| Pentose phosphate pathway (PPP) is one of important methoblism pathways in plants. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are both key and rate-limiting enzymes of pentose phosphate pathway. G6PDH and 6PGDH are widely present in cytosolic and plastidic compartments of higher plants. Based on the difference of amino acid sequence and protein immunization, plastidic G6PDH isoforms in plants are divided into the two classes: P1 and P2. With the plant growth and development or the adaptation to the change of surrounding environment, plants must accurately regulate this pathway.In order to isolate plastidic G6PDH and 6PGDH cDNAs from rice, Arabidopsis G6PDH gene (GenBank accession number AY099561) and rice OsG6PDH1 (accession number: AF486280) were used as query probes to search rice genome database in Genbank through BLAST algorithm program. It was found that there were two genomic sequences to be highly homologous with the relative probe respectively. Through splicing of extrons using FGENESH program it was obtained the two full-length cDNA sequences. By RT-PCR and the specific primers of assembled sequence, the two cDNA fragments were isolated from mRNAs from rice seedlings, and designated as 6PDH2 and Os6PGDH2 respectively, with accession number AY339367 and AY278362 in GenBank. The cytosolic G6PDH (OsG6PDH1) and 6PGDH (Os6PGDH1) genes were isolated from rice in this lab in 2002. Therefore all four types of G6PDH and 6PGDH genes were cloned from the same plant organism, which should be the first report in higher plants. It will be very helpful to further explore the sub-cellular distribution of two key enzymes and biochemical function of PPP in the plant growth and development and responses to the environmental stresses.Genomic organization analysis indicated that OsG6PDH genes contain multiple introns, with more introns in the cytosolic G6PDH than plastidic, whereas two Os6PGDH genes have no introns in their translated regions. Combined with the analysis of genomic structure and evolutionary of these two key enzyme genes of Arabidopsis, we suggested that G6PDH and 6PGDH genes might have very different evolutionary ways. Cytosolic G6PDHs in plants have a common ancestor with those in the animals and fungi, and plastidic G6PDHs of plants come from the prokaryotic. 6PGDH genes in plants, eithercytosolic or plastidic, probably originate from cyanobacterial through endosymbiosis.The OsG6PDH and Os6PGDH genes have divergent expression patterns in various rice tissues. The 0sG6PDHl mRNAs were constitutively expressed roots and immature spikes at lw after flowering; and the transcripts of Os6PGDH1 highly in stem, roots and immature spikes at lw after flowering. The OsG6PDH2 mRNA transcripts were a little showed in various rice tissues, with a higher expression in roots. In contrast, Os6PGDH2 were expressed highly in the leaves, which was agreed with the character of chloroplast genes.As a step toward understanding the functions of pentose phosphate pathway in plants under various environmental stresses, the expressions of four genes were investigated in the rice seedlings under various stresses including by semi-quantitative reverse transcription (RT)-PCR approach. The results showed that the expression 0sG6PDHl was not induced under the abiotic stresses with a constitutive level, and the OsG6PDH2 little detectable. However, the OsG6PDH2 was significantly induced by dark treatment. The transcript levels of both Os6PGDH1 and Os6PGDH2 were up-regulated to different extents by drought, cold, high salinity and ABA treatments. Meanwhile the enzymatic activities for G6PDH and 6PGDH were further evaluated in the rice seedlings under abiotic stresses. It was found that the activities of 6PGDH were steadily enhanced under the abiotic stresses like their mRNA expressions, but the G6PDH activities remained constant. Taken together, it suggested that the pentose phosphate pathway might play important roles in rice responses to abiotic stresses, and the 6PGDH might control the efficiency of P... |
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