多花黄精DREB基因家族鉴定及其在盐胁迫下的响应分析

doi:10.16420/j.issn.0513-353x.2024-0860

摘要/Abstract

摘要：

为研究多花黄精（Polygonatum cyrtonema）脱水反应元件结合蛋白（dehydration responsive element binding，DREB）基因家族的功能，基于多花黄精不同组织器官转录组、全长转录组数据对DREB家族成员进行鉴定，同时对其蛋白理化性质、系统进化特征、保守基序和蛋白结构进行分析；并结合FPKM值及qRT-PCR分析在不同器官、盐胁迫和PEG-6000模拟干旱胁迫下的表达量；通过农杆菌介导法转化洋葱，进行PcDREB10亚细胞定位；利用发根农杆菌诱导多花黄精毛状根，利用qRT-PCR分析过表达PcDREB10盐胁迫后PcDREB10/12/27的表达情况。研究结果表明：多花黄精DREB家族有35个成员，命名为PcDREB1 ~ PcDREB35，均为亲水蛋白，且稳定性较低。PcDREB家族成员在不同器官中的表达不一致，具有组织特异性，且在盐胁迫及干旱胁迫下表达模式不同。亚细胞定位结果显示PcDREB10在细胞核上。发根农杆菌介导的过表达PcDREB10后，经高盐溶液处理，根系中PcDREB10/27上调表达，而PcDREB12下调表达，提示PcDREB10/12/27在多花黄精抵御盐胁迫的过程中可能发挥不同作用。

关键词: 多花黄精, DREB, 基因家族鉴定, 盐胁迫, 干旱胁迫, 表达分析

Abstract:

To investigate the function of DREB（dehydration responsive element binding）gene family in Polygonatum cyrtonema in this study. The DREB family members were identified based on data including the full-length transcriptome and the transcriptome of different tissues and organs，and their protein physicochemical properties，phylogenetic characteristics，conserved motifs and protein structure were analyzed. The expression levels in different organs，salt stress and PEG-6000 simulated drought stress were analyzed by combining FPKM values and qRT-PCR. The subcellular localization of PcDREB10 was studied by Agrobacterium-mediated transformation of onion. The hairy roots of P. cyrtonema were induced by Agrobacterium rhizogenes，and the expression levels of PcDREB10/12/27 after overexpression of PcDREB10 under salt stress were analyzed by qRT-PCR. In this study，35 DREB family members were identified，named as PcDREB1-PcDREB35，all of which are hydrophilic proteins with low stability. The expression levels of PcDREB family members are inconsistent in different organs，with tissue specificity，and the expression levels are different under salt stress and drought stress. Subcellular localization results showed that PcDREB10 was located in the nucleus. After overexpression of PcDREB10 mediated by Agrobacterium rhizogenes，the expression of PcDREB10/27 were up-regulated while the expression of PcDREB12 was down-regulated in roots treated with high salt solution，indicating that PcDREB10/12/27 may play different roles in the process of P. cyrtonema resisting salt stress.

Key words: Polygonatum cyrtonema, DREB, gene family identification, salt stress, drought stress, expression analysis

王艳秋, 班景洁, 曾宇涵, 赖春旺, 黄晓铃, 周建金, 万奎, 赖钟雄, 林玉玲. 多花黄精DREB基因家族鉴定及其在盐胁迫下的响应分析[J]. 园艺学报, 2026, 53(1): 159-173.

WANG Yanqiu, BAN Jingjie, ZENG Yuhan, LAI Chunwang, HUANG Xiaoling, ZHOU Jianjin, WAN Kui, LAI Zhongxiong, LIN Yuling. Identification of the DREB Gene Family in Polygonatum cyrtonema and Analysis of Its Response to Salt Stress[J]. Acta Horticulturae Sinica, 2026, 53(1): 159-173.

