Cloning of Hexokinase Gene from Strongylocentrotus intermedius and Its Expression Response to High Temperature-acidification Stress

doi:10.13304/j.nykjdb.2021.1020

Abstract

Abstract:

In order to clarify the sequence information and expression pattern of hexokinase （HK）in Strongylocentrotus intermedius， and preliminarily understand the effects of high temperature-acidification stress on its expression and activity， Strongylocentrotus intermedius was as material. The full-length cDNA of SiHK in Strongylocentrotus intermedius was cloned and characterized usingcDNA ends rapid amplification （RACE） technology and bioinformatics software. And the expression and enzyme activity of SiHK were analyzed in different tissues.The results showed that the total cDNA length of SiHK gene was 2 041 bp， encoding 476 amino acids. The theoretical isoelectric point （pI） and molecular weight of SiHK protein were 6.44 and 52.72 kD， respectively. The bioinformatics analysis showed that the amino acid sequence of SiHK protein was the most similar to that of Strongylocentrotus purpuratus HK. The results of qRT-PCR showed that SiHK was expressed in all examined tissues with an obvious tissue specifity， which the relative expression levels in intestine and gonad were higher and the total enzyme activities of SiHK in coelomic fluid and tube feet were higher. Compared with CK， the relative expression of SiHK gene and total enzyme activity of SiHK in intestine and gonad of S. intermedius were changed after high temperature-acidification stress 60 d， which suggested that high temperature-acidification stress might affect metabolism of sea urchins by regulating the expression and enzyme activity of key enzymes of glucose metabolism.

Key words: Strongylocentrotus intermedius, hexokinase, gene cloning, high temperature-acidification stress, expression pattern, enzyme activity

摘要：

为明确中间球海胆己糖激酶的序列信息和表达规律，了解高温-酸化胁迫对其表达和生物活性的影响，以中间球海胆为研究对象，利用cDNA末端快速扩增技术获得中间球海胆已糖激酶基因的全长cDNA序列（SiHK），并利用生物信息学软件分析其序列特征，分析高温-酸化胁迫下中间球海胆肠和性腺组织中SiHK基因的表达及其酶活力变化。结果表明，SiHK基因的cDNA全长2 041 bp，编码476个氨基酸，SiHK蛋白的理论等电点为6.44，蛋白质分子质量52.72 kD。生物信息学分析显示，SiHK蛋白氨基酸序列与紫球海胆HK氨基酸序列相似最高。实时荧光定量PCR结果显示，SiHK基因的表达具有组织特异性，其中，肠和性腺组织中SiHK基因的相对表达量较高，而在体腔液和管足中SiHK酶活力较高。高温-酸化胁迫处理60 d后，与对照组相比，处理组中间球海胆的肠和性腺组织中SiHK基因的相对表达量和SiHK酶活性均发生改变。由此表明，高温-酸化胁迫可能通过调控糖代谢关键酶活性对海胆的物质代谢过程产生影响。

关键词: 中间球海胆, 己糖激酶, 基因克隆, 高温-酸化胁迫, 表达模式, 酶活力

CLC Number:

Q781

Boqiong WU, Dongyao CUI, Renhe JIAO, Jian SONG, Yaoyao ZHAN, Yaqing CHANG. Cloning of Hexokinase Gene from Strongylocentrotus intermedius and Its Expression Response to High Temperature-acidification Stress[J]. Journal of Agricultural Science and Technology, 2022, 24(7): 205-217.

武博琼, 崔东遥, 焦仁和, 宋坚, 湛垚垚, 常亚青. 中间球海胆己糖激酶基因克隆及高温-酸化胁迫对其表达影响的初步研究[J]. 中国农业科技导报, 2022, 24(7): 205-217.

