关键词: 高羊茅, 叶绿体表达载体, 定点整合, 重叠延伸PCR法, 瞬时表达, GFP荧光

Abstract:

【Objective】 The study was made to detect transient expression of the tall fescue chloroplast expression vector in chloroplasts of tall fescue and provide a strong foundation for genetic engineering drought-resistant tall fescue through chloroplast transformation in the future. 【Method】 Firstly, the homologous fragment 16S/trnI-trnA/23S was amplified for site-specific integration from tall fescue chloroplast genome by PCR. Secondly, the yeast trehalose synthase gene tps1 was placed under the 16S rRNA promoter (Prrn) from rice and 3’ regulatory region of psbA gene from tobacco to construct tps1 cassette, which is ligated with phosphinothricin acethltransferase gene bar cassette and fused gene nptII-gfp cassette consisting of neomycin phosphotransferase gene nptII and green fluorescent protein gene gfp. Finally, the three expression cassettes were inserted into the homologous fragment to obtain tall fescue chloroplast stable expression vector gTKGB. Then vector gTKGB was introduced into young leaves of tall fescue through particle bombardment. Expression of GFP was analyzed under confocal laser scanning microscope. 【Result】 Gene accession numbers of 16S/trnI-trnA/23S cloned from tall fescue were DQ490947, DQ490948, DQ490949 and DQ490950. Chloroplast expression vector gTKGB was introduced into young leaves of tall fescue, and strong green fluorescence was observed in the chloroplasts of bombarded leaves under confocal laser scanning microscope. 【Conclusion】 Tall fescue chloroplast stable expression vector gTKGB can be used in tall fescue chloroplast transformation.

Key words: tall fescue (Festuca arundinacea Schreb.), chloroplast expression vector, site-specific integration, splicing overlap extension by PCR, transient expression, GFP