天香百合、药百合黄酮醇合成酶FLS基因克隆和表达分析

doi:10.3969/j.issn.1004-1524.20230318

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (2): 344-357.DOI: 10.3969/j.issn.1004-1524.20230318

天香百合、药百合黄酮醇合成酶FLS基因克隆和表达分析

刘筱琳(), 孙婷婷, 杨捷, 何恒斌^*()

北京林业大学园林学院,花卉种质创新与分子育种北京市重点实验室,国家花卉工程技术研究中心,城乡生态环境北京实验室,北京 100083

收稿日期:2023-03-10 出版日期:2024-02-25 发布日期:2024-03-05
作者简介:刘筱琳(1996—),女,山东烟台人,硕士研究生,研究方向为花卉种质资源创新与育种。E-mail:742695401@qq.com
通讯作者: *何恒斌,E-mail:hengbinhe_1220@bjfu.edu.cn
基金资助:
国家自然科学基金(31672191);“十三五”国家重点研发计划(2019YFD1001002)

Cloning and expression analysis of FLS gene of flavonol synthetase in Lilium auratum and L.speciosum var. gloriosoides

LIU Xiaolin(), SUN Tingting, YANG Jie, HE Hengbin^*()

Beijing Key Laboratory of Ornamental Plants Germplasm Innovation and Molecular Breeding, National Engineering Research Center for Floriculture, Beijing Laboratory of Urban and Rural Ecological Environment, College of Landscape Architecture, Beijing Forestry University, Beijing 100083,China

Received:2023-03-10 Online:2024-02-25 Published:2024-03-05

摘要/Abstract

摘要：

以天香百合(Lilium auratum)和药百合(L. speciosum var. gloriosoides)为研究材料,分别克隆获得黄酮醇合成酶(flavonol synthase, FLS)基因,命名为LaFLS和LsFLS。实验结果表明,LaFLS和LsFLS基因均含完整的开放阅读框1 035 bp,均编码344个氨基酸,氨基酸序列高度保守,均具有DIOX-N结构域和2-酮戊二酸和铁(Ⅱ)依赖性双加氧酶结构域,属于2-酮戊二酸和铁(Ⅱ)依赖性双加氧酶超家族;系统进化分析表明,LaFLS和LsFLS除与东方系百合西伯利亚和索邦的FLS亲缘关系最近外,与百合科郁金香(Tulipa fosteriana)等亲缘关系较近;生物信息学分析显示,LaFLS和LsFLS蛋白无信号肽序列和跨膜结构域,均为亲水性蛋白,亚细胞定位结果显示二者主要定位在细胞质中。基因表达分析结果表明,在花蕾发育过程中,LaFLS和LsFLS随花蕾发育出现先上升后下降再上升的趋势,而且在花被片无色区的表达量显著高于有色区域。

关键词: 东方百合, 黄酮醇合成酶基因, 黄酮醇, 克隆, 基因表达

Abstract:

The flavonol synthetase gene FLS were cloned from Lilium auratum and L. speciosum var. gloriosoides, respectively, and named as LaFLS and LsFLS. The results showed that the LaFLS and LsFLS genes contained 1 035 bp complete open reading frame and encoded 344 amino acids. The amino acid sequences were highly conserved, and both genes had the DIOX-N domain and the 2-ketoglutaric acid and iron (Ⅱ) dependent dioxygenase domain, belonging to the 2-ketoglutaric acid and iron (Ⅱ) dependent dioxygenase superfamily. Phylogenetic analysis showed that LaFLS and LsFLS were closely related to Tulipa fosteriana, except for the FLS of oriental hybrids Siberia and Sorbonne. Bioinformatics analysis showed that LaFLS and LsFLS were hydrophilic proteins without signal peptide sequences and transmembrane domains. Subcellular localization results showed that they were mainly located in the cytoplasm. The results of gene expression analysis showed that LaFLS and LsFLS increased first, then decreased and then increased with the development of flower bud, and the expressions of LaFLS and LsFLS were significantly higher in the colorless region than in the colored region.