图/表 13

表1 引物名称及引物序列

Table 1 Primer name and primer sequence

用途 Purpose	引物名称 Primer name	引物序列（5′-3′） Primer sequence
载体构建Vector construction	PcDREB10-F	ACGGGGGACTCTTGACCATGGATGGAACCTATTCGAAACAACGAC
	PcDREB10-R	AAGTTCTTCTCCTTTACTAGTAAACCCCCATGTCCACTGAAATG
	GFP-F	AACATACGGAAAACTTACCCT
	GFP-R	ATAATGATCAGCGAGTTGCAC
实时荧光定量PCR	SYBR-PcDREB10-qF	CTGGGAAGGAGAAGCCTAAG
qRT-PCR	SYBR-PcDREB10-qR	CCCGTGAAATGCCTGTAA
	SYBR-PcDREB12-qF	GGCTTCACTCTATGCTCTG
	SYBR-PcDREB12-qR	TGGGCTTTCTTGGTTCTC
	SYBR-PcDREB27-qF	AGGGCATCGTCGCTCTAT
	SYBR-PcDREB27-qR	CCTGGTTCGGTTCTTTGG
	UBQ-qF	GGACCCAGAAGTACGCAATG
	UBQ-qR	AATTACCAGGGATACAGCACC

表2 多花黄精DREB家族成员蛋白理化性质分析

Table 2 Analysis of physicochemical properties of proteins from DREB family members of Polygonatum cyrtonema

家族 Family	基因 Gene	氨基酸数 Number of amino acid	分子质量/kD Molecular weight	等电点 pI	不稳定系数 Instability index	脂肪系数 Aliphatic index	总平均吸水性 Grand average of hydropathicity	亚细胞定位 Subcellular location
DREB-A1	PcDREB1	214	23.37	5.52	68.13	57.66	-0.545	细胞核 Nucleus
	PcDREB2	209	23.09	5.22	66.52	62.25	-0.517	细胞核 Nucleus
	PcDREB3	214	23.52	5.21	68.13	57.66	-0.574	细胞核 Nucleus
	PcDREB4	214	23.39	5.34	68.50	57.66	-0.550	细胞核 Nucleus
	PcDREB5	179	19.56	6.17	43.82	66.65	-0.430	细胞核 Nucleus
DREB-A2	PcDREB6	299	33.25	4.98	56.50	54.85	-0.906	细胞核 Nucleus
	PcDREB7	248	27.33	4.47	46.51	55.89	-0.779	细胞核 Nucleus
DREB-A2	PcDREB8	314	34.80	4.88	54.29	50.67	-0.971	细胞核 Nucleus
	PcDREB9	263	28.88	4.38	44.73	50.84	-0.863	细胞核 Nucleus
	PcDREB10	199	21.47	7.51	38.44	58.44	-0.458	细胞核 Nucleus
	PcDREB11	207	22.68	8.28	67.04	75.94	-0.478	细胞核 Nucleus
	PcDREB12	407	44.28	4.93	72.15	64.10	-0.426	细胞核 Nucleus
	PcDREB13	282	31.01	5.80	45.51	61.28	-0.526	细胞核 Nucleus
	PcDREB14	246	27.03	4.44	47.31	54.80	-0.620	细胞核 Nucleus
	PcDREB15	297	33.18	5.38	46.42	49.63	-0.895	细胞核 Nucleus
	PcDREB16	246	26.96	4.37	45.59	53.62	-0.651	细胞核 Nucleus
	PcDREB17	246	26.96	4.37	45.59	53.62	-0.651	细胞核 Nucleus
DREB-A4	PcDREB18	285	32.26	8.86	61.60	87.61	-0.212	叶绿体 Chloroplast
	PcDREB19	199	21.56	5.22	47.96	62.86	-0.384	细胞核 Nucleus
	PcDREB20	221	23.88	5.76	36.51	64.07	-0.534	细胞核 Nucleus
	PcDREB21	222	23.99	5.77	40.04	62.48	-0.528	细胞核 Nucleus
	PcDREB22	224	24.08	5.01	53.18	60.18	-0.546	细胞核 Nucleus
DREB-A5	PcDREB23	195	21.08	4.74	49.45	58.72	-0.371	细胞质Cytoplasm
	PcDREB24	172	18.48	5.95	59.10	72.15	-0.626	细胞核 Nucleus
	PcDREB25	172	18.51	6.61	57.28	61.40	-0.684	细胞核 Nucleus
	PcDREB26	202	21.68	5.78	54.66	59.11	-0.427	叶绿体 Chloroplast
DREB-A6	PcDREB27	481	52.26	8.60	61.01	49.88	-0.776	细胞核 Nucleus
	PcDREB28	482	52.40	7.95	61.51	50.58	-0.762	细胞核 Nucleus
	PcDREB29	481	52.26	8.60	61.01	49.88	-0.776	细胞核 Nucleus
	PcDREB30	352	39.06	6.66	59.27	74.06	-0.442	细胞核 Nucleus
	PcDREB31	354	38.74	5.86	68.70	72.60	-0.425	细胞核 Nucleus
	PcDREB32	422	47.14	5.84	51.95	77.01	-0.564	细胞核 Nucleus
	PcDREB33	365	40.10	9.22	63.35	72.16	-0.511	叶绿体 Chloroplast
	PcDREB34	353	39.12	7.05	58.53	76.06	-0.446	细胞核 Nucleus
	PcDREB35	308	34.73	5.80	63.56	71.98	-0.644	叶绿体 Chloroplast

