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狗蔷薇不定根可被噻苯隆(TDZ)高效诱导形成类原球茎,深入探讨其诱导阶段的分子生物学机制,有利于指导切花月季建立类似高效再生和遗传转化体系。快速、准确克隆和筛选狗蔷薇类原球茎发生的关键基因是进行分子生物学水平研究的一个重要前提。利用苹果(Malus×domestica)、桃(Prunus persica)和草莓(Fragaria vesca)高同源性基因5’UTR(非翻译区)的相对保守性,在其内保守区域设计上游简并引物,以M-MLV逆转录酶反转录合成cDNA为模板,直接扩增蔷薇科植物狗蔷薇(Rosa canina)RcPIN1(pin-formed)和RcPLT(plethora)基因5’末端,大大减少了获取众多目的基因全长的时间及成本。结果显示,在狗蔷薇RcPIN1和RcPLT基因与其对应苹果、桃和草莓高同源性基因的5’UTR内,很多区域表现出较高保守性,表明在5’UTR相对保守区域设计上游简并引物扩增目的基因5’末端的合理性。与常规5’CDNA末端快速扩增技术(5’RACE)相比,本方法无需对cDNA模板5’末端加尾同聚物和引入锚定引物,即不必使用昂贵的5’RACE试剂盒,具有快捷、节约等优点,是一种获取蔷薇科植物基因5’末端的可行途径。
Adventitious roots of dog rose can be efficiently induced by TDZ to form protocorm-like bodies, and the molecular biology mechanism in induction stage can be further explored, which is helpful to guide the researches on the establishment of similar efficient regeneration and genetic transformation system in cut rose. Rapid and Accurate Cloning and Screening of Key Genes Involved in the Protocorm-Like Growth of Dog Rose is an Important Prerequisite for Molecular Biology Research. Based on the relative conservation of 5 ’UTR (untranslated region) of homologous genes of Malus × domestica, Prunus persica and Fragaria vesca, upstream degenerate primers were designed in the conserved regions of them. M-MLV Reverse transcriptase reverse transcriptase cDNA as a template to directly amplify the 5 ’end of the Rosa canina RcPIN1 (pin-formed) and RcPLT (plethora) genes, greatly reducing the time for obtaining the full length of many target genes And cost. The results showed that many regions showed high conservation within the 5’UTR of high homology genes between R. solanacearum RcPIN1 and RcPLT genes and their corresponding apple, peach and strawberry genes, indicating that upstream degenerate primers were designed to be amplified in relatively conserved regions of 5’UTR The rationality of the 5 ’end of the target gene. Compared to conventional 5’CDNA rapid amplification (5’RACE), this method does not require the tailing of the 5 ’end of the cDNA template with the introduction of an anchor primer, ie without having to use an expensive 5’ RACE kit, with Fast, economical and other advantages, is a viable way to get the 5 ’end of Rosaceae gene.