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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。
The purpose of this study was to establish an efficient Agaricus bisporus genetic transformation method and to construct the 1-aminocyclopropane-1-carboxylic acid oxidase gene oxidase, ACO) RNAi mutant strains to study the function of the gene to explore Agaricus bisporus ethylene biosynthesis pathway. The double-stranded RNA expression plasmid p BHg-ds ACO of the liposome and constructed Agaricus bisporus ACO partial coding region was diluted 1 000 times with 1 μL: 2.5 μg and mixed with the bacterial pleat of the Agaricus bisporus fruiting bodies Tissue pieces (about 4 mm in length) were co-cultured at room temperature for 100 min and then cultured in regeneration complete medium (RCM) plates at 25 ° C for about 7 days. The germinated tissue pieces were picked and transplanted in a medium containing hygromycin PDA plates were selected for screening and subculture, and then identified by PCR to obtain transformants. The results showed that the RNAi transformants of ACO gene of Agaricus bisporus was genetically stable, the ACO gene expression was reduced by 47% -74% (P <0.01), the activity of ACO enzyme and ethylene production were reduced by 68% -86% and 27%, respectively % ~ 48% (P <0.05), indicating that Agaricus bisporus has the same ACC pathway for the synthesis of ethylene as the higher plants. The results of this study suggest that liposome-mediated transformation of Agaricus bisporus simple and efficient method for the genetic improvement of Agaricus bisporus provides a new means of efficient molecular biology.