Liposome-mediated Fruit Body Gill Tissue Transformation of Agaricus bisporus and Function Exploration of 1-aminocyclopropane-1-carboxylic Acid Oxidase Gene (ACO)
Abstract：To explore the function of 1-aminocyclopropane-1-carboxylic acid oxidase gene (ACO) and ethylene biosynthesis pathway in Agaricus bisporus, an efficient genetic transformation approach was developed for constructing the ACO-RNAi mutants. Cationic liposome was mixed with the dsRNA of ACO expression plasmid pBHg-dsACO at the ratio of 1 μL∶2.5 μg, then diluted 1 000-fold and co-incubated with fruit body gill tissue pieces (4 mm of length) of Agaricus bisporus at room temperature for 100 min. The transformation solution and tissue pieces were mixed with regeneration complete medium (RCM) and plated and cultured at 25 ℃ for about 7 d. The germinating tissue pieces were transplanted on PDA plates containing hygromycin for screening and subculturing. The transformants were obtained by PCR identification of putative transformants. Results showed that the ACO-RNAi transformants had genetic stability with the mRNA expression of ACO reduced 47%~74% compared with wild type (WT) (P＜0.01), ACO activity and ethylene production reduced 68%~86% and 27%~48% compared with WT (P＜0.05), respectively, indicating that the ethylene biosynthesis pathway in the Agaricus bisporus was ACC pathway, same as higher plants. The results demonstrated that the method of liposome-mediated fruit body gill tissue transformation of Agaricus bisporus was convenient and facile, and it provides an effective molecular biology approach for genetic improvement of Agaricus bisporus.
