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Preparation and application of citrus natural bacteriostatic protein CsLTP1

A protein and recombinant protein technology, applied in the directions of application, fungicide, botanical equipment and methods, etc., to achieve the effect of reducing the occurrence of green mildew, reducing harm, and having good application prospects

Active Publication Date: 2019-06-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on the development and application of antimicrobial peptides such as non-specific lipid transporters directly as plant-derived bacteriostatic agents in the prevention and control of postharvest diseases of fruits and vegetables

Method used

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  • Preparation and application of citrus natural bacteriostatic protein CsLTP1
  • Preparation and application of citrus natural bacteriostatic protein CsLTP1
  • Preparation and application of citrus natural bacteriostatic protein CsLTP1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Citrus natural antimicrobial protein CsLTP1 gene search, codon optimization, whole gene synthesis

[0048] 1.1 Citrus natural antibacterial protein CsLTP1 gene search

[0049]Through published articles (eg, Jinlong Wu, Lin Chen, Dingbo Lin, Zhaocheng Ma*, and Xiuxin Deng*. Development and Application of a Multiplex Real-Time PCR Assayas an Indicator of Potential Allergenicity in Citrus Fruits. Journal of agricultural and food chemistry, 2016,64(47):9089-9098.) Amino acid sequence of non-specific lipid transfer protein (GenBank XP_006429565.1), through sequence homology in sweet orange genome (http: / / citrus.hzau.edu.cn / orange / ) to search for the corresponding citrus non-specific lipid transfer protein and obtain the gene sequence, as shown in Table 1.

[0050] Table 1 Gene information of citrus non-specific lipid transfer protein CsLTP1

[0051]

[0052] 1.2 Allergen gene CsLTP1 sequence codon optimization and whole gene synthesis

[0053] The frequency...

Embodiment 2

[0057] Example 2 Prokaryotic expression vector construction

[0058] 2.1 Primer design

[0059] The primers in the present invention were designed using Primer 5.0 software, and the nucleotide sequences of the primers are shown in Table 3. All primers were synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.

[0060] Table 3 Primers

[0061]

[0062] Note: CsLTP1-F and CsLTP1-R in the table are forward and reverse primers. Bold text 15 bp indicates the linker used for vector construction. Italics indicate the 5'-end EcoRI and 3'-end XhoI restriction sites.

[0063] 2.2 Vector Construction

[0064] The present invention uses the homologous recombination one-step cloning kit (ClonExpress II One Step Cloning Kit, provided by Nanjing Novizan Biotechnology Co., Ltd.) to construct the prokaryotic expression vector pET-32a(+) / Trx-His-CsLTP1. Specifically, the synthetic CsLTP1 gene was used as a template, and CsLTP1-F and CsLTP1-R were used as primers to obtain a PCR p...

Embodiment 3

[0069] Example 3 Recombinant CsLTP1 protein expression, protein purification, mass spectrometry identification

[0070] 3.1 Induced expression of rCsLTP1 protein

[0071] After the positive plasmid with correct sequencing was transformed into E.coli BL21(DE3) competent cells, it was inoculated into 5 mL of LB culture medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C with shaking. The next day, they were respectively transferred to 50 mL of the above culture medium at a ratio of 1:100, cultured at 37°C until the logarithmic growth phase (A600=0.4-0.6), added with a final concentration of 0.1mmol / L IPTG, and induced at 20°C for 16h to 10000g Bacteria were collected by centrifugation for 5 min; washed with PBS, centrifuged, and resuspended in ice-cold lysis buffer (50mmol / L NaH 2 PO 4 , 300mmol / L NaCl, 0.1g / L lysozyme and 1mmol / L PMSF, pH 8.0), sonicated, centrifuged at 10000g for 10min, collected the supernatant, and carried out SDS-PAGE analysis ( image...

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Abstract

The invention discloses preparation and an application of a citrus natural bacteriostatic protein CsLTP1, specifically discloses a codon-optimized non-specific lipid transfer protein CsLTP1 gene in rutaceae plants, and is characterized in that the gene includes a sequence shown in SEQ ID NO:2. By optimization of a codon of the bacteriostatic protein CsLTP1, a prokaryotic expression vector is constructed, recombinant expression, purification and identification of rCsLTP1 are performed, and the obtained rCsLTP1 protein has good thermal stability and acid-base stability, and has obvious inhibitory effect on pathogenic bacteria penicillium digitatum of postharvest green mold of citrus under a living body condition. Compared with the prior art, the plant-derived bacteriostatic protein CsLTP1 isa natural bacteriostatic agent which can be produced on a large scale, can significantly reduce the occurrence of postharvest green mold of citrus fruits, reduces the harm caused by chemical bactericides and has good application prospects in the field of antisepsis and fresh keeping of the citrus fruits.

Description

technical field [0001] The invention belongs to the field of prevention and treatment of postharvest green mold of citrus, more specifically, relates to the preparation and application of a natural citrus antibacterial protein CsLTP1, which can be applied to the prevention and treatment of postharvest green mold of Rutaceae plant fruits (such as citrus), that is, using A method for preparing citrus natural bacteriostatic protein CsLTP1 by genetic engineering and the application of the extract prepared by the method as a botanical antibacterial agent in the prevention and treatment of citrus postharvest disease green mold. Background technique [0002] Citrus is the largest fruit in the world. During its harvesting, transportation and storage, the fruit is damaged due to improper measures, and it is susceptible to infection by various pathogenic bacteria, resulting in post-harvest diseases, resulting in fruit rot and deterioration, bringing huge economic benefits to the citrus...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C12N15/70C12P21/02C12Q1/18C07K14/415A01N63/04A01N47/44A01P3/00G01N27/62C12R1/19
Inventor 邓秀新马兆成程运江吴金龙
Owner HUAZHONG AGRI UNIV
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