转基因枳橙中rolABC基因荧光定量表达分析方法的建立  

Establishment of fluorescence quantitative RT-PCR assay on rolABC genes in transgenic Troyer citrange

作　　者：袁飞荣[1,2,3] 严佳文[1,2,3] 罗坤[1,2,3] Alessandra Gentile 王晓君[3] 刘东波[1,3] 邓子牛[1,2,3] 

机构地区：[1]湖南农业大学园艺园林学院,湖南长沙410128 [2]国家柑橘改良中心长沙分中心,湖南长沙410128 [3]湖南省作物种质创新与资源利用重点实验室,湖南长沙410128 [4]卡塔尼亚大学园艺与农产品加工系,意大利卡塔尼亚95123

出　　处：《湖南农业大学学报（自然科学版）》2010年第6期634-639,共6页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：国家科技支撑计划项目(2008BADB7B02)

摘　　要：以转rolABC基因枳橙B、D、E系及野生植株为材料,制备标准品,采用SYBR Green I染料,构建rol基因和β-actin基因的双标准曲线,校正引物扩增效率后,用2-△△Ct法对rol基因在嫩茎、嫩叶、功能叶、树皮和根中的mRNA水平进行相对定量,建立适合于rolA、rolB、rolC基因实时荧光定量RT-PCR分析方法.初步认为rolC基因在嫩茎、嫩叶、功能叶和树皮中表达量最高;其次是rolA基因,主要在嫩茎中表达;rolB基因表达量最低,主要在根系中表达.B,D,E transgenic clones with rolABC genes and wildtype plants were used as materials to construct binary standard curves of rol genes and β-actin gene with SYBR Green I fluorescent dye.After normalizing the influence of amplification efficiency with different primers in qRT-PCR,comparative Delta-delta Ct method was applied to develop suitable qRT-PCR method for analysising the expression of rolA,rolB,rolC genes in 5 tested tissues of B,D,E clones.The primary result showed that rolC expressed highest among 3 rol genes in tender stem,tender leaf,functional leaf and bark,rolA gene＇s expression in the middle level and mainly in tender stem,and rolB expressed lowest and mainly in root.

关 键 词：实时荧光定量RT-PCR rolABC基因 特洛亚枳橙 相对表达量 基因表达 

分 类 号：Q78[生物学—分子生物学]