Abstract:

CaNDR1a and CaNDR1b , 2 Arabidopsis NDR1 homologous genes were cloned from Chenopodium amaranticolor to confirm their subcellular location, gene expresion, and analyze their resiutance to virus infection, thus providing a technological basis for transgenic disease resistance breeding. According to the previous results of transcriptome sequencing, CaNDR1a and CaNDR1b were cloned by RT\|PCR. Then their gene expression level after virus infection was analyzed through Real\|time PCR. The plant transient expression vectors were constructed, and CaNDR1a and CaNDR1b were found locating on cell membrane. The transgenic tobacco were obtained. T1 plant’s resistance to tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) by enzyme\|linked immunosorbent assay（ELISA）. Bio\|informatic analysis revealed that CaNDR1a and CaNDR1b were sharing a striking similarity with Arabidopsis NDR1. CaNDR1a and CaNDR1b were up\|regulated remarkably after Chenopodium amaranticolor inoculated by TMV and CMV. CaNDR1a and CaNDR1b were located in the cell membrane, which was not affected by virus infection. ELISA result showed that the resistance of part transgenic lines to TMV and CMV was reinforced. This study indicated that CaNDR1a and CaNDR1b were functional orthologs of Arabidopsis NDR1 and played a role in plant endogenous immune response.

Key words: NDR1 , transgenic tobacco, tobacco mosaic virus, cucumber mosaic virus

摘要：

以抗病毒植物苋色藜为材料，克隆拟南芥NDR1的同源基因，确定其亚细胞定位及该基因表达和抗病毒特性分析，为转基因抗病育种奠定技术基础。根据苋色藜转录组测序分析结果，采用RT\|PCR克隆获得两个与拟南芥同源的NDR1基因，分别命名为CaNDR1a和CaNDR1b，并通过实时定量PCR分析病毒侵染后基因的表达情况；构建植物瞬时表达载体，发现CaNDR1a和CaNDR1b定位于细胞膜；构建CaNDR1a和CaNDR1b植物表达载体，遗传转化获得转基因烟草，经酶联免疫吸附测定（ELISA）分析T1代植株对烟草花叶病毒（TMV）和黄瓜花叶病毒（CMV）的抗性。生物信息学分析揭示CaNDR1a和CaNDR1b是NDR1的同源基因。CaNDR1a和CaNDR1b在苋色藜接种TMV和CMV后显著上调表达。CaNDR1a和CaNDR1b定位于细胞膜上，并且病毒对CaNDR1a和CaNDR1b的胞内定位没有影响。ELISA结果显示部分转基因株系对TMV和CMV的抗性增加。初步表明CaNDR1a和CaNDR1b是拟南芥NDR1的功能性同源基因，参与植物对病毒的内源免疫反应。

关键词: NDR1, 转基因烟草, 烟草花叶病毒, 黄瓜花叶病毒