Suppression Of RNA Silencing By P25 In Agrobacterium Inflitration System And Antiserum Preparation Of P25

RNA silencing can operate as a defense mechanism against virus infection involved in degradation of exogenous nucleic acid via sequence- specific interactions. Numerous plant viruses encode silencing suppressors, which are thought to have developed a response to RNA silencing. In principle, the Agrobacterium infiltration system can be applied on an industrial scale. However, the level of transgene expression usually peaks at 60–72 h post-infiltration and declines rapidly thereafter. In fact, RNA silencing is the key cause of this. Therefore, the protein was coexpressed with suppressor in order to acquire more useful protein. In our study, two suppressors, p25 encoded by PVX and p19 encoded by TBSV, were coexpressed with HPPR respectively using Agrobacterium infiltration system to study both the effect of suppressor on RNA silencing and the transient expressed HPPR on RA biosynthesis. The results are as follows:1. The HPPR protein can be detected in the tobacco leaves infiltrated by the Agrobacterium carrying transgenes for the 35S: HPPR solely. The content of HPPR protein reached the highest at 3 dpi and reduced in the following days. It could hardly be detected at 8dpi. But the expression of HPPR protein was enhanced in the present of suppressor. It can be improved 3-5 times in the present of p25 and 10 times in the present of p19 at 5dpi. At 8dpi the HPPR protein could be detected in present of RNA silencing suppressor and much higher in the present of p19 than that of p25.2. The C.blumei leaves infiltrated by the Agrobacterium carrying transgenes for the 35S: HPPR solely can accumulate 2.7% rosmarinic acid (RA) of the dry weight at 3dpi. The yield of RA reduced in the following days and equaled with its in untreated leaves (0.946% of the dry weight). When The C.blumei leaves were infiltrated by the Agrobacterium carrying transgenes for both 35S:HPPR and 35S:p25, the yield of RA reached to 4.05%,5.56%,2.83% at 3, 5, 8dpi, respectively. When The C.blumei leaves were infiltrated by the Agrobacterium carrying transgenes for both 35S:HPPR and 35S:p19, the yield of RA reached to 6.75%,7.65%,4.78% at 3, 5, 8dpi, respectively. The yield of RA seems to keep positive correlativity with the expression of HPPR protein by western blot and HPLC. When the HPPR protein expression was stronger, the production of RA was higher and the most highest could reach to 8 times.3. Constructed an E.coli expression vector pET-p25 and transform it into BL21 (DE3) pLysS. And the fusion proteins were injected into mice to prepare antiserum after being purified crudely. The titer of antibody reached 1∶4 000. The antiserum had special response with PVX (P/N>2) compared with no response with PVY and TMV. Using this antiserum, we examined PVX-p25 transgenic tobacco and proved that p25 gene had been stably expressed in some transgenic plants.