Preliminary study of calmodulin binding protein interacting with stylar S-RNase in apple ＇Rails Genet~ | 苹果‘国光’花柱中与S-RNase互作的钙调素结合蛋白研究

Chinese. 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。

English. The research was to investigate non-S factors interacting with S-RNase in SI through the yeast two-hybrid （Y2H）system. Yeast cDNA library was successfully constructed for apple ＇Rails Genet＇ style, and being inserted by inserting sequences of 300 - 2 000 bp in size. One cDNA fragment of 371 bp was obtained from the library screening by apple pGBKTT,SI-mat as bait through the yeast two-hybrid（Y2H）system. With comparison in NCBI and structure projection,the fragment had highly homology with calmodulin binding protein in Arabidopsis thaliana, and had the peculiar phosphodiesterase structure domain with calmodulin binding protein. The the calmodulin binding protein gene named as MdCaMBP could be considered as the segment, located on chromosome 9 of apple and interacted with calmodulin（CaM）. This gene had one complete ORF of 552 bp and expressed in leaf,sepal,petal,ovary and pollen,style of apple ＇Rails Genet）. The Y2H assay showed that MdCaMBP interacted with mature peptide area of S1-RNase, S2- RNase,Se-RNase. All these results indicated that apple MdCaMBP might be one of style non-S factors interacting with S- RNase nonspecifically in SI.