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戊糖磷酸途径是高等植物中重要的代谢途径,主要生理功能是产生NADPH以及供核酸代谢的磷酸戊糖。葡萄糖-6-磷酸脱氢酶(G6PDH)是戊糖磷酸途径的关键酶,广泛存在于高等植物细胞的细胞质和质体中。本研究首次从水稻(Oryza sativa L.)幼苗中分离了核编码的质体 G6PDH 基因 OsG6PDH2,序列分析表明 OsG6PDH2 编码一个具有 588 个氨基酸残基的多肽,等电点为8.5,分子量66 kDa。OsG6PDH2的N端有1个70个氨基酸的信号肽,推测的裂解位点为Gly55和Val56,表明 OsG6PDH2 编码产物可能定位于质体。多序列比较的结果表明 OsG6PDH2 与拟南芥、烟草、马铃薯质体 G6PDH 的一致性分别达81%、87%、83%。进化关系说明水稻OsG6PDH2与拟南芥(AtG6PDH3)、马铃薯(StG6PDH1)处于高等植物P2型质体G6PDH分支上,暗示了OsG6PDH2可能是一个P2型的质体蛋白。Matinspector程序分析表明,OsG6PDH2在起始密码子上游含有一个bZIP转录因子识别位点、一个ABA应答元件、一个CRT/DRE元件和1个W-box元件。半定量RT-PCR分析表明,OsG6PDH2在水稻根、茎、叶和幼穗组织中都呈低丰度组成型表达,在根部表达较高,在水稻幼苗中的表达显著受暗处理的诱导。将 OsG6PDH2 的完整开放阅读框构建到大肠杆菌表达载体 pET30a(+)中,pET30a(+)-OsG6PDH2 在大肠杆菌中得到了有效表达。酶活性测定证明,OsG6PDH2 的编码产物具有葡萄糖-6-磷酸脱氢酶的功能。
The pentose phosphate pathway is an important metabolic pathway in higher plants and the major physiological function is the production of NADPH and pentose phosphate for nucleic acid metabolism. Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme in the pentose phosphate pathway, widely found in the cytoplasm and plastids of higher plant cells. In this study, we first isolated OsG6PDH2, a nuclear-encoded plastid G6PDH gene, from rice (Oryza sativa L.) seedlings. Sequence analysis showed that OsG6PDH2 encoded a polypeptide of 588 amino acid residues with an isoelectric point of 8.5 and a molecular weight of 66 kDa. OsG6PDH2 has a 70 amino acid signal peptide at the N-terminus. The putative cleavage sites are Gly55 and Val56, indicating that the OsG6PDH2 encoding product may be located on the plastid. The results of multiple sequence comparison showed that the identities of OsG6PDH2 and Arabidopsis, tobacco and potato plastid G6PDH were 81%, 87% and 83% respectively. The evolutionary relationship between OsG6PDH2 and Arabidopsis (AtG6PDH3) and potato (StG6PDH1) on the G6PDH branch of higher plant type P2 suggests that OsG6PDH2 may be a type P2 plastid protein. Matinspector program analysis showed that OsG6PDH2 contains a bZIP transcription factor recognition site, an ABA response element, a CRT / DRE element and a W-box element upstream of the start codon. Semi-quantitative RT-PCR analysis showed that OsG6PDH2 was expressed in low abundance in root, stem, leaf and panicle of rice plants, and was highly expressed in roots. The expression of OsG6PDH2 in rice seedlings was significantly induced by dark treatment. The complete open reading frame of OsG6PDH2 was constructed into E. coli expression vector pET30a (+). PET30a (+) - OsG6PDH2 was efficiently expressed in E. coli. The enzyme activity assay demonstrated that the encoded product of OsG6PDH2 has the function of glucose-6-phosphate dehydrogenase.