水稻叶绿体基因组定点表达载体的构建及增强型绿色荧光蛋白原核表达分析

Construction of Rice Chloroplast-specific Expression Vectors and Detection on Prokaryotic Expression by EGFP

分别克隆了水稻叶绿体基因组同源重组片段trnI和trnA、具有3种不同核糖体结合位点(RBS)序列的增强型绿色荧光蛋白基因(egfp)、来自烟草叶绿体的16S rRNA基因启动子Prrn和psbA基因终止子TpsbA,将上述元件通过酶切位点依次连接到质粒pUC19上,构建了3种能编码增强型绿色荧光蛋白(EGFP)的水稻叶绿体基因组定点整合表达载体pIA-EGFP1、pIA-EGFP2和pIA-EGFP3。进行分子检测验证后,对上述载体进行了大肠杆菌EGFP原核表达检测,结果携带来源于λ噬菌体T7基因10的5'端非翻译区的载体pIA-EGFP3荧光最强,适合用于水稻叶绿体转化。

The elements for rice chloroplast-specific expression vectors were cloned.The fragments of TrnI and TrnA were obtained by PCR from rice,with 1 388 bp and 824 bp in length,respectively,by sequence analysis.Three enhanced green fluorescent protein genes(egfp) with different ribosome binding sites(RBS) were amplified by PCR from pEGFP-N2 plasmid as a template.The promoter Prrn and the terminator TpsbA were from tobacco and were cloned by PCR amplification of pLM21.Three rice chloroplast-specific expression vectors,named as pIA-EGFP1、pIA-EGFP2 and pIA-EGFP3,were constructed and a molecular analysis was conducted.The prokaryotic expression of vectors was tested in E.coli.The vector pIA-EGFP3 with RBS from 5′UTR of λ bacteriophage T7 gene 10 expressed the strongest enhanced green fluorescent protein,indicating that pIA-EGFP3 is a suitable vector for rice chloroplast transformation.