Genetic and molecular analyses of the S-locus in wild potato Solanum chacoense

自交不亲和性(SI)是开花植物用来促进物种间基因交换和避免近亲交配的最广泛的进化手段之一。在茄科植物中，自交不亲和性的雌蕊决定因素是一种高度多态的花柱胞外S核糖核酸酶。对SI的花粉决定因素已被鉴定为F-box家族成员(SLF或S-F-box)。有人提出，在亲和杂交中，泛素介导的S核糖核酸酶降解发生。然而，这一点并没有得到明确的证明。事实上，花粉排斥的生物化学和生理学似乎比之前怀疑的要复杂得多。我们利用遗传学、分子生物学和植物组织培养来解决关于SI的基本问题。这些问题包括：1)花粉管如何区分自体和异体S核糖核酸酶？2)除了S核糖核酸酶，还有哪些成分是进行SI反应所必需的？3)所有茄科植物S核糖核酸酶C2区保守的糖基化位点起什么作用？4)为什么S基因座上存在多个F-box基因，它们的功能是什么？5)花粉和雌蕊决定因素是如何共同进化来维持SI的？目的：1)克隆和鉴定花粉S的F-box基因和S基因座位上的其他F-box基因。2)我们将建立一个花粉转化系统，该系统包括具有适当S等位基因构成的高度可再生的基因型和花粉特有的启动子。这些正义和反义方向的结构应该有助于识别花粉-S F-box和其他F-box基因。3)我们将通过定点突变产生一个没有糖基化位点的S11-核糖核酸酶。我们预测，表达这种转基因的植物将拒绝所有类型的花粉。关于SI系统如何工作和进化的更多知识将为在杂交种子生产、果实育种等方面利用SI开辟道路。

Self-incompatibility (SI) is one of the most widespread evolutionary devices used by flowering plants to promote gene exchange within a given species and avoid inbreeding.  In Solanaceous plants like Solanum chacoense the pistillar determinant to SI is an extremely polymorphic stylar extracellular S-RNase. The pollen determinant to SI has been identified as an F-box family member (SLF or S-locus F-box). It has been proposed that in compatible crosses,ubiquitin-mediated S-RNase degradation takes place. This, however, has not been unambiguously demonstrated. Indeed, it appears that the biochemistry and physiology underlying pollen rejection is a far more complex than previously suspected.We use genetics, molecular biology and plant tissue culture to address basic questions about SI. These include: 1) How do pollen tubes discriminate between self and non self S-RNases? 2) Apart from the S-RNase, what other components are required for the SI reaction? 3) What is the role of the conserved glycosylation site present in the C2 region of all solanaceous S-RNases? 4) Why are multiple F-box genes present at the S-locus and what is their function? 5) How do the pollen and pistillar determinants coevolve to maintain SI?OBJECTIVES  1) Cloning and characterization of the pollen-S F-box gene and other F-box genes present at the S-locus of our genotypes. 2) We will develop a pollen transformation system involving highly regenerable genotypes of appropriate S-allele constitution and a pollen-specific promotor. These constructs in both sense and antisense orientation should help in identifying the pollen-S F-box, and other F-box genes. 3) We will produce by site-directed mutagenesis an S11-RNase without glycosylation sites. We predict that plants expressing such transgene will reject all types of pollen.SIGNIFICANCE Additional knowledge on how the SI system works and evolve should open the way to exploit SI in production of hybrid seeds, fruit breeding, etc.