Amaranth recombinant antibacterial protein as well as preparation method and application thereof

A technology of recombinant protein and protein, which is applied in the direction of botanical equipment and methods, biochemical equipment and methods, applications, etc., to achieve the effects of less spots, obvious antibacterial effect, and reduced harm

Active Publication Date: 2020-11-06
HUNAN AGRICULTURAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of amaranth ribosomal inactivating protein gene as a plant-derived bacteriostatic agent in the prevention and control of pepper phytophthora and rice sheath blight

Method used

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  • Amaranth recombinant antibacterial protein as well as preparation method and application thereof
  • Amaranth recombinant antibacterial protein as well as preparation method and application thereof
  • Amaranth recombinant antibacterial protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The present embodiment provides a kind of amaranth antibacterial protein AmaAAP:

[0037] The transmembrane region analysis, signal peptide analysis and hydrophobicity analysis of the amino acid sequence of amaranth antimicrobial protein Uniprot ID: Q96322 (gene name AAP, Gene ID: AAB67746.1) shows that the gene encodes 284 amino acids, and 6-26aa is the transmembrane region. In the membrane region, 1-31aa is the signal peptide sequence, the local hydrophilicity is good, and the expressed protein is expected to be soluble, and 32-284aa is selected for expression in Pichia pastoris. The amino acid sequence at positions 32-284 is the amino acid sequence shown in SEQ ID NO: 1, named as amaranth antibacterial protein AmaAAP.

Embodiment 2

[0039] This embodiment provides a gene encoding the above-mentioned amaranth antibacterial protein AmaAAP:

[0040] The amino acid sequence shown in SEQ ID NO: 1 was codon-optimized according to the codon preference of Pichia pastoris, and the optimized nucleotide sequence was SEQ ID No: 2, which was named the amaranth antibacterial protein AmaAAP gene.

Embodiment 3

[0042] The present embodiment provides a kind of expression vector containing amaranth protein AmaAAP gene, and described expression vector is constructed through the following steps:

[0043] The nucleotide sequence obtained in Example 2 is as shown in SEQ ID No: 2. After the two ends of the amaranth antimicrobial protein AmaAAP gene are added with restriction site EcoRI / NotI and His tag, it is carried out to the whole gene synthesis to obtain the gene Fragment; the whole gene synthesis of the present embodiment is entrusted to Sangong Bioengineering (Shanghai) Co., Ltd. (hereinafter referred to as Sangong) to complete;

[0044] Use EcoRI / NotI to double digest the vector pPIC9K and the gene fragment obtained in the above steps, recover and purify, connect the double digested gene fragment to the vector pPIC9K, obtain the ligated product and transform it into Escherichia coli XL10 competent, pick The positive clones were amplified by PCR with the general primers of the pPIC9K ...

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Abstract

The invention discloses an amaranth antibacterial protein AmaAAP. The amino acid sequence of the amaranth antibacterial protein AmaAAP is SEQ ID No: 1; the invention also discloses a gene for coding the amaranth antibacterial protein AmaAAP. The nucleotide sequence of the amaranth antibacterial protein AmaAAP gene is SEQ ID No: 2. The invention further discloses a recombinant antibacterial proteinrAmaAAP based on the amaranth antibacterial protein AmaAAP. The invention further discloses an application of the recombinant antibacterial protein rAmaAAP in preparation of a medicine for preventingand controlling phytophthora capsici. The invention also discloses an application of the recombinant antibacterial protein rAmaAAP in preparation of a drug for preventing and controlling rice sheathblight. The invention also discloses a preparation method of the recombinant antibacterial protein rAmaAAP. The pichia pastoris is used for producing the recombinant protein, the advantage of secretory expression is achieved, industrial production is easy, and more choices are provided for designing dosage forms of biopesticides.

Description

technical field [0001] The invention relates to the field of biological control of plant diseases, in particular to a recombinant antibacterial protein from amaranth and its preparation method and application. Background technique [0002] Capsicum phytophthora caused by Phytophthora capsici is a devastating disease that harms the growth of peppers, tomatoes, cucumbers and other crops. At present, the prevention and control of pepper Phytophthora mainly adopts chemical fungicides (metalaxyl, butylpyrroline and fluthiazolone, etc.) combined with cultivation management measures (planting disease-resistant varieties, crop rotation, etc.). Rice sheath blight is a worldwide fungal disease that commonly occurs in rice production. It is caused by Rhizoctonia solani Kühn. Seriously affect rice yield. Jinggangmycin is the main drug used to prevent and treat the disease, but long-term single use also causes the problem of increased drug resistance. Therefore, finding new plant-deri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/81C12N15/66A01N47/44A01P3/00C12R1/84
CPCC07K14/415C12N15/815C12N15/66A01N47/44C07K2319/21
Inventor 高杜娟廖石榴廖晓兰张亚
Owner HUNAN AGRICULTURAL UNIV
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