【摘要】 PCR扩增突变的 5′ 烯醇丙酮酸莽草酸 3 磷酸合成酶 (5′ enolpyruvylshikimate 3 phosphatesynthetase ,EPSPS)cDNA全长序列 ,插入 pLitmus2 8得到 pLEPSPS ,进而通过反向PCR在EPSPS 2 35 /2 36aa之间将其打断为无功能的片段。选用人工构建的具有顺式和反式剪接功能的mini型蛋白内含子SspDnaB和RmaDnaB ,插入被打断的aroA(抗除草剂基因 ) ,构建了质粒 pLEBC、pLEBT、pLERC和 pLERT。将 4种重组质粒中的aroA In(蛋白内含子Intein插入aroA)融合基因插入 pET 32得到表达载体 pETLEBC、pETLEBT、pETLERC和 pETLERT ,IPTG诱导后 ,SDS PAGE分析显示其能在DE3 中有效表达并发生相应的蛋白剪接。将aroA cisSspDnaB和aroA cisRmaDnaB融合基因分别插入 pLYM中进一步构建成植物表达载体 ,农杆菌叶盘法转化烟草。基因组PCR分析表明融合基因整合入植物核基因组 ;RT PCR分析显示其可在高等植物细胞中成功表达。结果说明蛋白内含子基因可以转化高等真核细胞 ,蛋白剪接技术可应用于高等植物细胞 ,从而为防止植物转基因扩散提供了一条新的途径更多还原

【Abstract】 Inteins are intervening protein sequences that undergo self-excision from precursor protein with concomitant joining of flanking sequences. Here,we demonstrated intein cis-splicing in Nicotiana tabacum nuclear genomes by using artificial cis Ssp DnaB and Rma DnaB intein.We want to test whether protein splicing can occur in higher eucaryotic cell,which would play an important role in transgene containment in transgenic plants.Glyphosate-resistant Salmonella typhimurium aroA gene was divided at position 235/236 aa within EPSPS by inverse PCR from pLEPSPS. Amplified gene products with artificial cis-Ssp DnaB/Rma DnaB intein and split-Ssp DnaB/Rma DnaB intein were inserted at position 235 of EPSPS respectively to construct plasmid pLEBC,pLERC,pLEBT and pLERT.Above four aroA-In gene fusions were ligated into pET-32 to obtain E.coli expression vectors termed pETLEBC,pETLEBT,pETLERC and pETLERT.E.coli DE₃ cells containing individual recombinant plasmids described above were induced by IPTG to produce corresponding protein products.Detectable spliced EPSPS and unspliced precursor demonstrated that splicing occurred in bacteria. aroA-cis SSp DnaB and aroA-cis Rma DnaB were ligated into Agrobacterium tumefaciens binary vector pLYM. Then A.tumefaciens containing EPSPS-（cis） intein cassettes were used for leaf disk transformation in N.tabacum.Integration of aroA-In gene into plant genome was confirmed by genomic PCR analyses. To verify the expression of fusion genes at transcriptional level,RT-PCR analyses were performed and the expected products were identified.These results suggested that plant cells support expression of S.typhimurium aroA-In fusion gene in nulear genomes.Thus,we speculated the existence of protein-splicing activity in plant cells.This opens the possibility of applying intein trans-splicing technique to reduce/prevent gene transfer by way of pollen in transgenic plants.更多还原