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Phytophthora capsici effector RxLR19781 gene and application thereof

A technology of Phytophthora capsicum and effector, applied in the field of molecular biology, can solve problems such as little known function

Inactive Publication Date: 2020-01-31
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, little is known about how most of the Phytophthora capsici RxLR effector molecules function in plants

Method used

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  • Phytophthora capsici effector RxLR19781 gene and application thereof
  • Phytophthora capsici effector RxLR19781 gene and application thereof
  • Phytophthora capsici effector RxLR19781 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Cloning and sequence analysis of Phytophthora capsici RxLR19781 gene

[0049] Using the whole genome cDNA of Phytophthora capsici SD33 as a template, amplify the RxLR19781 gene sequence according to the corresponding PCR reaction system: 25 μL 2×Phanta Max Master Mix, 4 μL DNA, 17 μL ddH2O, 2 μL ForwardPrimer (10 μM), 2 μL Reverse Primer (10μM), a total of 50μL system. The PCR reaction program is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30s, annealing at 55-65°C for 30s, extension at 72°C for several minutes (determined by the length of the cloned gene, about 1kb / min), 32 cycles; 72°C Extend for 10 minutes.

[0050] The products after PCR were electrophoresed on a 1% agarose gel ( figure 2 ), after 30 minutes, the target band was excised and recovered, and the recovered product was connected to the cloning vector pEASY-T3 carrier, and then sent to the company for sequencing to obtain the RxLR19781 gene sequence as shown in SEQ ID NO...

Embodiment 2

[0052] The construction of embodiment 2 plant expression vectors

[0053] In order to verify the function of the Phytophthora capsici effector molecule RxLR19781, according to the restriction site on the plant expression vector pBIN-GFP, gene-specific primers with corresponding restriction sites to remove the signal peptide were designed to T3 plasmid with RxLR19781 As a template, clone the gene sequence with the enzyme cleavage site after removing the signal peptide, construct the target gene gene into the plant expression vector by double enzyme cleavage method, and send the sample to the company for sequencing after the verification of bacterial liquid PCR After the sequencing is correct, the plasmid is extracted and transformed into Agrobacterium GV3101, and the inoculation experiment can be carried out after the successful transformation is verified. The result of PCR verification after pBIN-GFP recombinant vector transforms Agrobacterium GV3101 is as follows image 3 sh...

Embodiment 3

[0054] Example 3 Analysis of the expression pattern of Phytophthora capsici effector molecule RxLR19781

[0055] After the zoospores of Phytophthora capsici SD33 strain infected pepper leaves, samples were collected after 1.5h, 3h, 6h, 12h, 24h, 48h, and 72h, and RNA was extracted and reverse-transcribed into cDNA. Using this as a template, SD33 bacteria The cDNA in the silk stage was used as a control, and Phytophthora capsici Actin was used as an internal reference gene to detect the expression level of the effector molecule RxLR19781 in different stages by qRT-PCR. Take the average value of the three qRT-PCR results and analyze the variance, the results are as follows Figure 4 shown. Analysis of the expression pattern of RxLR19781 showed that the expression of this effector molecule was most obviously up-regulated at 1.5 h in the early stage of infection, and then the expression level gradually decreased, and then slightly increased after 24 h.

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Abstract

The invention provides a phytophthora capsici effector RxLR19781 gene and application thereof. By using multiple genetic engineering techniques, biological functions of the phytophthora capsici effector RxLR19781 (SEQ ID NO:2) gene and an interactions protein GME (guanosine diphosphatemannose mannose 3'5' epimerase (SEQ ID NO:4) are revealed, GME is a key factor in the process of regulating and controlling RxLR19781 to control zoospore infestation functions, RxLR19781 is capable of inhibiting necrosis caused by Bax, and results that RxLR19781 is further silenced in phytophthora capsici show that pathogenicity of the phytophthora capsici can be weakened by RxLR19781.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the Phytophthora capsici effector RxLR19781 gene and its application. Background technique [0002] Phytophthora capsici is a pathogenic oomycete with strong pathogenicity, wide parasitic range and the ability to produce a large number of RxLR effector molecules to infect hosts cooperatively. With the deepening of the research on the pathogenicity of pathogenic oomycetes, it has been found that the RxLR effector molecules of oomycetes play a key role in the interaction between pathogenic oomycetes and plants. However, little is known about how most P. capsici RxLR effector molecules function in plants. Therefore, research on the molecular pathogenic mechanism of RxLR effect and the prevention and treatment of oomycete diseases are particularly important. Contents of the invention [0003] The purpose of the present invention is to provide Phytophthora capsici effector RxLR197...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/37C12N15/84A01H5/00A01H6/82
CPCC07K14/37C12N15/8282
Inventor 梁涛朱春原李壮张修国
Owner SHANDONG AGRICULTURAL UNIVERSITY
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