Key words: oriental hybrids, FLS gene, flavonol, clone, gene expression

中图分类号:

S682.265

刘筱琳, 孙婷婷, 杨捷, 何恒斌. 天香百合、药百合黄酮醇合成酶FLS基因克隆和表达分析[J]. 浙江农业学报, 2024, 36(2): 344-357.

LIU Xiaolin, SUN Tingting, YANG Jie, HE Hengbin. Cloning and expression analysis of FLS gene of flavonol synthetase in Lilium auratum and L.speciosum var. gloriosoides[J]. Acta Agriculturae Zhejiangensis, 2024, 36(2): 344-357.

图/表 14

图1 试验材料 A,天香百合;B,药百合;C,天香百合4个阶段取材;D,药百合4个阶段取材。

Fig.1 Experimental materials A, L. auratum; B, L. speciosum var. gloriosoides; C, Four-stage materials of L. auratum; D, Four-stage materials of L. speciosum var. gloriosoides.

表1 引物序列表

Table 1 Primer sequences

引物名称 Primer name	引物序列Primer sequence(5'-3')
EF(F)	GGCACTAACTCGCTCCTTCTG
EF(R)	GGATTGAGAGGAGCTTCGGTGAGA
TIP41(F)	CGAAGCCAGAAACGGAGAAGAAT
TIP41(R)	GGGTAGGGTGGATTGGGAAGA
FLS(Clone)F	ATGTCTACCCGACATCACCTTG
FLS(Clone)R	CTATTATTGTGGAAGTTTGTTAATC
FLS(sub-clone)F	CCAAATCGACTCTAGAATGTCTACCCGACATCACCTTG
FLS(sub-clone)R	TACCGGATCCACTAGTTTGTGGAAGTTTGTTAATCTTGC
FLS(qRT-PCR)F	ACTTAGCACTTGGGGTGGTG
FLS(qRT-PCR)R	CGTCCCATTGCTCAAAATCT
FLS(RT-PCR)F	GAGGAGAAGGAGGCGATAGC
FLS(RT-PCR)R	CGTCTAACCCTAAACCAACTG

表2 序列比对基因信息

Table 2 Gene information of system sequence alignment

拉丁名Latin name	登录号Login ID	基因名Gene name
Allium cepa L.	AAO63023.1	AcFLS(H6)
A. cepa L.	AQR58516.1	AcFLS (HRB)
Arabidopsis thaliana (L.) Heynh.	AAB41504.1	AtFLS
Fagopyrum tataricum (L.) Gaertn.	AEC33116.1	FtFLS
Lilium regale E.H.Wilson	ASV46329.1	LrFLS
Narcissus tazetta L.	AFS63899.1	NtFLS
Ornithogalum longebracteatum Jacq.	QBQ58059.1	OlFLS2
Scutellaria baicalensis Georgi	AHA14501.1	SbFLS

表3 系统进化树基因信息

Table 3 Gene information of phylogenetic tree

拉丁名Latin name	登录号Login ID	基因名Gene name
Acacia confusa Merr.	AFA55179.1	AcFLS
Allium cepa L.	AAO63023.1	AcFLS (H6)
A. cepa L.	AQR58516.1	AcFLS (HRB)
A. cepa L.	AAT68476.1	AcFLS
Antirrhinum majus L.	ABB53382.1	AmFLS
Arabidopsis thaliana (L.) Heynh.	AAB41504.1	AtFLS
Camellia nitidissima	ADZ28516.1	CnFLS
C. sinensis (L.) O. Ktze.	ABM88786.1	CsFLS
Citrus unshiu Marcow.	BAA36554.1	CuFLS
Cyclamen purpurascens	BBA27024.1	CpurFLS2
Eustoma exaltatum subsp. russellianum	AAF64168.1	EeFLS
Fagopyrum tataricum (L.) Gaertn.	AEC33116.1	FtFLS
F. tataricum (L.) Gaertn.	AGE13752.1	FtFLS1
Gentiana trifloral Pall.	BAK09226.1	GtFLS
Ginkgo biloba L.	ACY00393.1	GbFLS1
Glycine max (Linn.) Merr.	BAF31231.1	GmFLS1
Lilium regale E.H.Wilson	ASV46329.1	LrFLS
Malus×domestica Borkh.	AAX89401.1	MdFLS(M9)
Narcissus tazetta	AFS63899.1	NtFLS
Nicotiana tabacum L.	BAF96939.1	NtFLS
Ornithogalum longebracteatum	QBQ58059.1	OlFLS2
Petroselinum crispum	AAP57395.1	PcFLS
Petunia×hybrida E.Vilm	CAA80264.1	PhFLS
Rosa rugosa Thunb.	AIS22436.1	RrFLS
Scutellaria baicalensis Georgi	AHA14501.1	SbFLS
Tricyrtis sp. Shinonome	BAU20368.1	TrFLS
Tulipa fosteriana W.Irving	AHY20032.1	TfFLS1
Vaccinium corymbosum	AKJ87100.1	VcFLS

