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利用农杆菌介导的遗传转化法将含有普通烟草Ubi.U_4启动子驱动NrCN基因表达的元件导入TMV敏感烟草品种K326中,对筛选鉴定出的T_0代转基因植株接种TMV,测定其接种前后不同时期的理化指标,以接种TMV的野生型植株为对照。结果显示,转基因植株和野生型植株接种TMV前叶绿素(Chl)和MDA含量及SOD、POD和CAT酶活力均无显著差异,但接种TMV后野生型植株Chl含量逐渐降低,转基因植株Chl含量先增后降,在接种TMV后5 d时转基因植株叶片Chl含量显著高于野生型植株。接种前期(0~3 d)转基因植株MDA含量略低于野生型植株,但差异不显著;但接种后期(3~5 d)前者的MDA含量显著低于后者。接种TMV后转基因植株中SOD、POD及CAT酶活性变化幅度较野生型植株高,以CAT的变幅最为显著。另外,Real-time PCR分析结果表明,接种TMV后3 d时转基因植株MAPK、PR-1a及NrCN表达量均显著高于野生型植株。以上结果表明,通过转基因技术提高烟草抗病基因NrCN的表达量,能提高防御酶活性及病程相关基因的表达量,从而延缓植株感染TMV的发病时间,增强敏感植株对TMV的抗性。
Agrobacterium tumefaciens-mediated genetic transformation was used to transfer the components of NrCN gene, which contains the Ubi.U_4 promoter of common tobacco, into TMV sensitive tobacco variety K326. TMV was screened from the T_0 transgenic plants screened and identified before and after inoculation Of the physical and chemical indicators, inoculated with TMV wild-type plants as a control. The results showed that the contents of chlorophyll (Chl) and MDA and the activities of SOD, POD and CAT in transgenic plants and wild type plants were not significantly different after inoculated with TMV, but the content of Chl in wild type plants decreased gradually after TMV vaccination, and the content of Chl in transgenic plants increased first After dropping, the content of Chl in leaves of transgenic plants was significantly higher than that of wild-type plants at 5 days after inoculation of TMV. The content of MDA in the transgenic plants was slightly lower than that of the wild type plants at the early stage of inoculation (0 ~ 3 d), but the difference was not significant. However, the content of MDA in the former (3 ~ 5 d) after inoculation was significantly lower than that in the latter. After TMV vaccination, the changes of SOD, POD and CAT activities in transgenic plants were higher than wild type plants, and the amplitude of CAT was the most significant. In addition, Real-time PCR analysis showed that the expression levels of MAPK, PR-1a and NrCN in transgenic plants were significantly higher than those in wild-type plants at 3 days after TMV vaccination. The above results indicated that increasing the expression level of tobacco resistance gene NrCN by transgenic technology can increase the expression of defense enzyme activity and disease-related genes, thus delay the onset of TMV infection and enhance the resistance of susceptible plants to TMV.