《植物生理学报》 2014, 50(4): 439-446

超量表达NrCN基因提高烟草植株对TMV的抗性

秦利军^1,2,3,*, 孙洪荣^2,3,4,*, 赵丹^2,3,4,*, 赵杰宏⁵, 赵德刚^1,2,3,4,**

贵州大学¹绿色农药与农业生物工程国家重点实验室培育基地, ²贵州省农业生物工程重点实验室, ³山地植物资源保护与种质创新省部共建教育部重点实验室, ⁴生命科学学院, 贵阳550025; ⁵贵州省烟草科学研究院行业烟草分子遗传重点实验室, 贵阳550081

通信作者：秦利军；E-mail: dgzhao@gzu.edu.cn；Tel: 0851-3863615

摘　要：

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。

关键词：TMV抗性; NrCN; 防御酶活性; 烟草

收稿：2013-11-18   修定：2014-02-27

资助：中国烟草总公司贵州省公司科技计划项目“基因组学指导的烟草定向突变育种新技术研究及应用” (201201)、中国烟草总公司贵州省公司科技项目“抗TMV、富钾转基因烟草育种研究” (合同号200910)和贵州省科学技术基金“去甲基烟碱生物合成调控分子机制研究” (黔科合J字[2011]2161)。* 同等贡献。

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

QIN Li-Jun^1,2,3,*, SUN Hong-Rong^2,3,4,*, ZHAO Dan^2,3,4,*, ZHAO Jie-Hong⁵, ZHAO De-Gang^1,2,3,4,**

¹The State Key Lab of Green Pesticide and Agricultural Biological Engineering, ²Guizhou Key Lab of Agro-Bioengineering, ³Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region, Ministry of Education, ⁴College of Life Science, Guizhou University, Guiyang 550025, China; ⁵The Key Laboratory of Molecular-Genetics on Industrial Tobacco, Research Institution of Tobacco Sciences in Guizhou Province, Guiyang 550081, China

Corresponding author: QIN Li-Jun; E-mail: dgzhao@gzu.edu.cn; Tel: 0851-3863615

Abstract:

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 method. 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 transgenic 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 enzyme 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-responded genes, which concluded the mitogen-activated protein kinase (MAPK) gene MAPK, pathogenesis-related proteins (PR) gene PR-1a 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-H2O. 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 H2O. The research results suggested that the over-expression of NrCN gene improved the disease-resistance ability in transgenic tobacco plants.

Key words: TMV-resistance; NrCN gene; defense enzymes; Nicotiana tabacum