金针菇Fvpal1基因的RNAi降低PAL酶活及菌丝色素分泌  

RNAi of the Flammulina filiformis Fvpal1 gene reduces PAL enzyme activities and mycelial pigment secretion

作　　者：陆欢[1] 沈玲 刘建雨[1] 尚晓冬 王瑞娟[1] 谭琦[1] 唐桂容[1] LU Huan;SHEN Ling;LIU Jianyu;SHANG Xiaodong;WANG Ruijuan;TAN Qi;TANG Guirong(Key Laboratory of Resources and Utilization of Edible Fungi(South),Ministry of Agriculture and Rural Affairs of the People’s Republic of China,National Engineering Research Center of Edible Fungi,Shanghai Key Laboratory of Agricultural Genetics and Breeding,Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences,Shanghai 201403,China;College of Food Science&Technology,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海市农业科学院食用菌研究所,农业农村部南方食用菌资源利用重点实验室,国家食用菌工程技术研究中心,上海市农业遗传育种重点实验室,上海201403 [2]上海海洋大学食品学院,上海201306

出　　处：《菌物学报》2025年第4期81-97,共17页Mycosystema

基　　金：上海市农业科学院卓越团队建设计划(沪农科卓[2022]001);上海市农业科学院助跑计划(ZP24161)。

摘　　要：本研究构建了金针菇Flammulina filiformis苯丙氨酸解氨酶基因1(F.filiformis PAL gene1,Fvpal1)的RNAi载体,以黄色的单核体菌株0990-(5)为受体,通过遗传转化获得5个基因沉默的单核转化子(RNAi-Fvpal11–5)。5个单核转化子再分别与白色的单核体菌株Dan3进行杂交,获得5个基因沉默的双核转化子(ZRNAi-Fvpal11–5)。考察并分析了单核转化子和双核转化子在PDA培养基上的菌丝生长速度、菌丝的PAL酶活、菌丝在培养基上的色素分泌及Fvpal1基因表达量情况,以验证Fvpal1基因具有调控金针菇颜色的功能。结果显示,10个转化子的Fvpal1基因表达较野生菌株相比都显著下调(P<0.05),其中转化子RNAi-Fvpal11–5分别下调83.41%、75.92%、79.69%、66.49%和43.22%,转化子ZRNAi-Fvpal11–5分别下调80.26%、45.24%、34.09%、84.05%和79.62%;除转化子ZRNAi-Fvpal14外的9个转化子的PAL酶活力都显著低于出发菌株(P<0.05)。10个转化子的菌丝在PDA培养基上的色素分泌都比出发菌株浅,双核转化子的菌丝在木屑培养基中的颜色也显著变浅,以及双核转化子的子实体颜色也比出发菌株浅。本研究构建了金针菇Fvpal1基因的RNAi体系,发现该基因对金针菇菌丝和子实体的颜色具有正调控作用,为进一步开展金针菇Fvpal1基因的功能基因研究提供了数据支撑。RNA interference for the phenylalanine deaminase gene 1 of Flammulina filiformis(F.filiformis PAL gene1,Fvpal1)was constructed,and five gene-silenced mononuclear transformants(RNAi-Fvpal11-5)were obtained through genetic transformation by using the mononuclear strain 0990-⑤(yellow)as the receptor.The five transformants were separately crossed with monokaryotic strain Dan3(white)to obtain five gene-silenced binuclear transformants(ZRNAi-Fvpal11-5).The Fvpal1 gene expression and traits of the transformants such as mycelial growth rate in PDA medium,PAL enzyme activity,and mycelial pigment secretion were further investigated to verify that Fvpal1 gene has the function of regulating the color of F.filiformis.The results showed that Fvpal1 gene expression was significantly down-regulated in all 10 transformants as compared with that in wild strain(P<0.05).Mononuclear transformants RNAi-Fvpal11-5 were down-regulated respectively by 83.41%,75.92%,79.69%,66.49%,and 43.22%,and binuclear transformants ZRNAi-Fvpal11-5 were down-regulated respectively by 80.26%,45.24%,34.09%,84.05%,and 79.62%.All nine transformants except the binuclear transformant ZRNAi-Fvpal14 had significantly lower PAL-ase activity than the original strain 0990-⑤(P<0.05).The mycelial pigment secretion of all the 10 transformants grown on PDA medium was lighter than that of the original strain,and the colour of the binucleate transformants became lighter even more significantly on woodchip medium.The fruiting body color of the binuclear transformants is also lighter than that of the strain 0990-⑤.In this study,the RNAi system of Fvpal1 gene of F.filiformis was constructed,and it was found that this gene had a positive regulatory effect on the color of mycelium and fruiting body of F.filiformis.The results provide preliminary evidence in support of further functional gene research of Fvpal1 of F.filiformis.

关 键 词：金针菇 Fvpal1基因 RNA干扰 颜色 

分 类 号：S646.1[农业科学—蔬菜学]