Construction And Genetic Transofrmation Of The Rice Chloroplast-specific Expression Vector

The chloroplast transformation approach offers a number of unique advantages. But now the extension of plastid transformation technology to monocotyledonous cereal crops including rice is a little difficult. Here, All elements for rice chloroplast-specific expression vector were cloned. The fragments of TrnI and TrnA were obtained by PCR from rice, with 1388bp and 824bp in length respectively by sequence analysis.3 enhanced green fluorescent protein genes(egfp) with different ribosome binding site(RBS) were amplified by PCR from pEGFP-N2 plasmid as a template. The promoter Prrn and the terminator TpsbA were from tobacco chloroplast and were cloned by PCR amplification of pLM21. 3 rice chloroplast-specific expression vectors, named as pIA-EGFPl,pIA-EGFP2 and pIA-EGFP3, were constructed and molecular analysis were be taken. Prokaryotic expression of vectors was tested in E.coli. The vector pIA-EGFP3 with RBS from 5'UTR ofλbacteriophage T7 gene 10 expressed the strongest enhanced green fluorescent protein(EGFP) Then we introduced the hygromycin phosphotransferase gene(hpt) into the pIA-EGFP3, constructing a new vector named pIA-EGFP3-HPT. Moreover, obvious transient egfp expression detected in onion (AIlium cepa) epidermis cell by microprojectile bombardment with vector pIA-EGFP3-HPT. The results showed that pIA-EGFP3-HPT is suitable vector for rice chloroplast transformation. Considering that there is a high conservation between rice chloroplast genome and the tobacco(Nicotiana tabacum) chloroplast genome and a well-developed system of tobacco chloroplast tansformation, We attempted to transformed the vector pIA-EGFP3-HPT into the tobacco chloroplast.