Hu Lei, Li Hao, Qin Ruiying, Xu Rongfang, Li Juan, Li Li, Wei Pengcheng, Yang Jianbo
Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, 230031, China.
School of Agronomy, Anhui Agricultural University, Hefei, 230036, China.
Sci Rep. 2016 May 13;6:25921. doi: 10.1038/srep25921.
The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering.
大肠杆菌磷酸甘露糖异构酶（EcPMI）基因在基于甘露糖（Man）选择的植物转化中被广泛用作选择标记基因（SMG）。尽管一些植物物种表现出显著的PMI活性，甚至在对甘露糖敏感的植物中也鉴定出了活性PMI，但植物PMI是否可用作选择标记基因仍不清楚。在本研究中，我们从可变小球藻和水稻中分离出四个新的PMI基因。在体外检测了它们的同工酶活性，并与EcPMI的活性进行了比较。将活性植物PMI分别构建到二元载体中作为选择标记基因，然后通过农杆菌转化到水稻中。在籼稻和粳稻亚种中，我们的结果表明，植物PMI能够以与EcPMI相似的方式筛选并产生转基因植物。转基因植物表现出植物PMI转录本的积累和体内PMI活性的增强。此外，利用植物PMI作为选择标记基因，成功地将一个目的基因转化到水稻中。因此，从植物中分离出了用于甘露糖选择的新型选择标记基因，我们的分析表明，编码活性酶的PMI在植物中可能很常见，并有可能在顺式基因工程中用作合适的遗传元件。
