【摘要】 为解决杂交大豆(Glycinemax)制种过程中不育系易与保持系混杂的问题,开展了大豆叶绿体转基因的研究。通过转基因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(GenBankX07675)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,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(Nicotianatobacum)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。更多还原

【Abstract】 In order to eliminate the maintain-line seeds from the cytoplasm male sterile line seeds a selectable marker gene was inserted 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,LHRR and RHRR, which were inserted into target sites 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 and 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 were successfully transformed into the tobacco (Nicotiana tobacum) chloroplast. The expression of the aadA gene was detected by PCR amplification and found that the transformed seedlings conferred green fluorescent activity.更多还原