关键词: RNA干扰, 载体构建, 反向重复, 测序

Abstract: Adjacent inverted repeats in DNA tend to form secondary structure in a single strand,which are difficult templates for sequencing. The objective of this work is to solve the difficulty in sequencing inverted repeats inserted in RNAi vectors,and thus to lay a foundation for the verification of such vectors through DNA sequencing. A routine method of molecular cloning was adopted to construct an RNAi vector expressing tandem reverse repetitive fragments of wheat TaATG2 gene. Colony PCR was used to preliminarily identify the constructed vector,and 2 sequencing strategies were designed in this study：One was to use intact plasmids as sequencing templates,and the other was to use linearized plasmids as sequencing templates in which one of the reverse repetitive fragments was removed by digestion. The results showed that the sequencing reaction of the first strategy was influenced by secondary DNA structures formed within the region of reverse repetitive fragments. The sequencing signals from the reverse repetitive fragments were very weak or displayed chaotic peaks. The second strategy eliminated the interference between the two reverse repetitive fragments. Under this strategy,clear sequencing signals and accurate sequences were obtained for each fragment retained on the vector. The two reverse repetitive fragments inserted in an RNAi vector can be separately and effectively sequenced by 2 times of digesting and sequencing under a strategy of sequencing one fragment in the vector after removing the other by digestion.

Key words: RNAi, vector construction, inverted repeats, DNA sequencing