Jia Hongge, Pang Yongqi, Chen Xiaoying, Fang Rongxiang
National Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, 100080 Beijing, China.
Transgenic Res. 2006 Jun;15(3):375-84. doi: 10.1007/s11248-006-0011-6.
Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus-host plant systems.
选择标记在植物转化过程中通常是不可或缺的，但一旦建立了转基因植物，它们就不再必要。Cre/lox位点特异性重组系统已被用于从转基因植物中消除选择标记基因。在此，我们描述了使用一种移动功能改进的烟草花叶病毒（TMV）载体m30B来表达Cre重组酶，以从转基因烟草植物中消除选择标记基因nptII。转基因烟草植物是通过农杆菌介导的转化产生的，使用了一种专门设计的二元载体pGNG，其T-DNA区域包含一个序列复合体：35S启动子-lox-绿色荧光蛋白（gfp）编码序列-红细胞生成素结合蛋白S（rbcS）终止子-胭脂碱合成酶（Nos）启动子-nptII-Nos终止子-lox-β-葡萄糖醛酸酶（gus）编码区-Nos终止子。通过将病毒载体置于35S启动子的控制下并进行农杆菌接种，实现了重组病毒载体m30B:Cre在植物细胞中的表达。在用农杆菌35S-m30B:Cre与pGNG叶盘共培养，然后在不进行任何选择的情况下进行芽再生后，通过对再生植株的组织化学GUS分析发现，获得了不含包括nptII在内的lox侧翼序列的植株，效率约为34%。通过Southern印迹分析，来自两个含有单拷贝pGNG T-DNA的独立转基因系的11个GUS表达再生植株中有3个被证明不含lox侧翼序列。这三株植物中lox侧翼序列的切除可归因于共培养早期病毒载体中Cre的瞬时表达，因为在植物中积累的病毒RNA分子及其基因组DNA中均未检测到cre序列。无标记的亲本基因型在其自交后代中得以遗传，并且所有后代均无病毒，显然是因为TMV不通过种子传播。因此，基于TMV的载体表达Cre可用于在不进行有性杂交和分离的情况下从转基因烟草植物中消除选择标记基因，并且该策略可扩展到其他受TMV感染的植物物种，并适用于其他兼容的病毒-宿主植物系统。
