遗传学报
遺傳學報
유전학보
ACTA GENETICA SINICA
2006年
5期
441-448
,共8页
侯夫云%黄骥%陆驹飞%王州飞%张红生
侯伕雲%黃驥%陸駒飛%王州飛%張紅生
후부운%황기%륙구비%왕주비%장홍생
水稻%葡萄糖-6-磷酸脱氢酶%基因克隆%表达分析
水稻%葡萄糖-6-燐痠脫氫酶%基因剋隆%錶達分析
수도%포도당-6-린산탈경매%기인극륭%표체분석
rice%glucose-6-phosphate dehydrogenase%gene cloning%expression analysis
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(Oryzasativa 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.
戊糖燐痠途徑是高等植物中重要的代謝途徑,主要生理功能是產生NADPH以及供覈痠代謝的燐痠戊糖.葡萄糖-6-燐痠脫氫酶(G6PDH)是戊糖燐痠途徑的關鍵酶,廣汎存在于高等植物細胞的細胞質和質體中.本研究首次從水稻(Oryzasativa L.)幼苗中分離瞭覈編碼的質體G6PDH基因OsG6PDH2,序列分析錶明OsG6PDH2編碼一箇具有588箇氨基痠殘基的多肽,等電點為8.5,分子量66 kDa.OsG6PDH2的N耑有1箇70箇氨基痠的信號肽,推測的裂解位點為Gly55和Val56,錶明OsG6PDH2編碼產物可能定位于質體.多序列比較的結果錶明OsG6PDH2與擬南芥、煙草、馬鈴藷質體G6PDH的一緻性分彆達81%、87%、83%.進化關繫說明水稻OsG6PDH2與擬南芥(AtG6PDH3)、馬鈴藷(StG6PDH1)處于高等植物P2型質體G6PDH分支上,暗示瞭OsG6PDH2可能是一箇P2型的質體蛋白.Matinspector程序分析錶明,OsG6PDH2在起始密碼子上遊含有一箇bZIP轉錄因子識彆位點、一箇ABA應答元件、一箇CRT/DRE元件和1箇W-box元件.半定量RT-PCR分析錶明,OsG6PDH2在水稻根、莖、葉和幼穗組織中都呈低豐度組成型錶達,在根部錶達較高,在水稻幼苗中的錶達顯著受暗處理的誘導.將OsG6PDH2的完整開放閱讀框構建到大腸桿菌錶達載體pET30a(+)中,pET30a(+)-OsG6PDH2在大腸桿菌中得到瞭有效錶達.酶活性測定證明,OsG6PDH2的編碼產物具有葡萄糖-6-燐痠脫氫酶的功能.
무당린산도경시고등식물중중요적대사도경,주요생리공능시산생NADPH이급공핵산대사적린산무당.포도당-6-린산탈경매(G6PDH)시무당린산도경적관건매,엄범존재우고등식물세포적세포질화질체중.본연구수차종수도(Oryzasativa L.)유묘중분리료핵편마적질체G6PDH기인OsG6PDH2,서렬분석표명OsG6PDH2편마일개구유588개안기산잔기적다태,등전점위8.5,분자량66 kDa.OsG6PDH2적N단유1개70개안기산적신호태,추측적렬해위점위Gly55화Val56,표명OsG6PDH2편마산물가능정위우질체.다서렬비교적결과표명OsG6PDH2여의남개、연초、마령서질체G6PDH적일치성분별체81%、87%、83%.진화관계설명수도OsG6PDH2여의남개(AtG6PDH3)、마령서(StG6PDH1)처우고등식물P2형질체G6PDH분지상,암시료OsG6PDH2가능시일개P2형적질체단백.Matinspector정서분석표명,OsG6PDH2재기시밀마자상유함유일개bZIP전록인자식별위점、일개ABA응답원건、일개CRT/DRE원건화1개W-box원건.반정량RT-PCR분석표명,OsG6PDH2재수도근、경、협화유수조직중도정저봉도조성형표체,재근부표체교고,재수도유묘중적표체현저수암처리적유도.장OsG6PDH2적완정개방열독광구건도대장간균표체재체pET30a(+)중,pET30a(+)-OsG6PDH2재대장간균중득도료유효표체.매활성측정증명,OsG6PDH2적편마산물구유포도당-6-린산탈경매적공능.
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 reading 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 glucose-6-phosphate dehydrogenase.
