超量表达NrCN基因提高烟草植株对TMV的抗性

Overexpression of NrCN Gene Improved the TMV Resistance in Nicotiana tabacum L.

利用农杆菌介导的遗传转化法将含有普通烟草Ubi．U4启动子驱动MrCN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T0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YLSOD、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5d时转基因植株叶片Chl含量显著高于野生型植株。接种前期（0-3d）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（3～5d）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-timePCR分析结果表明，接种TMV后3d时转基因植株删蹦、PR-1α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NNrCN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。

In this study, we transferred TMV-resistance NrCN gene drived by the Nicotiana tabacum Ubi. U4 promoter into TMV-sensitive tobacco variety K326 by using the Agrobacterium-mediated transformation meth- od. The content of chlorophyll （Chl） and malonaldehyde （MDA） and the activity of superoxide dismutase （SOD）, peroxidase （POD） and catalase （CAT） were determinated after part of transgenic tobacco plants were inoculated with TMV. There was no significant difference among these physiological indexes between the trans- genie and wild-type plants before inoculating the virus. However, the chlorophyll content became to decline in the transgenic plants and the wild-type plants after TMV-inoculation executed and the decreasing extent was so higher in the wild-type than the transgenic. Compared with the variable range of the chlorophyll content, the change of MDA content was more significant in the transgenic plants than the wild-type. In addition, the en- zyme activity of SOD, POD and CAT was analyzed among these TMV-inoculated plants. We found that there was a significant difference in enzyme activity of SOD and POD between the transgenic and wild-type tobacco plants after inoculation. Real-time PCR was executed to study the expression level of some pathogenesis-re- sponded genes, which concluded the mitogen-activated protein kinase （MAPK） gene MAPK, pathogenesis-re-lated proteins （PR） gene PR-1α and TMV-resistance N-like protein （CN） gene NrCN. The results showed that MAPK gene expression level in transgenic plants TMV-inoculated was nearly 3 folds than that in wild-type, while it was 2 folds than transgenic plants inoculated-H20. The highest expression level of NrCN gene was found in transgenic plants inoculated with TMV, which was about 9 folds higher than the wild-type and 4 folds highe than the transgenic inoculated with H20. The research results suggested that the over-expression of NrCN gene improved the disease-resistance ability in transgenic tobacco plants.