| Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing phenomenon discovered in recent years. With the forthcoming of post-genomics era, VIGS, as a fast, high-throughput method for gene’s functional research, has been applied in Solanaceae plants. PVX and TRV vectors has been used for gene silencing in potato, but there are some difficulties in application, such as potato genotypes, material status and environmental conditions which show greater effects on the gene silencing. In this study, tobacco rattle virus (TRV) was used as gene silencing vector. An ideal potato TRV mediated VIGS silencing system were established through screening of different genotypes and optimization of inoculation and environmental conditions. All this laid a technology platform for rapid functional identification of potato late blight resistance related genes. The results obtained are as follows:1. The potato PDS gene is successfully silenced in tobacco under the condition of temperature at 20-24℃, relative humidity 75%. Three ideal genotypes (AC285, AC079 and AC029) were identified from 131 potato genotypes based on tobacco VIGS system. The three different potato genotypes have no significant immunity after inoculation of TRV-PDS virus vector. The leaves showed different degrees of bleaching symptoms. The symptoms are relatively mild, means that three potato genotypes can be used for VIGS system.2. Leaf infiltration method is the most common method in TRV vector inoculation. In this study, stem injection method were used to inoculation potato plants and its gene silencing efficiency was compared with the leaf infiltration method. The results revealed that the stem injection method is better than the leaf infiltration method.3. The temperature and humidity are important factors which affects silencing efficiency of VIGS. Three different temperatures (5-9℃,20-24℃,28-30℃) and two different humidity (30% and 75%) were used in present experiments. The results demonstrated that bleaching symptom of the potato leaves were very clear at the temperature of 20-24℃and humidity of 75%. Under the condition of temperature at 20-24℃, humidity 30%, the plants has no bleaching symptom in 2 months; as well as plants grown in 5-9℃, humidity of 75% or 5-9℃, humidity 30% TRV-PDS and virus inoculation after 2 months there have no bleaching plants. When the plants grown under temperature at 26-30℃and humidity of 75% or 26-30℃and humidity 30%, the plants showed bleaching symptom 1 month after inoculation. But from then on, the bleaching symptom has no expansion, and the leaves gradually return to green.4. The in vitro plantlets, cultured for 20 days,30 days and 40 days in medium were planted in plastic pots filled with soil in greenhouse. Three weeks later, plants were inoculated by TRV-PDS virus vector. The results showed that up of plants from 20 days old in vitro plantlets showed necrotic; the leaves of plants from 30 days old in vitro plantlets showed photobleaching symptoms and expanded gradually; while just few leaves of plants from 40 days old in vitro plantlets showed partial photobleaching symptoms. One week later, the bleaching symptom disappeared.5. Same age seedlings and plantlets in vitro were planted in plastic pots filled with soil in greenhouse. Plants were inoculated with TRV-PDS virus vector three week after planting. Two months later, bleaching symptom still appeared on new leaves of plants from plantlets. Some new leaves of plants from seedlings shown bleaching symptom one month after inoculation. But two months later, new leaves stopped whitening, and the original bleaching symptom disappeared. These results suggested that plants grown from plantlets in vitro are more likely to silence gene, and the bleaching duration is more durable than seedlings.6. Based on the established gene silencing system,30 EST fragments were selected for VIGS. The results showed that 10 genes were silenced in different degree. After then, leaves of gene silenced plants were inoculated by P. infestans. The average lesion areas on the leaves silenced gene of W4, W6 and 08-H02 were bigger than the control. While average lesion areas on the leaves silenced gene of W2, W3,10-A04,05-E05,06-A01, 05-F11 and 08-E10W4 were smaller than that of control. From above results we can speculate that W2, W3,10-A04,05-E05,06-A01,05-F11,08-E10 genes may play a negative regulatory role in potato late blight resistance, while W4, W6,08-H02 genes play a positive regulatory role. |
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