番荔枝花器官发育基因AsAG的克隆、亚细胞定位及表达分析

doi:10.3864/j.issn.0578-1752.2016.01.013

摘要/Abstract

摘要： 【目的】从番荔枝中分离花器官特征决定基因AGAMOUS，并进行亚细胞定位及基因表达分析，为进一步研究该基因参与调控花发育调控的分子机理，及解决番荔枝花发育异常问题奠定基础。【方法】以番荔枝成熟花为材料，通过试剂盒提取总RNA，并以RACE方法克隆获得基因AG的全长序列。序列拼接和氨基酸序列分析采用DNAMAN软件；相似性分析通过BLASTn和BLASTp程序进行；进化树构建采用MEGA 5.1软件；蛋白质二级结构预测与3D结构建模分别采用ExPaSy的SOPMA和Phyre2程序进行；亚细胞定位表达采用烟草上皮细胞瞬时转染系统和基因枪轰击洋葱表皮细胞方法；AG在花发育不同时期、不同器官及不同激素信号分子处理下的表达特性分析，利用实时荧光定量RT-PCR方法。【结果】从番荔枝中克隆得到AGAMOUS，其cDNA全长ORF序列长度为669 bp，编码222个氨基酸，命名为AsAG，序列提交到GenBank（登录号为KT159768）。二级结构预测发现AsAG所编码蛋白由延伸链结构（俄extended strand）、α-螺旋（alpha helix）、β-转角（beta turn）和不规则卷曲（random coil）组成，四者比例分别为14.41%、59.46%、8.11%和18.02%。生物信息学分析显示AsAG编码的蛋白与海枣（XP 008781978.1）、芦笋（BAD83772.1）、拟南芥（AT4G18960）、油棕（XP 010912706.1）、山玉兰（AFH74390.1）、石斛（ABQ08574.1）、红花玉兰（AEO52692.1）等同源蛋白的相似度达79%—84%。ASAG蛋白含有一个高度保守的MADS-box结构域和一个次级保守的K区，该蛋白分子量为25.7 kDa，等电点为9.15，为稳定蛋白，无信号肽。亚细胞定位显示AsAG编码蛋白定位于细胞核。实时定量RT-PCR结果表明，AsAG在不同的器官中表达量存在差异，在花中表达量最高。AsAG在番荔枝花发育的整个过程中都有表达，而在花蕾期Ⅳ中表达量最高。进一步分析发现AsAG的表达水平呈现花器官特异性分布（雄蕊＞雌蕊＞萼片＞花瓣）。进一步研究表明，AsAG在畸形花中的表达量低于正常花。检测GA和ABA等不同信号分子分别处理番荔枝花芽2 h和4 h后AsAG的表达量，结果表明AsAG的表达受GA负调控，受ABA正调控。【结论】推测番荔枝AsAG可能参与雌蕊和雄蕊的发育及激素信号的响应。

关键词: 番荔枝, AG, 花发育, 表达模式

Abstract: 【Objective】This paper aims to isolate the cDNA of AGAMOUS from sugar apple (Annona squamosa L.) and to study their subcellular localization and expression level, in order to lay a foundation for further exploration of the flower development mechanism and solve the problem of flower abnormality. 【Method】 Total RNA was extracted from mature flowers of sugar apple. Polymerase chain reaction (PCR) combined with 5′ RACE, and 3′ RACE technology were used to clone the full length cDNA. The sequencing data were assembled by DNAMAN software. BLASTn and BLASTp in NCBI were used to conduct the similarity analysis. DNAMAN was used to analyze the amino acid sequences and MEGA 5.1 was used to create the phylogenetic tree; the protein secondary structure and the structure of 3D modeling was predicted by SOPMA and Phyre2 programs. Recombinant plasmid was introduced into onion epidermal cells and tobacco epithelial cells. Green fluorescence was monitored under a laser scanning confocal microscope. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern of AsAG in different developmental stages of flower and different flower organs. The expression levels of AsAG responsing to different hormone treatments were also determined by qRT-PCR. 【Result】 A full-length cDNA sequence of the homologous AGAMOUS gene was cloned from sugar apple. Sequence analysis showed that the AsAG gene contains a 669 bp open reading frame (ORF) encoding 222 amino acids. The sequence was submitted to GenBank, and the registration number is KT159768. The predictive secondary structure showed that AsAG protein was made up of 59.46% alpha-helix, 8.11% beta-turn, 14.41% extended strand and 18.02% random coil. The amino acid sequences shared 79%-84% in homology compared with Phoenix dactylifera (XP 008781978.1), Asparagus virgatus (BAD83772.1), Arabidopsis thaliana (AT4G18960), Elaeis guineensis (XP 010912706.1), Magnolia delavayi (AFH74390.1), Dendrobium nobile (ABQ08574.1) and Magnolia wufengensis (AEO52692.1). The deduced amino acids sequence contained a highly conserved MADS domain and a secondary conserved K domain. The molecular weight and an isoelectric point of this protein were 25.7 kDa and 9.15, respectively. The protein was stable protein and had no signal peptide. Subcellular localization assays showed that the AsAG protein was located in the nucleus. The qRT-PCR results suggested that AsAG gene showed a high transcription level in flower. The AsAG gene could be detected during the whole period of flower development. Interestingly, AsAG gene showed a high transcription level in the stage of flower bud Ⅳ. The expression level in the stamen was higher than that in the pistil, sepal and petal. The expression level of perfect flower was higher than deformity flower. After 2 h and 4 h treatments with GA and ABA signal molecules in flower buds, the expression patterns of AsAG showed negative regulation by GA, positive regulation by ABA. 【Conclusion】AsAG gene may participate in the pistil and stamen development and hormone signal response in sugar apple.

Key words: sugar apple, AG, flower development, expression pattern

刘锴栋，黎海利，钟舒婷，袁长春，陈燕，刘金祥，钟军弟. 番荔枝花器官发育基因AsAG的克隆、亚细胞定位及表达分析[J]. 中国农业科学, 2016, 49(1): 142-154.

LIU Kai-dong, LI Hai-li, ZHONG Shu-ting, YUAN Chang-chun, CHEN Yan, LIU Jin-xiang, ZHONG Jun-di. Cloning, Subcellular Localization and Expression Analysis of AsAG from Sugar Apple (Annona squamosa L.)[J]. Scientia Agricultura Sinica, 2016, 49(1): 142-154.

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