洋葱花器官B类MADS-box基因AcPI的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2012.23.002

摘要/Abstract

摘要： 【目的】克隆洋葱花器官B类PI/GLO家族MADS-box基因，分析其序列特征及时空表达模式，为探讨其在洋葱花发育过程中的分子遗传机制奠定基础。【方法】以洋葱花蕾总RNA为模板，根据同源克隆策略设计简并引物，利用RT-PCR结合RACE技术，获得AcPI的全长cDNA序列。用生物信息学方法对其基因序列特征进行分析；利用RT-PCR和Real-time PCR分析AcPI在花蕾整个生长过程中的时空表达模式。【结果】克隆获得洋葱AcPI基因（GenBank登录号：JX679083）的cDNA全长931 bp，包含615 bp的完整开放阅读框，编码205个氨基酸。蛋白分析表明，AcPI蛋白具有植物MADS-box蛋白典型的MADS和K结构域；与水仙的NTPI、风信子的HoMADS2有78%、75%的相似性，与金鱼草的GLO也有52%的相似性。系统进化树分析表明，AcPI属于B类MADS-box蛋白家族的PI亚家族。RT-PCR表达分析表明，AcPI只在生殖器官花蕾中表达，但主要在花的第一、二、三轮花器官中表达，而在营养组织根、茎和叶中不表达。Real-time PCR进一步分析表明，AcPI在花芽整个生长过程中，在心皮中微弱表达，但表达丰度呈递增趋势；而在外轮被片、内轮被片和雄蕊中强烈表达，其表达丰度除了在外轮被片中呈先增后减的趋势外，在内轮被片和雄蕊中都呈递增趋势。【结论】AcPI在洋葱第一轮花器官中的表达支持了van Tunen提出的修正的ABC模型；但AcPI在洋葱第四轮花器官中也有表达，这表明AcPI除了调控外轮被片、内轮被片和雄蕊发育外，还可能在心皮的形成发育过程中起着重要作用。

关键词: 洋葱 , 花器官 , MADS-box基因 , 基因克隆 , 表达分析

Abstract: 【Objective】The aim of this study is to clone the PI/GLO subfamily MADS-box gene of class B of the floral identity in onion, to analyze its sequence characteristics and the temporal and spatial expression pattern. The results will provide a foundation for further dissection of the molecular and genetics mechanisms of floral organ development in onion in detail.【Method】Using the total RNA from the floral buds of onion (Allium cepa) as the template, we design the degenerate primers based on homology cloning strategy, and then the full-length cDNA sequence of AcPI was obtained through a combined reverse transcription-PCR (RT-PCR) and RACE techniques strategy. The bioinformatics methods were used to analyze the sequence characteristics of this gene and the combination of RT-PCR and Real-time PCR methods were used to investigate the temporal and spatial expression pattern of AcPI gene.【Result】The cloned full-length cDNA of AcPI was 931 bp in length, containing a 615 bp open reading frame (ORF) which encodes 205 amino acids. Protein alignment analysis indicated that AcPI protein had typical structure domains of plants MADS-box protein, containing a MADS domain and a K domain. Protein sequence identity search showed that AcPI is similar to NTPI (Narcissus tazetta var. chinensis), HoMADS2 (Hyacinthus orientalis) and GLO (Antirrhinum majus) with similarities of 78%, 75% and 52% respectively. Phylogenetic tree reconstructed clearly showed that AcPI protein was classed into PI subgroup of B class MADS-box protein family. RT-PCR analysis indicated that expression of the AcPI gene was restricted to the flowers as its mRNA was not detected in vegetative tissues such as roots, stems, leaves. Real-time PCR analysis showed that AcPI gene mainly expressed in the first, second and third whorls of flower organ and only weak expression was detected in the fourth whorls of flower organ in all three flower bud developmental stages. Furthermore, the expression level of AcPI was increasing in all flower organs except in the first whorl flower organ is first increases and then decreases in the throughout growing process of flower buds.【Conclusion】The extended expression of AcPI in the first whorl floral organ supported the modified ABC model by van Tunen, but it also expressed in the fourth whorl floral organ, which showed that AcPI gene may play an important role in the development of carpels.

Key words: onion , floral organs , MADS-box gene , gene cloning , expression analysis

李洪有, 王婵, 李丽林, 王永勤, 赵瑞. 洋葱花器官B类MADS-box基因AcPI的克隆及表达分析[J]. 中国农业科学, 2012, 45(23): 4759-4769.

LI Hong-You, WANG Chan, LI Li-Lin, WANG Yong-Qin, ZHAO Rui. Cloning and Expression Analysis of A Putative B Class MADS-box Gene of AcPI in Onion[J]. Scientia Agricultura Sinica, 2012, 45(23): 4759-4769.

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