bacilli E.coliBL21, CCTCC M205136, PCR expanding hrp Z gene section to clone to pGEM-T carrier, ferment cutting and bonding by EcoR I / Xho I, inserting the hrp Z gene into the enteric bacilli expression carrier pET-32b(+) lower reaches of thioredoxin, constructing rebuilding expression particle pET-32b-hrp, and transferring into E.coliBL21, filtering to gain positive clone. The non-induced expression rebuilt albumen HrpZ, SDS-PAGE shows that high efficiently expresses APDZ albumen. The rebuilt albumen has good prevention effect to plant disease and could improves the yield.

Description

technical field [0001] The invention relates to the prevention and treatment of plant diseases, which can resist the harm of bacteria, fungi, viruses, nematodes and the like to plants. More specifically, the present invention relates to a non-induced expression Harpin gene working strain, and also relates to the construction method of the strain, and the application of the invention in the prevention and treatment of plant diseases. Background technique [0002] Allergic response is a kind of protective response that plants have, which is similar to the broad-spectrum anti-bacterial immune response of higher animals. Phytopathogenic fungi, bacteria and viruses can all cause hypersensitive response (HR). Anaphylaxis is a rapid, defense-related, plant cell death (programmed cell death, PCD) at the site of infection (Dangl et al., 1996; Klement et al., 1982), resulting in Pathogens are confined to the site of infection and cannot multiply. The main manifestation is localized...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195A01N63/02C12N1/21C12N15/31C12N15/70C12P21/02
Inventor 孟小林徐进平丁玲鲁伟王健
Owner WUHAN UNIV
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