无选择标记的植物表达载体的构建

以双元载体pBINPLUS为基础，在T-DNA侧翼区通过两次特异PCR、酶切和连接相结合的方法,构建了一个无标记载体pBINMF (marker-free vector)。通过酶切后测序分析，这个无标记载体在T-DNA左、右边界之间只有一个多克隆位点 (multiple cloned sites, MCS)。为了验证该载体的遗传稳定性，将gus基因克隆到该载体上并通过农杆菌介导的方法转化番茄栽培品种Moneymaker，特异PCR扩增目的基因和GUS组织染色结果表明，gus基因已经整合进入番茄基因组并得以表达。该载体为今后直接获得无标记基因、生物安全的转化体，尤其为象马铃薯、木薯等无性繁殖材料的无标记基因转化提供了可靠、有效的工具。

The Construction of a Binary Vector for Marker-free Transformation in Plants

A marker-free vector (pBINMF) derived from the binary vector pBINPLUS was generated, which only contained a multiple cloned site between left and right borders as validated by restriction analysis and sequencing. In order to identify the efficiency of pBINMF for transformation, the gus gene was cloned into the vector and was introduced into the tomato cultivar Moneymaker via Agrobacterium tumefaciens-mediated transformation. Gene-specific PCR and GUS stain assay showed that gus gene was integrated into the tomato genome and was expressed. This vector could become a reliable and efficient tool for direct generating marker-free transformants in plants, particularly useful for vegetatively propagated crops like potato and cassava.