芍药PlSVP基因的克隆及其花期调控功能分析

doi:10.16420/j.issn.0513-353x.2021-1253

摘要/Abstract

摘要：

从芍药（Paeonia lactiflora）中克隆SHORT VEGETATIVE PHASE（SVP）基因，研究其对花期的调控功能，为芍药分子育种储备重要基因资源。采用RT-PCR方法从‘大富贵’芍药中克隆了PlSVP基因，其开放阅读框包含687 bp碱基，编码228个氨基酸。系统进化树分析发现，PlSVP编码的蛋白与同属的牡丹PsSVP蛋白亲缘关系最近。qRT-PCR分析结果表明，PlSVP主要在营养器官中表达，在根中表达量最高，其次是叶片;在生殖器官花瓣中表达量极低。将PlSVP转化入哥伦比亚型野生型拟南芥，发现转PlSVP基因的株系较野生型的抽薹、开花时间分别延迟7和15 d;在拟南芥svp-41突变体中过表达PlSVP发现，其不再呈现早抽薹、早开花表型，即PlSVP基因回补了svp-41突变体的功能缺失。进一步比较研究表明，拟南芥转PlSVP基因植株中FT、SOC1、LFY、AP1等SVP下游开花促进基因的表达量均显著下调。以上结果表明，芍药PlSVP基因负调控植物开花时间，并且可能通过抑制FT、SOC1、LFY、AP1等基因表达而发挥其负调控功能。

关键词: 芍药, SVP基因, 过表达, 功能互补, 开花

Abstract:

Cloning SHORT VEGETATIVE PHASE（SVP）gene from peony and exploring its function in regulating flowering will provide gene resource for molecular breeding of peony. In this study，PlSVP gene was isolated from Paeonia lactiflora‘Da Fugui’by RT-PCR method. The open reading frame of PlSVP gene was 687 bp in length，encoding 228 amino acids. Phylogenetic analysis indicated that PlSVP protein had the closest relationship with the homologue from P. suffruticosa. The qRT-PCR results showed that the expression of PlSVP gene was generally confined to vegetative organs. The highest expression was detected in roots，followed by in leaves，and the expression level in the basal leaves was higher than that in the top leaves. Overexpression of PlSVP gene in Arabidopsis Col-0 indicated that both bolting and flowering of the transgenic plants were delayed，by 7 and 15 d，respectively. And the complementary overexpression study in Arabidopsis svp-41 verified the inhibition effect of PlSVP gene. Further study that the expression of the floral promotors FT，SOC1，LFY and AP1 in transgenic plants was significantly down-regulated. These results suggested that PlSVP gene distinctively and negatively control flowering time，and possibly via inhibiting the expression of FT，SOC1，LFY and AP1.

Key words: herbaceous peony, Paeonia lactiflora, SVP gene, overexpression, complementary function, flowering

中图分类号:

S682.12

籍凤娇, 马燕, 亓帅, 郭先锋, 陈俊强. 芍药PlSVP基因的克隆及其花期调控功能分析[J]. 园艺学报, 2022, 49(11): 2367-2376.

JI Fengjiao, MA Yan, QI Shuai, GUO Xianfeng, CHEN Junqiang. Cloning and Functional Analysis of Peony PlSVP Gene in Regulating Flowering[J]. Acta Horticulturae Sinica, 2022, 49(11): 2367-2376.

图/表 7

表1 所用引物列表

Table 1 List of primers

用途 Purpose	引物 Primer	核酸序列（5′-3′） Nucleotide sequence
基因克隆	PlSVP-F	ATGGCGAGAGAGAAGATTCAGAT
Gene cloning	PlSVP-R	TCAACCCGAATATGGCAGTC
载体构建Vector	pBI121-PlSVP-F	CGCGGATCCGCGATGGCGAGAGAGAAGATTCAGAT
construction	pBI121-PlSVP-R	CCGGAATTCCGGTCAACCCGAATATGGCAGTC
	35S	GACGCACAATCCCACTATCC
内参Internal	PlActin-F	ACTGCTGAACGGGAAATT
reference	PlActin-R	ATGGCTGGAACAGGACTT
qRT-PCR	qPlSVP-F	AGAACTGGCGGCTGAGACAA
	qPlSVP-R	TCCTTCCTCGCAAACCATG
内参Internal	Atactin-F	TCTCTATGCCAGTGGTCGTA
reference	Atactin-R	CCTCAGGACAACGGAATC
qRT-PCR	AtFT-F	TGGAACAACCTTTGGCAATG
	AtFT-R	GTCTTCTTCCTCCGCAGC
	AtAP1-F	CAGATCAAGGAGAGGGAA
	AtAP1-R	TTGATACAGACCACCCAT
	AtSOC1-F	CTCCAATATGCAAGATACCA
	AtSOC1-R	TATGCCTTCTCCCAAGAGTT
	AtLFY-F	CCCAAGAAGGGTTATCTGA
	AtLFY-R	AAACGGATGCTCCCTCTG

图1 PlSVP蛋白与其他SVP蛋白的系统发育聚类分析标尺表示序列间差异数值的单位长度。进化树分支处的数字表示该分支的置信度。

Fig. 1 Phylogenetic tree based on the amino acid alignment of PlSVP and SVP proteins from other plant species The scale indicates the reference length of the difference values between sequences. The number at each branch of the phylogenetic tree indicates the confidence level of the branch.

图2 ‘大富贵’芍药不同组织中PlSVP的相对表达量不同字母表示差异显著（P < 0.05）。

Fig. 2 Relative expression of PlSVP gene in different tissues of Paeonia lactiflora‘Da Fugui’ Different letters on bars indicate significant difference（P < 0.05）.

图3 拟南芥转PlSVP株系（a）和其svp-41突变体转PlSVP株系（b）的抽薹时间和开花时间不同字母表示差异显著（P < 0.05）。

Fig. 3 Bolting and flowering time of Arabidopsis thaliana svp-41：Arabidopsis svp-41 mutants;a：Transgenic wild type Arabidopsis plants;b：Transgenic Arabidopsis svp-41 mutants;Different letters on bars indicate significant difference（P < 0.05）.

图4 拟南芥转PlSVP株系（a1 ~ a4）和其svp-41突变体转PlSVP株系（b1 ~ b4）中PlSVP的表达量 **表示转基因株系与对照差异极显著（P < 0.01）。

Fig. 4 Expression of PlSVP gene in transgenic Arabidopsis a1-a4：Expression of PlSVP gene in different transgenic lines of wild type Arabidopsis plants;b1-b4：Expression of PlSVP gene in different transgenic lines of Arabidopsis svp-41 mutants;** represents significant difference between transgenic lines and the control（P < 0.01）.

图5 拟南芥转PlSVP株系（a2）和其svp-41突变体转PlSVP株系（b2）中SVP下游基因的表达量 **表示转基因株系与对照差异极显著（P < 0.01）。

Fig. 5 Expression of SVP downstream genes in transgenic Arabidopsis A：Expression of AtSVP downstream gene in PlSVP-transformed wild type Arabidopsis plants;B：Expression of AtSVP downstream gene in PlSVP- transformed Arabidopsis svp-41 mutants;** represents significant difference between transgenic lines and the control（P < 0.01）.

图6 PlSVP在芍药不同器官中的表达及其负向调控拟南芥开花时间示意图

Fig. 6 Schematic diagram of spatial expression of PlSVP gene in Paeonia lactiflora and the function of negatively regulating flowering time in Arabidopsis

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