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

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.

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。

中国是世界上澳洲坚果种植面积最大的国家，目前全国总面积已超过400万亩，澳洲坚果种植已成为云南、广西部分热带亚热带县市的农业支柱产业。自交不亲和性是影响澳洲坚果产量的重要因素，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，但澳洲坚果自交不亲和性形成的分子机制尚不清楚。本项目完成了36份澳洲坚果种质S等位基因的全长序列克隆，并将其划分为4种类型；通过AS-PCR鉴定体系建立实验，发现4种类型的S基因大小极为接近，在电泳条带上的位置几乎相同，须通过PCR+目的条带回收测序的方式进行准确鉴定；完成了S糖蛋白的双向电泳分析、差异位点筛选和质谱分析，初步预测了S蛋白的空间结构；完成了不同S蛋白对不同品种花粉萌发的离体干预和36个澳洲坚果品种自交亲和性的大田验证；并采用基因组、转录组、蛋白组等多组学手段对澳洲坚果自交不亲和性的分子机制进行了研究，在国际上首次报道了澳洲坚果粗壳种染色体水平的参考基因组、首次报道了澳洲坚果粗壳种及三叶种的叶绿体基因组和线粒体基因组；开展了以获得自交亲和种质为目的、基于人工诱变的澳洲坚果新种质创制；研发出了1套用于克服自交不亲和性的澳洲坚果辅助授粉方法，建立了1套基于自交亲和性的澳洲坚果园品种搭配方法；截止项目结题，发表研究论文6篇，申请发明专利4件，授权发明专利1件；通过项目实施，从分子生物学层面初步揭示了澳洲坚果自交不亲和性的形成机制，为澳洲坚果自交不亲和机制的继续深入研究和自交亲和性优良品种的选育奠定了坚实基础。