Study On The Impact On The Host Gene Expression Patterns Invoked By Citrus Exocortis Viroid Infection

Citrus exocortis viroid (CEVd) is one of the major pathogens that causes serious damage to citrus production.The resulting damages include dwarf, stem diameter reducing, scaling,declining,flower and fruit dropping off, leading to serious economic loss to the production of citrus. In this study, microarray technique was adopted to analyze the impact on gene expression profiles of the citrus infected by a severe variant of CEVd. It is expected to get insight into the genes involved in the interaction between CEVd and its host,and build basic information for understanding its pathogenetic mechanism. The main results are as follows:1.A CEVd variant ’CEVd.01’ with virulent molecular feature was used as test material. It was mechanically inoculated into the tender shoot of Citron861grafted on trifoliate seedlings after dimerized and transcribed. The results of RT-PCR identification at post inoculation of three months showed that the trifoliate seedlings were infected successfully by CEVd, in which sample ZK1,ZK2and ZK3could be detected with the expected target fragment,while the negative control sample CK1,CK2and CK3could not. The result was confirmed by sequencing the target fragment with original CEVd.2.Samples including negative control CK1, positive control CK+and ZK1inoculated by CEVd.01were subjected to differential expression analysis after the inoculation successfully. Leaves locating in top,middle and lower parts of the seedlings were collected and mixed from each sample at three time points with an interval of31days, and shipped to a biological company for microarray analysis. The result showed that the infection of CEVd caused significant changes in the level of gene expression of the trifoliate, with higher changes is in the positive control than in the inoculated one.The up-regulated expression genes mainly involved in the related genes of plant defense signal pathways. The down-regulated expression genes mainly included the genes involved into the metabolism of saccharides (fiber, pectin, xylose et al), protein (tubulin, extensin, fasciclin et al), hormone (ethylene, gibberellin and nicotinamide), cytochrome, oxidordeuctase, peroxidase,chitinase and et al. 3. The five obviously down-regulated expression genes that related to plant growth and cell wall synthesis were selected to confirm the microarray analysis results.These genes were Glycometabolism、Pectinesterase,ERF-like protein,Nicotianamine synthase1,Polygalacturonase. As performed in microarray analysis, leaves locating in top, middle and lower parts of the seedlings were collected from each sample at three time points with an interval of31days for analyzing the gene expression with semi-quantitative RT-PCR with18sRNA as internal reference. The results revealed that ERF-like protein,nicotianamine synthase1and polygalacturonase were down-regulated expressed at the three time points; glycometabolism and pectinesterase were up-regulated expressed at the first two time points while down-regulated expressed at the third time points.The results agreed with the results of microarray analysis on the whole.