A kind of method for producing mycelomycin with Artemisia annua

A technology of mycelia and Artemisia annua, applied in the fields of biotechnology and genetic engineering, can solve problems such as plasmid instability, decreased expression of exogenous mycelia gene, and decreased copy number

Active Publication Date: 2018-12-25
CHANGSHA ZHONGKEJINGBO BIOTECH
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Problems solved by technology

However, once the laboratory is expanded to industrial scale production, due to the disappearance of the selection pressure to maintain a high copy number of the plasmid in the culture medium, the plasmid becomes unstable and the copy number decreases, so the expression level of the exogenous plectasin gene also increases. decline

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  • A kind of method for producing mycelomycin with Artemisia annua
  • A kind of method for producing mycelomycin with Artemisia annua
  • A kind of method for producing mycelomycin with Artemisia annua

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specific Embodiment approach

[0119] Specific embodiments of the invention The sequences used in the examples are the sequences mentioned above

[0120] The method for producing Plectasin with Artemisia annua of the present invention, it comprises the following steps:

[0121] a. Transformation carrier T202;

[0122] b. Constructing the recombinant plasmid T202-Plec expressing plectasin gene in Artemisia annua cells;

[0123] c. Transform Artemisia annua with recombinant plasmid T202-Plec;

[0124] d. Screen positive transgenic Artemisia annua seedlings, and carry out real-time RT-PCR identification.

[0125] The specific operation process is:

[0126] 1. Transformation carrier T202

[0127] Clone the pRB fragment:

[0128] Using pCambia1300 as a template, design synthetic primers to amplify pRB ( figure 1 ). PCR reaction conditions: pre-denaturation at 94°C for 5 minutes, 30 cycles at 94°C for 30s-59°C for 30s-72°C for 70s, and 72°C for 7 minutes. After 2% agarose gel electrophoresis, the target ...

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Abstract

The invention discloses a method for producing plectasin through sweet wormwood herbs. The method includes the following steps of firstly, refitting and obtaining a carrier T202; secondly, optimizing and amplifying a plectasin gene; thirdly, establishing recombinant plasmid T202-Plec for expressing the plectasin gene in sweet wormwood herb cells; fourthly, converting agrobacterium EHA105 cells through the recombinant plasmid T202-Plec; fifthly, screening out positive strains, and conducting enzymatic detection; sixthly, transforming the sweet wormwood herbs to obtain sweet wormwood herb transformation plants with positive antimicrobial peptide plectasin; seventhly, quantitatively authenticating the expression amount of plectasin through PCR(qRT-PCR) in real time. An sweet wormwood herb expression system adopted for the method has the advantages that according to the codon preference of the sweet wormwood herbs, the nucleotide sequence of the plectasin gene is optimized, and the efficient and stable mRNA transcription, rapid and accurate translation and high external protein accumulation are ensured; plant expression plectasin is low in cost, safe and high in economic benefit; the expressed protein product can be directly used for being added without being purified.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and genetic engineering, in particular to a method for producing plectasin by using Artemisia annua. Background technique [0002] China is a large animal husbandry country, and the animal feed produced every year suffers huge losses due to corruption and deterioration during storage. Therefore, a large number of manufacturers add chemical preservatives to feed, but the chemical preservatives left in the animal body not only affect the quality of livestock and poultry products, but also threaten people's health, such as causing gastroenteritis and other diseases, affecting liver and kidney function etc. On the other hand, in order to prevent animal husbandry diseases, antibiotics have been added to animal feed in large quantities for a long time, and the problems of animal digestive tract disorders and drug resistance caused by this are also becoming more and more serious. [0003] Antiba...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/14
Inventor 夏新界崔延春余艳尹晟
Owner CHANGSHA ZHONGKEJINGBO BIOTECH
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