Studies of arginine-rich intracellular delivery peptides as the mediator for transformation of Antrodia cinnamomea

• Main Authors: Jyun-De Wu, 吳俊德
• Other Authors: Jyh-Ching Chou
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/87w8sf

碩士 === 國立東華大學 === 生命科學系 === 106 === Antrodia cinnamomea is an endemic fungus of Taiwan. It carrys several medicinal effects, such as anti-cancer, anti-intoxication, anti-inflammation…etc. With the in-depth studies of the active ingredients, it is important to establish the genetic transformation system for further genetic studies. In this study. I would like to optimize the protocol for protoplast preparation and regeneration of A. cinnamomea. I observed various factors of affection in the protoplast preparation, such as mycelial age, pH value of digestion and osmotic stabilizer…etc. In the protoplast regeneration, I investigated the effects of various conditions, including different media ingradients and osmotic pressure…etc. I found that the pH value is a key factor in the regeneration of A. cinnamomea protoplasts. This experiment provides an optimal system for A. cinnamomea regeneration. Various shortcomings have be found in fungal transformation experiments, such as high toxicity, poor transformation efficiency and expensive instrumentation. According to earlier studies, arginine-rich intracellular delivery peptides(AID peptides) have the property of penetrating cell membranes. The advantages of low cytotoxicity and high transfection efficiency make it widely used in animal cell transfection experiments. Thus, I am interested in applying the AID peptides for gene transformation in filamentous fungi. The fungal protoplasts and mycelia were first successfully expressed with the target fluorescence protein genes through AID peptide mediation. However, the mycelia of the transformation experiment could not grow in the media containing antibiotics indicating the permanent transgenic cells still unsuscessful. The transformed protoplasts did grow in media with antibiotics, but no target DNA detected with PCR screening. Further investigations are necessary to characterize these results.