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Tomato ubiquitin ligase gene and application thereof

A technology of ubiquitin ligase, tomato, applied in the field of plant genetic engineering

Active Publication Date: 2018-05-15
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in recent years, relevant research progress has mainly focused on the initiation and activation process of plant defense responses, and there is little research on how plants actively down-regulate and shut down this defense effect, especially how to carry out systematic and effective negative regulation of hypersensitivity reactions that produce rapid and severe tissue necrosis. There are reports

Method used

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  • Tomato ubiquitin ligase gene and application thereof
  • Tomato ubiquitin ligase gene and application thereof
  • Tomato ubiquitin ligase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: S1SINAL gene cloning and Escherichia coli expression vector construction

[0015] Total RNA was extracted from tomato leaves, reverse transcription-PCR (reverse transcription-PCR, RT-PCR), and cloned to obtain the S1SINAL gene.

[0016] 1. Reagents

[0017] Plant RNA extraction reagent Trizol was purchased from Invitrogen; DNase I (Dnase I) was purchased from Takara; reverse transcriptase (TransScript Reverse Transcriptase), Pfu high-fidelity DNA polymerase, and T4 DNA ligase were purchased from Beijing Quanshijin Technology Co., Ltd.; restriction endonucleases EcoR I and Sa1 I were purchased from Fermentas; plasmid extraction kits and gel recovery kits were purchased from Omega; primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.; other reagents were imported. packaged or domestically produced analytically pure products.

[0018] 2. Escherichia coli strains, vectors and plant material

[0019] Escherichia coli (Escherichia coli) strain BL...

Embodiment 2

[0102] Example 2: Expression and purification of S1SINAL tagged protein

[0103] The Escherichia coli with pMAL-SINAL vector obtained in Example 1 was subjected to induced expression of S1SINAL gene, and then the induced expressed S1SINAL protein was purified to obtain S1SINAL recombinant protein with MBP (maltose binding protein, maltose binding protein) tag .

[0104] 1. Reagents

[0105]Starch resin was purchased from NEB Company; maltose and IPTG (Isopropyl β-D-Thiogalactoside) were purchased from Sigma Company; other reagents were imported or domestically produced analytically pure products.

[0106] 2. Medium and solution

[0107] The preparation method of LB medium and 1000× ampicillin (Amp) is as described in Example 1.

[0108] Extraction buffer: 1M Tris (pH 7.5) 2mL, 5M NaCl 4mL, 0.5M EDTA 200μL, add sterilized deionized water to 100mL.

[0109] Elution buffer: Add 36 mg maltose to 10 mL extraction buffer.

[0110] 5×SDS-PAGE loading buffer: 0.6mL 1M Tris-HC1 (p...

Embodiment 3

[0130] Example 3: In vitro ubiquitination activity detection of S1SINAL protein

[0131] The purified S1SINAL protein prepared in Example 2 above was added to the reagents required for the ubiquitination reaction to detect the ubiquitin ligase activity of the S1SINAL protein.

[0132] 1. Reagents

[0133] Creatine phosphocreatine, creatine phosphokinase, ATP (inosine triphosphate), flag-labeled ubiquitin molecule Flag-Ub were purchased from Sigma; ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2 ) was purchased from R&D Systems; PVDF membrane was purchased from Merck Millipore; mouse Flag tag monoclonal antibody anti-Flag and anti-mouse antibody anti-mouse were purchased from Sigma; ECL western blotting substrate was purchased from GE; other reagents All are imported repackaged or domestically produced analytically pure products.

[0134] 2. Solution

[0135] 20× reaction buffer: 9.52mg MgCl, 24.2mg ATP, 7.65mg phosphocreatine, 1 mg phosphocreatine kinase,...

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Abstract

The invention discloses a tomato ubiquitin ligase gene and application thereof. The nucleotide sequence of the tomato gene S1SINAL is as shown in SEQ ID NO.1, and the amino acid sequence of the gene coding protein is as shown in SEQ ID NO.2. The tomato gene S1SINAL has ubiquitin ligase activity, has an inhibition effect on plant resistant protein mediated hypersensitivity reaction, and can inhibithypersensitivity reaction caused by pathogen effect protein. The tomato gene exerts negative regulation and control effects in plant immune reaction and can be used for adjusting the disease resistance of plants.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a tomato ubiquitin ligase activity gene S1SINAL and the application of the gene in regulating plant disease resistance. Background technique [0002] Pathogenic microorganisms cause the breakdown of plant defense system to form diseases, which will cause huge losses in agriculture. Bacterial diseases cause about 20% economic loss of crops every year. There is a complex interaction between plants and pathogenic invaders, and whether the disease occurs is also the result of the attack and defense game between plants and pathogenic microorganisms (Plant Signaling & Behavior, 2009, 4: 283-293). For example, in the offensive and defensive battle between Pseudomonassyringae pv. Inject toxic effector proteins into plants to order the closed stomata to open again, breaking through the physical barrier of the plant surface; breaking through the protective barrier of pl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/82A01H5/00A01H6/82
CPCC12N9/93C12N15/8281C12Y603/02
Inventor 牛向丽周宇张政王洋苗敏冯国栋刘永胜岳俊阳陈丹阳
Owner HEFEI UNIV OF TECH
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