Figures/Tables 9

Table1 Seawater parameters of each treatment

处理 Treatment	温度 Temperature/℃	pH_NBS	盐度 Salinaty/PPT	总碱度 TA/（µmol·kg^-1）	二氧化碳分压 pCO₂/kPa
CK	20.10±0.30	8.10±0.03	31.21±0.20	2 360.45±24.18	53.44±2.55
HC	23.20±0.30	8.10±0.03	31.33±0.21	2 363.32±25.34	54.35±3.26
LO	20.30±0.30	7.62±0.04	31.25±0.12	2 360.88±20.33	179.87±5.44
HO	23.00±0.30	7.61±0.03	31.18±0.13	2 349.54±26.42	189.17±7.00

Table 2 Primers used in this study

引物名称 Primer name	引物序列 Primer sequences（5’-3’）	用途 Application	退火温度Temperature/℃
SiHK-F1	AATGCCTGGCGAACAAAATACAG	SiHK-1	54
SiHK-R1	ATCACTGCTACTATGTCAACTCG	SiHK-1	54
SiHK-F2	ATTCCCCTCGGTTTCACCTTTTC	SiHK-2	60
SiHK-R2	ATACCCGAGGCAATCAGTCTGGC	SiHK-2	60
SiHK-F3	GCCAGACTGATTGCCTCGGGTAT	SiHK-3	57
SiHK-R3	GTACGTCCTTACAGTCCAGCAGA	SiHK-3	57
SiHK-5-out	TCACCCACATCACACGGAAGTTG	5’RACE	60
SiHK-5-in	GCATTGAGAGCAGCCTTCAGACC	5’RACE	61
SiHK-3-out	CAATACAACCATCTGCTGGACTG	3’RACE	56
SiHK-3-in	TTCAATAAGCAAGAACATCAGGC	3’RACE	53
UPM	CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGAGT	RACE	67
NUP	CTAATACGACTCACTATAGGGC	RACE	52
SiHK-F	ACTCCATCGTCTCCGAATGC	qPCR	60
SiHK-R	CAACGCCTGCTACATGGAAG	qPCR	60
β-actin-F	ACAGGGAAAAGATGGCACAGA	qPCR	60
β-actin-R	AGAGGCGTAGAGGGAAAGCAC	qPCR	60

Fig. 1 Nucleotide and deduced amino acid sequence of SiHK in Strongylocentrotus intermediusNote：Letter in the frame is the starting codon （ATG）； * indicates the terminating codon （TGA）； underline represents the amino acid sequence encoded by the SiHK protein； grey region represents the COG5026 domain； double underlined area （ AATAA） is the unstable factor in 3’-UTR.

Fig. 2 Secondary structure prediction and comparison of hexokinase betweenA：Predicted secondary structure of SiHK in Strongylocentrotus intermedius； B：Comparison of HK secondary structure among S. intermedius （SiHK）， Strongylocentrotus purpuratus （SpHK） and Apostichopus japonicus （AjHK）

Fig. 3 3D structure prediction of SiHKA：SiHK protein from Strongylocentrotus intermedius；B：HK protein from Homo spains；in the yellow framed area， the club structure represents the phosphate group recognition site， the surrounding protein structure is the enzyme active center， and the yellow dotted line represents the salt bridge.

Fig. 4 Phylogenetic analysis of deduced amino acid sequences of SiHKNote：SiHK is marked with black arrow， the numbers at the tree nodes indicate the percentage of bootstrapping after 1 000 replicates.

Fig. 5 Relative expression of SiHK and total SiHK activities in different tissues of Strongylocentrotus intermediusNote：Different lowercase letters indicate significant differences between different tissues at P<0.05 level.

Table 3 Two-way ANOVA of effects of high temperature-acidification stress on relative expression and total enzyme activities of SiHK

指标 Index	组织 Organ	因子Factor
指标 Index	组织 Organ	高温 High temperature	酸化Acidification	高温×酸化 High temperature×acidification
SiHK表达量 Relative expression level of SiHK	肠 Intestine	77.918^**	85.696^**	117.932^**
SiHK表达量 Relative expression level of SiHK	性腺 Gonad	0.320	4.173^*	29.133^**
总SiHK酶活力 Total SiHK activity	肠 Intestine	9.217^*	2.553	3.690
总SiHK酶活力 Total SiHK activity	性腺 Gonad	13.046^*	0.486	101.034^**

Fig. 6 Relative expression level of SiHK and the total SiHK activities under different treatmentsNote：* and ** indicate significant differences between treatment CK at P<0.05 and P<0.01 levels， respectively.

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