| Plant viral diseases affect the growth and reproduction of plants seriously. Tobacco mosaic virus is a plant pathogenic virus in the Virgaviridae family and a type member of the Tobamovirus genus. Tobacco mosaic virus infects a wide range of plants, especially tobacco and other members of the Solanaceae family. Cucumber mosaic virus is a plant pathogenic virus in the Bromoviridae family Cucumovirus genus. Cucumber mosaic virus infects a variety of momocots and dicots and has a wordwide distribution. Tobacco mosaic virus and Cucumber mosaic virus are two important pathogenic viruses that cause virus diseases in plants resulting in serious loss of agricultural production every year. Due to the special structure and the special biological characteristics of viruses, the effects of traditional methods of diseases prevention is not obvious. Currently, cultivation of transgenic antiviral plant varieties through the methods of genetic engineering has become an important means of controlling plant virus diseases.Functional analysis of resistance genes is a crucial step of plant genetic engineering for diseases resistance. Non-host disease resistance is a resistance type against the vast majority of potentially pathogenic microorganisms. Non-host disease resistance is the most common form of plant disease resistance. Non-host disease resistance is stable and lasting and has important potential value in crop breeding for diesease resistance. Arabidopsis thaliana AtNHO1 is a non-host resistance gene and can be induced by flagellin and other pathogen-associated molecules. AtNHO1 encodes a glycerol kinase and adjusts the glycerol metabolism in Arabidopsis thaliana. AtNHO1 is involved in the resistance against Pseudomonas syringae and other pathogens. However, few studies about AtNHO1 and its homologous genes to plant viruses have been reported.In this study, Arabidopsis thaliana AtNHO1 homologous gene, named CaNHO1, was cloned from Chenopodium amaranticolor which is a broad-spectrum antiviral plant. We known the full length cDNA sequence of CaNHO1 through the technology of RACE and performed the nucleotide sequence analysis and the amino acid sequence analysis of CaNHO1. We analyzed the expression changes of CaNHO1 through real-time quantitative PCR technology after inoculating Tobacco mosaic virus and Cucumber mosaic virus on Chenopodium amaranticolor respectively. We obtained transgenic tobacco and tomato plants through the Agrobacterium mediated genetic transformation method. Finally, we detected the resistance of transgenic tobacco T1 generation to Tobacco mosaic virus and Cucumber mosaic virus respectively through the enzyme-linked immuno sorbent assay and tried to reveal CaNHO1 antiviral properties. The main results are as follows:1) Through nucleotide sequence analysis, we found that CaNHO1 shares a high similarity with the homologous genes from other species and we speculated that CaNHO1 encodes a glycerol kinase. Through protein sequence analysis, we found that CaNHO1 have the N-terminal and C-terminal conserved domains of FGGY carbohydrate kinase family.2) Through real-time quantitative PCR, we found that CaNHO1 was up-regulated obviously after Chenopodium amaranticolor inoculated by Tobacco mosaic virus and Cucumber mosaic virus respectively. It is indicated that CaNHO1 may play an important role in plant virus resistance.3) We constructed the pBI121-CaNHO1 plant expression vector through the In-Fusion technology and obtained 15 transgenic tobacco lines and 3 transgenic tomato lines through Agrobacterium mediated genetic transformation method.15 transgenic tobacco lines were screened by kanamycin and only 5 lines can grow normally.4) Through ELISA anti-virus detection, we found that some transgenic tobacco lines delayed the accumulation of Tobacco mosaic virus in the early stages of infection and delayed the accumulation of Cucumber mosaic virus in the late stages of infection. It is indicated that CaNHO1 renders resistant to certain plant viruses. |
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