葡萄HCT基因家族鉴定及其对低温胁迫的响应

doi:10.13560/j.cnki.biotech.bull.1985.2024-0597

摘要/Abstract

摘要：

目的羟基肉桂酰转移酶（hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase, HCT）是双子叶植物中催化绿原酸合成的关键酶，广泛参与植物非生物胁迫应答。在葡萄全基因组中鉴定VvHCT基因家族成员，分析相关基因低温胁迫下表达模式，为后续研究提供理论基础。方法基于葡萄全基因组信息，利用生物信息学方法对VvHCT家族进行鉴定，并对家族成员理化性质、染色体分布、基因结构、蛋白保守结构、系统进化、启动子顺式作用元件以及组织表达特性进行分析，进一步利用荧光定量PCR检测VvHCT基因在低温胁迫下表达模式，最后通过瞬时转化方法对VvHCT8功能进行验证。结果在葡萄全基因组中鉴定到VvHCT的11个家族成员，编码398-457个氨基酸，分布于4条染色体上；按系统发育特征分为3个亚族；启动子顺式作用元件分析显示，VvHCT家族成员启动子上含有光响应元件、激素响应元件、应激响应元件、组织特异性元件和昼夜节律响应元件等；不同VvHCT家族成员表达模式存在较大差异；低温胁迫处理后VvHCT5表达量下调，其他10个家族成员表达量均上调；瞬时过表达葡萄叶片结果显示，VvHCT8能通过降低丙二醛和活性氧含量，缓解低温对葡萄叶片叶绿素和细胞膜的损伤。结论 VvHCT8参与葡萄抵御低温过程，可作为葡萄抗寒候选基因。

关键词: 葡萄, VvHCT基因家族, 生物信息学, 低温

Abstract:

Objective Hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase (HCT) is a key enzyme for catalyzing chlorogenic acid synthesis in dicotyledonous plants and is widely involved in plant abiotic stress response. We identified members of the VvHCT gene family in the whole grape genome and analyzed the expression pattern of the related genes under low-temperature stress, which may provide a theoretical basis for subsequent studies. Method Based on the whole genome information of grapevine, bioinformatics methods was sued to identify the VvHCT family, and analyze the physical and chemical properties, chromosome distribution, gene structure, protein conserved structure, systematic evolution, promoter cis-acting elements and tissue expression characteristics of the family members. Fluorescence quantitative PCR was adapted to detect the expression pattern of VvHCT gene under low-temperature stress, and transient transformation method to verify the function of VvHCT8 gene. Result Eleven family members of VvHCT were identified in the whole genome of grapevine, encoding 398–457 amino acids, distributed on four chromosomes. They were classified into three subfamilies according to their phylogenetic characteristics. The analysis of the promoter cis-acting elements showed that the promoters of the VvHCT family members contained light-responsive elements, hormone-responsive elements, stress-responsive elements, tissue-specific elements and circadian response elements. The expression patterns of different VvHCT family members differed greatly. The expression of VvHCT5 was down-regulated after low-temperature stress treatment, while the expressions of the other 10 family members were up-regulated; the transient overexpression results of grape leaves showed that VvHCT8 alleviated the damages to chlorophyll and cell membranes of grape leaves by reducing the content of MDA and ROS. Conclusion VvHCT8 is involved in the process of low-temperature resistance in grapes and can be used as a candidate gene for cold resistance in grapes.

Key words: grape, VvHCT gene family, bioinformatics, low temperature

颜伟, 陈慧婷, 叶青, 刘广超, 刘新, 侯丽霞. 葡萄HCT基因家族鉴定及其对低温胁迫的响应[J]. 生物技术通报, 2025, 41(2): 175-186.

YAN Wei, CHEN Hui-ting, YE Qing, LIU Guang-chao, LIU Xin, HOU Li-xia. Identification of the Grape HCT Gene Family and Their Responses to Low-temperature Stress[J]. Biotechnology Bulletin, 2025, 41(2): 175-186.

图/表 12

表1 引物序列

Table 1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence （5′-3′）
VvActin-F	ATAGAAGCAGCAAGGGA
VvActin-R	TGAGGCTCTTACTAATG
VvHCT1-F	CCACCTTCGCCATTTCCAAC
VvHCT1-R	TAGGGAACACCCTGGTCAGT
VvHCT2-F	CCTCTCCAACCCCACATTCC
VvHCT2-R	CCCCTTATCATTGCATGGGTT
VvHCT3-F	TGAGTGCAACGATGAGGGAG
VvHCT3-R	TGCCGTGGAGATCAGAAACC
VvHCT4-F	GGAGGTTTGTGTTTGATGGGG
VvHCT4-R	TGGGCGCAAGTTTATGCTGT
VvHCT5-F	ACTTCCCTTGCGGTCCATTC
VvHCT5-R	TTATCGCAGTCGGGAACACA
VvHCT6-F	GGTTGTAAGCCGAGCGAAAC
VvHCT6-R	TTGCCACTGTCCGCCATAAA
VvHCT7-F	CTGCCAACTTCTTCGACCCT
VvHCT7-R	GTAGGGGCGAAATCACCGAA
VvHCT8-F	CGACAGGCAATAGAGAAGGCA
VvHCT8-R	AGATGGGAAGTCTAGCCCAG
VvHCT9-F	CTAGCGGGATGGTTGCACT
VvHCT9-R	CATCAGTCATGATATGCAATGCTC
VvHCT10-F	AGCAATTGGCTACGATGGGA
VvHCT10-R	AGCAATTGGCTACGATGGGA
VvHCT11-F	ATCGGAGCCCACTTTCGATG
VvHCT11-R	AGCGGGTAGAAATGCACCAA

