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通过酵母双杂交的方法寻找苹果‘国光’花柱中与S-RNase互作的非S因子。以苹果‘国光’花柱为试材,构建了酵母cDNA文库,检测插入片段大小在300～2 000bp之间,符合库容要求。将S1-RNase成熟区cDNA序列S1-mat构建到pGBKT7载体上作为诱饵,筛选‘国光’花柱酵母cDNA文库。经文库筛选,获得一个大小为371bp的片段,与苹果全基因组序列比对后发现,该片段位于第9号染色体,其全长序列为552bp。NCBI BLAST比对及蛋白结构域分析显示其与拟南芥钙调素结合蛋白的同源性最高,且具有钙调素结合蛋白特有的磷酸二酯酶结构域。同时,酵母互作实验显示其与‘国光’花柱钙调素(CaM)有强烈互作,故认为此基因是苹果钙调素结合蛋白基因,命名为MdCaMBP。半定量RT-PCR结果显示其在‘国光’叶片及花的各组织中均有表达,与苹果花柱S1-、S2-、S9-RNase成熟多肽区均有互作且作用强烈。推测MdCaMBP可能作为一种S-RNase辅助因子参与了自交不亲和反应。
Search for Non - S Factor Interacting with S - RNase in Apple ’Guoguang’ Style by Yeast Two - Hybrid Method. The apple ’Guoguang’ style was used as the test material to construct the yeast cDNA library. The size of the inserted fragment was detected between 300-2000bp, which accorded with the requirement of library capacity. S1-RNase mature cDNA sequence S1-mat was constructed into pGBKT7 vector as a bait to screen the ’Guoguang’ column cDNA library. The library was screened to obtain a fragment of 371bp in size. Comparing with the whole apple genome sequence, it was found that the fragment was located on chromosome 9 with a full-length sequence of 552bp. The NCBI BLAST alignment and protein domain analysis revealed that it has the highest homology with the Arabidopsis calmodulin binding protein and has the phosphodiesterase domain unique to calmodulin binding proteins. At the same time, the yeast interaction experiment showed that there was a strong interaction with ’Guoguang’ style CaM (CaM), so this gene is considered as an apple calmodulin binding protein gene and named as MdCaMBP. The results of semi-quantitative RT-PCR showed that it was expressed in all tissues of ’Guoguang’ leaves and flowers, and interacted strongly with the mature polypeptide regions of apple type S1-, S2- and S9-RNase. It is speculated that MdCaMBP may participate in the self-incompatibility reaction as an S-RNase cofactor.