胼胝质酶对大白菜核不育小孢子胼胝质沉积的调控机制

Callase regulation to callose deposition is an important scentific problem in pollen development. However, at this time, little is known about the mechanism by which callase affects callose degradation during microspore development. In order to study this process, this project will utilize near-isogenic lines containing of multiple allelic genes for GMS Chinese cabbage. Based on a positive regulation to callose deposition,we will start to study on the callase itself. Callase structural composition and its activity during microspores development will be systemical studied by employing Native-PAGE/activity gel assays, SDS-PAGE and mass spectrometer analysis (MALDI-TOF-MS). Through analysing and comparing the callase data between fertile and sterile microspores we hypothesize that we will see a genetic difference between the callase enzymes present in each type. To elucidate the ralationship between the callase and callose degradation, subcellular localization of callase will be investigated via colloidal gold immunohystochemisty. The purpose of this is to show the transport pathway from the tapetum to the locule of the anther in which the microspores development. To profile the expression level of callase, we will investigate callase transcription and translation by Real-Time PCR and Western blot analysis, respectively, thereby further revealing the callase regulatory mechanism of callose degradation in the sterile microspores. In summary, the collective analysis of these experiments will allow us to determine the nature of the critical role callase plays in microspore development at the genetic and physiologic level, including callase synthesis, callase activity and its transport pathway.

胼胝质酶对胼胝质沉积调控机制是花粉发育研究的重要科学问题，目前对小孢子发育过程中胼胝质酶调控小孢子胼胝质壁沉积的机制仍缺少深入的认识。本项目以大白菜核不育复等位基因的近等基因系为材料，基于胼胝质酶对胼胝质沉积的正调控，从胼胝质酶本身入手，运用非变性电泳技术、胶内酶活性检测电泳技术、变性电泳及质谱技术对小孢子发育过程中花药胼胝质酶的结构组成、生物活性和含量进行系统研究，通过与不育小孢子败育过程中的胼胝质酶进行比较分析，找到调控败育小孢子胼胝质沉积的差异蛋白；利用免疫金技术对不同小孢子发育时期的胼胝质酶进行亚细胞定位，研究胼胝质酶从绒毡层到药腔的转运途径及其与小孢子败育胼胝质沉积的关系；应用Real-time PCR和Western blot对胼胝质酶基因在转录水平和翻译水平的表达水平和规律进行研究，进而揭示胼胝质酶从基因、蛋白、酶、酶的转运对败育小孢子胼胝质沉积的调控机制。

胼胝质的沉积和适时降解是决定花粉发育的重要过程,胼胝质的异常沉积将导致花粉败育,发生雄性不育。胼胝质沉积受胼胝质合成酶（GSL）和endo-1,3-β-葡萄糖苷酶(BG)直接调控,本项目以大白菜核不育近等基因系为试材，以花粉发育过程中的胼胝质沉积为切入点，采用超微形态学、组织化学及分子生物学的研究手段，对大白菜核不育小孢子发育过程中胼胝质异常沉积现象进行研究，以期能够解释大白菜核不育小孢子的败育机制。研究获得如下结果：1、大白菜核不育小孢子减数分裂正常，能够发育形成四分体；小孢子败育与胼胝质的异常沉积有关，不育花药胼胝质持续沉积直至小孢子完全败育，四分体胼胝质延迟降解，小孢子发育受阻，直至完全败育。2、四分体和单核期，大白菜核不育花药中胼胝质合成酶与胼胝质酶活性与小孢子胼胝质沉积表型相吻合，败育花药中GSL、BG活性在小孢子发育过程中变化趋势基本一致，但在四分体之后，GSL、BG活性分别显著高于、低于可育花药，这期间败育花药中胼胝质的沉积也明显高于可育花药。3、在四分体及单核期，胼胝质合成酶BRaA1004525的高表达以及降解酶BRaA4000861的低表达直接调控不育花药中胼胝质的异常沉积，导致雄性不育。4、超微形态学研究发现，可育花药绒毡层组织结构可以持续到花粉成熟，细胞降解逐个发生，而败育花药在四分体时期绒毡组织结构向腔内扩展，细胞空泡化，细胞质染色浅，细胞降解早，且具有爆发性，使得小孢子进一步发育所需的胼胝质酶得不到供应，四分小孢子不能释放单核小孢子，小孢子发育受阻而导致败育。.结论：大白菜核不育四分小孢子周围的胼胝质不能及时降解，四分体不能释放单核小孢子，小孢子进一步发育受阻时大白菜核不育小孢子败育的直接原因，四分体之后败育花药中胼胝质的沉积与胼胝质合成代谢增强、降解代谢不足有关，这一过程涉及胼胝质合成酶基因BRaA1004525的高表达以及胼胝质酶基因BRaA4000861的低表达，基因的异常表达可能与绒毡层的异常发育有关。