东方百合LoAS1和LoAS2基因的克隆、亚细胞定位及表达分析

Cloning, subcellular localization and expression analysis of LoAS1 and LoAS2 genes in Lilium oriental hybrids

关键词:  东方百合‘索邦’，LoAS1，LoAS2，亚细胞定位，表达分析

Key words:  Oriental lily 'Sorbonne', LoAS1, LoAS2, subcellular localization, expression analysis

摘要:

AS1和AS2基因在植物叶片发育过程中具有重要作用，然而东方百合LoAS1和LoAS2是否在鳞茎低温休眠过程中起调控作用尚未见报道。为研究LoAS1和LoAS2与鳞茎低温休眠过程中侧生原基发育的相关性，通过同源克隆得到‘索邦’百合MYB转录因子LoAS1和LBD家族基因LoAS2，并进行系统进化树构建，经实时荧光定量PCR、亚细胞定位等分析其表达特征。结果表明：（1）LoAS1基因编码序列长度为1 035 bp，共编码344个氨基酸，LoAS2基因编码序列长度为717 bp，共编码238个氨基酸。（2）系统进化分析显示，LoAS1与老鸦瓣、卷丹亲缘关系较近，与拟南芥、玉米、水稻等AS1同源基因亲缘关系最远；LoAS2与油棕、椰子亲缘关系较近，与玉米、水稻亲缘关系较远。（3）保守基序和结构域分析显示，LoAS1、LoAS2分别与其他植物中的同源蛋白含有相同的保守基序和N端结构域。（4）亚细胞定位结果显示，LoAS1蛋白定位于细胞核，LoAS2蛋白定位于细胞质和细胞核。（5）LoAS1和LoAS2在低温贮藏过程中，整体表达量显著高于常温组，二者均响应低温在茎尖中上调表达，并在30~40 d表达量上升到最高。（6）石蜡切片结果显示，低温贮藏45 d时，百合鳞茎侧生原基明显增大，与LoAS1和LoAS2表达特征相吻合。该研究表明，LoAS1和LoAS2可能对百合鳞茎休眠解除过程中侧生原基的发育具有重要作用，为百合鳞茎休眠解除机制研究提供了分子依据。

Abstract:

AS1 and AS2 genes have critical roles in plant leaf development, however, whether Oriental lily LoAS1 and LoAS2 play a regulatory role in bulb low-temperature dormancy has not been reported. In order to investigate the correlation between LoAS1 and LoAS2 and the development of lateral primordia during bulb low-temperature dormancy, we obtained the 'Sorbonne' lily MYB transcription factor LoAS1 and the LBD family gene LoAS2 by homologous cloning, constructed a phylogenetic tree, and analyzed their expression characteristics by RT-qPCR and subcellular localization. The results were as follows: (1) The coding sequence length of LoAS1 was 1 035 bp, encoding a total of 344 amino acids, while the coding sequence length of LoAS2 was 717 bp, encoding a total of 238 amino acids. (2) Phylogenetic analysis revealed that LoAS1 was more closely related to homologous proteins in Amana edulis and Lilium lancifolium, and most distantly related to Arabidopsis thaliana, Oryza sativa and Zea may; LoAS2 was more closely related to homologous proteins in Elaeis guineensis and Cocos nucifera, and more distantly related to Oryza sativa and Zea may. (3) Conserved motifs and structural domains analysis revealed that LoAS1 and LoAS2 share conserved motifs and N-terminal domains with their homologous proteins in other plants. (4) The results of subcellular localization revealed that LoAS1 protein was localized in the nucleus and LoAS2 protein in the cytoplasm and nucleus. (5) The overall expression levels of LoAS1 and LoAS2 were significantly higher than those in the control group stored at room temperature., and both of them were up-regulated in the shoot apex in response to the cold temperature, with their expression peaking at 30~40 days of storage. (6) The results of paraffin sections revealed that the lateral primordia of lily bulbs were significantly enlarged at 45 days of low-temperature storage, which was consistent with the expression patterns of LoAS1 and LoAS2. The study indicates that LoAS1 and LoAS2 may play an important role in the development of lateral primordia during the dormancy release process of lily bulbs, which provides a molecular basis for further research into the dormancy release mechanism of lily bulbs.