甜瓜CmHXK基因家族的鉴定及响应非生物胁迫的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0418

摘要/Abstract

摘要：

【目的】明确甜瓜己糖激酶（hexokinase）家族的基因特征和潜在功能。【方法】从全基因组水平鉴定甜瓜HXK基因家族，分析其蛋白质理化性质、亚细胞定位、系统进化、基因结构、染色体定位、保守基序、蛋白结构、启动子顺式作用元件等，同时，基于转录组数据挖掘CmHXK基因在雄花、雌花、根、叶、果实及果实发育过程中的表达模式，通过RT-qPCR检测CmHXK基因在脱落酸、干旱、盐、低温胁迫下的表达模式。【结果】共鉴定到6个CmHXK基因家族成员，分别分布在第2、4、5和8染色体上，含有6-9个外显子，5-8个内含子，平均氨基酸个数为462，平均分子量为50.2 kD，等电点为4.99-6.34。系统进化分析表明，甜瓜HXK基因家族成员与黄瓜的亲缘关系最近。甜瓜CmHXK启动子中主要顺式作用元件为光响应元件、激素响应元件和胁迫响应元件。CmHXKs在甜瓜不同器官中的表达具有特异性，其中，CmHXK1、CmHXK2和CmHXK5在雄花、雌花、根、叶、果实等组织和果实发育成熟过程中均处于较高表达水平，而CmHXK3、CmHXK4和CmHXK6的表达随着果实的发育和成熟呈现降低趋势。CmHXKs不同程度地响应4种非生物胁迫（脱落酸、干旱、盐、低温），其中，CmHXK1、CmHXK3和CmHXK4受ABA强烈诱导，CmHXK1、CmHXK3和CmHXK6受盐胁迫强烈诱导，CmHXK1、CmHXK2和CmHXK3受干旱胁迫强烈诱导；CmHXK1和CmHXK6受低温强烈诱导。【结论】CmHXK家族成员高度保守，在甜瓜生长发育及响应非生物胁迫过程中扮演着重要角色。其中，CmHXK1对4种非生物胁迫最为敏感。

关键词: 甜瓜, 己糖激酶, 生物信息学, 非生物胁迫, 表达分析

Abstract:

【Objective】 To clarify the genetic characteristics and potential functions of the hexokinase family in melon（Cucumis melo）. 【Method】 The melon HXK gene family was identified at the whole genome level, and its protein's physicochemical properties, subcellular localization, systematic evolution, gene structure, chromosome localization, conserved motifs, protein structure, promoter cis acting elements, etc., were analyzed. Based on transcriptome data, the expression patterns of CmHXK gene in male flowers, female flowers, roots, leaves, fruits, and fruit development processes were analyzed. In addition, the expression patterns of CmHXK gene under abscisic acid, drought, salt, and low temperature stress were measured by RT-qPCR. 【Result】A total of six CmHXK gene family members were identified on chromosomes 2, 4, 5 and 8, containing 6-9 exons, 5-8 introns, with an average amino acid number of 462, an average molecular weight of 50.2 kD and an isoelectric point of 4.99-6.34. Systematic evolutionary analysis showed that the melon HXK members had the closest homology with cucumber. The main cis-acting elements in the melon CmHXK promoter were light responsive elements, hormone responsive elements, and stress responsive elements. The expression of CmHXKs was specific in different organs. Among them, CmHXK1, CmHXK2, and CmHXK5 expressed at high level in male flowers, female flowers, roots, leaves, and fruits during the development and maturation of fruits. However, the expressions of CmHXK3, CmHXK4, and CmHXK6 decreased with the development and maturation of fruits. The responses of CmHXKs to the four abiotic（abscisic acid, drought, salt, and cold）stresses differed. Among them, CmHXK1, CmHXK3, and CmHXK4 were strongly induced by ABA. CmHXK1, CmHXK3, and CmHXK6 were strongly induced by salt stress, and CmHXK1, CmHXK2, and CmHXK3 were strongly induced by drought stress. CmHXK1 and CmHXK6 were strongly induced by cold stress. 【Conclusion】 The CmHXK family are highly conserved and play important roles in the growth, development, and abiotic stress in melon. Among them, CmHXK1 is the most sensitive to four abiotic stresses.

Key words: melon, hexokinase, bioinformatics, abiotic stress, expression analysis

张帅博, 尹金鹏, 王吉庆, 肖怀娟, 李猛. 甜瓜CmHXK基因家族的鉴定及响应非生物胁迫的表达分析[J]. 生物技术通报, 2024, 40(12): 102-112.

ZHANG Shuai-bo, YIN Jin-peng, WANG Ji-qing, XIAO Huai-juan, LI Meng. Identification of the HXK Gene Family in Cucumis melo and Their Expression Analysis under Abiotic Stresses[J]. Biotechnology Bulletin, 2024, 40(12): 102-112.

