澳洲坚果自交不亲和性S-RNase基因及其表达产物S糖蛋白的鉴定与作用机制研究

澳洲坚果引入中国40余年，目前种植面积已达180万亩，自交不亲和性是限制其产量的重要因素。本研究组发现：绝大多数澳洲坚果品种表现为部分自交不亲和性，极少数品种为完全自交不亲和性，澳洲坚果具有配子体自交不亲和性的典型现象及与蔷薇科植物同源性较高的花柱S-RNase基因保守序列；但其S-RNase基因全长序列和表达产物S糖蛋白尚未获得，作用机制尚不清楚。本项目拟通过S-RNase基因的全长RACE和序列分析，获得澳洲坚果S-RNase基因全长序列，建立不同类型S-RNase等位基因AS-PCR鉴定技术；采用柱层析分离不同类型花柱S糖蛋白，对花粉离体萌发进行量化干预；通过双向电泳和质谱分析，研究不同类型S糖蛋白的空间结构和功能；结合大田试验，对品种亲和性进行鉴定。从细胞和分子层面揭示澳洲坚果自交不亲和性的产生机制，为制定品种搭配规范和创制自交亲和种质奠定基础。

Macadamia have been introduced into China 40 years ago, the planting area has reached one 120,000 hectares. Self-incompatibility was an important factor which influence the yield of macadamia. The results of our research showed that the vast majority of macadamia varieties were partial self-incompatibility, but a few varieties were completely self-incompatibility and have the typical phenomenon of gametophytic self-incompatibility and high homology conservative sequence of S-RNase alleles of Rosaceae plants, but the full length sequence of S-RNase alleles and S-glycoprotein of macadamia have not yet been received, the self-incompatibility mechanism of macadamia were still unclear. This project intends to establish the AS-PCR identification system of S-RNase alleles by cloning, sequencing and rapid-amplification of cDNA ends (RACE) of S-RNase alleles. The macadamia pollen germination in vitro would been intervened by different types of S-glycoprotein which extracted by chromatography from different macadamia varieties. Two-dimensional electrophoresis and matrix/assisted laser desorptionion ization/time of flight mass spectrometry (MALDI-TOF MS) would been used to separate and identificate the structure and function of S-glycoprotein. The project will revealed the molecular and cellular mechanisms of self-incompatibility of macadamia and lay the foundation for macadamia variety arrangement and self affinity germplasm creation.