【作者】 武军凯；

【导师】 李天忠；

【作者基本信息】 中国农业大学 ， 果树学， 2013， 博士

【摘要】 植物自交不亲和性是指功能正常的雌雄同花植物在自花授粉时不能产生合子的现象和机制,其广泛存在于植物界中,对植物的遗传多样性以及适应自然选择的能力具有很重要的意义。早期遗传学研究认为大多数植物自交不亲和机制由单一的复等位基因位点来控制,即自交不亲和位点—S-位点(S-locus),该位点至少包含两类基因,即花柱S基因和花粉S基因。在茄科和蔷薇科植物的自交不亲和中,花柱S基因编码了一类T2家族的核酸酶(S-RNase),花粉S基因编码了一类F-box蛋白(S-locus F-box,SLF/S-locus F-box brothers,SFBB).最新研究表明,在茄科及蔷薇科中,花柱与花粉S因子通过一个相似的自交不亲和识别体统,即‘多因子协同参与的非自我识别系统’,进行‘自己’与‘异己’的识别进而实现授粉的亲和与否,因此识别系统中自交不亲和因子的突变往往导致了自交亲和性突变体产生。在本研究中,我们首先对‘鸭梨’的一个自交亲和性突变体‘金坠梨’进行了分子遗传分析;其次,为了研究蔷薇科及茄科中自交不亲和系统的保守性,我们选取‘富士’苹果中两个花粉S基因MdSFBB9α和MdSFBB9β转化茄科植物进行了功能研究。实验结果包括以下两个方面：1.为了研究‘鸭梨’花粉部分突变体‘金坠梨’的自交亲和性突变机制,首先,从‘鸭梨’中同源克隆得到了5个F-box基因,氨基酸序列分析显示,它们均具有典型的F-box结构域；特异性RT-PCR显示这五个F-box基因均为花粉组织特异性表达；遗传连锁分析表明它们均为S位点的SLF基因；核酸序列比对分析表明,‘鸭梨’中共7个SLF基因均在‘金坠梨’梨中正常表达。其次,通过分子遗传学方法,进一步研究了‘鸭梨’花粉部分突变体‘金坠梨’自交亲和的原因,实验结果表明,‘金坠梨’自交亲和性突变不是由花粉s因子突变导致的,而是非S位点的因子突变导致的；同时,细胞学观察表明‘金坠梨’花粉母细胞的减数分裂行为异常,其花粉败育率明显高于‘鸭梨’。本研究首次在蔷薇科苹果亚科中发现了由非S因子突变导致的自交亲和性突变体,为蔷薇科自交不亲和机制的研究提供了重要的实验依据。2.为了验证蔷薇科植物的花粉S基因的功能以及蔷薇科和茄科植物自交不亲和系统的相似性,从‘富士’苹果中克隆到了与S-RNase紧邻的两个SFBBs基因—MdSFBB9α和MdSFBB9β,将克隆得到的‘富士’SFBBs基因转化到茄科矮牵牛中。授粉实验结果显示,MdSFBB9β作为花粉S基因能够打破转基因矮牵牛的自交不亲和性,这表明‘富士’MdSFBB9β在矮牵牛中具有花粉S因子的功能,花粉S因子在茄科及蔷薇科植物自交不亲和系统中的功能相对保守,同时该实验为研究茄科及蔷薇科之间自交不亲和系统的保守性提供了重要的实验证据。综上所述,本研究通过对‘金坠梨’自交亲和性机制的研究,首次在苹果亚科中发现了非S因子突变导致的自交亲和性突变体,为蔷薇科自交不亲和机制的研究提供了重要数据;其次,蔷薇科与茄科间的转基因实验为茄科和蔷薇科自交不亲和系统在进化上的同起源提供了证据。更多还原

【Abstract】 In many flowering plants, Self-incompatibility (SI) is a genetic mechanism that prevents self-fertilization and promotes cross-fertilization. In Solanaceae, Plantaginaceae and Rosaceae, SI is controlled by a single multi-allelic S locus, which is comprised of pistil-S gene and pollen-S gene. The pistil-S gene encodes a polymorphic ribonuclease (S-RNase), and the pollen-S gene is comprised of a cluster of pollen-expressed S-locus F-box genes named SLF/SFBB in different species. In Solanaceae and Rosaceae, the pollen S genes were recently proposed to involve in a’non-self recognition by multiple factors’ system. In this study,’Jin Zhui’, a self-compatible spontaneous mutant of ’Ya Li’ was studied. The results suggest that mutant non-S factor is responsible for self-compatible ’Jin Zhui’. And meanwhile, the similarity of self-incompatible system was studied by transgenic method between Solanaceae and Rosaceae.The main results of this study are as follows:1. To investigate the pollen part mutant ’Jin Zhui’,5SFBB genes which located in S-locus, were indentified from ’Ya Li’(Pyrus bretschneideri Rhed.). Genetic linkage analysis revealed that they belong to the pollen S gene. No nucleotide differences were found in the SLF genes of ’Jin Zhui’. Further genetic analysis suggested that non-S factor mutation is responsible for the change from self-incompatibility to self-compatibility. These findings support the hypothesis that loss of function of S-locus unlinked PPM expressed in pollen leads to SI breakdown in ’Jin Zhui’. Secondly, abnormal meiosis was observed in quite a few ’Jin Zhui’ pollen mother cells (PMCs), while not found in ’Ya Li’. These and other interesting findings are discussed.2. To investigate the function of two SFBB alleles, MdSFBB9a and MdSFBB9β, which are tightly linked to S-RNase, were isolated from ’Fuji’(Malus pumila.cv.’Fuji’), and were transferred into Petunia hybrida (S3S3L). The results indicated the breakdown of self-incompatibility was only observed in MdSFBB9β transgenic P. hybrida (S3S3L), which indicates that the apple MdSFBB9β may play the role of pollen S factor in P. hybrida, and also imply its pollen S factor role in’Fuji’, It was the first time to find the self-incompatibility breakdown between the Solanaceae and Rosaceae, and is also consistent with the idea that the two families exhibit the same type’non-self recognition by multiple factors’in self-incompatibility mechanism. However no homozygote was found in the progeny. These interesting findings are discussed.Taken together, it is the first time that we find the breakdown of self-incompatibility in Maloideae was due to S-locus unlinked PPM. The identification of S-locus unlinked PPM in Pyrus give a more complete picture of the self-incompatible mechanism. The transgenic results between Solanaceae and Rosaceae support MdSFBB9β was a pollen S and that the two families exhibit the same type ’non-self recognition by multiple factors’ in self-incompatibility mechanism.更多还原