图2 天香百合和药百合的FLS基因编码区序列扩增片段 M,DNA Marker;A1、A2,LaFLS的扩增产物;B1、B2,LsFLS的扩增产物。

Fig.2 PCR products of the cDNA of LaFLS in L. auratum and LsFLS in L. speciosum var. gloriosoides M, DNA Marker; A1, A2, Product of the LaFLS amplification in cDNA; B1, B2, Product of the LsFLS amplification in cDNA.

图3 LaFLS 和LsFLS核苷酸及编码氨基酸序列 A,LaFLS核苷酸及编码氨基酸序列;B, LsFLS核苷酸及编码氨基酸序列。方框表示LaFLS 和LsFLS核苷酸及编码氨基酸序列中的差异。

Fig.3 Nucleotide and amino acid sequences of LaFLS and LsFLS A, Nucleotide and amino acid sequences of LaFLS; B, Nucleotide and amino acid sequences of LsFLS. The frames show the differences between the nucleotide and amino acid sequences of LaFLS and LsFLS.

图4 LaFLS和LsFLS氨基酸多序列比对分析下划线位置是DIOX-N结构域,红色方框表示2-酮戊二酸和铁(Ⅱ)依赖性双加氧酶结构域;DHQ底物特异结合位点、2-酮戊二酸和Fe2+位点分别用黑色三角、星号及箭头表示。

Fig.4 Amino acid multiple sequence alignment analysis of LaFLS and LsFLS The underlined position is the DIOX-N domain, and the red box represents the 2-ketoglutaric acid and iron (Ⅱ) dependent dioxygenase domain; DHQ substrate specific binding sites, 2-ketoglutaric acid and Fe2+ sites are indicated by black triangles, asterisks and arrows, respectively.

图5 FLS基因系统进化树分析

Fig.5 The phylogenetic tree analysis of FLS

图6 FLS蛋白的信号肽分析

Fig.6 Analysis of FLS signal peptide

图7 FLS蛋白跨膜结构分析

Fig.7 Trans-membrane domain analysis of FLS protein

图8 FLS蛋白的亲疏水性分析

Fig.8 Hydrophilic-hydrophobic analysis of FLS protein

图9 LaFLS和LsFLS蛋白的亚细胞定位分析

Fig.9 Subcellular localization analysis of LaFLS and LsFLS proteins

图10 天香百合和药百合各组织部位FLS的半定量PCR分析 1,上部叶;2,下部叶;3,花柱;4,柱头;5,子房;6,花丝;7,花药;8,花被片;LaEF,天香百合内参基因;LsTIP,药百合内参基因。

Fig.10 RT-PCR expression of FLS in L. auratum and L. speciosum var. gloriosoides different tissues 1, Upper leaf; 2, Lower leaf; 3, Style; 4, Stigma; 5, Ovary; 6, Perianth; 7, Anther; 8, Perianth; LaEF, Reference gene in L. auratum; LsTIP, Reference gene in L. speciosum var. gloriosoides.

图11 天香百合(A)和药百合(B)花蕾不同发育阶段FLS基因的表达不同小写字母表示差异显著(P<0.05)。

Fig.11 Expression of FLS in L. auratum (A) and L. speciosum var. gloriosoides (B) at different development stages of flower buds Different lowercase letters indicate significant differences (P<0.05).

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天香百合、药百合黄酮醇合成酶FLS基因克隆和表达分析

Cloning and expression analysis of FLS gene of flavonol synthetase in Lilium auratum and L.speciosum var. gloriosoides

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