| Genome sequencing has provided a molecular basis for comparing the structures of homologous genes among the species. In this study, three BAC clones were cloned from BAC library (Oryza sativa indica) by hybridization with the probe R896, which is specific to rice chromosome 4. The sequence of BAC 4949, which was selected to sequence by random from the identified three BACs, has been completely determined and used for analyzing the function of two unknown genes through molecular approaches. There were nine genes predicted in this 61.79-kb BAC sequences, revealing a high gene density region. Only one gene was failed to have a matched EST sequence from databank. Analyzing the BAC 4949 sequence by BLASTX in protein database indicated that a sequence might be involved in encoding Gag-Pol protein of retrotransposon. This sequence was therefore named as a RIRE10. It is a big Ty3-like retrotransposon with long LTR sequence. The copy numbers of RIRE10 internal region and LTR were detected as 65+8 (SE) and 1100+8 (SE) separately by dot blot hybridization with genomic DNA. Different transcripts in length from LTR region were detected by Northern blot analysis. Both the transcription of LTR region and about 900 RIRE10 solo-LTRs indicated that there might be an interaction mechanism between rice genome and the members of RIRE10 family. The transcriptional regulation site in LTR region was speculated to take part in the expression regulation of the nearby rice gene and influence the read-out transcription of RIRE10. The homology recombination or other mechanism may be used by rice genome to make the retrotransposited elements lost the internal region and one LTR and the remained LTR is a remnant of transposition. Besides, we cloned a novel rice gene, named as rml1, by RACE and RT-PCR from the same BAC. The results from both Southern blot analysis and database searching showed that it is a single copy gene in rice genome. BLASTX analysis indicated that rml1 gene encoded a protein with single trihelix DNA-binding domain, which is a putative transcriptional regulation protein. Northern blot hybridizations showed that rml1 gene expression was down-regulated obviously by continuous white light. And the red light had stronger effects on rml1 transcription comparing with other single-wave light. There is different influence on calli and etiolated seedling under the same red light fluence. This result indicated that rml1 RNA levels in calli and etiolated seedling were regulated by different phytochrome under various light-responsive mechanisms. Under the same time of illumination, the rml1 RNA level in etiolated seedling was strongly repressed by white light, to a less extent by red light and blue light has little effecter. Based on these results, we speculated that other photoreceptor might be involved in regulating rml1 RNA level in etiolated seedling besides phytochrome. So it is a new member of light signal transduction pathway. Moreover, rml1 was identified to be an induced gene in the early stage of Magnaporthe grisea infection and the tissue specific expression is observed. We also detected the clear rhythm expression of rml1 gene and found this circadian regulation is dependent on light/dark cycles. As a gene encoding the putative transcription regulation protein, the rml1 gene was involved in the rice reaction responding to the environmental signal (such as light, pathogen) and may be involved in the regulation other gene expression in the rice life cycle. We have cloned the promoter region and part CDS sequence of rml1 in pCAMBIA vectors separately to try to understand the transcriptional regulation and the subcellular localization of RML protein.
|
PDF Full Text Request