油菜叶绿体多顺反子定点整合表达载体的构建

根据拟南芥叶绿体基因组序列设计PCR引物,从我国甘蓝型油菜栽培品种F4叶绿体基因组中克隆了长度为1.5kb的trnI和trnA2个基因序列,核酸序列分析表明它们与拟南芥基因的同源性高达94%和99%,这两个克隆的油菜叶绿体基因组序列trnI和trnA被用于构建叶绿体定点整合表达载体。利用烟草叶绿体基因强启动子Prrn和终止子TpsbA,以及筛选标记基因aadA和目的基因HSA,构建了多顺反子表达盒Prrn-aadA-HSA-TpsbA,将表达盒置于油菜叶绿体trnI和trnA序列之间,最后构建成油菜叶绿体多顺反子定点整合表达载体pIPaHTA。酶切鉴定及序列分析证实,构建的表达载体具有预期的调控元件及结构,这为后期油菜叶绿体转化体系的建立奠定了基础。

Construction of A Polycistronic Expression Vector for Site-Specific Integration into Rapeseed Chloroplast

Based on the highly conserved features of plastomes in plants, two pairs of primers were designed according to the chloroplast sequences of A rabidopsis tha!iana and used for amplifying two adjacent functional genes, trnI and trnA, from rapeseed （Brassica napus） chloroplast genome by polymerase chain reaction （PCR）. Sequencing analyses revealed that the PCR-amplified trnI and trnA sequences displayed 94% and 99% similarity to that of A rabidopsis thaliana. The rapeseed chloroplast-derived sequences were then used for construction of a polycistronic expression vector, pIPaHTA. This vector contains Prm promoter and psbA at the 3＇ untraslated region from tobacco. A selection marker gene oadA and a HSA gene were constructed in a tandem manner, Prm-aadA-HSA-TpsbA, in one cassette. Restriction endonuclease digestions and sequencing analyses confirmed the construction. The constructed rapeseed chloroplast expression vector is suitable for use in rapeseed chloroplast transformation.