摘要
利用叶绿体基因组进化中高度保守的特点,根据烟草叶绿体基因组全序列设计引物,从甘薯(Ipomoeabatatas)叶绿体基因组中克隆了2个相邻的功能基因rbcL(GenBank登录号为AY942199)和accD(GenBank登录号为AY942200),并以此作为定点整合外源基因的同源重组片段。以来自叶绿体基因组的强启动子Prrn和Rps-bA-pro分别驱动选择标记基因aadA及phaC-gfp融合基因,构建成表达盒prrn-aadA-TpsbA-ter与RpsbA-pro-phaC-gfp-RpsbA-ter,然后将这2个表达盒串联在一起克隆进甘薯叶绿体同源片段中,获得甘薯叶绿体定点整合表达载体pSC-GFP。酶切分析证明,所构建的载体符合预期设计;采用该载体对甘薯叶片进行基因枪转化,结果显示,phaC-gfp融合基因可在叶绿体特异启动子和终止子的调控下在甘薯幼嫩叶片中瞬间表达,证明构建的载体pSC-GFP可用于甘薯叶绿体转化。
Since chloroplast genomes were highly conservative in evolution,PCR primers used in amplifying chloroplast homologous fragments of sweet potato were designed based on the corresponding sequences in the tobacco chloroplast genome.Two chloroplast homologous fragments containing both rbcL and accD genes were cloned from sweet potato chloroplast genome.Sequencing analysis indicated that these two fragments were adjacent.Using these two cloned fragments as the homologous targeting fragments,the sweet potato specific chloroplast expression vector named pSC-GFP that carries both the expression cassettes of prrn-aadA-TpsbA-ter and RpsbA-pro-phaC-gfp-RpsbA-ter was constructed.The results of restriction analysis were in accord with the desired.The vector pSC-GFP was transformed into chloroplasts of sweet potato using PDS-1000/HE biolistic particle delivery system.The green fluorescence was observed in the chloroplasts of some bombarded leaves under both inverse fluorescence microscope and confocal laser scanning microscope.The results indicated that the transient expression of phaC-gfp gene in young leaves of sweet potato was in high efficiency under the regulation of chloroplast specific promoters and terminaters terminators,which indicated that the chloroplast expression vector pSC-GFP can be used in sweet potato chloroplast transformation.
出处
《西北植物学报》
CAS
CSCD
北大核心
2009年第9期1747-1755,共9页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金(30900914)
陕西省教育厅专项(09JK777)
西北大学重点实验室开放基金资助
关键词
甘薯
叶绿体同源片段
叶绿体表达载体
瞬间表达
中长链聚羟基脂肪酸酯
sweet potato
chloroplast homologous fragments
chloroplast expression vector
transient expression
medium-chain-length-polyhydroxybutyrates
