【作者】 尹昌喜；

【导师】 蔡庆生； 夏凯；

【作者基本信息】 南京农业大学 ， 植物学， 2007， 博士

【摘要】 细胞质雄性不育（Cytoplasmic male sterile，CMS）水稻是生产杂交种过程中广泛应用的不育系。CMS水稻普遍存在包穗问题，包穗是指倒一节间（穗下第一节间）伸长不足而导致穗子不能完全抽出倒一叶的叶鞘。包穗会引起作为母本的CMS水稻不能正常接受花粉，从而使杂交制种产量大幅度下降。目前为止，包穗的分子和生理机制还不清楚。本文利用广为应用的CMS水稻珍汕97A（Zhenshan 97A，ZS97A）及其保持系珍汕97B（Zhenshan 97B，ZS97B）作为实验材料，从分子和生理水平对CMS水稻包穗的原因进行了研究。在本试验中，我们比较分析了ZS97A和ZS97B穗分化及节间伸长的差异。结果显示：在ZS97A花粉败育之前，ZS97A和ZS97B幼穗分化和节间伸长都没有明显的差异；而伴随着花粉发育受阻至花粉败育之后，ZS97A的节间伸长速率下降，最终导致倒一节间缩短而引起包穗。内源激素分析揭示，ZS97A倒一节间中赤霉素A1（Gibberellin A1，GA₁）的含量只有ZS97B倒一节间中GA₁含量的三分之一左右。在抽穗早期，施用外源GA₁能够促进ZS97A倒一节间伸长，从而能解除包穗；而且，施用赤霉素合成抑制剂烯效唑（Uniconazole）能够抑制ZS97B倒一节间伸长，致使倒一节间缩短引起包穗。这些结果提示了GA₁控制着水稻倒一节间伸长生长，GA₁的亏缺是导致ZS97A倒一节间缩短的原因。植物体中活性赤霉素的水平不仅受控于合成还受控于钝化。为了找出ZS97A倒一节间缺乏GA₁的原因，我们分析了赤霉素合成及其钝化途径中一些关键基因的表达水平。结果表明：在整体植株中，ZS97A和ZS97B倒一节间中OsGA2ox1（促使活性赤霉素钝化）表达水平没有差异；然而，ZS97A倒一节中OsGA3ox2表达水平显著降低。这些结果暗示了ZS97A倒一节中GA₁的亏缺很有可能是OsGA3ox2表达水平下降，导致赤霉素合成最后一个步骤受阻引起的。为了找出ZS97A花粉败育与倒一节间亏缺GA₁的关系，我们分析了穗子及其倒一节间中内源吲哚乙酸（Indole-3-acetic acid，IAA）的含量。结果表明ZS97A的穗子和倒一节间中的IAA含量都显著低于ZS97B相应部位的IAA含量。生长素极性运输抑制剂2，3，5-三碘苯甲酸（2，3，5-triiodobenzoic acid，TIBA）能够显著抑制穗中IAA向倒一节间运输，从而导致倒一节间IAA含量显著下降，并且引起GA₁含量及倒一节伸长速率的下降；去顶（去除穗子）处理能够阻断穗中IAA向倒一节间的运输，导致倒一节间中IAA含量大幅度下降，也引起GA₁的含量及其倒一节间伸长速率的下降。上述结果提示，倒一节间中的IAA主要由穗中IAA极性运输而来，穗源IAA是倒一节间正常伸长所必需的。ZS97A倒一节间GA₁的亏缺可能是由穗源IAA亏缺引起的。进一步分析了GA₁和穗源IAA在调控倒一节间伸长生长中的相互关系。去顶处理引起倒一节间中IAA含量下降，并引起OsGA3ox2表达水平下降以及OsGA2ox1表达水平上升，致使GA₁含量下降；在去顶的切口处补充外源IAA能够促进倒一节间中OsGA3ox2表达并抑制OsGA2ox1表达，从而提高GA₁含量。这一结果揭示：穗中IAA极性运输至倒一节间中，通过上调OsGA3ox2并下调OsGA2ox1的表达水平，来维持较高水平的GA₁，从而调控倒一节间正常的伸长生长。我们还探讨了ZS97A穗中IAA亏缺的可能原因，结果表明：在花粉发育时期以及抽穗期，ZS97A穗中IAA氧化酶的活性都显著高于同时期ZS97B穗中IAA氧化酶的活性，暗示着ZS97A穗中IAA氧化酶活性的升高是导致穗中IAA亏缺的原因之一。更多还原

