Cloning of SiPFK Gene from Strongylocentrotus intermedius and the Effect of Acidification-High Temperature Stress on Its Expression
JIAO Ren-He, CUI Dong-Yao, WU Bo-Qiong, SONG Jian, CHANG Ya-Qing, ZHAN Yao-Yao*
Key Laboratory of Mariculture ＆ Stock Enhancement in North China's Sea, Ministry of Agriculture and Rural Affairs, Dalian Ocean University, Dalian 116023, China
Abstract：The key enzymes of glycolysis pathway play an important role in regulation and adaptation of echinoderms to the changes of marine environment.In order to clarify the sequence information and expression pattern of phosphofructokinase (PFK) in Strongylocentrotus intermedius, as well as, to understand the effect of "acidification-high temperature" stress on its expression and biological activity, in this study, the rapid-amplification of cDNA ends (RACE) technique was used to clone the full-length cDNA sequence of PFK gene in S. intermedius (SiPFK)(GenBank No. OM780114), and then, the characteristics of SiPFK were analyzed by bioinformatics software, at last, the relative expression pattern of SiPFK and enzyme activity of SiPFK in intestines and gonads of S. intermedius under "acidification-high temperature" stress were investigated. The results showed that, the full- length cDNA of SiPFK gene was 2 787 bp, encoding 840 amino acids, the theoretical isoelectric point of SiPFK protein was 7.48 with a predicted molecular weight of 92.01 kD; The amino acid sequence of SiPFK protein was the most similar to that from S. purpuratus (similarity: 96.20 %); The results of qPCR showed that SiPFK gene specificly expressed in all examined tissue, the relative expression level of SiPFK and total SiPFK enzyme activities in intestines and gonads of S. intermedius were changed after 60 d of "acidification-high temperature" stress, suggesting that "acidification-high temperature" might affect the metabolic process of sea urchins by regulating the expression and activity of key enzymes of glucose metabolism. This study provides an important reference basis for exploring the response of echinoderms to future marine environmental changes.
焦仁和, 崔东遥, 武博琼, 宋坚, 常亚青, 湛垚垚. 中间球海胆SiPFK基因的克隆及酸化-高温胁迫对其表达的影响[J]. 农业生物技术学报, 2022, 30(10): 1962-1975.
JIAO Ren-He, CUI Dong-Yao, WU Bo-Qiong, SONG Jian, CHANG Ya-Qing, ZHAN Yao-Yao. Cloning of SiPFK Gene from Strongylocentrotus intermedius and the Effect of Acidification-High Temperature Stress on Its Expression. 农业生物技术学报, 2022, 30(10): 1962-1975.
[1] 常亚青, 丁君, 宋坚, 等. 2004. 海参、海胆生物学研究与养殖[M]. 北京: 海洋出版社, pp. 5-40. (Chang Y Q, Ding J, Song J, et al.2004. Research and Breeding of Sea Cucumber and Sea Urchin Biology[M]. Beijing: Ocean Press, pp. 5-40; 211-216.) [2] 常亚青, 王子臣, 王国江. 1999 .温度和藻类饵料对虾夷马粪海胆摄食及生长的影响[J]. 水产学报, 23(1): 69-76. (Chang Y Q, Wang Z C, Wang G J.1999. Effect temperature and algae on feeding and growth in sea urchin, Strongylocentrotus intermedius[J]. Journal of Fisheries of China, 23(1): 69-76.) [3] 崔东遥, 任丽媛, 邢冬飞, 等. 2019.中间球海胆乳酸脱氢酶基因克隆及其对海水酸化的响应[J]. 水产学报, 43(6): 1423-1437. (Cui D Y, Ren L Y, Xing D F, et al.2019. Identification and characterization of LDH gene and its response to seawater acidification in the sea urchin (Strongylocentrotus intermedius)[J]. Journal of Fisheries of China, 43(6): 1423-1437.) [4] 丁君, 常亚青, 孙巍. 2011. 