洋葱AcGAI的克隆及其在开花途径的功能分析

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

摘要/Abstract

摘要：

以长日照型洋葱品系SB2为材料，克隆获得DELLA家族基因AcGAI。序列分析显示AcGAI编码区全长1 575 bp，推测编码524个氨基酸，序列比对表明AcGAI具有典型的DELLA结构域和GRAS结构域。qRT-PCR结果显示AcGAI在洋葱生殖生长期叶片、叶鞘、鳞茎、花茎和花中均有表达，且在花中表达量较高。过表达AcGAI拟南芥转基因株系呈现开花时间延迟；AcGAI在拟南芥gai及della突变体中异源过表达，不同程度地抑制了其早花表型。利用qRT-PCR检测AcGAI过表达植株中光周期核心基因CO和开花调控关键基因FT的表达，发现CO、FT表达量均显著下降。酵母双杂交和双分子荧光互补试验表明AcGAI和AcCO存在互作，说明AcGAI通过调控CO的表达进而延迟植物开花。

关键词: 洋葱, AcGAI, 异源过表达, 开花调控

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

赵静一, 吴小旭, 胡云捷, 盖淑婷, 朱志浩, 秦蕾, 王勇. 洋葱AcGAI的克隆及其在开花途径的功能分析[J]. 园艺学报, 2024, 51(8): 1792-1802.

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.

图/表 8

表1 本研究中所用引物序列

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

图1 洋葱AcGAI与其他植物GAI蛋白的氨基酸序列比对 Vv：葡萄；Sl：番茄；St：马铃薯；Ps：豌豆；Ca：鹰嘴豆；Mt：蒺藜苜蓿；Gm：大豆；Va：赤豆；Pv：菜豆；Si：芝麻；Gh：陆地棉；Pp：沙梨；Md：苹果；Rc：蓖麻；Cs：黄瓜；Aa：豚草；At：拟南芥；Ac：洋葱。

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.

图2 洋葱AcGAI与其他植物GAI蛋白的系统发育树分析 Vv：葡萄；Aa：豚草；At：拟南芥；Ac：洋葱；Si：芝麻；Sl：番茄；St：马铃薯；Ps：豌豆；Ca：鹰嘴豆；Mt：蒺藜苜蓿； Gm：大豆；Va：赤豆；Pv：菜豆；Cs：黄瓜；Gh：陆地棉；Rc：蓖麻；Pp：沙梨；Md：苹果。

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.

图3 AcGAI在洋葱不同器官中的表达分析不同小写字母表示单因素方差（ANOVA）分析，P < 0.05。

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.

图4 拟南芥野生型（WT）和过表达AcGAI植株（WT-AcGAI-OE）的表型和相关内源基因表达量变化同时期处理与对照的差异显著性分析用t-test法统计，**P < 0.01。下同。

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.

图5 过表达AcGAI拟南芥突变体株系的表型和相关内源基因表达量变化

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

图6 AcGAI与AcCO的酵母共转化结果

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

图7 AcGAI与AcCO的BiFC验证

Fig. 7 Verification of AcGAI and AcCO by BiFC

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