大豆叶绿体转化载体pJY系列的构建及其转化研究

杂交大豆是我国具自主知识产权的科研成果，具有广阔的应用前景。为解决杂交大豆制种过程中不育系易与保持系混杂的问题，我们开展了大豆叶绿体转基因的研究，目的是通过转基因的方法在不育系的细胞质中，植入筛选标记基因，以区分不育系和保持系。根据已发表的大豆叶绿体序列（GenBank X07675）设计引物，用PCR方法获得两段大豆叶绿体同源重组序列LHRR和RHRR，克隆到质粒pUC19中，并将来源于质粒pTF102的壮观霉素抗性基因aadA克隆到两段同源重组序列之间，以构建大豆叶绿体转化表达载体pJY01。 aadA基因的启动子Prrn和终止子psbA基因3’序列，分别从烟草叶绿体基因组中扩增而来。在此基础上，我们还将草丁膦抗性基因或绿色荧光蛋白与GUS的融合蛋白基因克隆到该载体上，得到pJY03与pJY04，并初步应用于烟草的叶绿体转化，获得了具壮观霉素抗性的绿色愈伤和幼苗，PCR扩增出aadA基因的条带，并在激光共聚焦扫描下检测到GFP的表达，由此证实了这一实验思路的可行性，为后期大豆叶绿体转化工作的顺利进行打下了良好的基础。

Construction of Soybean Chloroplast Transformation Vector pJY Series and Transformation Research

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.