脂质体介导遗传转化双孢蘑菇菌褶及1-氨基环丙烷-1-羧酸氧化酶基因(ACO)功能初探  

Liposome- mediated Fruit Body Gill Tissue Transformation of Agaricus bisporus and Function Exploration of 1-aminocyclopropane-1-carboxylic Acid Oxidase Gene(ACO)

作　　者：张岩[1] 张朝辉[1] 刘冬忍[1] 王小飞[1] 王景冒 邱立友[1] 

机构地区：[1]河南农业大学生命科学学院/农业部农业微生物酶工程重点实验室,郑州450002

出　　处：《农业生物技术学报》2015年第8期1112-1120,共9页Journal of Agricultural Biotechnology

基　　金：河南省教育厅重点项目(No.13A180483)

摘　　要：本研究旨在建立一种高效的双孢蘑菇（Agaricus bisporus）遗传转化方法,并以此方法构建双孢蘑菇1-氨基环丙烷-1-羧酸氧化酶基因（1-aminocyclopropane-1-carboxylic acid oxidase,ACO）的RNAi突变株,研究该基因的功能,探索双孢蘑菇的乙烯生物合成途径。将脂质体和构建的双孢蘑菇ACO部分编码区序列的双链RNA表达质粒p BHg-ds ACO按1μL∶2.5μg混匀后稀释1 000倍,与幼嫩双孢蘑菇子实体的菌褶组织细块（长约4 mm）在室温下共培养100 min,然后在再生培养基（regeneration complete medium,RCM）平板中25℃培养7 d左右,挑取萌发的组织块移植于含潮霉素的PDA平板中进行筛选和继代培养,再经PCR鉴定获得转化子。结果表明,构建的双孢蘑菇ACO基因RNAi转化子遗传稳定,ACO基因表达量比出发菌株降低47%~74%（P〈0.01）,ACO酶活力和乙烯产量分别降低68%~86%和27%~48%（P〈0.05）,表明双孢蘑菇具有和高等植物相同的合成乙烯的ACC途径。本研究结果提示,脂质体介导转化双孢蘑菇菌褶方法简便高效,为双孢蘑菇的遗传改良提供了新的高效分子生物学手段。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） ofAgaricus 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 reduced68%-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 ofAgaricus bisporus.

关 键 词：脂质体介导转化 双孢蘑菇 菌褶 RNA干扰(RNAI) 1-氨基环丙烷-1-羧酸氧化酶基因(ACO) 

分 类 号：S513.03[农业科学—作物学]