Cre／lox介导剔除转双价抗虫sck＋cryIAc基因籼稻恢复系明恢86材料的选择标记基因

Cre/lox系统通过其Cre重组酶对lox序列进行切割和重新连接,介导lox序列发生特异性重组。利用重组报告基因系统Pactin-lox-hpt-lox-gusA,对Cre/lox系统在水稻(Oryza　sativa　L.)中介导转基因的剔除进行了研究。Pactin-lox-hpt-lox-gusA系统中选择标记hpt基因侧翼含两个同向lox位点,并位于水稻actin1启动子和gusA基因之间。当hpt在Cre酶作用下被剔除时,actin1启动子可以和gusA基因融合在一起从而驱动GUS表达。通过农杆菌介导获得了分别转cre基因、Pactin-lox-hpt-lox-gusA结构和双价抗虫基因lox-hpt-lox-sck-cryIAc结构的水稻。利用有性杂交方法将cre基因导入到转化lox结构的植株中。在4个转Pactin-lox-hpt-lox-gusA　T0植株×转cre　T0植株所配组合的30个杂交F1植株中,12个植株表达GUS活性,9个表现潮霉素敏感,表明hpt基因被剔除。研究进一步通过Cre/lox介导剔除转双价抗虫sck　 cryIAc基因籼稻恢复系明恢86材料基因组中的选择标记hpt基因。在9个转lox-hpt-lox-sck-cryIAc　T2代纯合植株×转creT2代纯合植株所配组合的77个杂交F1植株中,　56个植株表现潮霉素敏感。分子分析证实在这些对潮霉素敏感的植株中hpt基因已经被剔除。

Cre/lox-mediated Marker Gene Excision in Elite Indica Rice Plants Trans- formed with Genes Conferring Resistance to Lepidopteran Insects

Cre/lox -mediated gene excision in commercial rice (Oryza sativa L.) plants was studied with a recombination-reporter gene system, in which the selectable marker hygromycin phosphotransferase gene (hpt) flanking by two directly oriented lox sites was located between the rice actin1 promoter and a promoterless gusA gene. This system allows visualization of GUS expression by activating promoterless gusA after site-specific recombination. The crossing strategy was used to introduce the cre gene into the lox plants. In 30 hybrid plants from four crossesmade from T0 actin1 promoter-lox -hpt -lox -gusA plant with T0 cre plant, 12 expressed GUS and 9 showed hygromycin-sensitive. We furthermore demonstrated the utility of the Cre/lox in excision of hpt marker gene in an elite indica rice restorer Minghui 86 transformed with both insecticidal modified cowpea trypsin inhibitor gene sck and Bacillus thuringiensis endotoxin gene cryIAc. In 77 hybrid plants from nine crosses made from T2 homozygous lox -hpt -lox -sck -cryIAc plant with T2 homozygous cre plant, 56 showed hygromycin-sensitive. Molecular analyses confirmed the excision of hpt in all hygromycin-sensitive plants.