Abstract：Hybrid soybean is one of the most significant scientific achievements that Chinese scientists are holding the intellectual property. While the invention improved the soybean yield greatly, the difficulty to eliminate the maintain-line seeds from the cytoplasm male sterile line seeds is often a problem limiting the application of the new technology. To solve the problem, we initiated the research. The purpose of the research is to insert a selectable marker gene into the cytoplasmic genome of the male sterile line through chloroplast transformation. Based on the published soybean chloroplast genome sequence (GenBank X07675), two pairs of primers were synthesized to amplify two adjacent soybean plastome sequences allowing its targeted insertion between the trnV gene and the rps12/7 operon of soybean plastid. Within the targeting region, an aadA gene encoding the Aminoglycoside Adenylyltransferase was inserted . The gene was flanked by the rrn promoter and the 3’ sequence of psbA gene. The constructed vector was designated as pJY01. Expression cassettes of bar gene and GFP-GUS fusion gene were then inserted into the pJY01 vector to construct two new vectors: pJY03 and pJY04, which we successfully transformed in the tobacco chloroplast. We detected the expression of the aadA gene by PCR amplification and found that the transformed seedlings conferred green fluorescent activity. This research build a solid base for the future soybean chloroplast transformation.