表2 多花黄精DREB家族成员蛋白理化性质分析

Table 2 Analysis of physicochemical properties of proteins from DREB family members of Polygonatum cyrtonema

家族 Family	基因 Gene	氨基酸数 Number of amino acid	分子质量/kD Molecular weight	等电点 pI	不稳定系数 Instability index	脂肪系数 Aliphatic index	总平均吸水性 Grand average of hydropathicity	亚细胞定位 Subcellular location
DREB-A1	PcDREB1	214	23.37	5.52	68.13	57.66	-0.545	细胞核 Nucleus
	PcDREB2	209	23.09	5.22	66.52	62.25	-0.517	细胞核 Nucleus
	PcDREB3	214	23.52	5.21	68.13	57.66	-0.574	细胞核 Nucleus
	PcDREB4	214	23.39	5.34	68.50	57.66	-0.550	细胞核 Nucleus
	PcDREB5	179	19.56	6.17	43.82	66.65	-0.430	细胞核 Nucleus
DREB-A2	PcDREB6	299	33.25	4.98	56.50	54.85	-0.906	细胞核 Nucleus
	PcDREB7	248	27.33	4.47	46.51	55.89	-0.779	细胞核 Nucleus
DREB-A2	PcDREB8	314	34.80	4.88	54.29	50.67	-0.971	细胞核 Nucleus
	PcDREB9	263	28.88	4.38	44.73	50.84	-0.863	细胞核 Nucleus
	PcDREB10	199	21.47	7.51	38.44	58.44	-0.458	细胞核 Nucleus
	PcDREB11	207	22.68	8.28	67.04	75.94	-0.478	细胞核 Nucleus
	PcDREB12	407	44.28	4.93	72.15	64.10	-0.426	细胞核 Nucleus
	PcDREB13	282	31.01	5.80	45.51	61.28	-0.526	细胞核 Nucleus
	PcDREB14	246	27.03	4.44	47.31	54.80	-0.620	细胞核 Nucleus
	PcDREB15	297	33.18	5.38	46.42	49.63	-0.895	细胞核 Nucleus
	PcDREB16	246	26.96	4.37	45.59	53.62	-0.651	细胞核 Nucleus
	PcDREB17	246	26.96	4.37	45.59	53.62	-0.651	细胞核 Nucleus
DREB-A4	PcDREB18	285	32.26	8.86	61.60	87.61	-0.212	叶绿体 Chloroplast
	PcDREB19	199	21.56	5.22	47.96	62.86	-0.384	细胞核 Nucleus
	PcDREB20	221	23.88	5.76	36.51	64.07	-0.534	细胞核 Nucleus
	PcDREB21	222	23.99	5.77	40.04	62.48	-0.528	细胞核 Nucleus
	PcDREB22	224	24.08	5.01	53.18	60.18	-0.546	细胞核 Nucleus
DREB-A5	PcDREB23	195	21.08	4.74	49.45	58.72	-0.371	细胞质Cytoplasm
	PcDREB24	172	18.48	5.95	59.10	72.15	-0.626	细胞核 Nucleus
	PcDREB25	172	18.51	6.61	57.28	61.40	-0.684	细胞核 Nucleus
	PcDREB26	202	21.68	5.78	54.66	59.11	-0.427	叶绿体 Chloroplast
DREB-A6	PcDREB27	481	52.26	8.60	61.01	49.88	-0.776	细胞核 Nucleus
	PcDREB28	482	52.40	7.95	61.51	50.58	-0.762	细胞核 Nucleus
	PcDREB29	481	52.26	8.60	61.01	49.88	-0.776	细胞核 Nucleus
	PcDREB30	352	39.06	6.66	59.27	74.06	-0.442	细胞核 Nucleus
	PcDREB31	354	38.74	5.86	68.70	72.60	-0.425	细胞核 Nucleus
	PcDREB32	422	47.14	5.84	51.95	77.01	-0.564	细胞核 Nucleus
	PcDREB33	365	40.10	9.22	63.35	72.16	-0.511	叶绿体 Chloroplast
	PcDREB34	353	39.12	7.05	58.53	76.06	-0.446	细胞核 Nucleus
	PcDREB35	308	34.73	5.80	63.56	71.98	-0.644	叶绿体 Chloroplast