【Abstract】 Cytoplasmic male sterile （CMS） lines are widely used in hybrid rice seed production. However, most of the CMS lines suffer from panicle enclosure. Panicle enclosure means that the panicle is partly or fully enclosed within the flag leaf sheath, which blocks normal pollination and greatly reduces seed production of hybrid rice seed.Up to now, the cause of panicle enclosure is still unclear. In this study, Zhenshan 97A （ZS97A）, a widely used CMS line, and its maintainer line Zhenshan 97B （ZS97B） were used to investigate the cause of panicle through molecular and physiological methodsIn this study, we compared differences in panicle differentiation and internode elongation between ZS97A and ZS97B. Our result indicated that, before ZS97A pollen abortion, no difference in panicle differentiation and internode elongation between ZS97A and ZS97B was observed. But after the abortion, the development of ZS97A pollen was impaired and the elongation rate decreased, leading to panicle enclosure by shortening the uppermost internode （UI） length.Analysis result of endogenous hormone indicated that gibberellin A1 （GA₁） level in ZS97A UI was only one-third of that in ZS97B UI. At the early heading stage, exogenous GA₁ application could eliminate panicle enclosure in ZS97A by promoting UI elongation, while exogenous uniconazole （GA biosynthesis inbhibitor） application could cause panicle enclosure in ZS97B by shortening the UI length. These results suggested that GA₁ plays an important role in UI elongation, and that GA₁-deficiency is the caused of panicle enclosure in ZS97A.Active GA level in plant was regulated not only by biosynthesis but also by catabolism. In order to find out the caused of GA₁-deficiency in ZS97A UI, expression levels of some genes involving in GA metabolism pathway were observed. The result showed that, in intact plant, no difference of OsGA2ox1 （converts bioactive GA to inactive GA） transcript level was detected between ZS97A and ZS97B UI, but, OsGA3ox2 transcript level in ZS97A UI decreased obviously. This result suggested that GA₁-deficiency in ZS97A UI might be resulted from the impaired GA biosynthesis by down-regulating OsGA3ox2 transcript level.In order to find out the relationship between ZS97A pollen abortion and GA₁-deficeciency in UI, Indole-3-acetic acid （IAA） level in panicle and UI was analyzed. Our result indicated that IAA level in panicle and UI of ZS97A was much lower than that of ZS97B. Treatment of auxin transport inhibitor 2,3,5-triiodobenzoic acid （TIBA） could inhibited IAA transport from panicle to UI, which caused a strong decrease in IAA level in UI, leading to decreases in GA₁ level and in UI elongation rate. Decapitation （remove the panicle） could inhibit IAA transport from panicle to UI completely, which caused a significant decrease in IAA level in UI, leading to significant decreases in GA₁ level and in UI elongation rate. These results indicated that IAA in UI is mainly transported from panicle, and suggested that IAA-deficiency might be the cause of GA₁-deficiency in ZS97A UI.Moreover, we analyzed the relationship between IAA and GA₁ in regulation of UI elongation. Decapitation caused a decrease in IAA level in UI, which led to down-regulation of OsGA3ox2 and up-regulation of OsGA2ox1, resulting in a decrease of GA₁ level. In decapitated plant, exogenous IAA application could up-regulate OsGA3ox2 transcript level and down-regulated OsGA2oxl transcript level, leading to an increasing in GA₁ level.In this study, we also analyzed IAA oxidase activity. The result indicated that, IAA oxidase activity in ZS97A panicle was much higher than that in ZS97B panicle either at pollen development stage or at heading stage. This result suggested that higher IAA oxidase activity might be contributed to IAA-deficiency in ZS97A panicle.更多还原