水稻质体葡萄糖-6-磷酸脱氢酶基因的克隆与表达研究

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。木研究首次从水稻（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66kDa。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。

Isolation and Expression Analysis of Plastidic Glucose-6-phosphate Dehydrogenase Gene from Rice (Oryza sativa L.)

Glucose-6-phosphate dehydrogenase is a rate-limiting enzyme of pentose phosphate pathway, existing in cytosolic and plastidic compartments of higher plants. A novel gene encoding plastidic glucose-6-phosphate dehydrogenase was isolated from rice (Oryza sativa L.) and designated OsG6PDH2 in this article. Through semiquantitative RT-PCR approach it was found that OsG6PDH2 mRNA was weakly expressed in rice leaves, stems, immature spikes or flowered spikes, and a little higher in roots. However, the expression of OsG6PDH2 in rice seedlings was significantly induced by dark treatment. The complete opening read-ing frame (ORF) of OsG6PDH2 was inserted into pET30a (+), and expressed in Escherichia coli strain BL21 (DE3). The enzyme activity assay of transformed bacterial cells indicated that OsG6PDH2 encoding product had a typical function of glu-cose-6-phosphate dehydrogenase.