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High-efficiency biocontrol recombinant streptomyces lydicus as well as construction method and application thereof

A technology of Streptomyces lydidii and its composition, applied in the field of high-efficiency bio-control recombinant Streptomyces lydida and its construction and application, can solve problems such as environmental pollution, pesticide residues, threats to human health and the ecological environment

Inactive Publication Date: 2015-04-01
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the amount of chemical pesticide application accounts for more than 80% of the amount of pesticide use, which causes serious environmental pollution, pesticide residues and other problems, and seriously threatens human health and the ecological environment.

Method used

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  • High-efficiency biocontrol recombinant streptomyces lydicus as well as construction method and application thereof
  • High-efficiency biocontrol recombinant streptomyces lydicus as well as construction method and application thereof
  • High-efficiency biocontrol recombinant streptomyces lydicus as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Embodiment 1, construct recombinant Streptomyces lydidii

[0100] 1. Preparation of DNA fragments

[0101] The synthetic nucleotide sequence is the DNA fragment of SEQ ID No.1 in the sequence listing. Among them, the DNA fragment shown in SEQ ID No.1, its name is P-SlnM-vhb, the DNA fragment from the 5' end to the 3' end is: Nde I recognition site, natamycin biosynthesis normal The coding gene of regulatory factors, the recognition site of Xba I, the promoter of erythromycin resistance gene, the coding gene of hemoglobin and the recognition site of EcoR I. SEQ ID No.1 is made up of 1237 nucleotides, the 1st-6th nucleotide of SEQ ID No.1 is the recognition site of Nde I, and the 7th-585th nucleotide of SEQ ID No.1 is The coding gene of the positive regulator of natamycin biosynthesis, the 586-591 nucleotide of SEQ ID No.1 is the recognition site of Xba I, and the 598-788 nucleotide of SEQ ID No.1 is Erythromycin resistance gene promoter, the 789-1229th nucleotide of S...

Embodiment 2

[0126] Embodiment 2, AMV02 and AVM02 produce the ability of natamycin

[0127] One, the preparation of natamycin fermented liquid

[0128] The experiment was repeated three times, and the specific steps of each repeated experiment were as follows:

[0129] Inoculate the AMV02 in Example 1 onto Gao’s No. 1 slant medium, cultivate at 28°C for 7-10 days, wait for it to produce sufficient spores, scrape the spores with a sterile platinum loop and inoculate 2-3 loops in 250ml Put the 50ml seed culture medium in the triangular flask on a temperature-controllable shaker, under the conditions of 28°C and 200rpm (rotation radius: 13mm), shake and cultivate at a constant temperature for 24h-30h; then inoculate it in 60 In the fermentation medium A (YSG medium) in the 500ml Erlenmeyer flask (the volume of each bottle is 100ml), the OD of each bottle after inoculation 600 The values ​​were all 0.1; the inoculated shake flasks were shaken at a speed of 240rpm (rotation radius 13mm) at 31...

Embodiment 3

[0173] The antibacterial activity of embodiment 3, AMV02 and AVM02

[0174] The experiment was repeated three times, and the specific steps of each repeated experiment were as follows:

[0175] 1. Preparation of plant fungal disease inhibitors

[0176] The AMV02 of Example 1 was inoculated on Gao’s No. 1 slant medium, and cultured at 28°C for 7-10 days. After it produced a sufficient amount of spores, the spores were scraped with a sterile platinum loop and inoculated in 2-3 loops. In a 250ml Erlenmeyer flask with 50ml of seed medium, under the conditions of 28°C and 200rpm (rotation radius: 13mm), shake and cultivate at constant temperature for 24h-30h; In the fermentation medium B in the 500ml Erlenmeyer flask, the OD of each bottle after inoculation 600 Values ​​are all 0.1; Shake flask after inoculation is shaken and cultured 96h under the condition of 31 ℃, 240rpm (radius of rotation 13mm), obtains AMV02 fermented liquid, contains the highly concentrated bacteriostatic ...

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Abstract

The invention discloses high-efficiency biocontrol recombinant streptomyces lydicus as well as a construction method and application thereof. The construction method of the high-efficiency biocontrol recombinant streptomyces lydicus comprises the steps of: transferring the encoding gene of hemoglobin and the encoding gene of a positive natamycin-biosynthesis regulation factor into streptomyces lydicus which serves as host bacteria, and screening to obtain the recombinant streptomyces lydicus, wherein the hemoglobin is a protein A1) which is formed by amino acids from an amino acid sequence of SEQ ID No.3 as shown in an amino acid sequence table or a protein A2) which has the functions of hemoglobin and is derived from the protein A1), and the positive natamycin-biosynthesis regulation factor is a protein B1) which is formed by amino acids from an amino acid sequence of SEQ ID No.4 as shown in an amino acid sequence table or a protein B2) which has the functions of the positive natamycin-biosynthesis regulation factor and is derived from the protein B1).

Description

technical field [0001] The invention relates to a high-efficiency biocontrol recombinant Streptomyces lydidis in the field of biotechnology, a construction method and application thereof. Background technique [0002] With the increase of world population and the decrease of arable land, food supply is becoming more and more severe. However, the perennial occurrence of crop diseases and insect pests has brought huge losses to the world economy (Chen Jun et al., 2010). my country is a large agricultural country based on planting. The use of pesticides can reduce economic losses by about 30 billion yuan per year. Among them, the amount of chemical pesticide application accounts for more than 80% of the amount of pesticide use, which causes serious environmental pollution, pesticide residues and other problems, and seriously threatens human health and the ecological environment. Therefore, it is urgent to develop new, environment-friendly and efficient microbial pesticides. A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/76C12N1/21C12P19/62A01N63/00A01N63/02A01P3/00C12R1/465
Inventor 吴慧玲刘伟成李锦锦田兆丰董丹刘霆张涛涛卢彩鸽张殿朋刘德文
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES