Molecular Genetic Basis of Self-Incompatibility in Petunia

自交不亲和的矮牵牛（Petunia inflata）和自交不亲和的矮牵牛（Petunia hybrida）将作为模型系统，用于配子体自交不亲和的分子、生化和遗传基础的研究。第一个目标是为s基因（编码雌蕊s蛋白的基因）在识别和排斥自交花粉中的作用提供直接证据。将采用两种方法：a)分别将s1 -基因导入S3S3基因型和s3 -基因导入S1S1基因型获得的转基因油松植株，分别观察转基因油松是否具有拒绝携带s1 -等位基因和s3 -等位基因花粉的能力；b)将反义s1基因引入S1S3基因型的膨胀松植株，观察转基因植株对s1花粉的拒斥能力是否被取消。第二个目标是通过瞬时实验和稳定转化子检测一系列启动子gus构建子的表达，确定S-基因和rnx2基因（编码RNase X2）雌蕊特异性表达的调控序列。第三个目标是识别和描述参与自不相容相互作用的其他组件。PCR技术将用于研究s基因在花粉原位生长过程中是否在花粉中表达。染色体行走实验将用于鉴定与s基因相关的序列，这些序列可能编码自交不亲和系统的其他组成部分。一种基于免疫沉淀的方法将用于检查授粉雌蕊是否含有与s蛋白相互作用的蛋白质。这些实验可能有助于鉴定s位点长期寻找的花粉成分。第四个也是最后一个目的是确定自交亲和品种杂交自交不亲和破裂的遗传基础。设计了遗传杂交来测试一个模型，该模型暗示了一个修饰位点在自交不亲和相互作用中调节s等位基因的活性。

Self-incompatible Petunia inflata and a self-compatible cultivar of Petunia hybrida will be used as model systems in an ongoing study of the molecular, biochemical, and genetic bases of gametophytic self-incompatibility. The first objective is to provide direct evidence for the role of the S-gene (the gene encoding the pistil S-protein) in the recognition and rejection of self pollen. Two approaches will be taken: a) transgenic P. inflata plants obtained by introducing S1-gene into S3S3 genotype, and S3-gene into S1S1 genotype, will be tested to see whether the ability to reject pollen bearing S1-allele and S3-allele respectively has been conferred by the transgenes; b) antisense S1-gene will be introduced into P. inflata plants with S1S3 genotype to see whether the ability to reject S1-bearing pollen is annulled in the transgenic plants. The second objective is to determine the regulatory sequences for the pistil-specific expression of the S- gene and rnx2 gene (encoding RNase X2) by examining the expression of a series of promoter-GUS constructs via transient assays and in stable transformants. The third objective is to identify and characterize other components that participate in self- incompatibility interactions. PCR techniques will be used to investigate whether the S-gene is expressed in pollen during its growth in situ. Chromosome walking experiments will be used to identify sequences linked to the S-gene that might encode other components of the self-incompatibility system. An approach based on immunoprecipitation will be used to examine whether pollinated pistils contain any proteins that interact with S-proteins. These experiments may lead to identification of the long sought pollen component of the S-locus. The fourth and final objective is to determine the genetic basis for the breakdown of self- incompatibility in the self-compatible cultivar of P. hybrida. Genetic crosses have been designed to test a model implicating a modifier locus in the modulation of S-alleles activity in self- incompatibility interactions.