Fw2.2同源基因调控库尔勒香梨果实大小的分子机理研究

The regulation of pear fruit size is an important scientific issue, and has the vital significance for farmers to increase their income and develop breeding plans. Although this research group has concentrated on the development of Korla fragrant pear fruit for long and found that it was closely related with the expression of FWL (fw2.2-like) in pear, how it is regulated by FWL is still unknown. In this project, in order to interpret this regulation mechanism, Korla fragrant pear was selected as the experimental material, while the large-size Zaomeixiang pear (one bud mutation variety of Korla fragrant pear), the large-size Yali pear and the wild small-size Duli pear were taken as the control. And during the flowering period and the young fruit expansion period, these material were collected to study the relationship between FWL gene family and cell division and fruit size of Kolar pear with methods of cytology, molecular biology and bioinformatics, exporing the expression pattern of FWL gene and FWL interacting protein in Korla fragrant pear and interpreting the functions of FWL gene based on the analysis of heterologous transformation and phenotype of FWL. The implementation of this project could play an important role in elucidating the mechanism of FWL gene regulating fruit size of Korla fragrant pear and revealing the development pattern of pear fruit, which lays foudation for higher yeild and breeding of Korla fragrant pear and provides theoretical basis for variation and evaluation of pear.

梨果实的大小调控是一个重要的科学问题，对于农民增收与育种计划的制定有着重要的意义。本课题组长期关注香梨果实发育过程，前期研究发现，梨FWL（fw2.2-like）的表达与果实大小发育密切相关，但FWL如何调控梨果实大小的机制依然未知。为了解析FWL调控梨果实大小的机制，本项目以库尔勒香梨为研究材料，大果型库尔勒香梨芽变早美香、大果型鸭梨和小果型野生杜梨为对照。在花期和幼果膨大期，通过细胞学、分子生物学以及生物信息学方法对梨FWL基因家族、细胞分裂与果实大小发育之间的关联进行研究，探索香梨FWL基因表达模式和FWL互作蛋白，结合异源转化FWL和表型分析，对其功能进行解析。本项目对阐明FWL基因调控香梨果实大小的机理和揭示梨果实大小发育规律具有重要意义。研究结果可为香梨增产育种提供基础，为梨果实大小变异进化提供理论依据。

果实大小是决定香梨市场经济价值的主要因素之一，果重120 g的库尔勒香梨比果重80 g的香梨贵了3-4倍。在实际生产栽培过程中，香梨平均单果重多不足100 g，且可食部分少，影响了其经济价值和香梨产业的发展。本项目聚焦到花期与幼果膨大期这两个细胞分裂最旺盛时期，选取香梨（中果型）为研究材料，早美香（大果型香梨芽变）、鸭梨（大果型）、杜梨（野生小果型）为对照，对梨FWL基因家族进行深入发掘。通过细胞学、分子生物学以及生物信息学等方法对梨FWL基因家族、细胞分裂与果实大小发育之间的关联进行研究，明确梨果实发育的生物学基础，探索FWL基因调控香梨果实大小的分子机理，旨在为香梨增产育种，梨果实大小变异进化提供理论依据。研究结果表明：梨细胞分裂的最关键时期不是在花期，而是在幼果期，为10 DAFB-20 DAFB。库尔勒香梨脱萼果和宿萼果果实大小差异是由果肉部分细胞体积和细胞数目共同造成的，其中细胞数目的影响比细胞体积的影响大。芽变早美香的大果特性不是由倍性改变引起，也是受果肉部分细胞体积和细胞数目影响，而葡萄糖、山梨醇、ZR和ABA的含量以及内源激素的比例与库尔勒香梨和早美香的细胞分裂有关。共有共14个序列被鉴定为梨FW2.2-like蛋白，分布在9条梨染色体上，荧光定量结果表明：PbFWL1，PbFWL3和PbFWL5与细胞分裂相关，且在小果中表达水平高。PbFWL1，PbFWL2和PbFWL5均为膜蛋白，定位在果心到果肉的部位，且在小果中的杂交信号比大果要强。利用VIGS技术对库尔勒香梨幼果期的果实进行侵染，发现侵染10 d后，仅有梨FWL5处理侵染的生长指标与对照相比差异显著。PbFWL1，PbFWL2和PbFWL5在转基因拟南芥中能够使植株器官变小。构建梨幼果FWL膜系统酵母双杂交三框cDNA文库，筛选出的272个互作蛋白，其中collagen and calcium-binding EGF domain-containing protein 1和metallothionein-like protein可能与梨果实大小发育有关。