Method for improving gene knockdown conversion rate of talaromyces marneffei

Abstract

The invention discloses a method for improving the gene knockdown conversion rate of talaromyces marneffei, and relates to the field of biotechnology. The method comprises the steps of (1) talaromycesmarneffei strain preparation; (2) protoplast preparation, wherein a novel wall breaking enzyme is mixed with the strain, and cell walls of the strain are dissolved and digested for 1.5-2h; (3) protoplast transformation; (4) protoplast rejuvenation, wherein transformed protoplasts are resuspended in a STC buffer, an SD culture medium containing 1-1.5 mol / L of sorbitol is added, shaking culture isconducted at 23-28 DEG C in a shaker at 80-120 rpm / min for 1-2 days for growth resuming, then spore suspension is inoculated into an ANM-U solid medium, and positive transformants are screened out tocomplete cloning screening. Through the method, the gene conversion efficiency can be improved, and a solid guarantee for researches on gene functions of the talaromyces marneffei is provided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel gene knockout method for T. marneffei. Background technique [0002] T. marneffei is prevalent in southern my country and Southeast Asia. It is one of the most common opportunistic infections among AIDS patients in endemic areas. It is reported that 19% of AIDS patients in Guangxi are infected with T. marneffei, and the mortality rate is as high as 80%, is an important factor leading to the death of local AIDS patients. In recent years, due to the widespread use of immunosuppressants, T. marneffei infection has become more frequent in patients without overt immunodeficiency. However, little is known about the pathogenic mechanism of T. marneffei at home and abroad. [0003] Gene knockout technology is the basis for studying the infection mechanism, immune escape mechanism and gene function of T. marneffei. The cell wall of T. marneffei is hard to digest; The medium is easil...

AI Technical Summary

Problems solved by technology

However, little is known about the pathogenic mechanism of T. marneffei at home and abroad

[0003] Gene knockout technology is the basis for studying the infection mechanism, immune escape mechanism and gene function of T. marneffei. The cell wall of T. marneffei is hard to digest; The medium is easily broken and difficult to survive, etc., resulting in low transformation efficiency of this method. How to improve the preparation efficiency of T. marneffei protoplasts in a short period of time and how to promote the survival of the gene transformation has become a key technical problem.

The existing protoplast transformation method gene knockout technology in China uses an enzymatic hydrolysis solution (including cellulase, helicase, lysozyme) to prepare complicated and difficult to preserve, and it takes 4-6 hours to break the wall, which takes a long time and is not Ease of protoplast regeneration

After gene transformation, the existing transformation method directly spreads the suspension on the solid medium, which is prone to morphological imbalance and rupture, resulting in a decrease in the regeneration rate

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