马铃薯质体表达载体构建及GFP基因在块茎中的瞬时表达

利用高等植物质体基因组在进化中高度保守的特点, 根据烟草质体基因组全序列设计合成引物, PCR扩增并克隆了马铃薯质体的trnI-trnA基因片段。将测序正确的trnI基因和trnA基因作为定点整合外源基因的同源重组片段, 构建成包含Prrn-gfp-aadA-TpsbA表达盒的马铃薯质体定点转化载体pBMLSIA-GFP, 酶切鉴定表明, 所构建载体符合预期设计。采用该载体对马铃薯块茎进行基因枪法转化, 结果表明, GFP基因可在质体特异性启动子Prrn及终止子TpsbA的调控下在马铃薯块茎中瞬时高量表达, 经基因枪轰击后马铃薯块茎在紫外投射仪下产生很强的绿色荧光, 在距离为6 cm, 压力1 100 psi轰击2枪的条件下产生的绿色荧光最强。马铃薯块茎可溶性蛋白SDS-PAGE电泳分析表明, GFP蛋白表达量约占总可溶性蛋白的15.4%~30.2%, 均达到了较高的表达水平。该载体对后期马铃薯质体转化体系的建立和其他功能基因导入马铃薯质体进行性状改良具有重要应用价值。

Construction of Potato Plastid Transformation Vector and Transient Expression of GFP Gene in Tuber

Plastid transformation in higher plants offers several advantages over nuclear transformation, including maternal inheritance of transgenic, lack of position effects and gene silencing, and high levels of transgenic expression, because the high ploidy level of the plastome in cells and the genes of interest are integrated into the plastome via homologous recombination. Based on the highly conservative features of trnI and trnA genes during the plastid genome evolution of higher plants, we described a distinct construction protocol of species-specific transformation vector of potato. We designed the primers and PCR-amplified the trnI-trnA targeting region from total genomic DNA of potato line of ‘Hui-2’. After sequencing and digesting the amplifed 2.7 kb fragment, which was used as homologous targeting sequences, we constructed the potato-specific plastid expression vector named pBMLSIA-GFP that carries the expression cassette of Prrn-gfp-aadA-TpsbA. Then, verified by digestion with restriction enzymes, the vector pBMLSIA-GFP was transformed into potato tubers using PDS-1000/He biolistic particle delivery system. The fluorescence upon excitation with 365 nm light 2 days after bombardment was observed, the results indicated that the transient expression of GFP gene was in high efficiency under the regulation of plastid promoter Prrn and signals TpsbA. Tubers bombarded twice at 1 100 psi pressure and a target distance of 6 cm emitted the most intensive fluorescence. The analysis results of SDS-PAGE electrophoresis showed that GFP protein in high-level expression was up to 15.4–30.2% of the total soluble protein in potato tubers. It indicated that this potato-specific plastid expression vector pBMLSIA-GFP is highly effective and desirable to be applied in traits improvement of potato via plastid genetic transformation.