高羊茅叶绿体表达载体构建及绿色荧光蛋白基因瞬时表达检测

 【目的】验证高羊茅叶绿体表达载体在高羊茅叶绿体中的瞬时表达情况，为今后在高羊茅叶绿体中稳定表达该载体，获得耐旱高羊茅的叶绿体转基因株系奠定基础。【方法】首先从高羊茅草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。然后将耐旱相关基因酵母海藻糖合酶基因tps1与水稻叶绿体16S rRNA基因的强启动子Prrn、烟草叶绿体基因psbA的终止子构建表达盒（Prrn-tps1-TpsbA-ter），连同草丁膦抗性基因bar表达盒、卡那霉素抗性基因nptII和绿色荧光蛋白基因gfp构建的融和基因表达盒一起克隆到定点整合同源片段之间，构建高羊茅叶绿体的稳定表达载体gTKGB。通过基因枪轰击法将gTKGB转化到高羊茅幼嫩叶片中，用激光共聚焦扫描显微镜对GFP的表达情况进行检测分析。【结果】克隆的高羊茅叶绿体基因16S-trnI-trnA-23S在NCBI登录号为：DQ490947－DQ490950。构建的叶绿体表达载体gTKGB转化高羊茅幼嫩叶片，在叶绿体中有很好的GFP表达。【结论】高羊茅叶绿体表达载体gTKGB可用于高羊茅叶绿体转化。

Construction of Tall Fescue Chloroplast Expression Vector and Transient Expression in Tall Fescue Chloroplasts by Detecting GFP

【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.