【作者】 侯兴亮；

【导师】 吴平；

【作者基本信息】 浙江大学 ， 遗传学， 2004， 博士

【摘要】 世界范围内土壤中有效磷的缺乏已经不同程度地影响了农作物的生长与产量，因此研究植物在磷胁迫环境下的逆境反应分子机制，并对其进行遗传改良具有重大的现实意义。本研究通过对水稻日本晴cDNA扣除杂交库中ESTs的筛选结果进行分析，克隆了磷饥饿诱导基因OsIPS1与OsIPS2，其中OsIPS2(OsPI1)已于2003年被Wasaki等报道。对OsIPS1与OsIPS2基因的序列特征与在不同条件下的表达模式分析，以及转基因研究，我们得到以下结论： 1．通过对OsIPS1的基因全长克隆，OsIPS1和OsIPS2基因序列及表达特征的分析，我们确认二者属于TPSI1／Mt4家族，并具有TPSI1／Mt4家族的共同特征。同时，我们利用序列比较分析分别在玉米、小麦、大麦中找到OsIPS1和OsIPS2的同源基因ESTs，并分别命名为ZmIPS1，ZmIPS2：只aIPS1，TaIPS2；HvIPS1，HvIPS2。分析结果显示，TPSI1／Mt4家族内部存在两个亚类。Ⅰ类基因包括存在双子叶植物中的已知基因TPSI1，Mt4，At4，AtIPS1，与单子叶中的OsIPS1，ZmIPS1，TaIPS1，HvIPS1基因；Ⅱ类基因包括OsIPS2，ZmIPS2，TaIPS2，HvIPS2基因，仅在单子叶植物中发现。拟南芥中新发现的At4-1、At4-2基因不属于上述两类。 2． 水稻OsIPS1与OsIPS2基因在磷饥饿胁迫下的表达特征： 1)在时间上主要分为两步，即在磷胁迫开始一段时间的低丰度诱导表达和持续胁迫后的高丰度诱导表达。2)在表达部位上，无磷条件下OsIPS1基因主要在初生根的中柱和侧根中表达，地上部的表达极弱；OsIPS2基因根部的表达模式与OsIPS1一致，但在地上部的表达则明显高于OsIPS1基因。3)在调控模式上可分两个层次，即依赖于植株整体磷水平的系统调控和依赖于局部磷供应状况的局部调控。两种调控方式相互独立，在短期外界磷浓度变化时局部信号可以迅速调节基因表达。在长期低磷胁迫中， 系统调控起主导作用，但局部调控仍然存在。 3．水稻OsIPS1与OsIPS2基因在磷饥饿的系统与局部调控下，激素对其表达的影响： 无论全株还是局部磷胁迫条件下，1)外源生长素能够在一定程度上扩大 OsIPS1与OsIPS2基因的诱导表达水平；2)外源细胞分裂素能够显著抑制 OsIPS1与OsIPS2基因的诱导表达：3)外源ABA在一定程度上降低了水稻oslp51与劣五只52基因磷饥饿信号及激素调控模式的比较研究 口占护5了与口‘尸5夕基因的诱导表达;4)外源乙烯对磷胁迫下口‘护Sj与 口占护召2基因的诱导表达无显著影响。 在供磷条件下，上述激素都不能诱导口了护召了与乙七护犯基因的表达。植物体内证明PIBS元件为磷饥饿反应的结合元件，且是水稻Os1PSI基因磷饥饿下高度特异表达调控的关键元件。OsIPsZ与osIPSZ基因之间在转录水平上不存在功能性的反馈调节。更多还原

【Abstract】 A worldwide shortage of available phosphate (Pi) in soil has limited the growth and yield of crops .So it is very significant to study the plant stress responsible molecular mechanism under phosphate starvation and improve the genetic background of crops. With the analysis of ESTs from rice Nipponbare cDNA library screened by a method of Suppression subtracted hybridization (SSH), the phosphate-induced genes OsIPSl and OsIPS2 were cloned in this research. OsIPS2 has been reported as OsPIl by Wasaki et al. on 2003. To investigate the gene sequence characters of OsIPSl, OsIPS2 and expression pattern between them under different treatments, as well as a transgenic research, we concluded as following:1. By cloning the full-length gene of OsIPSl and analyzing the characters of the gene sequences and expressions of OsIPSl, OsIPS2, we confirmed that the two genes belong to TPSIl/Mt4 family. Moreover, with comparison of nucleic acid sequences, we found the homologous genes of OsIPSl, OsIPS2 in maize, wheat and Barley, which were named as ZmlPSl, ZmIPS2 (maize); TalPSl, TaIPS2 (wheat), HvIPSL HvIPS2 (barley) respectively. The analysis results suggest that it exists two subgroup I and II in TPSIl/Mt4 family. I subgroup includes the dicotyledon genes TPSI1, Mt4, At4, AtIPSI and the monocotyledon novel genes OsIPSl, ZmlPSl, TalPSl, HvIPSl, while II subgroup includes the novel genes OsIPS2, ZmIPS2, TaIPS2 and HvIPS2 which were found only in monocotyledon. The genes At4-l, At4-2 found in Arabidopsis recently don’t belong to the above two subgroups.2. The expression pattern of rice OsIPSl and OsIPS2 under phosphate starvation:1) A temporal two-step expression involves the low transcriptional abundance induced during the early stage of P stress and the high transcriptional abundance during a continuous severe P stress. 2) Under P starvation condition, the expression of OsIPSl gene mainly locates on lateral roots and the stele of primary roots, but scarcely on shoots; OsIPS2 expression in roots is similar with OsIPSl, but much more in shoots than OsIPSl. 3)It exists two expression modes under P-starvation, namely the systemic up-regulation depending on the P status of whole plant and the local inducement due to the local P available of plantroots. The two modes are independent each other. Under the early stage of P stress, the local signal aroused by external P concentration varieties can regulate the genes expression rapidly; during the continuous severe P stress, systemic regulation is dominant while the local inducement still exists. 3. The effects of phytohormone on rice OsIPSl and OsIPS2 genes under the systemic and local regulation of P-starvation:Under both the systemic and local P-starvation, 1) exogenous auxins can enlarge the induced levels of OsIPSl and OsIPS2 to a certain extent; 2) exogenous cytokinins can repress the expression of OsIPSl and OsIPS2 remarkably; 3) exogenous ABA can depress the induced levels of OsIPSl and OsIPS2 to a certain extent; 4) exogenous ethylenes have no visible effects on the expressions of OsIPSl and OsIPS2.The above phytohormone can’t induce the expressions of OsIPSl and OsIPS2 under normal nutrition conditions.4. Through the transform experiments in vivo, It suggests that the element P1BS is the P-starvation response binding site and the indispensable component for the drastic and specific inducement expression of gene OsIPSl by P-starvation.5. It doesn’t exist the a functional positive regulatory feedback on the level of mRNA between OsIPSl and OslPS2.更多还原