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利用已构建的月季花朵转录组数据库,筛选得到了22个与乙烯受体同源性较高的Uni Gene片段,并利用这些片段从月季花瓣中克隆得到了3个乙烯受体基因全长,分别为RhETR1、RhETR3和RhETR5。其中,RhETR1全长为2814bp,编码一个595aa的肽链;RhETR3全长为2468bp,编码一个765aa的肽链;RhETR5全长2740bp,编码一个742aa的肽链。这3个基因推定的编码蛋白分别与枣(Ziziphus jujuba)中的ZjETR1、杨树(Populus trichocarpa)中的PtETR2和砂梨(Pyrus pyrifolia)中的PpERS2同源性最高,依次为85.2%、75.8%和80.3%。蛋白同源性及结构分析表明,RhETR1和RhETR5分别与拟南芥中的ERS1和ETR1结构相似,属于第1亚家族成员,RhETR3与ERS2结构相似,属于第2亚家族成员。在花朵自然瓶插过程中,这3个基因分别表现为下调、上调和组成型表达。利用原核表达载体,选择和月季花朵开放最为相关的RhETR3,获得了和理论分子量一致的重组蛋白,表明克隆得到的RhETR3具有完整的读码框。
Twenty-two Uni Gene fragments with high homology with ethylene receptors were screened by using the constructed rose flower transcriptome database. Three full-length ethylene receptor genes were cloned from rose petals using these fragments, RhETR1, RhETR3 and RhETR5. Among them, RhETR1 is 2814bp in length and encodes a 595aa peptide chain; RhETR3 is 2468bp in length and encodes a 765aa peptide; and RhETR5 is 2740bp in length and encodes a 742aa peptide chain. The deduced proteins encoded by these three genes had the highest homology with PpERS2 in ZjETR1, PtETR2 and Pyrus pyrifolia respectively in Ziziphus jujuba, followed by 85.2%, 75.8% And 80.3%. Protein homology and structural analysis showed that RhETR1 and RhETR5 are similar to ERS1 and ETR1 in Arabidopsis, respectively, belong to the first subfamily member. RhETR3 and ERS2 have similar structures and belong to the second subfamily member. In the process of flower natural bottle insertion, these three genes showed down-regulation, up-regulation and constitutive expression respectively. The prokaryotic expression vector was used to select RhETR3, which was the most closely related to the opening of the rose flowers. The recombinant protein with the same theoretical molecular weight was obtained, indicating that the cloned rhETR3 has a complete reading frame.