Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (9): 1771-1782.doi: 10.3864/j.issn.0578-1752.2018.09.014

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Functional Verification of AINTEGUMENTA Gene in Chrysanthemum

WEN LiZhu, SUN Xia, FAN HongMei, GUO YunHui, YU YuanYuan, REN Hong, WANG WenLi, ZHENG ChengShu   

  1. College of Horticulture Science and Engineering, Shandong Agricultural University/Chrysanthemum Research Center of China, Japan and Korea in Shandong Province, Tai’an 271018, Shandong
  • Received:2017-09-20 Online:2018-05-01 Published:2018-05-01

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Abstract: 【Objective】 To understand the role of AINTEGUMENTA gene in chrysanthemum inflorescence development, we cloned AINTEGUMENTA gene from chrysanthemum, analyzed its sequence, and characterized its temporal and spatial expression pattern. We further analyzed the impact of AINTEGUMENTA silence on inflorescence development and its possible regulation mode. This study was carried to reveal the potential mechanism of AINTEGUMENTA in governing inflorescence development, which in turn can provide a theoretical basis for chrysanthemum inflorescence diameter adjustment. 【Method】 The chrysanthemum AINTEGUMENTA gene was cloned by RACE method and its sequence was analyzed with DANMAN software. Its expression in different developmental stages and organs of chrysanthemum was detected by real-time fluorescent quantitation PCR. The plasmid of 35S::CmANT-GFP was constructed for the subcellular localization. The silence vector of TRV2-CmANT was constructed to infect chrysanthemums. The statistics of phenotypic changes in CmANT silenced chrysanthemums were analyzed via SPSS software. The ray florets petal epithelial cells were observed with optical microscope. The expression of CmANT and related genes were detected by real-time fluorescent quantitation PCR. 【Result】 The full-length of CmANT was cloned from chrysanthemum. It encodes 540 amino acids and contains two AP2 conserved function domains and VYL modification sites. The theoretical isoelectric point of CmANT is 7.39 and its molecular weight is 60.4 kD. The phylogenetic tree composed of ANT proteins from various plants species showed that CmANT and AaANT had been grouped together. The real-time fluorescent quantitation results showed that: (1) CmANT was expressed most in floral buds, followed by roots, stems and leaves. (2) Gene expression in different parts of inflorescence indicated that CmANT was expressed the most in ray florets followed tubular florets and the lowest in the sepal. (3) The expression of CmANT declined during the development of ray florets. (4) The expression of CmANT under 2,4-D treatment increased between 3 h to 6 h. WoLF PSORT prediction and 35S::CmANT-GFP fusion protein localization in onion epithelial cells indicated that CmANT protein was located in cell nucleus. Compared with the control, the mean inflorescence diameters of chrysanthemums in TRV-CmANT-1 and TRV-CmANT-2 lines were decreased by 18.93% and 27.47% respectively and the numbers of ray florets were decreased by 11.39% and 14.66% respectively, among which the difference between TRV-CmANT-2 lines and the control was statistically significant (P＜0.05 ). The numbers of tubular florets were decreased by 14.55% and 36.56%, the mean length of ray florets in top inflorescences was decreased by 34.17% and 54.68%, and the width was decreased by 24.05% and 10.13% respectively in TRV-CmANT-1 and TRV-CmANT-2 lines compared with the control, the mean fresh weight of leaves was decreased by 13.19% and 21.98% respectively in TRV-CmANT-1 and TRV-CmANT-2 lines and the correlation between leaf fresh weight and tubular floret number was significant (P＜0.05 ). The microscopic observation of epithelial cells in ray floret petals indicated that the cell length and width of petals in the silenced lines and the control had no visible differences. The expression of CmLAX3 in two silenced chrysanthemums lines were 1.8 and 1.78 times higher than the control, respectively. Meanwhile the expression of CmCYCD3 was decreased by 32.28% and 38.19% in two silenced lines and the expression of CmXTH4 and CmEXPA1 also decreased at most developmental stages. 【Conclusion】 Due to the phenotypic changes and CmANT expression pattern in silenced chrysanthemums lines, we speculated that the silence of CmANT might relieve the repression on CmLAX3, facilitate the transport and accumulation of auxins, indirectly repress the activation of CmCYCD3, limit the cell division, and cause the decrease of cell number, combined of which would eventually lead to the smaller organ sizes in silenced chrysanthemums lines.

Key words: Chrysanthemum morifolium, AINTEGUMENTA, inflorescence diameter, gene expression, gene silence

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