虾夷马粪海胆谷胱甘肽过氧化物酶、谷胱甘肽硫转移酶基因的克隆与表达分析[J]. 海洋科学进展, 29(A01): 67-79. (Ding J, Chang Y Q, Sun W.2011. Molecular cloning and expression of glutathione peroxidase and glutathione S-transferases genes from sea vrchin (Strongylocentrotus intermedius)[J]. Advances in Marine Science, 29(A01): 67-79.) [5] 卢羽洁. 2017. 海洋酸化及变暖对刺参主要生理生态过程和免疫的影响[D]. 硕士学位论文, 大连海洋大学, 导师:邢坤, pp. 33. (Lu Y J.2014. Effects of ocean acidification and warming on physio-ecological process and immune responses of sea cucumber Apostichopus japonicus (Selenka)[D]. Thesis for M.S., Dalian Ocean University, Supervisor: Xing K, pp. 33.) [6] 马红悦, 李玲, 李艳艳, 等. 2020. 沙葱萤叶甲己糖激酶基因的克隆, 相对表达量及RNA干扰效应[J]. 植物保护学报, 47(6): 1211-1218. (Ma H Y, Li L, Li YY, et al.2020. Cloning, relative expression, and RNAi effects of the hexokinase gene in Galeruca daurica (Coleoptera: Chrysomelidae)[J]. Journal of Plant Protection, 47(6): 1211-1218.) [7] 马正伟, 汪仕良. 2002. 缺氧状态下大鼠肝细胞糖酵解变化的实验研究[J]. 中华烧伤杂志, 18(4): 238-241. (Ma Z W, Wang S L.2002. An experimental study of the changes of rat hepatocytic glycolysis during hypoxia[J]. Chinese Journal of Burns, 18(4): 238-241.) [8] 莫小燕, 张晓宏, 郭俊明. 2016. 非编码RNA在肿瘤细胞糖代谢中的调控作用[J]. 中国生物化学与分子生物学报, 32(1): 10-16. (Mo X Y, Zhang X H, Guo J M.2016. Regulation of non-coding rnas on glucose metabolism in tumor cells[J]. Chinese Journal of Biochemistry Molecular Biology, 32(1): 10-16.) [9] 聂琴. 2013. 饲料糖类物质对大菱鲆糖代谢酶活性和基因表达量的影响[D]. 硕士学位论文, 中国海洋大学, 导师: 张文兵, pp. 15-17. (Nie Q.2013. Effects of dietary carbohydrates on the activities and gene expression of carbohydrate metabolic enzymes in turbot (Scophthalmus maximus)[D]. Thesis for M.S., Ocean University of China, Supervisor: Zhang W B, pp. 15-17.) [10] 孙菊燕, 黄鹤忠. 2005. 环境因子对海洋藻类酶活性的影响及其应用[J]. 海洋科学进展, 23(B12): 74-79. (Sun J Y, Huang H Z.2005. Effect of environmental factors on enzyme activities in seaweed and its application[J]. Advances in Marine Science, 23(B12) :74-79.) [11] 王茜. 2018. 研究表明海洋食物链可能因环境的改变而崩溃[J]. 渔业信息与战略, 33(1): 70. (Wang Q.2018. Research shows that the marine food chain may collapse due to environmental changes[J]. Fishery Information And Strategy, 33(1): 70.) [12] 姚南瑜, 张英泽, 蔡淑频, 等. 1985. 近海底栖海藻对介质渗透压变化的适应研究——Ⅱ.海水浓度对潮间带底栖褐藻光合和呼吸活性的影响[J]. 海洋与湖沼, 16(1): 51-56. (Yao N Y, Zhang Y Z, Cai S P, et al.1985. Studies on adaptation of the marine benthic algae in coastal areas to the changes in osmotic pressure-Ⅱ. The effects of sea water concentration on photosynthesis and respiration of intertidal benthic brown algae[J]. Oceanologia Et Limnologia Sinica, 16(1): 51-56.) [13] 尹文露, 崔东遥, 李莹莹, 等. 2020. 中间球海胆丙酮酸激酶(PK)基因克隆及其对海水酸化的响应[J]. 大连海洋大学学报, 35(03): 360-367. (Yin W L, Cui D Y, Li Y Y, et al.2020. Cloning and response of pyruvate kinase (PK) gene to seawater acidification in sea urchin Strongylocentrotus intermedius[J]. Journal of Dalian Ocean University, 35(03): 360-367.) [14] 余舜武, 刘鸿艳, 罗利军. 2007. 利用不同实时定量PCR方法分析相对基因表达差异[J]. 作物学报, 33(7): 1214-1218. (Yu S W, Liu H Y, Luo L J.2007. Analysis of relative gene expression using different real-time quantitative PCR[J]. Acta Agronomica Sinica, 33(7): 1214-1218) [15] 湛垚垚, 黄显雅, 段立柱, 等. 2013. 