表2 VvHCT基因家族成员理化性质分析

Table 2 Physicochemical properties analysis of VvHCT gene family members

基因名称 Gene name	基因 ID Gene ID	染色体位置 Chromosome location	编码区 CDS/bp	氨基酸 Amino acid/bp	分子量 Molecular weigh/kD	等电点 Theoretical isoelectric points	不稳定系数 Instability index	总平均亲水性 Grand average of hydropathicity	KEGG	亚细胞定位 Subcellular localization
VvHCT1	Vitvi01g01514	Chr1：20494476：20495771	1 296	431	48.45	6.04	38.66	-0.018	2.3.1.133	细胞质
VvHCT2	Vitvi01g01517	Chr1：20532349：20533650	1 197	398	44.78	5.76	41.1	-0.096	2.3.1.133	细胞质
VvHCT3	Vitvi01g02214	Chr1：20566690：20568324	1 290	429	47.97	6.87	40.5	-0.139	2.3.1.133	细胞质
VvHCT4	Vitvi03g00077	Chr3：1057145：1058434	1 290	429	47.75	6.84	42.89	-0.257	2.3.1.133	细胞质
VvHCT5	Vitvi03g01800	Chr3：16887244：16888554	1 311	436	47.96	6.39	35.16	-0.128	2.3.1.133	细胞质
VvHCT6	Vitvi03g01816	Chr3：17430346：17432051	1 284	427	47.51	7.6	44.02	-0.171	2.3.1.133	细胞质
VvHCT7	Vitvi09g01229	Chr9：19655320：19657669	1 290	429	47.86	6.13	47.54	-0.166	2.3.1.133	细胞质
VvHCT8	Vitvi11g00730	Chr11：8730745：8732578	1 338	445	49.41	6.33	44.21	-0.273	2.3.1.133	细胞质
VvHCT9	Vitvi11g00735	Chr11：8783989：8786081	1 374	457	50.73	6.51	49.25	-0.224	2.3.1.133	细胞质
VvHCT10	Vitvi11g00742	Chr11：8868521：8869858	1 338	445	49.63	7.67	46.99	-0.252	2.3.1.133	细胞质
VvHCT11	Vitvi11g01099	Chr11：15978906：15980276	1 371	456	50.25	5.33	44.68	-0.163	2.3.1.133	细胞质

图1 VvHCT基因家族染色体定位分析

Fig. 1 Chromosomal localization analysis of VvHCT gene family

图2 VvHCT基因家族成员的基因结构（A）、保守结构域（B）和保守基序（C）分析

Fig. 2 Analysis of gene structure (A), conserved domains (B), and conserved motifs (C) of VvHCT gene family members

图3 VvHCT蛋白系统进化树分析

Fig. 3 Phylogenetic analysis of VvHCT proteins

图4 VvHCT基因启动子顺式作用元件分析A：顺式作用元件类型和数量；B：顺式作用元件分布模式

Fig. 4 Analysis of cis-acting elements in VvHCT gene promotersA: Types and quantity of cis-acting elements. B: Distribution pattern of cis-acting elements

图5 VvHCT组织表达模式分析

Fig. 5 Analysis of tissue-specific expression patterns of VvHCT

图6 VvHCT基因低温胁迫下表达模式分析与0 h对照相比，*P<0.05，**P<0.01，***P<0.001

Fig. 6 Analysis of the expression patterns of VvHCT genes under low-temperature stressCompared with 0 h control, *P<0.05, **P<0.01, ***P<0.001

图7 pSuper1300-VvHCT8过表达载体的构建A：VvHCT8基因CDS序列PCR产物；B：pSuper1300-VvHCT8转化DH5α菌落PCR；C：pSuper1300-VvHCT8转化GV3101菌落PCR

Fig. 7 Construction of pSuper1300-VvHCT8 over-expression vectorA: PCR product of the VvHCT8 gene CDS sequence. B: Colony PCR of DH5α transformed with pSuper1300-VvHCT8. C: Colony PCR of GV3101 transformed with pSuper1300-VvHCT8

图8 低温胁迫下VvHCT8瞬时过表达对葡萄叶片表型和光合指标的影响A：表型；B：VvHCT8表达量；C：叶绿素含量；D：Fv/Fm；E：ΦPS Ⅱ；*P<0.05，**P<0.01，***P<0.001；#代表对照。下同

Fig. 8 Effects of transient overexpression of VvHCT8 on the leaf phenotype and photosynthetic indexes of grapeA: Phenotype. B: Relative expression of VvHCT8. C: Chlorophyll content. D: Fv/Fm. E: ΦPS Ⅱ. *P<0.05, **P<0.01, ***P<0.001. #indicate control. The same below

图9 VvHCT8瞬时过表达对葡萄叶片细胞膜相对透性和丙二醛含量的影响

Fig. 9 Effects of transient overexpression of VvHCT8 on relative membrane permeability and malondialdehyde content in grape leaf cells

图10 VvHCT8瞬时过表达对葡萄叶片活性氧含量的影响

Fig. 10 Impact of transient overexpression of VvHCT8 on the content of active oxygen in grape leaves

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