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不定根发生相关加氧酶基因(adventitious rooting related oxygenase,ARRO-1)被认为是木本植物不定根形成的分子标记之一,属不定根发生起始阶段的特异表达基因。本实验以牡丹‘乌龙捧盛’为材料,运用RT-PCR和RACE相结合的方法克隆得到一个ARRO-1的全长cDNA序列,命名为PsARRO-1(GenBank登录号KJ620008)。PsARRO-1 cDNA序列的开放阅读框长度为900 bp,编码299个氨基酸。氨基酸序列同源性分析结果显示与拟南芥、毛果杨、苹果、梅花等植物有较高的相似性。利用实时荧光定量PCR对该基因在牡丹‘凤丹白’试管苗及实生苗的表达情况分析表明,PsARRO-1在试管苗及实生苗的根、茎、叶、花中均有不同程度的表达,根中的平均表达量大于其他部位,且实生苗中的整体表达量一般高于试管苗中的表达量。试管苗中,PsARRO-1在根中的表达量变化趋势明显,生根诱导初期表达量平稳且微弱,第10天开始上调表达,第15天和第40天分别出现两次峰值;实生苗中,PsARRO-1在根中的表达量在取样初期就开始快速上升,第10天达到峰值,之后迅速回落。这与牡丹不定根的发生过程基本一致,说明PsARRO-1与牡丹不定根的形成密切相关。
Adventitious rooting related oxygenase (ARRO-1) is considered as one of the molecular markers of adventitious root formation in woody plants, which is a specific gene expressed at the initial stage of adventitious root formation. In this experiment, the full-length cDNA sequence of ARRO-1 was cloned by RT-PCR and RACE, and named PsARRO-1 (GenBank accession number KJ620008). The open reading frame of PsARRO-1 cDNA was 900 bp, encoding 299 amino acids. Amino acid sequence homology analysis showed that Arabidopsis, Populus trichocarpa, apple, plum and other plants have a higher similarity. The results of real-time PCR showed that PsARRO-1 was expressed in roots, stems, leaves and flowers of seedlings and seedlings in different degrees , The average expression level in roots was greater than that in other sites, and the overall expression level in seedlings was generally higher than that in in vitro plantlets. The results showed that PsARRO-1 had obvious changes in the root of test-tube plantlets, the initial expression of PsARRO-1 was stable and weak, the expression of PsARRO-1 was up-regulated from the 10th day, and the peak appeared on the 15th day and the 40th day respectively. The expression of PsARRO-1 in the roots began to increase rapidly at the beginning of the sampling period, reached the peak on the 10th day and then dropped rapidly. This is consistent with the adventitious rooting process of peony, indicating that PsARRO-1 is closely related to adventitious root formation.