彩色马铃薯花青素合成相关ERF基因筛选及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0460

摘要/Abstract

摘要：

【目的】乙烯响应因子（ethylene responsive factor，ERF）是一类重要的转录因子，参与调控植物花青素的生物合成。筛选可能参与调控马铃薯块茎花青素合成的StERFs基因，为开展彩色马铃薯花青素相关StERFs基因功能研究奠定基础。【方法】利用生物信息学方法对其理化性质、亚细胞定位、保守基序、进化关系、蛋白质二级结构、启动子顺式作用元件及蛋白互作关系进行分析，同时采用RT-qPCR方法对不同颜色马铃薯薯肉中StERFs进行表达模式分析。【结果】基于不同颜色马铃薯块茎的转录组表达谱，获得7个差异表达ERF转录因子。7个StERFs属于亲水性蛋白，均预测定位于细胞核。Motif 1（RWLG）和Motif 2（YRG）是7个StERFs中共同的保守基序，属于AP2的结构域特征序列。StERFs基因启动子序列含有响应激素作用元件，以及响应防御与胁迫、低温和光照等的顺式作用元件。7个StERFs在紫色薯肉中表达量显著高于黄色薯肉，其中StERF72和StERF110与已知的花青素相关ERFs（IbERF71和PyERF3）亲缘关系较近，且7个StERFs与56个转录因子存在蛋白互作关系。【结论】7个StERFs基因可能参与调控马铃薯块茎中花青素的合成，其中StERF72和StERF110可作为候选基因进一步验证其功能。

关键词: 彩色马铃薯, 花青素, ERF转录因子, 生物信息学分析, 表达模式分析

Abstract:

【Objective】Ethylene responsive factor（ERF）is an important transcription factor involved in the regulation of anthocyanin biosynthesis in plants. Screening of StERF genes that may be involved in the regulation of anthocyanin synthesis in potato tubers lays the foundation for the next step of research on the function of anthocyanin-related StERFs genes in colored potato.【Method】Bioinformatics methods were used to analyze their physicochemical properties, subcellular localization, conserved motifs, evolutionary relationships, protein secondary structure, promoter cis-acting elements, and protein interactions, while RT-qPCR was used to analyze the expression patterns of StERFs in potato flesh of different colors.【Result】Seven differentially expressed ERF transcription factors were obtained based on the transcriptome expression profiles of potato tubers of different colors. The seven StERFs are hydrophilic proteins, all of which are predicted to be localized in the nucleus. Motif 1（RWLG）and Motif 2（YRG）are the common conserved motifs in the seven StERFs, which belong to the structural domains of the characteristic sequences of AP2. The promoter sequences of the StERFs genes contain hormone-responsive action elements, as well as cis-action elements in response to defense and stress, low temperature and light, etc. The expressions of the seven StERFs were significantly higher in purple potato flesh than in yellow potato flesh, among which StERF72 and StERF110 were closer to known anthocyanin-associated ERFs（IbERF71 and PyERF3）. The existence of protein interactions between the seven StERFs and 56 transcription factors.【Conclusion】Seven StERFs may be involved in the regulation of anthocyanin synthesis in potato tubers, among which StERF72 and StERF110 can be candidated genes to further validate their functions.

Key words: colored potato, anthocyanin, ERF transcription factor, bioinformatics analysis, expression pattern analysis

吴娟, 武小娟, 王沛捷, 谢锐, 聂虎帅, 李楠, 马艳红. 彩色马铃薯花青素合成相关ERF基因筛选及表达分析[J]. 生物技术通报, 2024, 40(9): 82-91.

WU Juan, WU Xiao-juan, WANG Pei-jie, XIE Rui, NIE Hu-shuai, LI Nan, MA Yan-hong. Screening and Expression Analysis of ERF Gene Related to Anthocyanin Synthesis in Colored Potato[J]. Biotechnology Bulletin, 2024, 40(9): 82-91.

图/表 12

图1 转录组测序材料马铃薯“黑金刚”和“后旗红”

Fig. 1 Potato（Solanum tuberosum L.）“Heijingang” and “Houqihong” as transcriptome sequencing materials

表1 RT-qPCR引物序列

Table 1 Primer sequences for RT-qPCR

基因序号Gene ID	基因名称Gene name	引物序列Primer sequence（5'-3'）
Soltu.DM.05G020900	StERF92-1	F-ATGGAGTTAGGGTTTGGCTAGGAAC R-CCCTCATTGATAATGCGGCTTGATC
Soltu.DM.09G021200	StERF92-2	F-CTGAAGAAGCTGCGTTAGCGTATG R-ACCGGCGAACAATTCTCATCAAATC
Soltu.DM.09G023730	StERF72	F-GAAAAGGGGTTAGGGTCTGGTTAGG R-AGTAGTGGTCGTCGTCTTCATTGG
Soltu.DM.04G021630	StERF059	F-TGCATCAACAACAACAGAGCTTAGG R-GTTTGCTTCATCGGGACTGGTTTC
Soltu.DM.04G027450	StEF110	F-TGATGACGCCGCTCTTAGATTCAG R-TGGTGCTGGTGCTGTGGTTG
Soltu.DM.03G031310	StERF3	F-TGCCACATGACATCTCTCACTGC R-TCTCACTCTGCCTTTCTTCTCCATC
Soltu.DM.04G033390	StERF10	F-CAGAGGAATACGGATGAGGAAGTGG R-GTAAGCACGAGCAGCAGCAAC
	Actin	F-CCTTGTATGCTAGTGGTCG R-GCTCATAGTCAAGAGCCAC

图2 “黑金刚”vs“后旗红”差异表达ERF基因火山图

Fig. 2 A volcano plot of the differentially expressed ERF genes in “Heijingang” vs “Houqihong”

表2 同源序列比对

Table 2 An alignment of the homologous sequences

基因序号 Gene ID	基因名称 Gene name	NCBI数据库BLAST序列ID NCBI database BLAST sequence ID	PlantTB数据库BLAST序列ID PlantTB database BLAST sequence ID	相似度 Identities/%
Soltu.DM.05G020900	StERF92-1	XP_006360378.1	PGSC0003DMP400025705	100
Soltu.DM.09G021200	StERF92-2	XP_006351652.2	PGSC0003DMP400018171	100
Soltu.DM.09G023730	StERF72	NP_001275232.1	PGSC0003DMP400055026	92
Soltu.DM.04G021630	StERF059	XP_006355363.1	PGSC0003DMP400035842	100
Soltu.DM.04G027450	StEF110	XP_006363442.1	PGSC0003DMP400011298	100
Soltu.DM.03G031310	StERF3	XP_006341371.1	PGSC0003DMP400024835	92
Soltu.DM.04G033390	StERF10	XP_006354856.1	PGSC0003DMP400013982	100

表3 马铃薯StERFs理化性质分析及亚细胞定位预测

Table 3 Analysis of physicochemical properties and prediction of subcellular localization in potato StERFs

蛋白名称 Protein name	氨基酸数目 Number of amion acids/aa	相对分子质量 Molecular weight/ kD	理论等电点 pI	分子式 Molecular formula	脂肪系数 Aliphatc index	总平均亲水性 GRAVY	亚细胞定位 Subcellular localization
StERF92-1	225	25 615.43	5.4	C₁₁₂₅H₁₇₄₉N₃₁₁O₃₆₄S₅	70.18	-0.738	细胞核
StERF92-2	197	22 839.51	4.89	C₁₀₀₃H₁₅₅₄N₂₇₆O₃₁₉S₈	68.78	-0.652	细胞核
StERF72	278	31 714.03	5.1	C₁₃₈₅H₂₁₂₉N₃₉₁O₄₄₈S₉	57.55	-0.905	细胞核
StERF059	370	40 579.22	5.61	C₁₇₈₅H₂₇₆₂N₄₈₀O₅₇₆S₁₃	66.84	-0.465	细胞核
StEF110	441	48 254.51	5.97	C₂₀₈₄H₃₁₈₆N₆₁₂O₆₉₅S₁₀	46.78	-0.834	细胞核
StERF3	159	18 282.48	6.91	C₇₈₂H₁₂₅₀N₂₃₄O₂₅₄S₉	59.62	-0.899	细胞核
StERF10	145	16 663.74	9.04	C₇₂₂H₁₁₃₉N₂₁₉O₂₂₂S₇	56.62	-0.974	细胞核

图3 马铃薯StERFs保守基序

Fig. 3 StERFs conserved motif in potato

图4 马铃薯StERFs蛋白二级结构图中蓝色为α-螺旋；紫色为无规则卷曲；红色为延伸链；绿色为β-折叠

Fig. 4 Secondary structure of the potato StERFs protein The figure shows α-helices in blue; irregular coiling in purple; extended chains in red; and β-folding in green

图5 马铃薯StERFs及其他已知的花青素相关ERFs的氨基酸序列比对图中红色的方框为AP2结构域中典型的YRG和WLG单元

Fig. 5 Amino acid sequence alignment of potato StERFs and other known anthocyanin-related ERFs The red boxes in the figure show typical YRG and WLG units in the AP2 structural domains

图6 马铃薯StERFs及其他已知的花青素相关ERFs的系统进化树图中红色五角星为转录组筛选到的7个马铃薯StERFs

Fig. 6 Phylogenetic tree of potato StERFs and other known anthocyanin-related ERFs The red pentagons in the figure show the seven potato StERFs screened by the transcriptome

图7 马铃薯StERFs启动子顺式作用元件

Fig. 7 Cis-acting elements of the potato StERFs promoter

图8 彩色马铃薯StERFs与其他转录因子家族间蛋白互作网络图

Fig. 8 Plot of the protein interaction network between colored potato StERFs and other transcription factors families

图9 彩色马铃薯StERFs表达模式柱形代表通过RT-qPCR测定的基因相对表达量，折线代表通过转录组测序的基因FPKM值。不同小写字母表示P<0.05

Fig. 9 Expression pattern of StERFs in colored potato Bars indicate gene relative expression as determined by RT-qPCR, and broken lines indicate gene FPKM values by transcriptome sequencing. Different lower letters indicate significant difference at 0.05 level

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