Cloning And Functional Analysis Of Leaf-Specific Expressed Genes And Malate Dehydrogenase Genes In Rice (Orysa Sativa. L.)

In order to study the leaf-specific expressed genes in Oryza sativa L., the subtracted library of rice leaf was established by suppression subtractive hybridization (SSH). After screening the library, 171 clones differentially expressed were obtained. All these inserts were put into GenBank separately. It was shown that 85 clones belonged to the cloned genes in rice, 45 clones belonged to the known rice gene that were not cloned and the other 41 clones belonged to unknown genes. The known genes were involved in the procedures of photosynthesis, metabolisms, membrane transduction, signal transduction and the resistance in the rice. Using the DNA fragments isolated from SSH, the full-length cDNAs of rice phosphoenolpyruvate carboxylase (Ospepc), invertase (Osinv) and Lon protease (Oslon) were cloned, separately. Their gene sequences and functions were also studied. After sequence alignment, it was found that Ospepc was highly homologous to those known PEPCs (84%-86%). Besides, some PEPC-specific conserved domains were detected in Ospepc. The expressing pattern of Ospepc was verified by quantitative PCR analysis that its signal was obviously stronger in leaf tissues than in roots. Meanwhile, it was shown that Osinv was homologous to the neutral/basic invertase in other plants (71%-80%). It was indicated from the RT-PCR results that the expression of Osinv was increased in leaves, and its expression level could be improved under salt and cold treatments. A putative Lon protease, which degraded the abnormal proteins in mitochondria, was translated from Oslon. It was shown from the alignment results that Oslon was homologous to other Lon proteases (77%-92%). From the conserved domain search, it was found that Oslon has all the important conserved domains found in other Lon proteases. It was convinced from Northern blot analysis that the expression of Oslon was increased in leaves. An alternative splicing of this gene was found in the reproductive organs (young panicles and anthers) in rice sterile line ZHENSHAN 97A. By RNA in situ hybridization analysis, it was shown that this alternativesplicing was located probably in the tapetum, noting that it may be related to rice cytoplasmic male sterility. Oslon was also expressed as a fusion protein in prokaryotic system, which showed that the protein could be expressed as a normal protein with the correct size. At the same time, two homologous genes of Oslon were found in rice genome. Moreover, the cytoplasmic and mitochondrial malate dehydroxyase genes were cloned by screening the rice seedlings cDNA library. It was shown from the RT-PCR results that the expression patterns of them were according to each other. The highest expressions were detected in the young panicles and the premature embryos, while they were much lower in leaves and roots. Both of them could be expressed in prokaryotic system in the correct size. Furthermore, the MDH activity of OscMDH was detected.