摘要
Tps1是生物保护物质海藻糖的关键合酶基因。构建含Tps1的玉米叶绿体表达载体,首先从玉米叶绿体基因组中克隆16S/trnI-trnA/23S片段,作为定点整合同源片段;然后将编码酵母海藻糖合酶基因Tps1表达盒(Prrn-Tps1-TpsbA-ter)、草丁膦抗性基因Bar表达盒(RpsbApro-Bar-RpsbAter)、卡那霉素抗性基因Npt II和绿色荧光蛋白基因Gfp构建的融和基因表达盒(Prrn-Npt II/Gfp-RrbcLter)一起克隆到定点整合同源片段之间,构建了玉米叶绿体的稳定表达载体mTKGB。通过基因枪轰击法将mTKGB转化到玉米幼嫩叶片中,培养2 d后,在激光扫描共聚焦显微镜下观测到玉米一些细胞的叶绿体中有很强的GFP绿色荧光,结果表明构建的载体可在玉米叶绿体中表达。
Tps1 encodes a key enzyme in trehalose synthesis. Maize chloroplast expression vector harboring the yeast Tps1 is constructed in this paper. Firstly,the homologous fragment 16S/trnI-trnA/23S was amplified from maize chloroplast genorne by PCR for site-specific integration. Secondly, the yeast Tpsl cassette （Prrn-Tpsl-TpsbA-ter）, phosphinothricin-resistant gene Bar cassette（RpsbApro-Bar-RpsbAter） and Npt /Gfp cassette consisting of kanarnycin-resistant gene Npt. and green fluorescent protein gene Gfp （Prrn-Npt/Gfp-RrbcLter ） were constructed respectively. Finally, the three expression cassettes were cloned into the homologous fragrnent to obtain chloroplast stable expression vector rnTKGB. Then rnTKGB was introduced into young leaves of maize through particle bombardment. Two days later, strong green fluorescence was observed in the chloroplasts of some bombarded leaves under confocal laser scanning microscope, indicating that rnTKGB could be expressed in maize chloroplasts. The results of this paper provide a strong foundation for genetic engineering drought-resistant maize through chloroplast transformation in the future.
出处
《华北农学报》
CSCD
北大核心
2008年第4期72-76,共5页
Acta Agriculturae Boreali-Sinica
基金
北京市自然科学基金项目(5062012)
国家自然科学基金项目(30400228
30600318)
