【作者】 胡利伟；

【导师】 蔡伟明；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 植物学， 2007， 博士

【摘要】 植物的向重性反应主要是指重力反应器官响应重力刺激而发生的不对称生长的过程。生长素、乙烯、赤霉素等植物激素以及Ca2+、质子、IP3等信号分子参与了植物的向重性反应。有学者曾采用基因芯片技术来研究向重性对拟南芥基因表达的影响,虽然分离到很多与重力相关的基因,但由于向重性弯曲是一个不对称生长的过程,而拟南芥株形较小不易纵切,所以并不清楚这些基因在向重性弯曲中的作用。我们采用株形较大的水稻为材料,采用RT-PCR、SSH、基因芯片、Realtime PCR等技术进行相关的研究,克隆了在水稻叶鞘负向重性反应中上下两半部不对称表达的基因,对这些基因的功能进行了研究,并测定了相关的生理指标。另外,我们利用一次空间搭载的机会,在我国的一颗返回式科学实验卫星上搭载了水稻和拟南芥种子,并进行相关的实验。我们的研究发现扩张蛋白OsEXPA4和水孔蛋白OsRWC3参与了水稻叶鞘的负向重性弯曲。在水稻叶鞘的负向重性反应中,OsEXPA4和OsRWC3在水稻叶鞘基部的下半部特异表达,它们也都被GA3诱导,而且OsEXPA4的不对称表达早于OsRWC3的不对称表达。此外我们又采用转有OsRWC3启动子-GUS的水稻植株,对负向重性反应中OsRWC3的表达进行了验证,GUS活性在重力刺激的水稻叶鞘的下半部明显增高,并且GUS染色主要分布在微管束。GA合成抑制剂Ancymidol能够减弱OsEXPA4和OsRWC3在上下两半部的不对称表达。研究还发现,负向重性反应中下半部的渗透势明显降低,水孔蛋白抑制剂HgCl2和phloretin也都能抑制水稻的负向重性反应。这些结果表明水稻叶鞘基部负向重性反应中不对称分布的GA诱导了OsEXPA4和OsRWC3的不对称表达,促进了水稻叶鞘基部下半部的细胞壁松弛和通过水孔蛋白的水分内流,最终导致了下半部细胞膨胀,使叶鞘向上弯曲。综合运用抑制差减杂交法(SSH)和基因芯片等技术,以负向重性反应中的水稻叶鞘基部上下两半部互为对照,并采用Realtime PCR进行验证,我们发现有11个基因在负向重性反应中水稻叶鞘基部的下半部特异表达,其中包括乙醛脱氢酶( Aldehyde dehydrogenase,ALDH1)、反复糖基化多肽(Reversibly glycosylated polypeptide,RGP1)、蔗糖合成酶(Sucrose synthase,SUS)、己糖转运蛋白(Hexose transporter)、核糖体结合蛋白(Ribophorin II)、染色体结构维持蛋白(Structural maintenace of chromosomes 3,SMC3)、尿卟啉原脱羧酶(Uroporphyrinogen decarboxylase,UROD)、鲨烯合酶(Squalene synthase,SQS)等,以及3个未知功能的基因。而胱硫醚γ-合成酶(cystathionine gamma-synthase,CGS1)在水稻叶鞘基部的上半部特异表达。我们研究了这些基因在负向重性反应中的不对称表达是否受到不对称分布的生长素的诱导,IAA处理实验结果表明RGP1、SUS、UROD、SMC3等基因的不对称表达受到了IAA的诱导,而且IAA对这几个基因的调控呈现剂量依赖效应,TIBA处理能够抑制RGP1、SUS、SMC3、UROD等基因在负向重性反应中4小时和6小时的不对称表达,说明水稻叶鞘负向重性反应中这4个基因的不对称表达受到生长素的诱导。另外我们测定负向重性反应中水稻叶鞘基部上下两半部己糖含量,结果证明负向重性反应中下半部积累了较多的己糖,并且上下两半部的己糖含量出现差异是在蔗糖合成酶和己糖运输蛋白的不对称表达出现之后,说明负向重性反应中这种己糖的不对称分布是由于蔗糖合成酶和己糖运输蛋白的不对称表达造成的。向重性过程中表达的基因参与了细胞壁物质的代谢,己糖合成和己糖的运输,能量物质的合成,甾醇类物质的代谢,蛋氨酸的合成,以及蛋白质的翻译等重要过程。利用空间搭载的机会,我们在我国于2003年11月3日发射的一颗返回式科学与技术试验卫星上搭载了拟南芥和水稻的种子。在水稻中发现了1棵个体矮小的致死型突变体,而在拟南芥中发现了3株稳定遗传的突变体,1株第1代出现表型突变的突变体,第2代表型恢复正常的突变体。在拟南芥的3株稳定遗传的突变体中,有1株呈伞房花序(Corymb-like inflorescence),而拟南芥野生型植株是总状花序(raceme),所以我们将这个突变体命名为cli,并对cli突变体进行了相关的研究。与野生型拟南芥相比,cli突变体的叶子向下卷曲,果荚末端圆滑,植株高度较低,茎和花序轴较为粗壮。黑暗处理能够显著促进野生型拟南芥下胚轴的生长,但是我们发现黑暗处理对cli突变体下胚轴生长的促进作用较弱。在未来的工作中,我们打算进行图位克隆,最终确定其中发生突变的基因。更多还原

