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特定物种内基因交换和避免近亲繁殖的最广泛的进化手段之一。 在茄科植物如Solanum chacoense中，SI的雌蕊决定因子是极其多态的花柱细胞外S-RNase。S1的花粉决定子被鉴定为F-box家族成员（SLF或S-locus F-box）。有人提出，在亲和杂交中，发生泛素介导的S-RNase降解。然而，这一点并没有得到明确的证明。事实上，花粉排斥背后的生物化学和生理学似乎比以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有关SI的基本问题。这些问题包括：1）花粉管如何区分自身和非自身S-RNases？2)除了S-RNase，SI反应还需要哪些其他成分？3)所有茄科S-RNase的C2区保守糖基化位点的作用是什么？4)为什么多个F-box基因存在于S位点，它们的功能是什么？5)花粉和雌蕊决定子是如何共同进化以维持SI的？1）花粉-S F-box基因和存在于我们的基因型的S-位点的其它F-box基因的克隆和表征。2)我们将开发一个花粉转化系统，包括高度可再生的基因型适当的S-等位基因的宪法和花粉特异性启动子。这些构建体在正义和反义方向应有助于确定花粉S F盒，和其他F盒基因。3)我们将通过定点突变产生一种没有糖基化位点的S11-RNase。我们预测表达这种转基因的植物将拒绝所有类型的花粉。意义关于SI系统如何工作和进化的额外知识应该为在杂交种子生产、水果育种等中利用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.