图1 多花黄精DREB家族成员结构域片段的序列比对

Fig. 1 Sequence alignment of the structural domain fragments of the DREB family members in Polygonatum cyrtonema

图2 多花黄精与拟南芥、水稻、石刁柏的系统发育进化树 Pc：多花黄精；At：拟南芥；Os：水稻，Ao：石刁柏

Fig. 2 Phylogenetic and phylogenetic trees of Polygonatum cyrtonema and Arabidopsis thaliana，Oryza sativa and Asparagus officinalis Pc：Polygonatum cyrtonema；At：Arabidopsis thaliana；Os：Oryza sativa；Ao：Asparagus officinalis

图3 多花黄精DREB家族成员的基因结构及保守基序分析

Fig. 3 Gene structure and conserved motifs analysis of DREB family members of Polygonatum cyrtonema

图4 PcDREB家族成员在不同器官（A）和盐胁迫转录组（B）下的表达 R：根；T：根状茎；F：果实（果肉组织及种子）；L：叶片；S：茎秆。下同

Fig. 4 Expression of PcDREB family members under transcriptomes of different organs（A）and salt-stressed（B） R：Root；T：Rhizome；F：Fruit（Flesh tissue and seeds）；L：Leaf；S：Stem. The same below

图5 PcDREB家族成员在不同器官中的表达分析图中小写字母代表 0.05 水平差异显著

Fig. 5 Expression analysis of PcDREB family members in different organs Different lowercase letters represent a significant difference at the 0.05 level

图6 PcDREB家族成员在不同浓度NaCl处理48 h后的表达分析不同小写字母代表 0.05 水平差异显著

Fig. 6 Expression analysis of PcDREB family members treated with different concentrations of NaCl for 48 h Different lowercase letters represent a significant difference at the 0.05 level

图7 PcDREB家族成员在不同浓度PEG6000处理72 h后的表达分析不同小写字母代表 0.05 水平差异显著

Fig. 7 Expressionanalysis of PcDREB family members following treatment with varying concentrations of PEG6000 for 72 h Different lowercase letters represent a significant difference at the 0.05 level

图8 PcDREB10在洋葱内表皮中亚细胞定位

Fig. 8 Subcellular localization of PcDREB10 protein in onion

图9 过表达多花黄精PcDREB10的阳性根系红色箭头代表新长的PcDREB10的阳性根，白色箭头代表保留的野生型根

Fig. 9 PcDREB10 overexpressing roots of Polygonatum cyrtonema The red arrows represent the newly grown positive roots of PcDREB10，while the white arrows indicate the retained wild-type roots

图10 多花黄精过表达PcDREB10的阳性根系的共聚焦显微镜观察

Fig. 10 Confocal microscopy observations of positive root system of OE-PcDREB10 in Polygonatumcyrtonema

图11 过表达PcDREB10对多花黄精盐胁迫的响应不同小写字母代表0.05水平差异显著

Fig. 11 Response of PcDREB10 overexpression to salt stress in Polygonatum cyrtonema Different lowercase letters represent a significant difference at the 0.05 level

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