Molecular Basis of Self-Incompatibility in Petunia

Teh-hui KaoPetunia inflata具有配子体型自交不亲和性，该配子体型自交不亲和性由一个多态位点s座控制。花粉携带的s等位基因与雌蕊携带的s等位基因匹配，抑制花柱中花粉管的生长。在s位点上发现了一个多态基因，称为S-RNasegene，它控制着SI中雌蕊的功能。然而，控制花粉在SI相互作用中的功能的基因，称为花粉基因，尚未被确定。本课题的目的是利用功能基因组学方法鉴定膨松花粉s基因。Obj。利用s位点和S-RNase基因的14个cdna标记作为探针，从已构建的S2S2 BAC文库中分离克隆。Obj。II是构建S1S1基因型的BAClibrary，并使用与Obj相同的探针。我想分离阳性克隆。Obj。III .对从Obj中分离的所有BAC克隆进行表征。I和II通过脉冲场凝胶电泳和荧光原位杂交来确定它们之间的任何重叠，在s位点对它们进行排序，并估计它们之间的间隙大小。将从两个BAC文库中分离更多的克隆，以尽可能广泛地覆盖整个s位点。Obj。四是将所有BAC无性系分别导入S1S2基因型的膨松植株中，确定是否含有花粉s基因。如果一个BAC克隆含有花粉s2基因，则非转基因植株产生的花粉粒中有四分之一携带花粉s1等位基因和花粉s2转基因基因，并且由于竞争互作，它们与S1S2雌蕊相容。将研究具有自我亲和性的转基因植物，以确定导致表型的转基因。花粉s基因候选基因的功能将通过功能缺失实验进一步确定。本研究的完成将促进对基于rase的自我/非自我识别机制的理解。分离的BAC克隆可用于研究其他S-locus基因的功能，并可用于S1和S2两种不同s -haplotype的S-locus的比较研究。在实践方面，人们可以探索将SItrait恢复为自相容栽培物种以促进杂交种子生产的可能性。如果成功，这将对农艺学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.