Effector protein from botryosphaeria dieback and encoding gene and application thereof

A grape canker bacteria and effector protein technology, applied in the field of molecular biology, can solve problems such as environmental pollution and increasing the burden on farmers

Inactive Publication Date: 2017-03-08
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And for a long time, at home and abroad, the main use of spraying pesticides to control the prevalence and harm of grape canker has failed to achieve the desired control effect, which not only increases the burden on farmers, but also causes environmental pollution.

Method used

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  • Effector protein from botryosphaeria dieback and encoding gene and application thereof
  • Effector protein from botryosphaeria dieback and encoding gene and application thereof
  • Effector protein from botryosphaeria dieback and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Cloning of embodiment 1, BrCRE1 protein and its coding gene

[0049] The BrCRE1 gene studied in this paper is an effector protein gene that may induce host resistance in plants, which was screened from the gene expression profile data of the infection of 'Summer Black' grape by the grape canker bacterium CSS-01s strain. Using the total RNA extracted from grape canker sore CSS-01s strain as a template, specific primers BrCRE1-F and BrCRE1-R were designed, and the full-length cDNA fragment of BrCRE1 gene was amplified by RT-PCR. The primer sequences are as follows:

[0050] BrCRE1-F: 5'-ATGAAGGCTTCCGGTCTC-3' (sequence 5 in the sequence listing);

[0051] BrCRE1-R: 5'-GAAGTACTGCAGCAACTG-3' (sequence 5 in the sequence listing).

[0052] The total RNA was extracted and cDNA was obtained by reverse transcribing the hyphae of P. cankeratum CSS-01s grown on PDA medium for 7 days. Using cDNA as a template, PCR amplification was performed with a primer pair composed of BrCRE1-...

Embodiment 2

[0056] Example 2. Analysis of BrCRE1 gene expression pattern during grape canker infection of grape hosts

[0057] 1. Inoculation of grape branches with grape canker

[0058] The CSS-01s mycelium block stored in a 4°C refrigerator was placed on a PDA solid medium for activation, and cultured at 28°C for 24-36h. Select the semi-lignified green branches of the annual 'Xiahei' grape, and disinfect the surface with 70% ethanol. Use a hole puncher to punch a hole in the middle of the branch internode, put the CSS-01s mycelium block in the hole, and fix it with a parafilm. Place the inoculated shoots in flower pots in a culture room for cultivation, humidity ≥ 80%, temperature at 26-28°C, take the skins of grape shoots at 0h, 12h, 24h, 36h and 48h after inoculation, and store them in -80°C after freezing in liquid nitrogen. ℃ refrigerator for RNA extraction.

[0059] 2. RNA extraction and expression verification of inoculated samples

[0060] Grape branch RNA was extracted with ...

Embodiment 3

[0066] Embodiment 3, BrCRE1 protein tobacco virulence assay

[0067] 1. Carrier construction

[0068] The primers BrCRE1-SIF and BrCRE1-BIR were paired, and the plasmid pMD18-T / BrCRE1 was used as a template, and the amplified fragment was connected to the T vector, named pMD18-T / BrCRE1t; it was digested with BamH Ⅰ and Sal Ⅰ pMD18-T / BrCRE1t, the recovered small fragment was inserted into the same restriction site of pEDV, and the resulting recombinant vector was named pEDV / BrCRE1. Colony PCR and enzyme digestion results showed that the expression vector pEDV / BrCRE1 was successfully constructed. The primer sequences used are as follows:

[0069] BrCRE1-SIF: 5'-TGTCGACCGTTCCCATGCTTAAC-3';

[0070] BrCRE1-BIR: 5'-TGGATCCGAAGTACTGCAGC-3'.

[0071] 2. Transformation of Rice Grain Blight Bacteria (Burkholderia glumae) with Recombinant Plasmids

[0072] 1. Preparation and transformation of competent rice blight bacteria

[0073] Transfer 10μL rice blight bacteria into 50mL liqu...

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Abstract

The invention discloses effector protein from botryosphaeria dothidea and an encoding gene and application thereof. The effector protein is the protein having one of the following amino acid residue sequences: (1) the protein formed by an SEQ ID No. 1 amino acid residue sequence in a sequence list; (2) the protein which is derived from SEQ ID No. 1 by carrying out substitution and / or deficiency and / or addition of one or several amino acid residues on the SEQ ID No. 1 amino acid residue sequence, and has a pathopoiesis-related effector protein function. Through a transgenosis experiment of the effector protein shows that the encoding gene has a function of motivating anti-disease genes in tobacco, and a hypersensitive necrosis reaction of the tobacco can be caused; in addition, the pathogenicity of the botryosphaeria dieback can be enhanced after overexpression, and the effector protein is an important target for researching the anti-disease genes.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to an effector protein derived from grape canker bacterium, its coding gene and application. Background technique [0002] Grape (Vitis vinifera L.) has always been one of the most important fruits in the world. Its economic value is very high. It can not only be eaten fresh but also processed into raisins and wine. At present, the area of ​​grape cultivation in the world is very large, and the cultivation area accounts for at least 10% of all fruit types. By the end of 2013, the cultivation area of ​​grapes in my country has reached 714,600 hm 2 , grape production 11.55 million tons. [0003] Grape disease is one of the most important factors affecting grape yield and quality. The occurrence of grape disease is closely related to human factors such as management methods, variety selection, and climate and environment. Most of my country's grapes are planted in temperate and subtrop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C12N1/15A01N47/44A01P3/00C12R1/645
CPCA01N47/44C07K14/37
Inventor 燕继晔邢启凯李兴红张玮刘梅
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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