Predicted promoter regions of Milk vetch dwarf virus (MDV) components (C1-C11) were isolated and fused with a b-glucuronidase (GUS) reporter gene and the characteristics of the promoters were examined. In transgenic tobacco calli, promoters of MDV C4 (encoding a cell-cycle link protein), C5 and C7 (both encoding unknown proteins), C6 (encoding a nuclear-shuttle protein) and C8 (encoding a movement protein) generated a stronger level of GUS expression than the Cauliflower mosaic virus 35S RNA promoter (P35S). In leaves of transgenic tobacco plants, the promoters of C5 and C8 conferred a level of GUS activity comparable to that of P35S. Histochemical GUS analysis showed that the promoters of C4-C9, the latter encoding a capsid protein, were active in phloem and meristematic tissue. The promoter of C8 was also active in mesophyll and cortex cell types. A low level of activity was found for the promoters of C11, which encodes a master replication-initiator protein (Rep), and C1, C2, C3 and C10, which encode additional Reps, in both transgenic tobacco calli and plants.
The activity of a predicted promoter, PMC8, from Milk vetch dwarf virus was evaluated by comparing it with the cauliflower mosaic virus 35S RNA promoter (P35S) and PNCR, a promoter from Soybean chlorotic mottle virus. When the GUS fusion gene was introduced into tobacco, PMC8 showed a similar expression profile to P35S but with a more intense expression in proliferating tissues. The usefulness of PMC8 was confirmed by driving NPTII for selection of kanamycin-resistant tobacco plants with improved transformation efficiency. PMC8 was also effective in transgenic rice plants. Thus, PMC8 is useful as an alternative to P35S in both dicotyledonous and monocotyledonous plants, especially for gene expression in proliferating tissues.
Transgenic tobacco plants that overproduce luciferase (Luc) frequently exhibit post-transcriptional gene silencing (PTGS) of luc. The silencing was observed over five generations and found not to be inherited but acquired by the next generation at a certain frequency. Luc imaging analysis of silenced plants revealed Luc activity only in proliferating tissues such as shoot meristem and developing flower. The luc gene expression has been recovered from silencing before development of germ cells, excluding a possible recovery from the PTGS at meiosis. A systemic silencing signal transferred from older tissue likely induces gene silencing of younger tissues in which cell proliferation has been completed. Only seeds maintained Luc activity, probably because of isolation from the silencing signal by a possible partition from the parent placenta. Calli newly induced from the leaf pieces of silenced plants recovered from the silencing and exhibited strong Luc activity similar to nonsilenced leaves, further indicating that the silencing cannot be maintained in proliferating cells. Thus release from PTGS in proliferating cells is a possible mechanism for noninheritance of silencing.
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