胼胝质酶对大白菜核不育小孢子胼胝质沉积的调控机制

项目名称： 胼胝质酶对大白菜核不育小孢子胼胝质沉积的调控机制

项目编号： No.31460524

项目类型： 地区科学基金项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 岳艳玲

作者单位： 云南农业大学

项目金额： 52万元

中文摘要： 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。

中文关键词： 胼胝质酶；胼胝质沉积；大白菜核不育；小孢子发育；调控机制

英文摘要： 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.

英文关键词： Callase;Callose deposition;GMS of Chinese cabbage;microspores development;Regulatory mechanism