Resistance Test And Expression Study Of Transgenic Tobaccos With PtDRG01 Gene

PtDRG01 gene had been cloned from resistant lines of Populus tomentosa Carr., according to the conservation of NBS/LRR region of plant resistance genes. Upon bioinformatics analysis, it has been confirmed that PtDRG01 gene was highly homologous with many poplar resistance sequences. However, it is still necessary to identify the function of PtDRG01 gene. Therefore, this research was directed to the resistance test and expression study of transgenic tobaccos, which had been transformed with PtDRG01 gene, in order to identify the gene function. Through this research, it is respected to provide basis for the further resistance mechanism and genetic engineering studies of PtDRG01 gene.In this research, 23 positive transgenic tobacco lines with PtDRG01 gene which were previously obtained were inoculated with tobacco mosaic viruses (TMV) for resistance test. Observation results showed that disease severity of transgenic tobacco line TG-11 was obviously lighter than non-transgenic tobaccos one week and six weeks after inoculation. By real-time PCR, we found that quantity of virus in TG-11 was much fewer than non-transgenic tobaccos, and transcription level of gene PtDRG01 in TG-11 was significantly higher than non-transgenic tobaccos. These results revealed that PtDRG01 gene has obvious resistance function,and its efficient expression could enhance tobaccos resistance to TMV. Furthermore, change of catalyse activity and expression of PR-1 gene (encoding pathogenesis related protein) in reisistance line TG-11 after inoculation with TMV were studied. Results were as follows: after inoculation, reducing extent of catalyse activity was obviously larger than non-transgenic tobaccos and catalyse activity value in TG-11 was much lower than non-transgenic tobaccos, the reducing of catalyse activity to a large extent could indicate the increase of H2O2, thus it could be presumed that H2O2 functioned in resistance response mediated with PtDRG01 gene as a signaling molecule; besides, expression of PR-1 gene in resistance line TG-11 was distinguishly higher than nontransgenic tobaccos after inoculation, on one hand, it could indicate that PR-1 gene participated in the resistance response against TMV as a defence gene, on the other hand, since PR-1 gene was Marker gene of salisylic acid, its high expression also refered to the increase of salisylic acid, thus the role of salisylic acid as a signaling molecule in the resistance response could also be presumed. Finally, PCR detection was carried out on autocopulatory filial generation T1 of resistance line TG-11, and it proved that separation ratio was three to one, indicating the genetic stability of foreigngene(PtDRG01).This study finished the resistance identification of PtDRG01 gene, found out two signaling molecules in resistance response mediated with PtDRG01 gene,and proved the genetic stability of PtDRG01 gene in tobaccos. It could underlie further functional identification and resistance mechanism research of PtDRG01 gene in poplars. It also could provide references for other genes functional identification and resistance mechanism researches.