Site-independently integrated transgenes in the elite restorer rice line Minghui 63 allow removal of a selectable marker from the gene of interest by self-segregation

In this study, we have demonstrated that two independent loci are involved in the integration of the insecticidal protein gene cryIAb/cryIAc and selectable marker gene hph in the recipient genome of the elite Chinese CMS restorer line Minghui 63. We have also documented the structural organization of these transgenes in each locus by restriction enzyme digestion and Southern blot analysis. The independent locus integration of different transgenes allowed us to remove the selectable marker gene hph from the gene of interest simply by self-segregation. Not having the selectable marker gene will enhance the commercial value of our transgenic line TT51-1, which showed a consistently high level of resistance against repeated infestations of yellow stem borers and natural outbreaks of leaf-folders, without a reduction in yield potential.     

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(Oryzasativa 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actinl启动子和gusA基因之间.当hpt在Cre酶作用下被剔除时,actinl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-cryIAcT2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。     

cryIAc基因在转基因玉米中的遗传规律及对抗虫性影响

旨在研究目的基因在转基因植株和后代植株(株系)中的遗传规律及其对转化植株抗虫性的影响。以花粉介导法将cryIAc基因导入玉米自交系‘郑58’和‘昌7-2’,对转化植株及其后代株系进行分子检测和田间抗虫鉴定。结果表明:(1)转化‘郑58’和‘昌7-2’,T1代分别获得转基因植株24和41个,转化率高达20%以上;(2)转基因T2代、杂交F2代及回交1代(B1)的分子检测结果证明,外源基因的遗传符合孟德尔的3∶1、3∶1和1∶1的遗传分离规律;(3)连续多带的分子检测结果还表明,外源基因可稳定遗传并有效表达,表达水平在9.8-14.3 ng/g叶片鲜重之间;(4)抗虫鉴定结果显示,在阴性对照全部感虫情形下,转基因纯合株系仍表现出较高抗虫活性;(5)此外,回交试验结果还证明外源基因通过杂交可传递给下一代;(6)最终经筛选得到SZ003、SZ005、SC001、SC004和SC007五个高抗虫转基因株系。结果表明,花粉介导法是一种高效、快捷的转化方法,cryIAc基因导入玉米自交系植株后赋予和提高了转基因植株的抗虫活性。