Wünsch A, Hormaza J I
Unidad de Fruticultura, S.I.A.-D.G.A., Campus de Aula Dei, Apdo. 727, 50080, Zaragoza, Spain.
Theor Appl Genet. 2004 Jan;108(2):299-305. doi: 10.1007/s00122-003-1418-6. Epub 2003 Sep 4.
Gametophytic self-incompatibility (GSI) in sweet cherry is determined by a locus S with multiple alleles. In the style, the S-locus codifies for an allele-specific ribonuclease ( S-RNase) that is involved in the rejection of pollen that carries the same S allele. In this work we report the cloning and genomic DNA sequence analysis including the 5' flanking regions of four S-RNases of sweet cherry ( Prunus avium L., Rosaceae). DNA from the cultivars Ferrovia, Pico Colorado, Taleguera Brillante and Vittoria was amplified through PCR using primers designed in the conserved sequences of sweet cherry S-RNases. Two alleles were amplified for each cultivar and three of them correspond to three new S-alleles named S23, S24 and S25 present in 'Pico Colorado', 'Vittoria' and 'Taleguera Brillante' respectively. To confirm the identity of the amplified fragments, the genomic DNA of these three putative S-RNases and the allele S12 amplified in the cultivar Ferrovia were cloned and sequenced. The nucleotide and deduced amino-acid sequences obtained contained the structural features of rosaceous S-RNases. The isolation of the 5'-flanking sequences of these four S-RNases revealed a conserved putative TATA box and high similarity among them downstream from that sequence. However, similarity was low compared with the 5'-flanking regions of S-RNases from the Maloideae. S6- and S24-RNase sequences are highly similar, and most amino-acid substitutions among these two RNases occur outside the rosaceous hypervariable region (RHV), but within another highly variable region. The confirmation of the different specificity of these two S-RNases would help elucidate which regions of the S-RNase sequences play a role in S-pollen specific recognition.
甜樱桃的配子体自交不亲和性（GSI）由具有多个等位基因的S位点决定。在花柱中，S位点编码一种等位基因特异性核糖核酸酶（S-RNase），该酶参与排斥携带相同S等位基因的花粉。在本研究中，我们报道了甜樱桃（蔷薇科李属欧洲甜樱桃）4种S-RNase的克隆及基因组DNA序列分析，包括其5'侧翼区。利用根据甜樱桃S-RNase保守序列设计的引物，通过PCR扩增了品种费罗维亚、皮科科罗拉多、塔莱格拉布利兰特和维多利亚的DNA。每个品种扩增出两个等位基因，其中三个分别对应于皮科科罗拉多、维多利亚和塔莱格拉布利兰特中存在的三个新的S等位基因，命名为S23、S24和S25。为了确认扩增片段的身份，对这三个推定的S-RNase的基因组DNA以及在费罗维亚品种中扩增的S12等位基因进行了克隆和测序。获得的核苷酸和推导的氨基酸序列包含蔷薇科S-RNase的结构特征。这4种S-RNase的5'侧翼序列的分离揭示了一个保守的推定TATA框，并且在该序列下游它们之间具有高度相似性。然而，与苹果亚科S-RNase的5'侧翼区相比，相似性较低。S6-RNase和S24-RNase序列高度相似，这两种核糖核酸酶之间的大多数氨基酸替换发生在蔷薇科高变区（RHV）之外，但在另一个高变区内。确认这两种S-RNase的不同特异性将有助于阐明S-RNase序列的哪些区域在S花粉特异性识别中起作用。
