柱状田头菇遗传转化体系启动子的筛选  

Screening Promoters for Genetic Transformation of Cyclocybe aegerita

作　　者：崔祥华 陶南[2] 程波普 赵永昌[2] 陈卫民[2] 李靖 CUI Xiang-hua;TAO Nan;CHENG Bo-pu;ZHAO Yong-chang;CHEN Wei-min;LI Jing(College of Life Sciences,Southwest Forestry University,Kunming 650224;Biotechnology and Germplasm Resources Institute,Yunnan Academy of Agricultural Sciences,Kunming 650223)

机构地区：[1]西南林业大学生命科学学院,昆明650224 [2]云南省农业科学院生物技术与种质资源研究所,昆明650223

出　　处：《生物技术通报》2021年第5期259-266,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31960017);现代农业(食用菌)产业技术体系项目(CARS20)。

摘　　要：柱状田头菇(茶树菇)Cyclocybe aegerita是一种美味的食用菌,具有很高的经济价值。构建高效表达的遗传转化体系,可以深入揭示其遗传特征及基因功能。以柱状田头菇YSG为实验材料,使用多片段重组克隆构建载体,PEG介导的原生质体转化,并以qRT-PCR为测定方法,对actin、gpd和Pumgpd不同长度5个启动子片段驱动靶标基因的表达量进行分析。依据actin基因启动子构建的载体pAa-actin-1和pAa-actin-2获得转化子中,靶标基因表达量为对照3倍以上转化子数量分别为50%和33.33%,最高表达量分别为对照的10.45和6.23倍;携带pAa-gpd-1、pAa-gpd-2和pAa-Pumgpd启动子的载体获得的转化子中,表达量为对照3倍以上的数量分别为0、28.57%和25%,最高表达量分别为对照的2.93、7.75和4.31倍。actin启动子获得高表达转化子得率较gpd和Pumgpd启动子片段高,适用于柱状田头菇转化体系构建。Cyclocybe aegerita is a delicious fungus with high economic value.The genetic characterization and gene function of C.aegerita can be uncovered through the construction of highly expressed genetic transformation system.C.aegerita strain YSG was employed in this study,and the multi-fragments recombination and cloning were used to construct a plasmid,and protoplast was transformed by PEG mediation.The expression levels of targeting gene driving by 5 varied-length promoter fragments of actin,gpd and Pumgpd were analyzed via qRT-PCR.Among the transformants carrying plasmid pAa-actin-1 and pAa-actin-2 constructed by promoter elements of actin,the numbers of the transformants with the targeting gene expression level more than 3 times were 50.00%and 33.33%respectively,with the highest expressions to 10.45 and 6.23 times of the control,respectively.Accordingly,the numbers of the transformants carrying pAa-gpd-1,pAa-gpd-2 and pAa-Pumgpd with expression level more than 3 times were 0,28.57%and 25.00%with the highest expression levels to 2.93,7.75 and 4.31 times of the control,respectively.In conclusion,the yield of transformants with high expression level of targeting gene driven by actin promoter is higher than that by gpd and Pumgpd,indicating that actin promoter is suitable for the construction of genetic transformation system of C.aegerita.

关 键 词：柱状田头菇 启动子筛选 遗传转化 载体构建 过量表达 

分 类 号：S646[农业科学—蔬菜学]