【Abstract】 Gravitropism results from the asymmetric growth or curvature of plant organs in response to gravistimulation. Differential growth of plants in response to changes in the gravity vector requires a complex signal transduction cascade. Ca2+, proton, IP3, auxin, Gibberellin and ethylene involved in the process. Using the whole seedlings or root tips as material, many gravitropism-regulated genes in Arabidopsis are identified by microarray. Because there is asymmetric growth between upper side and lower side in the gravitropic bending, the function of these genes in the gravitropism is not clear. Using rice as material, we identified genes related with asymmetrical growth in the gravitropism by RT-PCR, SSH, Microarray, Realtime PCR, etc. The function of genes in the gravitropism are explored, related physiological research are done. In addition, we sent the dry seeds of rice and Arabidopsis to the space on board of a recoverable satellite, and did related experiments after landing.We find that an expansin gene (OsEXPA4) and a plasma membrane aquaporin (OsRWC3) are involved in gravitropic bending of rice leaf sheath bases. Expression of OsEXPA4 and OsRWC3 were induced in the lower side of rice leaf sheath bases during gravitropism. Expression of both of these genes was induced by GA3.β-glucuronidase (GUS) activity in transgenic rice expressing a GUS reporter gene under the control of the gibberellin (GA)-responsive OsRWC3 promoter was investigated during gravitropic curvature and found to be enhanced in the lower side of graviresponding leaf sheath bases. Ancymidol, a GA biosynthesis inhibitor, reduced the expression gradient of OsEXPA4 and OsRWC3. The osmotic potential was transiently lower on the lower side of gravistimulated leaf sheath bases, and the gravitropic curvature was inhibited by HgCl2 and phloretin, two known inhibitors of aquaporins. These data suggest that asymmetric GA redistribution following gravistimulation results in the localized expression of OsEXPA4 and OsRWC3, which in turn might bring about differential cell wall loosening, cell expansion and water influx via aquaporins, thus leading to gravitropic bending.In order to further study the molecular mechanism in the gravitropic bending, we identify 12 genes differently expressed between upper and lower halves of rice leaf sheath bases by combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques. 11 genes specially expressed in the lower side of rice leaf sheath bases, which included Aldehyde dehydrogenase(ALDH1),Reversibly glycosylated polypeptide(RGP1), Sucrose synthase(SUS), Hexose transporter, Ribophorin II, Structural maintenace of chromosomes 3(SMC3), Uroporphyrinogen decarboxylase(UROD), Squalene synthase(SQS) and 3 unknown protein. Cystathionine gamma-synthase (CGS1) specially expressed in the upper side. In addion, IAA treatment could induce the expression of RGP1, SUS, UROD and SMC3. TIBA (an inhibitor of auxin transport) could abolish the differential expression of these genes in the gravitropism. These results suggested that asymmetrical auxin induced the asymmetrical expression of RGP1, SUS, UROD and SMC3 in gravitropic bending of rice leaf sheath beases. The hexose content of rice leaf sheath base was measured, and there was more hexose in the lower halves after 12h gravi-stimulation. The asymmetrical hexose content occurred later than that of SUS and Hexose transporter. It suggested that the higher hexose content in the lower side of rice leaf sheath bases results from the higher expression of SUS and Hexose transporter. These gravitropism-regulated genes involved in some important process: metabolism of cell wall substance, hexose synthesis and hexose transport, energy substance synthesis, metabolism of steroids, the methionine synthesis and protein translation, etc.Dry seeds of rice and Arabidopsis were carried to the space on board of a recoverable satellite for 18 days.Some related experiments were done after landing. There was one dwarfish lethal mutant in rice, and three mutants in Arabidopsis. In addtiton, the aberrance of one Arabidopsis plant occured in SP1, and resumed normal in SP2. In the three Arabidopsis mutants, one had the phenotype of corymb-like inflorescence, different from raceme of Arabidopsis wild type, so this mutant was nominated cli (Corymb-like inflorescence). Compared with wild type, cli mutant had curled leaf, circinal pod bottom, strong inflorescence stem, lower plant height. Compared with Arabidopsis wild type, dark treatment had less effect on growth of cli mutant’s hypocotyls. In further study, we plan to clone the mutant gene in cli mutant by map-based cloning strategy.更多还原