Tao R, Yamane H, Sassa H, Mori H, Gradziel T M, Dandekar A M, Sugiura A
Faculty of Agriculture, Kyoto University, Japan.
Plant Cell Physiol. 1997 Mar;38(3):304-11. doi: 10.1093/oxfordjournals.pcp.a029167.
Stylar proteins of 13 almond (Prunus dulcis) cultivars with known S-genotypes were surveyed by IEF and 2D-PAGE combined with immunoblot and N-terminal amino acid sequence analyses to identify S-RNases associated with gametophytic self-incompatibility (SI) in this plant species. RNase activities corresponding to Sa and Sb, two of the four S-alleles tested, were identified by IEF and RNase activity staining. The Sa-RNase band reacted with the anti-S4-serum prepared from Japanese pear (Pyrus serotina); no reaction with the antiserum was observed with the Sb-RNase band. When the Sa-RNase band was excised from an IEF gel stained for RNase activity, subjected to SDS-PAGE, and detected by immunoblotting, it appeared that this band consisted of a single protein that reacted with the anti-S4-serum with M(r) of about 28 kDa. With 2D-PAGE and silver staining of the stylar extracts, all four S-proteins could be successfully distinguished from each other in the highly basic zone of the gel. Although Sb-, Sc-, and Sd-proteins had roughly the same M(r) of about 30 kDa, the Sc-protein seemed to be slightly smaller than the Sb-protein and slightly larger than the Sd-protein. In 2D-PAGE profiles as well, the Sa-protein had M(r) of about 28 kDa, apparently smaller than the other three proteins. A bud sport, in which one of the two S-alleles of the original cultivar is impaired, was visualized as a loss of Sc-protein, which is consistent with the previous pollination study. All four S-proteins reacted with the anti-S4-serum, probably because of the differing conformations of these S-proteins in the IEF and 2D-PAGE gels. The Sa-protein in 2D-PAGE appeared to be identical to Sa-RNase in IEF; both had the same M(r) and were reactive with the anti-S4-serum. N-terminal amino acid sequence analysis of the four S-proteins revealed that they were highly homologous to each other and similar to the S-RNases of Malus, Pyrus, Scrophulariaceae, and Solanaceae. Taken together, RNases in the style are strongly suggested to be associated with the gametophytic SI of almond. This is the first report identifying and characterizing S-RNase in almond.
通过等电聚焦（IEF）和二维聚丙烯酰胺凝胶电泳（2D-PAGE），结合免疫印迹和N端氨基酸序列分析，对13个已知S基因型的扁桃（Prunus dulcis）品种的花柱蛋白进行了检测，以鉴定与该植物物种配子体自交不亲和性（SI）相关的S-RNases。通过IEF和RNase活性染色，鉴定出了与所测试的四个S等位基因中的两个（Sa和Sb）相对应的RNase活性。Sa-RNase条带与由日本梨（Pyrus serotina）制备的抗S4血清发生反应；未观察到Sb-RNase条带与该抗血清发生反应。当从经RNase活性染色的IEF凝胶中切下Sa-RNase条带，进行SDS-PAGE并通过免疫印迹检测时，发现该条带由一种单一蛋白质组成，其与抗S4血清发生反应，相对分子质量（M(r)）约为28 kDa。通过对花柱提取物进行2D-PAGE和银染，在凝胶的高碱性区域中可以成功区分所有四种S蛋白。尽管Sb-、Sc-和Sd-蛋白的M(r)大致相同，约为30 kDa，但Sc-蛋白似乎比Sb-蛋白略小，比Sd-蛋白略大。在2D-PAGE图谱中，Sa-蛋白的M(r)约为28 kDa，明显小于其他三种蛋白。一个芽变品种（其中原始品种的两个S等位基因之一受损）表现为Sc-蛋白缺失，这与之前的授粉研究结果一致。所有四种S蛋白都与抗S4血清发生反应，可能是因为这些S蛋白在IEF和2D-PAGE凝胶中的构象不同。2D-PAGE中的Sa-蛋白似乎与IEF中的Sa-RNase相同；两者具有相同的M(r)，并且都与抗S4血清发生反应。对四种S蛋白的N端氨基酸序列分析表明，它们彼此高度同源，并且与苹果属、梨属、玄参科和茄科的S-RNases相似。综上所述，强烈表明花柱中的RNases与扁桃的配子体自交不亲和性有关。这是关于在扁桃中鉴定和表征S-RNase的首次报道。
