《植物生理学报》 2014, 50(8): 1151-1158

牡丹不定根形成相关基因PsARRO-1的克隆及表达分析

贺丹¹, 李睿¹, 纪思羽², 吴静¹, 王政¹, 刘艺平¹, 何松林^1,*

¹河南农业大学林学院, 郑州450002; ²郑州市园林局经纬广场, 郑州450002

通信作者：何松林；E-mail: hsl213@163.com；Tel: 0373-6355-8809

摘　要：

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。

关键词：牡丹; 不定根; PsARRO-1; 基因克隆; 表达分析

收稿：2014-04-22   修定：2014-06-27

资助：国家自然科学基金(31272189)、国家科技成果转化项目(2012D0001018)和郑州市创新团队项目(10CXTD147)。

Cloning and Expression Analysis of Adventitious Rooting Related Gene PsARRO-1 of Tree Peony

HE Dan¹, LI Rui¹, JI Si-Yu², WU Jing¹, WANG Zheng¹, LIU Yi-Ping¹, HE Song-Lin^1,*

¹College of Forestry, Henan Agricultural University, Zhengzhou 450002, China; ²Jing Wei Square, Zhengzhou Bureau of Parks, Zhengzhou 450002, China

Corresponding author: HE Song-Lin; E-mail: hsl213@163.com; Tel: 0373-6355-8809

Abstract:

A cDNA clone encoding a novel 2-oxoacid-dependent dioxygenase (adventitious rooting related oxygenase, ARRO-1) may prove a reliable molecular marker for adventitious root formation in woody plants. ARRO-1 is a specifically expressed gene of adventitious root in the starting stage. In this study, the full-length cDNA of ARRO-1 gene was cloned by RT-PCR and RACE from Paeonia suffruticosa ‘Wu long peng sheng’, and named as PsARRO-1 (GenBank accession number KJ620008). The cDNA sequence of PsARRO-1 contained a 900-bp open reading frame, and encoding 299 amino acids. The results of amino acid sequence homology analysis showed that the sequence had high homology with other plant 2-oxoacid-dependent dioxygenase gene. Quantitative real-time PCR analysis of the gene’s expression in P. suffruticosa ‘Feng dan bai’ in vitro plantlets and seedlings showed that it had different gene expression level in their roots, stems, leaves and flowers. The average expression of root was higher than other parts and it had higher expression in seedlings than in in vitro plantlets. In in vitro plantlets, the expression of PsARRO-1 in root had an obvious variation trend. At the beginning of root induction, the expression was smooth and slender. Then it was up-regulated at the 10th day and appeared two significant peaks at the 15th and 40th day. In seedlings, the expression of PsARRO-1 in root increased rapidly at the beginning of the sampling and peaked at the 10th day, then fell back quickly. These results are corresponding with the way of adventitious roots forming. It suggests that PsARRO-1 is closely related to the peony adventitious root formation.

Key words: Paeonia suffruticosa; adventitious rooting; PsARRO-1; gene cloning; expression analysis