摘要
本研究旨在构建1个适用于农杆菌介导真菌遗传转化(ATMT)的RNA干扰载体pCamhybgfp4,为系统研究植物病原真菌基因功能提供便利。通过基因工程及分子生物学技术,将pSilent-1载体的RNAi发夹结构与潮霉素抗性基因(HygR)整合到能被根癌农杆菌识别的T-DNA序列中。以eGFP为靶基因,构建eGFP RNAi载体,并将eGFP RNAi载体转入根癌农杆菌LBA4404,通过ATMT方法,获得eGFP RNAi菌株。绿色荧光检测和定量PCR分析结果显示,eGFP RNAi菌株eGFP无表达或表达量明显降低,表明RNA干扰载体pCamhybgfp4适用于ATMT介导的苹果炭疽叶枯病菌遗传转化。RNAi载体的成功构建为进一步通过RNA干扰技术研究苹果炭疽叶枯病菌致病机制研究奠定了基础。
This study is aimed to construct one RNA interference(RNAi)vector pCamhybgfp4 that applies to the genetic transformation of the fungus by ATMT,to facilitate the research in the gene function of plant pathogenic fungi.RNAi hairpin structures of pSilent-1 vector and hygromycin resistance gene(HygR)were integrated into the T-DNA sequence recognized by Agrobacterium tumefaciens,using biology and genetic engineering theory and technology.The eGFP RNAi vector was constructed with eGFP as the target gene,transferred into the Agrobacterium tumefaciens LBA4404.We obtained the eGFP RNAi strain using the ATMT method.The result of green fluorescence detection and quantitative PCR analysis showed that the eGFP is not expressed or that the expression level of the eGFP is significantly decreased in RNAi strain,indicating that RNAi vector pCamhybgfp4 was suitable for ATMT-mediated genetic transformation of Colletotrichum gloeosporioides.Successful construction of RNAi vector will lay a foundation for further research on the pathogenic mechanism of C.gloeosporioides by using RNA interference technology.
作者
徐杰
王美玉
王娜
周宗山
张俊祥
Xu Jie;Wang Meiyu;Wang Na;Zhou Zongshan;Zhang Junxiang(Key Laboratory of Horticulture Crops Germplasm Resources Utilization,Ministry of Agriculture,Xingcheng,125100;Research Institute of Pomology,Chinese Academy of Agricultural Sciences,Xingcheng,125100)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2020年第9期4053-4057,共5页
Genomics and Applied Biology
基金
中央级公益性科研院所基本科研业务费专项资金(Y2019PT12)
中国农业科学院科技创新工程(CAASASTIP)共同资助
作者简介
通信作者:张俊祥,zhangjunxiang@caas.cn
