森林草莓FvbHLH130转录因子调控植株提前开花

doi:10.16420/j.issn.0513-353x.2021-1246

摘要/Abstract

摘要：

分析草莓碱性螺旋—环—螺旋（basic helix-loop-helix，bHLH）转录因子家族，鉴定到森林草莓（Fragaria vesca）花特异表达的转录因子基因FvbHLH130。FvbHLH130编码区序列长度为960 bp，编码319个氨基酸，保守结构域预测结果显示第247 ~ 297 aa是bHLH结合域，其属于bHLH家族成员。FvbHLH130启动子含有生长素等激素响应、逆境胁迫和低温响应元件，推测基因表达可能受到生物和非生物胁迫诱导。FvbHLH130蛋白氨基酸序列与月季（Rosa chinensis）的相似性高达93.70%，与其他蔷薇科bHLH蛋白聚在一个分支上，与拟南芥AtFBH4是同源蛋白。农杆菌介导的FvbHLH130拟南芥转基因株系提前7 d开花，且促进开花相关基因AtAP1、AtFT、AtFUL和AtCO的表达。酵母双杂交实验结果表明FvbHLH130与FvARF4和FvARF6互作，可能作为蛋白复合体共同参与开花调控。

关键词: 森林草莓, bHLH转录因子, FvbHLH130, 开花调控

Abstract:

The basic helix-loop-helix（bHLH）transcription factor family of strawberry was analyzed，and the transcription factor FvbHLH130 was identified in Fragaria vesca. qRT-PCR analysis in different tissues showed that FvbHLH130 was flower-specifically expressed. Overexpression of FvbHLH130 in Arabidopsis thaliana activated flowering in transgenic lines. The full-length coding sequence of FvbHLH130 was 960 bp，encoding a 319-amino-acid protein. The prediction of conserved domain of FvbHLH130 showed that 247-297 amino acids were bHLH binding domain. The promoter of FvbHLH130 contained hormone，stress- and low temperature-response elements，which indicated FvbHLH130 might be induced by biotic and abiotic stress. The amino acids sequence of FvbHLH130 revealed up to 93.70% similarity with RcbHLH130. FvbHLH130 sequence was more closely related to Rosaceae bHLH proteins and was homologous to AtFBH4. Agrobacterium-mediated transformation of Arabidopsis thaliana showed that the FvbHLH130 overexpression lines flowered seven days earlier than the control plants，and activated the expression of flowering-related genes AtAP1，AtFT，AtFUL and AtCO. The yeast two-hybrid results showed that FvbHLH130 interacted with FvARF4 and FvARF6，which may form protein complexes in the regulation of flowering.

Key words: Fragaria vesca, bHLH transcription factor, FvbHLH130, flowering

中图分类号:

S668.4

宋艳红, 陈亚铎, 张晓玉, 宋盼, 刘丽锋, 李刚, 赵霞, 周厚成. 森林草莓FvbHLH130转录因子调控植株提前开花[J]. 园艺学报, 2023, 50(2): 295-306.

SONG Yanhong, CHEN Yaduo, ZHANG Xiaoyu, SONG Pan, LIU Lifeng, LI Gang, ZHAO Xia, ZHOU Houcheng. The Transcription Factor FvbHLH130 Activates Flowering in Fragaria vesca[J]. Acta Horticulturae Sinica, 2023, 50(2): 295-306.

