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

doi:10.13304/j.nykjdb.2021.1020

摘要/Abstract

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

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

中图分类号:

Q781

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.

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.

图/表 9

表1 各试验组的海水参数

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

表2 本研究中使用的引物

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

图1 中间球海胆SiHK的核苷酸序列及其所编码的氨基酸序列注：方框中的字母为起始密码子（ATG），*表示终止密码子（TGA）；下划线表示SiHK蛋白编码的氨基酸序列；灰色区域表示COG5026结构域；双下划线区域字母（AATAA）为3’-UTR中的不稳定因素。

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.

图2 中间球海胆SiHK二级结构预测及其与紫球海胆、仿刺参HK的二级结构比较A：中间球海胆SiHK二级结构；B：中间球海胆SiHK、紫球海胆SpHK、仿刺参AjHK的二级结构比对

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）

图3 中间球海胆SiHK蛋白质的三级结构A：中间球海胆SiHK蛋白；B：人HK蛋白；黄色框住区域中，球棍结构表示磷酸基团识别位点，周围蛋白质构成的结构为酶活性中心，黄色虚线代表盐桥。

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.

图 4 中间球海胆SiHK氨基酸序列系统进化分析注：黑色箭头指出中间球海胆SiHK，左侧数字代表迭代次数。

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.

图5 中间球海胆不同组织SiHK基因表达量和总SiHK酶活力注：不同小写字母表示不同组织间在P<0.05水平差异显著。

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.

表3 高温和酸化对SiHK基因表达和总SiHK酶活力影响的双因素方差分析

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^**

图6 不同处理下肠和性腺中SiHK基因的表达和总SiHK酶活力注：*和**分别表示处理与对照间差P<0.05和P<0.01水平差异显著。

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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