桑树miR166f的表达分析及双元超表达载体的构建

Analysis on Expression Pattern of Mulberry miR166f and Construction of Its Binary Over-Expression Vector

MicroRNA(miRNA)在生物生理生化进程、信号传导、细胞调亡、响应生物和非生物胁迫等方面发挥重要功能,本试验克隆桑树miR166f及其前体序列,分析其时空表达特性,并构建用于瞬时表达的miR166f双元超表达载体,为桑树miR166f抗旱功能机制的进一步探究奠定基础。克隆获得了桑树miR166f及其前体序列,pre-miR166f长91 nt,miR166f长21 nt,miR166f在前体的3′臂上,pre-miR166f最小折叠自由能(MEF)为-217.14 kJ/mol,具有典型的植物miRNA前体二级结构特征;miR166f的5′端上游除了包含典型的具有转录起始功能的TATA-box和CAAT-box外,还含有多个胁迫响应顺式作用元件和一些激素响应元件;qRT-PCR分析表明miR166f在桑树不同组织中的相对表达量有较大差异,其中在茎中表达量最高,在木质部中表达量最低;miR166f在高盐和干旱胁迫处理条件下均上调表达,表明该基因参与了桑树的抗逆响应过程;成功构建了用于瞬时转化表达的miR166f的双元超表达载体pCAMBIA-35S-GUS-miR166f,为桑树miR166f抗逆功能机制的进一步探究奠定基础。

MicroRNA(miRNA)plays an important role in organisms in the processes like physiological and biochemical process,signal transduction,cell apoptosis,and biotic and abiotic stress responses.Therefore,miR166f and its precursor(pre-miR166f)were cloned,its spatio-temperal expression features was analyzed,and a binary over-expression vector for transient transformation was constructed for in-depth research on drought resistance of mulberry miR166f in this study.miR166f and pre-miR166f were cloned and identified,whose length were 21 nt and 91 nt respectively.Mature miR166f was at the 3′-arm of the precursor,and pre-miR166f had a minimum energy folding(MEF)of-217.14 kJ/mol,exhibiting typical secondary structural features of plant miRNA precursor.Besides,5′upstream region of miR166f contained not only TATA-box and CAAT-box which functioned in transcription initiation,but also many cis-acting elements in response to stress and some elements in response to hormone.qRT-PCR analysis revealed that relative expression level of miR166f varied in different tissues of mulberry with the highest expression in stem and the lowest expression in xylem.Expression level of miR166f was up-regulated under drought and salt stress,indicating that this gene participated in stress response.Finally,binary overexpression vector pCAMBIA-35 S-GUS-miR166f used for transient transformation was successfully constructed,which would promote further study on functional mechanism of miR166f in mulberry response to stress.