图/表 7

表1 在本研究中使用的引物列表

Table 1 A list of primers used in this study

用途 Usage	引物名称 Primer name	序列（5′-3′） Sequence
FvbHLH130基因编码区克隆 Cloning the CDS of FvbHLH130	FvbHLH130-CDS-F	ATGGATTCAAATACCCATCTCA
FvbHLH130基因编码区克隆 Cloning the CDS of FvbHLH130	FvbHLH130-CDS-R	TCAAACTATCTGATTTTGAA
FvbHLH130基因启动子克隆 Cloning the promoter of FvbHLH130	FvbHLH130-Pro-F	GACATAATAACCTTGCATTT
FvbHLH130基因启动子克隆 Cloning the promoter of FvbHLH130	FvbHLH130-Pro-R	TATACACAACTCTGCTTGCC
构建FvbHLH130过表达载体 Cloning onstruction of FvbHLH130-overexpression vector	FvbHLH130-121-F	gagaacacgggggactctagaATGGATTCAAATACCCATCTCA
	FvbHLH130-121-R	gcccttgctcaccatggatccAACTATCTGATTTTGAACCGG
转基因阳性苗鉴定 Identification of transgenic positive seedlings	35S	GACGCACAATCCCACTATCC
转基因阳性苗鉴定 Identification of transgenic positive seedlings	GFP-R	TTACTTGTACAGCTCGTCCATG
草莓荧光定量内参基因 Reference genes for qRT-PCR in Fragaria vesca	Fv28S-qF	TAACCGCATCAGGTCTCCAA
草莓荧光定量内参基因 Reference genes for qRT-PCR in Fragaria vesca	Fv28S-qR	CTCGAGCAGTTCTCCGACAG
拟南芥荧光定量内参基因 Reference genes for qRT-PCR in F. vesca	At18S-qF	GAGAAGTTACTCCGCAACCT
拟南芥荧光定量内参基因 Reference genes for qRT-PCR in F. vesca	At18S-qR	GAATGATGCGTCGCCAGCACAAAGG
FvbHLH130不同组织表达模式分析 Expression pattern of FvbHLH130 in different tissues	FvbHLH130-qF	ATCCCCGAAGCATTGCAGAA
	FvbHLH130-qR	GCTCGAACATCGCTCAGACT
拟南芥开花相关基因表达分析 Expression of flowering-related genes in Arabidopsis thaliana	AtAP1-qF	CAATGAGCCCTAAAGAGCTT
	AtAP1-qR	GGGAGGCATATTGTGGCCTT
	AtFT-qF	CCCTGCTACAACTGGAACAA
	AtFT-qR	TGACAATTGTAGAAAACTGCG
	AtFUL-qF	GGAGAAGAAAACGGGTCAGC
	AtFUL-qR	ACTCGTTCGTAGTGGTAGGAC
	AtCO-qF	CTACAACGACAATGGTTCCATTAAC
	AtCO-qR	CAGGGTCAGGTTGTTGC
酵母双杂交载体构建 Construction the vector of yeast two-hybrid system	FvARF4-AD-F	gtaccagattacgctcatatgATGGAATTTGATCTGAACCA
	FvARF4-AD-R	cagctcgagctcgatggatccTCAGACCCTAATTGCTGTTG
	FvARF6-AD-F	gtaccagattacgctcatatgATGAGGCTTTCTTCTTCGTC
	FvARF6-AD-R	cagctcgagctcgatggatccTTAGTAGTCAAGGGAGCCCA
	FvARF8-AD-F	gtaccagattacgctcatatgATGAAGCTTTCCACATCAGG
	FvARF8-AD-R	cagctcgagctcgatggatccTCAATACTCAAGTGAGCCGA

表1 在本研究中使用的引物列表

Table 1 A list of primers used in this study

用途 Usage	引物名称 Primer name	序列（5′-3′） Sequence
FvbHLH130基因编码区克隆 Cloning the CDS of FvbHLH130	FvbHLH130-CDS-F	ATGGATTCAAATACCCATCTCA
FvbHLH130基因编码区克隆 Cloning the CDS of FvbHLH130	FvbHLH130-CDS-R	TCAAACTATCTGATTTTGAA
FvbHLH130基因启动子克隆 Cloning the promoter of FvbHLH130	FvbHLH130-Pro-F	GACATAATAACCTTGCATTT
FvbHLH130基因启动子克隆 Cloning the promoter of FvbHLH130	FvbHLH130-Pro-R	TATACACAACTCTGCTTGCC
构建FvbHLH130过表达载体 Cloning onstruction of FvbHLH130-overexpression vector	FvbHLH130-121-F	gagaacacgggggactctagaATGGATTCAAATACCCATCTCA
	FvbHLH130-121-R	gcccttgctcaccatggatccAACTATCTGATTTTGAACCGG
转基因阳性苗鉴定 Identification of transgenic positive seedlings	35S	GACGCACAATCCCACTATCC
转基因阳性苗鉴定 Identification of transgenic positive seedlings	GFP-R	TTACTTGTACAGCTCGTCCATG
草莓荧光定量内参基因 Reference genes for qRT-PCR in Fragaria vesca	Fv28S-qF	TAACCGCATCAGGTCTCCAA
草莓荧光定量内参基因 Reference genes for qRT-PCR in Fragaria vesca	Fv28S-qR	CTCGAGCAGTTCTCCGACAG
拟南芥荧光定量内参基因 Reference genes for qRT-PCR in F. vesca	At18S-qF	GAGAAGTTACTCCGCAACCT
拟南芥荧光定量内参基因 Reference genes for qRT-PCR in F. vesca	At18S-qR	GAATGATGCGTCGCCAGCACAAAGG
FvbHLH130不同组织表达模式分析 Expression pattern of FvbHLH130 in different tissues	FvbHLH130-qF	ATCCCCGAAGCATTGCAGAA
	FvbHLH130-qR	GCTCGAACATCGCTCAGACT
拟南芥开花相关基因表达分析 Expression of flowering-related genes in Arabidopsis thaliana	AtAP1-qF	CAATGAGCCCTAAAGAGCTT
	AtAP1-qR	GGGAGGCATATTGTGGCCTT
	AtFT-qF	CCCTGCTACAACTGGAACAA
	AtFT-qR	TGACAATTGTAGAAAACTGCG
	AtFUL-qF	GGAGAAGAAAACGGGTCAGC
	AtFUL-qR	ACTCGTTCGTAGTGGTAGGAC
	AtCO-qF	CTACAACGACAATGGTTCCATTAAC
	AtCO-qR	CAGGGTCAGGTTGTTGC
酵母双杂交载体构建 Construction the vector of yeast two-hybrid system	FvARF4-AD-F	gtaccagattacgctcatatgATGGAATTTGATCTGAACCA
	FvARF4-AD-R	cagctcgagctcgatggatccTCAGACCCTAATTGCTGTTG
	FvARF6-AD-F	gtaccagattacgctcatatgATGAGGCTTTCTTCTTCGTC
	FvARF6-AD-R	cagctcgagctcgatggatccTTAGTAGTCAAGGGAGCCCA
	FvARF8-AD-F	gtaccagattacgctcatatgATGAAGCTTTCCACATCAGG
	FvARF8-AD-R	cagctcgagctcgatggatccTCAATACTCAAGTGAGCCGA

