转基因甜橙定量PCR分析体系的建立

Expression of PthA-nls Gene in Sweet Orange Analyzed by Quantitative Polymerase Chain Reaction

以转pthA-nls基因甜橙45d叶龄叶片、枳橙的根、茎、叶不同器官以及非转基因枳、枸橼、温州蜜柑、沙田柚的45d叶龄叶片为试材,采SYBR GreenⅠ荧光染料法,检验不同柑橘类型以及枳橙不同器官中β-actin基因表达的稳定性,并以β-actin基因作内参基因对pthA-nls基因pthA-nls基因甜橙中的表达量进行分析。结果表明:β-actin基因在柑橘的不同类型及同一类型的不同组织中表达均较恒定,可作为内参基因用于柑橘基因的定量PCR分析;同时,试验还应用建立的方法体系分析转pthA-nls基因甜橙,基本明确各转基因株系中目的基因表达水平。

45 days old leaves from transformed sweet orange withpthA-nls gene, different organs （roots, shoots, leaves and buds） from Citrange, and 45 days old leaves from untransformed Trifoliate, Satsuma Mandarin, Citron, Pomelo, Bingtang sweet orange, Citrange were tested. The stability of expression ofβ-actin gene was identified by SYBR Green I fluorescent dye method. The results demonstrated the expression ofβ-actin gene in different varieties and tissues of citrus in a constant states, β-actin gene can be used as reference gene for quantitative PCR analysis in citrus and the relative expression level of pthA-nls gene in different transgenic sweet orange was detected with β-actin as reference gene.