Molecular Basis of Self-Incompatibility in Petunia

IBN-9982659PI：德惠矮牵牛具有配子体型自交不亲和性，受S基因座控制。花粉和雌蕊携带的S等位基因的匹配导致花柱中花粉管的生长受到抑制。在S基因座上发现了一个控制雌蕊功能的多态基因S-RNasegene。然而，在SI互作中控制花粉功能的基因称为花粉-基因，目前还没有被鉴定出来。本项目的目的是利用功能基因组学方法来鉴定膨胀松花粉S基因。OBJ。我将用S基因和S核糖核酸酶基因的14个cDNA标记作为探针，从已构建的S2S2BAC文库中分离克隆。OBJ。II是构建S1S1基因的BAC文库，并使用OBJ中使用的相同探针。我要分离阳性克隆。OBJ。III是对分离在OBJ中的所有BAC克隆进行鉴定。用脉冲场凝胶电泳法和荧光原位杂交法确定它们之间的重叠，对它们在S基因座上进行排序，并估计它们之间的间隙大小。将从两个BAC文库中分离出更多的克隆，以尽可能广泛地覆盖整个S基因座。OBJ。四是将所有的BAC克隆分别导入S1S2基因型的膨胀杨植株，以确定其中是否含有花粉S基因。如果BAC克隆含有花粉S2基因，转基因植株产生的四分之一的花粉粒将携带花粉S1等位基因和花粉S2转基因，并且由于竞争相互作用，它们将与S1S2雌蕊相容。将对自交不亲和的转基因植物进行研究，以确定导致表型的转基因。花粉S基因的候选基因(S)的功能将通过功能丧失实验进一步确定，这项研究的完成将促进对基于核糖核酸酶的自我/非我识别机制的理解。分离的BAC克隆可用于研究附加的S基因的功能，也可用于两个不同的S单倍型S1和S2的S基因的比较研究。在实用方面，可以探索将该性状恢复到自交亲和栽培种以利于杂交制种的可能性。如果成功，这将对农业产生非常重要的影响。

IBN-9982659PI: Teh-hui KaoPetunia inflata possesses gametophytic type self-incompatibility which iscontrolled by a polymorphic locus, the S-locus. Matching of S-allelescarried by the pollen and pistil results in inhibition of pollen tubegrowth in the style. A polymorphic gene at the S-locus, termed the S-RNasegene, that controls pistil function in SI has been identified. However, thegene that controls pollen function in SI interactions, termed the pollenS-gene, has not been identified.The goal of this proposed project is to use a functional genomic approachto identify the pollen S-gene of P. inflata. Obj. I is to use 14 cDNAmarkers for the S-locus and the S-RNase gene as probes to isolate clonesfrom an S2S2 BAC library already contsructed. Obj. II is to construct a BAClibrary of the S1S1 genotype and use the same probes employed in Obj. I toisolate positive clones. Obj. III is to characterize all the BAC clonesisolated in Obj. I and II by pulse-field gel electrophoresis andfluorescence in situ hybridization to determine any overlap between them,to order them at the S-locus, and to estimate the size of the gaps betweenthem. Additional clones will be isolated from both BAC libraries to coveras extensively as possible the entire S-locus. Obj. IV is to introduce allthe BAC clones separately into P. inflata plants of S1S2 genotype todetermine whether any of them contains the pollen S-gene. If a BAC clonecontains the pollen S2-gene, a quarter of the pollen grains produced by atransgenic plant will carry the pollen S1-allele and the pollenS2-transgene, and they will be compatible with S1S2 pistils due tocompetitive interaction. Transgenic plants that have becomeself-compatible will be studied to identify the transgene that isresponsible for the phenotype. The function of the candidate(s) for thepollen S-gene will be further ascertained by loss-of-function experiments.Accomplishment of this proposed research will advance the understanding ofan RNase-based self/non-self recognition mechanism. The BAC clones isolatedcan be used for studying the functions of additional S-locus genes, and forcomparative study of the S-locus of two different S-haplotypes, S1 and S2.On the practical side, one can explore the possibility of restoring the SItrait to self-compatible cultivated species to facilitate hybrid seedproduction. If successful, this will have a very important agronomicimpact.