Sequence Analysis Of The BAC 1365 Of Rice Chromosome 4 And Structural And Functional Research On OsALDH1, OsRLK, RPA1

A rice genome BAC clone 1365 was anchored on the 82.9cM of rice chromosome 4 by probe G282. We completely sequenced the 127563bp of this BAC clone and analyzed the sequence by combining many kinds of prediction programs. There are 15 genes in this BAC. Sequence analysis showed that there was an aldehyde dehydrogenase gene in this BAC and two EST sequence matched perfectly to the prediction result. Further identifications were carried out on analysis of this gene. First, we cloned the full-length cDNA of this gene by RT-PCR, and the gene was designated as OsALDH1; Southern blot analysis indicated that this gene exists in a 7.9kb HindIII enzyme restriction fragment in the rice genome as a single copy; Northern blot analysis of total RNA showed that the OsALDH1 gene was induced in rice seedlings by submergence. An in vitro ALDH assay using protein extracts from E.coli cells that overproduced OsALDH1 indicated that OsALDH1 functions in the oxidation of acetaldehyde in cytosol. A 965bp fragment containing the 5'-flanking region of the first exon of OsALDH1 with the GUS reporter gene showed specific promoter activity in rice cells. Sequencing analysis of a 323-kb contig of rice chromosome 4 revealed that 14 predicted S-locus glycoprotein (SLG) -related protein kinase genes are tandemly clustered in the 108-kb fragment. Nine putative genes were identified by reverse transcription-polymerase chain reaction (RT-PCR) and revealed that they had the different expression patterns of these genes: two genes are expressed predominantly in reproductive tissues while the other seven genes are expressed in both reproductive and vegetative tissues. Four cDNA clones encoding S domain-related receptor-like kinase (RLK) have been isolated from Oryza sativa indica Guanaluai4. Analysis of the predicted amino acid sequences demonstrated that the extracellular receptor domains are highly homologous to SLG of Brassica, whereas the cytoplasmic kinase domains contain conserved amino acids present in serine/threonine kinases. In the earlier research work, a cDNA of 2225-bp in length, designated as RPA1, was isolated from a Oryza sativa green seedling cDNA library, which was proved by homologous andstructure analysis to encode A regulatory subunit of protein phosphatase 2A (PP2A). Genomic DNA containing the gene was also obtained by hybridization with the cDNA as probe from the BAC library of the rice genome. Southern blot analysis indicated that there was only a single copy for the RPA1 in rice genome. Sequence analysis of the genomic DNA revealed that RPA1 gene contained eleven introns. Because BAC clones containing RPA1 gene were anchored on rice chromosome 3 or 7 by probe RG409 (X), we deduced that RPA1 located in rice chromosome 3 or 7. Comparative study showed that 88% of amino acids of the protein were identical with that of its counterpart in Arabidopsis, showing its conservation among different classes. RPA1 gene could complement the yeast tpd3 mutant.