Molecular Cloning and Functional Analysis of AcGAI in Onion Flowering Regulation

doi:10.16420/j.issn.0513-353x.2023-0385

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (8): 1792-1802.doi: 10.16420/j.issn.0513-353x.2023-0385

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Molecular Cloning and Functional Analysis of AcGAI in Onion Flowering Regulation

ZHAO Jingyi, WU Xiaoxu, HU Yunjie, GAI Shuting, ZHU Zhihao, QIN Lei^*(), WANG Yong^*()

Key Laboratory of Biology and Genetic Improvement of Horticultural Crops in Northeast Region，Ministry of Agriculture and Rural Affairs，College of Horticulture and Landscape Architecture，Northeast Agricultural University，Harbin 150030，China

Received:2024-04-17 Revised:2024-06-21 Online:2024-08-25 Published:2024-08-21
Contact: QIN Lei, WANG Yong

Abstract

Abstract:

AcGAI of DELLA family was cloned from a long-day ecotype onion inbred line SB2. Sequence analysis showed that the full length of AcGAI coding region was 1 575 bp，which was estimated to encode 524 amino acids. AcGAI has typical DELLA and GRAS domain. qRT-PCR results showed that AcGAI was expressed in the leaves，leaf sheaths，bulbs，flower stems and flowers of onion during the reproductive growth stage，and the expression level was higher in the flowers. AcGAI overexpressed Arabidopsis thaliana transgenic lines showed delayed flowering time. AcGAI was heterogeneously overexpressed in the gai and della mutants of Arabidopsis thaliana，and the early flowering phenotypes of the gai and della mutants were inhibited to varying degrees. qRT-PCR was used to detect the expressions of the photoperiodic core gene CO and the flowering regulation key gene FT in AcGAI overexpressed plants，and it was found that the expressions of CO and FT were significantly decreased. AcGAI interact with AcCO by yeast two-hybrid system and bimolecular fluorescence complementation technique. This study suggests that GAI involved in flowering regulation by regulating the expression of CO.

Key words: Allium cepa, AcGAI, heterologous overexpression, flowering regulation

ZHAO Jingyi, WU Xiaoxu, HU Yunjie, GAI Shuting, ZHU Zhihao, QIN Lei, WANG Yong. Molecular Cloning and Functional Analysis of AcGAI in Onion Flowering Regulation[J]. Acta Horticulturae Sinica, 2024, 51(8): 1792-1802.

Figures/Tables 8

Table 1 Primer sequence used in this study

用途 Use	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因克隆 Gene of cloning	GAI-S	CGGGATCCATGATCGATCCATTTCTGTTC
基因克隆 Gene of cloning	GAI-X	CGAGCTCCTACAGCAGGTTACTGACATTG
酵母双杂交	GAI-JS	CCGGAATTCATGATCGATCCATTTCTGTTC
Yeast two-hybrid assay	GAI-JX	CGGGATCCCTACAGCAGGTTACTGACATTG
	CO-JS-AD	GCCATGGAGGCCAGTGAATTCATGTATGCTGAGCCTAGGCC
	CO-JX-AD	AGCTCGAGCTCGATGGATCCTTACTTATTTTGTGATTTTGTGAC
双分子荧光互补	C-GAI-F	TGGCGCGCCACTAGTGGATCCATGATCGATCCATTTCTGT
Bimolecular fluorescent complimentary	C-GAI-R	CCCGGGAGCGGTACCCTCGAGCAGCAGGTTACTGACATT
	N-CO-F	TGGCGCGCCACTAGTGGATCCATGTATGCTGAGCCTAGG
	N-CO-R	CCCGGGAGCGGTACCCTCGAGCTTATTTTGTGATTTTGTGACTT
qRT-PCR	AtGAI-F	GGACCTGACCGAGTTGAGCG
	AtGAI-R	AGTTTCCAAGCCGAGGTGGC
	AtCO-F	ACTACTCACCACCAAAGCGA
	AtCO-R	GAAGCAACCTCCTTGGCATC
	AtFT-F	TGGAGACGTTCTTGATCCGT
	AtFT-R	CCTGAGGTCTTCTCCACCAA
	AcActin-F	ACACGGCCTGGATAGCAACAT
	AcActin-R	AGAGCAGTATTCCCAAGCATT

Fig. 1 Amino acid sequence alignment of AcGAI from Allium cepa and GAI proteins of other plants Vv：Vitis vinifera；Sl：Solanum lycopersicum；St：Solanum tuberosum；Ps：Pisum sativum；Ca：Cicer arietinum；Mt：Medicago truncatula；Gm：Glycine max；Va：Vigna angularis；Pv：Phaseolus vulgaris；Si：Sesamum indicum；Gh：Gossypium hirsutum；Pp：Pyrus pyrifolia；Md：Malus × domestica；Rc：Ricinus communis；Cs：Cucumis sativus；Aa：Ambrosia artemisiifolia；At：Arabidopsis thaliana；Ac：Allium cepa.

Fig. 2 Phylogenetic analysis of AcGAI from Allium cepa and GAI proteins of other plants Vv：Vitis vinifera；Aa：Ambrosia artemisiifolia；At：Arabidopsis thaliana；Ac：Allium cepa；Si：Sesamum indicum；Sl：Solanum lycopersicum；St：Solanum tuberosum；Ps：Pisum sativum；Ca：Cicer arietinum；Mt：Medicago truncatula；Gm：Glycine max；Va：Vigna angularis；Pv：Phaseolus vulgaris；Cs：Cucumis sativus；Gh：Gossypium hirsutum；Rc：Ricinus communis；Pp：Pyrus pyrifolia；Md：Malus × domestica.

Fig. 3 Expression analysis of AcGAI at different tissues in Allium cepa Different lowercase letters indicate one-way analysis of variance（ANOVA），P < 0.05.

Fig. 4 Changes in phenotype and expression of related endogenous genes in Arabidopsis thaliana wild type（WT）and overexpressing AcGAI（WT-AcGAI-OE） Signifcant difference between treatment and control at same time point were analyszed by using t-test. **P < 0.01. The same below.

Fig. 5 Relative expression levels and changes of endogenous genes of AcGAI mutant Arabidopsis lines

Fig. 6 Yeast co-transformation results of AcGAI and AcCO

Fig. 7 Verification of AcGAI and AcCO by BiFC

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Molecular Cloning and Functional Analysis of AcGAI in Onion Flowering Regulation

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