Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (1): 142-154.doi: 10.3864/j.issn.0578-1752.2016.01.013

• HORTICULTURE • Previous Articles     Next Articles

Cloning, Subcellular Localization and Expression Analysis of AsAG from Sugar Apple (Annona squamosa L.)

LIU Kai-dong, LI Hai-li, ZHONG Shu-ting, YUAN Chang-chun, CHEN Yan, LIU Jin-xiang, ZHONG Jun-di   

  1. Life Science and Technology School, Lingnan Normal University, Zhanjiang 524048, Guangdong
  • Received:2015-07-31 Online:2016-01-01 Published:2016-01-01

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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

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