实验室模拟海水酸化系统[P]. 中国: 201320267332.7. (Zhan Y Y, Huang X Y, Duan L Z, et al.2013. Laboratory simulation of seawater acidification system[P]. China: 201320267332.7.) [16] 张中青, 李春, 刘宁. 2011. NaCl刺激对乳杆菌冻干存活率及胞内磷酸果糖激酶的影响[J]. 微生物学通报, 38(10): 1554-1560. (Zhang Z Q, Li C, Liu N.2011. Effect of NaCl stimulation on the survival rate of freeze-dried cells and intracellular posphofructokinase in Lactobacillus[J]. Bulletin of Biology, 38(10): 1554-1560.) [17] 周遵春, 包振民, 董颖, 等. 2008. MYP基因在中间球海胆及杂交海胆生殖腺不同发育时期的转录表达差异[J]. 遗传, 30(11): 1453-1458. (Zhou Z C, Bao Z M, Dong y, et al.2008. MYP gene expressions at transcription level in different stages of gonad of sea urchin Strongylocentrotus intermedius and hybrids[J]. Hereditas, 30(11): 1453-1458.) [18] Cheng L, Abraham J, Hausfather Z, et al.2019. How fast are the oceans warming[J]. Science, 363(6423): 128-129. [19] Deutscher J, Saier M H.1983. ATP- dependent protein kinase-catalyzed phosphorylation of a seryl residue in HPr, a phosphate carrier protein of the phosphotransferase system in Streptococcus pyogenes[J]. Proceedings of the National Academy of Sciences of the USA, 80(22): 6790-6794. [20] Fraenkel D G.1992. Genetics and intermediary metabolism[J]. Annual Review of Genetics, 26(1): 159-177. [21] Freed J M.1971a. Temperature effects on muscle phosphofructokinase of the Alaskan king crab, Paralithodes camtschatica[J]. Comparative Biochemistry & Physiology Part B Comparative Biochemistry, 39(4): 765-774. [22] Freed J M.1971b. Properties of muscle phosphofructokinase of cold and warm-acclimated Carassius auratus[J]. Comparative Biochemistry & Physiology B Comparative Biochemistry, 39(4): 747-764. [23] Freed J M, Kirk C R.1976. Phosphofructokinase activity of temperature-acclimated crayfish[J]. Comparative Biochemistry & Physiology Part B Comparative Biochemistry, 54(4): 515-517. [24] Hu M Y, Katharina M, Kreiss C M, et al.2016. Temperature modulates the effects of ocean acidification on intestinal ion transport in Atlantic Cod, Gadus morhua[J]. Frontiers in Physiology, 7: 198. [25] Kandler O.1983. Carbohydrate metabolism in lactic acid bacte-ria[J]. Antonie van Leeuwenhoek, 49(3): 209-224. [26] Mayewski P A, Meredith M P, Summerhayes C P, et al.2009. State of the Antarctic and Southern Ocean climate system[J]. Reviews of Geophysics, 47: RG1003. [27] Riser S C, Freeland H J, Roemmich D, et al.2016. Fifteen years of ocean observations with the global Argo array[J]. Nature Climate Change 6(2): 145-153. [28] Stocker T F, Qin D, Plattner G K, et al.2013. The physical science basis. Contribution of working group I to the fifth assessment report of the intergovernmental panel on climate change[J]. Computational Geometry, 18(3): 95-123. [29] Shiau S Y, Liang H S.1995. Carbohydrate utilization and digestibility by tilapia, Oreochromis nilocitus x O. aureus, are affected by chromic oxide inclusion in the diet[J]. Journal of Nutrition, 125(4): 976-982. [30] Zhan Y Y, Cui D Y, Xing D F, et al.2020. CO2-driven ocean acidification repressed the growth of adult sea urchin Strongylocentrotus intermedius by impairing intestine function[J]. Marine Pollution Bulletin, 153(3): 110944.
