生长素抑制梨果肉石细胞形成机理研究

The presence of stone cells is one of the direct causes limiting the quality of pear. Therefore, elucidating the mechanism of stone cells formation is critical to improve pear fruit quality. Previous studies have demonstrated that application of exogenous IAA could reduce stone cells in pear, however, the functional mechanism is not yet clear. Therefore, based on conclusions on the developmental patterns of stone cells in the pear cultivar 'Xiangnan' and optimized external growth regulator application conditions, we propose to conduct the following research: Firstly, we propose to compare and analyze the developmental patterns of stone cells under exogenous IAA or NAP treatment via microscopic observation and in situ hybridization so as to elucidate the effects of IAA on stone cell formation. Then, using immune-electron microscopy and histochemical localization methods, we propose to explore the relationship between IAA-regulated stone cells formation and the distribution of Ca2+ in the apoplastic translocation system. Furthermore, we propose to utilize HPLC-MS and transmission electron microscopy to determine hormone content and subcellular morphology in the pulp cells, in combination with qRT-PCR analysis for expression level determination of marker genes related to the formation of secondary cell wall in stone cells, and eventually clarify the mechanism of IAA regulating the development of pear stone cells. The implementation of this project has important theoretical and practical significance for the further use of biotechnology to reduce the content of pear stone cells and achieve high-quality and stable industrial cultivation.

石细胞是导致梨果肉粗糙、口感多渣的直接因素。揭示影响石细胞形成的机理对于改良梨果实品质具有重要意义。研究证实外源施加IAA能降低梨石细胞含量，然而作用机理尚不清楚。为此，本项目在已总结‘湘南’梨果实石细胞发育规律并筛选了适宜的外施生长调节剂条件的基础上，拟进行以下研究：1）采用显微观察和原位杂交技术对比分析外源施加IAA或及其抑制剂NAP处理后的梨石细胞发育规律，明确IAA对石细胞形成的作用；2）通过胶体金免疫电镜观察和组织化学定位方法，探究IAA调控石细胞发生与Ca2+在质外体运输系统分布的联系；3）利用HPLC-MS和透射电镜进行果肉激素含量测定及亚细胞形态学观察，结合qRT-PCR分析石细胞次生壁形成相关Marker基因的表达水平检测，最终初步阐明IAA调控梨石细胞发育的机理。本项目的实施对将来进一步利用生物技术手段降低梨石细胞含量，实现优质稳定的产业化栽培具有重要的理论和实践意义。

本项目以厘清生长素和钙元素与石细胞发育之间的内在联系为目标，开展了系统性研究工作，结果如下：1）总结了‘湘南’梨果实石细胞发育规律，筛选了适宜的外施生长调节剂IAA和NPA的有效浓度；2）采用显微观察和原位杂交技术对比分析外源施加IAA或NAP处理后的梨石细胞发育规律，明确了IAA对石细胞形成的作用；3）通过组织化学定位方法，探究IAA调控石细胞发生与包含Ca在内的多种矿质元素的关系，证明施加外源IAA能引起幼果期钙含量降低，同时改变果肉中石细胞的极性分布，引起赤道部位果肉石细胞含量显著降低；4）通过果肉激素含量测定及亚细胞形态学观察，结合qRT-PCR分析结果，初步阐明IAA调控梨石细胞发育的机理。本项目对将来进一步利用生物技术手段降低梨石细胞含量，实现优质稳定的产业化栽培具有重要的理论和实践参考价值。