图/表 11

表1 本研究所用RT-qPCR引物序列

Table 1 Sequences of primers used for RT-qPCR in this study

基因Gene	正向引物序列Forward primer sequence（5'-3'）	反向引物序列Reverse primer sequence（5'-3'）
CmHXK1	GTTGGACAAGATGTGGTAGG	CCAGTTCCTAGAATCACAGC
CmHXK2 CmHXK3 CmHXK4 CmHXK5 CmHXK6	CAGGTGGTTGATGCTATGG GACAGATTCCACCTTTCTCC ATGGAGGAGAGTAGTTAGGG TTGCTGCTGTGATATTAGGC ATGGTTGATGATACTGTTGGG	CTTCAAAGTAAGAGACCTCTGG CAAGCAACAACATCTTTCCC CAGGGAGATTATCAACGTAGG CTTCTTCAGCCATCTTGACC GACTCTATATAGGCAGCATTGG
CmActin7	GTGATGGTGTGAGTCACACTGTTC	ACGACCAGCAAGGTCCAAAC

图1 CmHXKs基因家族染色体定位分析

Fig. 1 Chromosome location analysis of the CmHXKs gene family

图2 植物HXK基因家族系统发育分析 Cm：甜瓜；At：拟南芥

Fig. 2 Phylogenetic tree of HXK gene family in plants Cm: Cucumis melo; At: Arabidopsis thaliana

图3 甜瓜CmHXK基因家族氨基酸序列多重比对结果不同阴影显示不同的同源性，黑色：100%；粉色：≥75%；蓝色：≥50%

Fig. 3 Amino acid sequence multiple alignment of the CmHXK gene family in C. melo Different shadows show different homology, black:100%; pink: ≥75%; blue: ≥50%

图4 甜瓜HXK家族基因结构（A）和蛋白基序（B）

Fig. 4 Gene structure（A）and protein motifs（B）of HXK family in melon

表2 CmHXKs基因启动子顺式作用调控元件分析

Table 2 Analysis of cis-acting regulatory elements in the promoter of CmHXKs genes

功能Function	元件Element	CmHXK1	CmHXK2	CmHXK3	CmHXK4	CmHXK5	CmHXK6
脱落酸ABA	ABRE	0	0	0	3	0	3
生长素Auxin	TGA-element	1	1	0	0	1	0
乙烯ETH	ERE	1	0	2	6	6	3
赤霉素GA	P-box	0	1	1	0	0	1
赤霉素GA	w box	1	0	0	2	1	0
茉莉酸甲酯MeJa	CGTCA-motif	1	2	0	0	1	2
茉莉酸甲酯MeJa	TGACG-motif	1	2	0	0	2	2
水杨酸SA	As-1	1	1	0	1	1	0
	TCA	1	0	1	0	0	1
	TCA-element	2	0	1	0	0	0
	DRE	0	0	0	3	1	0
非生物胁迫Abiotic stresses	LTR	0	0	0	0	1	0
	STRE	2	1	0	3	1	2
	WRE3	1	0	1	1	0	0
	WUN-motif	2	2	0	0	0	0
	TC-rich repeats	0	0	0	0	1	0
光响应Light	3-AFlbinding site	0	0	0	1	0	1
	AE-box	0	0	0	0	1	2
	ATCT-motif	1	0	0	0	0	0
	Box 4	3	3	2	3	2	2
	Box II	0	1	0	0	0	0
	chs-CMAla	0	0	0	1	0	0
	GA-motif	0	0	0	0	0	1
	GATA-motif	1	1	0	2	1	1
	G-Box	0	0	0	1	0	2
	G-box	0	1	0	2	1	2
	GT1-motif	2	0	0	2	1	0
	I-box	1	0	0	0	2	0
	Sp1	0	0	1	0	0	0
	TCCC-motif	0	3	0	0	0	0
	TCT-motif	1	1	3	0	1	0

表3 CmHXKs蛋白二级结构组成

Table 3 Secondary structure composition of CmHXKs proteins

蛋白名称 Protein name	α-螺旋 α-helix/%	延伸链 Extended strand/%	β转角 β-turn/%	无规卷曲 Random/%
CmHXK1	47.39	14.06	5.22	33.33
CmHXK2	43.00	15.78	5.52	35.70
CmHXK3	44.51	13.57	4.79	37.13
CmHXK4	43.46	13.29	5.49	37.76
CmHXK5	47.59	12.85	5.62	33.94
CmHXK6	51.52	12.12	6.4	29.97

图5 甜瓜CmHXK蛋白质三维结构

Fig. 5 Three -dimensional structure of CmHXK protein in C. melo

图6 甜瓜CmHXK基因家族蛋白互作

Fig. 6 Protein-protein interaction of CmHXK gene family in C. melo

图7 甜瓜CmHXKs基因家族的组织表达模式分析 Green：绿色果肉的甜瓜；Orange：橙色果肉的甜瓜；DAA：授粉后天数；Mature：成熟期

Fig. 7 Analysis of tissue expression pattern of CmHXKs family in C. melo Green: Melons with green flesh. Orange: Melons with orange flesh. DAA: Day after anthesis. Mature: Mature period

图8 不同非生物胁迫下CmHXKs相对表达水平

Fig. 8 Relative expressions of CmHXKs in C. melo under different abiotic stresses

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