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Phytophthora capsici cell division protein as well as encoding gene and application thereof

A technology of Phytophthora capsici and coding, applied in the field of genetic engineering, can solve problems such as soil pollution and pesticide residues

Inactive Publication Date: 2015-03-25
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the case of slow progress in disease-resistant breeding, we mainly rely on chemical agents for prevention and control, but this has also led to a certain degree of negative impact, such as soil pollution and pesticide residues.

Method used

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  • Phytophthora capsici cell division protein as well as encoding gene and application thereof
  • Phytophthora capsici cell division protein as well as encoding gene and application thereof
  • Phytophthora capsici cell division protein as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Cloning of Phytophthora capsicum PcCDP gene

[0073] 1. Extraction of total RNA from Phytophthora capsicum

[0074] The hyphae of Phytophthora capsicum strain SD33 were collected and ground into powder with liquid nitrogen in a mortar. Then use the kit (QIAGEN RNA Kit) for RNA extraction, the specific steps are as follows:

[0075] (1) Weigh 100 mg of mycelial samples and freeze them in liquid nitrogen.

[0076] (2) Grind into a fine powder in a pre-cooled mortar and transfer to a 2ml centrifuge tube. Note that incomplete grinding can result in severe yield loss.

[0077] (3) After the liquid nitrogen is volatilized (but not thawed), add 450 ml of extraction buffer RLT, vortex to mix, and incubate at 56°C for 1-3 minutes. Note: It is best to add 1 / 100 volume of 14.5M β-mercaptoethanol before RLT is used, and the storage time after adding should not exceed 1 month. RLT solution should be used within 1 month. For high starch content materials, the temperat...

Embodiment 2

[0094] Example 2. Analysis of expression patterns of PcCDP gene in different developmental stages of Phytophthora capsici

[0095] The total RNA of Phytophthora capsici strain SD33 at different developmental stages (mycelium (MY), sporangia (SP), zoospores (ZO), resting spores (C), germinated resting spores (CG)) (extraction method) Refer to Example 1) The cDNA synthesized by reverse transcription is used as a template, and the PcCDP gene is detected by fluorescence quantitative PCR. The β-Actin, β-Tublin and Ubc genes of Phytophthora capsicum were used as internal reference. details as follows:

[0096] Reaction system: 2.5×realMasterMix 8μL; 20×SYBR solution 1μL; forward primer (10μM) 0.5μL; reverse primer (10μM) 0.5μL; cDNA template 2μL; RNase-free dd H 2 O make up to 20 μL. Among them, 2.5×realMasterMix, 20×SYBR solution and RNase-free dd H 2 O is the product in the SuperReal PreMix Plus (SYBR Green) kit (catalog number FP205) produced by Tiangen Biochemical Technology...

Embodiment 3

[0103] Example 3. Obtainment of silencing transformants and overexpression transformants of Phytophthora capsicum PcCDP gene

[0104] 1. Construction of silent expression vector and overexpression vector of Phytophthora capsicum PcCDP gene

[0105] The genomic cDNA of Phytophthora capsici strain SD33 was used as the template, and the primers CDP-SmaI-F and CDP-SmaI-R were used for PCR amplification.

[0106] CDP-SmaI-F: 5'-TCC CCCGGG ATGGCTTCTCGTTGTGCTTC-3' (the underlined part is the recognition site of SmaI, and the subsequent sequence is the 1-20th position of sequence 2);

[0107] CDP-SmaI-R: 5'-TCC CCCGGG TTAAAGCGAGAAAAAGCGGC-3' (the underlined part is the recognition site of SmaI, and the following sequence is the reverse complement of the 1400-1419th position of sequence 2).

[0108] The reaction product was electrophoresed in a 1% agarose gel, and the block containing the target band was recovered. The recovered product was cloned into the pEASY-T3 vector and sent...

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Abstract

The invention discloses a phytophthora capsici cell division protein as well as an encoding gene and application thereof. The phytophthora capsici cell division protein can be a protein (a) which is formed by amino acid sequences 1 or a protein (b) which is formed by substituting amino acid sequences 1 by one or more amino acid residues and / or deleting amino acid sequences 1 and / or adding one or more amino acid residues, is related to plant salt resistance and is derived from amino acid sequences 1. Experiments prove that the phytophthora capsici cell division protein plays a role during the growth and development of phytophthora capsici, and the obtained silence transformants are obviously different from the wild phytophthora capsici in growth and development. The inoculation of hot pepper leaves against zoospores in vivo finds that the diseased spot areas of the inoculated silence transformants are obviously smaller than those of the wild phytophthora capsici. The research carried out on development of spores during staining by adopting the aniline blue staining technology proves that the spores of the silence transformants germinate late and the germinal tubes are short. The conclusions are helpful to the research on the development process and the molecular pathogenic mechanism of the phytophthora capsici to some extent.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a cell division protein of Phytophthora capsicum and its encoding gene and application. Background technique [0002] Pepper blight is a worldwide devastating soil-borne disease caused by the phytopathogenic oomycete Phytophthora capsicum, a facultative parasite. In addition to capsicum and other nightshade plants, its host can also infect cucumber, pumpkin and other cucurbit crops. Pepper blight was first discovered in New Mexico, USA, and later found in other countries, causing huge economic losses to vegetable production worldwide. With the spread of Phytophthora capsicum in the world, the pepper blight in vegetable growing areas in my country has become increasingly serious since the 1980s, and has become one of the important diseases that endanger the quality and yield of vegetable products in my country. Therefore, the prevention and control of pepper blight is of great s...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/11C12N1/14A01N61/00A01P3/00
CPCA01N63/30C07K14/37
Inventor 朱春原张梦妍张修国
Owner SHANDONG AGRICULTURAL UNIVERSITY
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