Optimization Of The System Of Virus-induced Gene Silencing And Function Analysis Of Tobacco (N. Benthamiana) DHS

With the progress in plant genomics research, the study direction is turned into the discovery and functional analysis of important genes. But an effective method is needed to investigate the biological function of cDNA sequences in large-scale. With the newly developed technique— virus-induced gene silencing (VIGS), plants infected with virus vector carrying host-derived sequence inserts will show loss-of-function or reduced-expression mutants in the host gene. The symptoms will tell the functional information of the gene. Normally the process of constructing a virus vector and monitoring symptoms on infected plants can be completed within a few weeks, such that VIGS provides a simple, rapid and high throughput means of analyzing the function of sequenced genes.In order to expend the application of VIGS widely and effectively in plants for analysis of gene function in large-scale, and to learn whether VIGS could be used in analyses of developmental gene function, we used PDS as a reporter gene to optimize the agroinfection system used in VIGS; Moreover a PVX-based VIGS approach was used to investigate the function of the Nicotiana benthamiana Deoxyhypusine Synthase (NbDHS) in this study. The following presents the main progress.1. Firstly the effects of Agrobacterium concentration, leaf age of plant seedlings and the homology of the inserts with the corresponding endogenous genes on VIGS efficiency were studied. The results showed: the Agrobacterium concentration (OD₆00=0.6-2.0) had no obvious effects on VIGS efficiency; six-leaf old seedling is the most suit for agroinfection, and when being more than six-week old the infected plants was not able to be induced VIGS; the similarity and length of the longest identical fragment of inserts with endogenous genes dramatically affected the ability to induce VIGS in infected plants, and the four Prunus dulcis cDNA fragments tested, sharing identity 82-88% with corresponding endogenous gene, were not able to induce VIGS of target genes in Nicotiana benthamiana.2. In the present study, we also obtained function-loss phenotype of Nicotiana benthamiana DHS (NbDHS) by using VIGS in N. benthamiana. The suppression of DHS resulted in pleiotropic effects in N. benthamiana. At beginning DHS silenced plants exhibited an increase in leaf biomass, loss of green in the vicinity of vein and decrease in leaf chlorophyll content. Later plants showed different silencing phenotypes. Some displayed delay in natural leaf senescence and flowering time, and others lost green in the upper leaves, and a few had a drawf phenotype. We think that the pleiotropic effects by DHS silencing in N. benthamiana are related to the extent and duration of DHS silencing in different silenced plants by VIGS system on the one hand, and on the other hand to differential functions of target protein eIF-5A isoforms. The results showed that DHS mediates several physiological processes and plays important roles in plant growth, development and senescence and has potentially important application in genetic modification of crops, woods and horticultural plants. It was also proved that VIGS coulde be used in analyses of developmental gene function.