Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cott...Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.展开更多
Web MicroRNA Designer(WMD3)是一个专门应用于植物人工miRNA设计和前体序列设计的专业网站,其中利用重叠PCR法合成amiRNA前体序列,涉及多个PCR和纯化回收过程,步骤繁琐,周期较长。本研究利用WMD3设计了麻竹转录因子DlSCL6基因的人工mi...Web MicroRNA Designer(WMD3)是一个专门应用于植物人工miRNA设计和前体序列设计的专业网站,其中利用重叠PCR法合成amiRNA前体序列,涉及多个PCR和纯化回收过程,步骤繁琐,周期较长。本研究利用WMD3设计了麻竹转录因子DlSCL6基因的人工miRNA序列和其前体序列,采用改进的重叠PCR方法,快速克隆获得了该前体序列,并以改造的pCMABIA1301载体为框架,构建了该前体的植物表达载体,为进一步研究利用amiRNA调控SCL6基因的表达奠定了基础。展开更多
RNA干扰作为植物抗病毒免疫的核心机制,其调控网络中SGS3(suppressor of gene silencing)基因在RDR6介导的双链RNA合成过程中扮演关键角色。为深入探究SGS3基因功能,对本氏烟草SGS3基因引入新型多靶点基因沉默系统——PTA(poly-tRNA-ami...RNA干扰作为植物抗病毒免疫的核心机制,其调控网络中SGS3(suppressor of gene silencing)基因在RDR6介导的双链RNA合成过程中扮演关键角色。为深入探究SGS3基因功能,对本氏烟草SGS3基因引入新型多靶点基因沉默系统——PTA(poly-tRNA-amiRNA)技术,并与传统单个amiRNA进行RNAi效率的系统性比较。首先针对SGS3基因的锌指(ZF)、XS和卷曲螺旋(coiled coil,CC)等三个结构域各设计合成一个特异性amiRNA,通过分子克隆技术构建pBI121-SGS3-amiRNA1/2/3重组表达载体。利用农杆菌介导的瞬时表达系统,对三个重组载体进行功能验证,结果显示amiRNA2对SGS3基因的沉默效率显著高于其他两个靶点。基于此,采用Golden Gate克隆技术,将上述三个amiRNA与三个tRNA串联组装,成功构建PTA-SGS3表达盒,并整合至pBI121植物表达载体。通过与携带绿色荧光蛋白标记的大豆花叶病毒侵染性克隆SMV-GFP共侵染本氏烟草,系统检测PTA系统的RNAi效率及抗病毒活性。RTqPCR分析表明,相较于单一amiRNA,PTA系统可使SGS3基因沉默效率提升44.8%,显著增强靶基因沉默效果。通过GFP荧光强度定量分析及DAB组织化学染色实验,发现经PTA表达盒侵染的本氏烟草中,SMV病毒积累量较单amiRNA处理组增加50.6%。展开更多
文摘Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.
