番茄ARF4基因果实特异RNAi载体的构建及遗传转化

Construction and Genetic Transformation of Tomato ARF4 RNA Interference Expression Vector with Fruit Specific Promoter

构建ARF4基因果实特异表达的RNA干涉载体,对转基因番茄果实进行初步分析,可为采用基因工程方法改良番茄果实品质做出新尝试.利用RT-PCR技术从番茄果实cDNA中扩增SlARF4基因全长,将番茄ARF4基因正反向重复序列片段导入到植物表达载体pBI121上,启动子是番茄果实特异表达的TFM7.将构建的ARF4基因果实特异RNA干涉载体pBI121-TFM7-A4Ri通过根癌农杆菌介导转入到野生型番茄中,进而对转化获得的植株进行了阳性鉴定.分别以转基因番茄和野生型番茄为材料,分析ARF4在果实中的表达水平,测定绿熟期果实叶绿素含量、果实的单果重量和果皮厚度.酶切证实pBI121-TFM7-A4Ri果实特异表达载体构建成功,而且,PCR检测也得到阳性转基因株.半定量RT-PCR分析显示,转基因番茄果实中ARF4的表达量明显低于野生型果实.转基因番茄果实的叶绿素含量、单果重量和果皮厚度都比野生型有提高.因此,ARF4果实特异表达的RNAi方法能够改良番茄果实品质.

SlARF4 RNA interference vector expressing only in tomato fruits was constructed to investigate its effect on fruit characteristics of tomato.The full-length cDNA sequence of SlARF4 gene was amplified by RT-PCR and a plant RNAi expression vector harboring inversely repeated sequence of ARF4 driven by a fruit-specific promoter TFM7 was constructed,and transferred into tomato plants through a reproducible Agrobacterium-mediated transform method.Double digest assay confirmed the expression vector pBI121-TFM7-A4Ri was successfully constructed.The transgenic plants were obtained through PCR assay.The analysis of semi-quantitative RT-PCR showed significant reduction of ARF4 expression levels in transgenic tomatoes.The content of chlorophyll in green mature fruit,the weight per fruit and pericarp thickness of the transgenic lines were higher than those of the control plants.These results suggested that fruit-specific expression of SlARF4 interference in tomato could improve tomato fruit quality.