ABSTRACT
Recent studies demonstrated that PNZIP and its homologs encode a special cyclase and play an important role in chlorophyll biosynthesis in higher plants. To investigate the molecular mechanism governing the PNZIP gene, the PNZIP promoter was isolated and analyzed. Deletion analysis indicated that G-box is an important element in the regulation of the reporter gene expression. Further mutation assay demonstrated that G-box and GATACT elements are necessary and sufficient for the high and tissue-specific expression of the GUS gene. Using yeast one-hybrid screening, we have isolated a novel tobacco bZIP protein, NtbZIP, which can specifically recognize the G-box of the PNZIP promoter. The NtbZIP protein shares a limited amino acid homology to Arabidopsis ABI5 and AtAREB1 and very low homology to other bZIP proteins. Northern blot analysis showed that the NtbZIP gene is not induced by exogenous ABA and is expressed in different tobacco organs. Cotransformation assays showed that the NtbZIP protein could activate the transcription of the GUS gene driven by the PNZIP promoter. Transgenic tobaccos analysis demonstrated that constitutively expressing antisense NtbZIP gene resulted in a lower NTZIP synthesis and reduced chlorophyll levels. We suggest that NTZIP is a target gene of NtbZIP, which is involved in the regulation of chlorophyll biosynthesis.
Subject(s)
Nicotiana/genetics , Oxygenases/genetics , Plant Proteins/genetics , Promoter Regions, Genetic , Trans-Activators/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , DNA, Complementary/isolation & purification , DNA-Binding Proteins/genetics , Gene Expression Regulation, Plant , Genes, Reporter , Molecular Sequence Data , Nuclear Proteins/analysis , Oxygenases/biosynthesis , Photosynthesis/genetics , Plant Proteins/biosynthesis , Plant Proteins/metabolism , Response Elements , Sequence Analysis , Tissue Distribution , Nicotiana/enzymology , Nicotiana/metabolism , Trans-Activators/analysisABSTRACT
The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.
Subject(s)
Nicotiana/genetics , RNA Interference , RNA-Dependent RNA Polymerase/genetics , Blotting, Northern , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Plants, Genetically Modified , Potexvirus/genetics , Potexvirus/growth & development , RNA-Dependent RNA Polymerase/metabolism , Temperature , Nicotiana/virology , Tobacco Mosaic Virus/genetics , Tobacco Mosaic Virus/growth & developmentABSTRACT
In previous study, we have proved that the resistance of the transgenic tobacco plants containing untranslatable PVYN CP gene was mediated by PVYN CP transgene RNAs, and the resistance mechanism was similar to PTGS. In this paper, T0 progeny transgenic lines with different resistant levels were selected to further study the transgene inheritance and resistant stability of transgenic plants. Results showed that T0 progeny susceptible lines, which contained 1-2 transgene copies, displayed a 3:1 segregation ratio in T1 progeny lines; T0 progeny middle resistant lines, which contained 3-4 transgene copies, revealed a 15:1 segergation ratio in T1 progeny lines; T0 progeny highly resistant lines, which contained 5-7 transgene copies, followed 15:1 or 63:1 segregation pattern in T1 progeny lines. Southern blot analysis revealed the resistance in most T1 and T2 progeny transgenic lines was related to copy numbers of the transgene. Northern blot analysis indicated that PVYN CP transgenes were expressed at transcription level in most T1 and T2 progeny transgenic lines, and the transgene mRNA accumulation in cytoplasm varied among transgenic lines. There was an inverse correlation between transgene transcript accumulation and virus resistance. Resistance of transgenic lines was inheritable over at least two generations, and the resistance of T2 progeny transgenic lines, which containing untranslatable PVYN CP gene, was (1) not overcome by PVYN particle or PVYN RNA; (2) independent of inoculum levels; (3) resistant to either aphid or mechanically transmitted PVYN; (4) not dependent on plant development stages; (5) PVY-specific (i.e., broad-spectrum resistance was not observed).
