| WRKY proteins are involved in responses to biotic and abiotic stresses, and in regulation diverse developmental processes. Two WRKY cDNA, designated OsWRKY89 and OsWRKY52, respectively, were isolated from a cDNA library. Their functions in plants were explored preliminarily in this study. The results are summarized as follows:1 Isolation of OsWRKY89 gene and analyses for its functions OsWRKY89 encodes a polypeptide of 263 amino acid residues containing only one conserved WRKY domain with a zinc finger motif of C2HC, belonging to the WRKY subgroup III. It shared low identity with other WRKY proteins.The recombinant OsWRKY89 was found to bind specially to sequences harboring W box cis elements. The leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions. OsWRKY89 is a nuclear regulator.OsWRKY89 possessed transactivating activity and the C-terminal acidic region was responsible for the activity. The lecucine zipper enhanced the activity.OsWRKY89 were expressed at different levels in various organs, with the highest abundance in stems, next in leaves, roots and just detectable in flowers. However, it was also responsive to various stresses and hormone treatments. A striking observation was a rapid and strong UV-B, methyl jasmonate (MeJA) inducibility of OsWRKY89 transcripts. Also, mechanical wounding, salt, cold (4℃), heat (37℃), ethphon and abscisic acid (ABA) induced its expression. In other hand, UV-A irradiation, salicyclic acid (SA) and gibberellin GA3 had little effect on its expression.An overexpression vector for OsWRKY89 (Ubi::OsWRKY89) and a double-stranded RNA interfering vector (35S::ds89) were constructed and transformed into rice calli by Agrobacterium-mediated method. 5 T1 and 5 T0 independent lines carrying Ubi::OsWRKY89, and 9 lines of RNAi regenerated TO plants were generated. In the next T2 generation of Ubi::OsWRKY89-canying plants, the mRNA levels in each line were constitutively higher than those in wild-type plants. OsWRKY89-overexpressing plants exhibited more or less severe dwarfism, of which the intensity was dependent on the levels of OsWRKY89 transcripts. Plants contained relatively higher levels of OsWRKY89 transcript were more dwarfed, and plants contained lower levels of OsWRKY89 transcripts were less dwarfed. In addition, significantly less adventitious roots were observed in transgenic plants.OsWRKY89-overexpressing plants showed enhanced UV-B tolerance, indicating that it was involved in defense responses to UV-B in plant. Constitutively higher expression of OsBBPI, a UV-B-responsive gene regulated by JA, suggests that JA signaling pathways might play roles in elevated UV-B tolerance in transgenic plants.2 Isolation of OsWRKYS2 gene and analyses for its functions This gene encodes deduced polypeptide contained 572 amino acids, sharing 54 % identity with WRKY1 protein from Avena sativa. The protein possessed two WRKY domain and C2H2 zinc fingers, falling into the group I of WRKY. OsWRKY52 could bind specially to DNA elements containing W box. OsWRKY52 was localized to chloroplasts and the first N-terminal 20 aa was transit peptide. In view of this gene was expressed in no photosynthetic tissues, OsWRKY52 may be a plastid-localized protein.OsWRKY52 had transactivating ability. The N-terminal serine-rich island, threonine-rich island and C-terminal acidic region were narrowed down to be transactivating domains.Expression of OsWRKY52 gene was observed in roots, stems, and leaves, whereas the expression level in flowers was much lower comparing with that in other organs. Continuous dark significantly inhibited the expression of OsWRKY52,whereas evidently increased when transferred to continuous light, suggesting that it may be regulated by light. Among abiotic stresses, cold (4℃), MeJA and ethphon stimulated its expression. However, salt, SA, ABA and GA3 exerted no effects on its expression.The mRNA accumulation of OsWRKY52 was increased rapidly and strongly after inoculation with an incompatible strain of M. grisea. How... |
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