图1 FvbHLH130表达模式与启动子元件预测分析 A：SGR数据库中FvbHLH130对应基因的表达水平（柱头1：始花期的花柱和柱头；花药9：花后9 d的花药；心皮9：花后9 d心皮组织；皮层1：始花期皮层；Yw-15D和Yw-22D代表森林草莓‘Yellow Wonder’花后15 d和花后22 d果实）。B和C：FvbHLH130蛋白结构和基因启动子顺式作用元件分布；D：森林草莓不同组织FvbHLH130相对表达量。

Fig. 1 The gene expression pattern and prediction of the promoter cis-elements of FvbHLH130 A：Expression pattern of selected samples searched on SGR.（Style1：Styles and stigmas from just open flower；Anther9：Anthers from 9 days post-anthesis；Carpel9：Carpels from 9 days post-anthesis；Cortex1：Cortex from just open flower；Yw-15D and Yw-22D，15 days post-anthesis and 22 days post-anthesis of Fragaria vesca Yellow Wonder）；B and C：Protein structure of FvbHLH130 and prediction of cis-elements in the promoter of FvbHLH130；D：The relative expression of FvbHLH130 in different tissues of F. vesca.

图2 bHLH130的氨基酸序列比对 At：拟南芥；Fv：森林草莓；Pt：胡杨；Pd：扁桃。

Fig. 2 Amino acid sequence alignment of bHLH130 At：Arabidopsis thaliana；Fv：Fragaria vesca；Pt：Populus trichocarpa；Pd：Prunus dulcis.

图3 FvbHLH130与其他物种bHLH130氨基酸序列进化树分析

Fig. 3 Phylogenetic tree of FvbHLH130 with other bHLH130 proteins from difference plant species

图4 FvbHLH130过表达拟南芥株系（OE）提前开花 A：转基因阳性株系鉴定，对照为转化空载质粒的拟南芥株系；B：单株莲座叶数量；C：转基因阳性株系开花时间。** 表示在P < 0.01水平上差异显著。

Fig. 4 Overexpression of FvbHLH130 accelerates flowing A. Identification of transgenic lines，the control was Arabidopsis plants with empty vector；B. The number of rosette leaves；C. Phenotypes of flowing of transgenic lines. ** indicates significantly difference at P < 0.01 level.

图5 FvbHLH130过表达（OE）拟南芥开花相关基因的表达 * 表示在P < 0.05水平上差异显著；** 表示在P < 0.01水平上差异显著。

Fig. 5 Flowering-related genes expression analysis of FvbHLH130-overexpression lines in Arabidopsis * indicates significantly difference at P < 0.05 level；** indicates significantly difference at P < 0.01 level.

图6 FvbHLH130与FvARF4和FvARF6蛋白互作

Fig. 6 FvbHLH130 interacted with FvARF4 and FvARF6 proteins BD：pGBKT7；AD：pGADT7.

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