Abstract：Myo-inositol-1-phosphate synthase (MIPS) is one of the key enzyme in biological inositol metabolism. In order to explore the role of MIPS in the stress response of Larimichthys crocea under cold treatments, MIPS gene was cloned and then its sequence were analyzed and the expression changes were detected under chronic and acute cold stress. The full length of MIPS cDNA (GenBank accession No. MG760436) was 2 599 bp which contained a 1 659 bp open reading frame (ORF) and encoded 553 amino acids, the molecular weight of putative MIPS protein was 60.5 kD. Sequence alignment showed that the amino acid sequence of L. crocea MIPS shared high similarity (85.5%~88.4%) to other teleost and all of these MIPS had 4 highly conserved core regions. In the phylogenetic tree, L. crocea MIPS gathered with MIPS of other teleost and was most closely related to the MIPS of Fugu rubripes (Takifugu rubripes). During chronic cold stress (water temperature slowed decreased from 12 ℃ to 6 ℃), the MIPS expression in gill, skin and heart significantly increased at first and then significantly decreased (P＜0.05), and the maximum appeared at 8 ℃ which were respectively 13.71, 89.50 and 50.83 fold, respectively, compared to 12 ℃. The expression continuously elevated in brain and muscle during chronic cold stress and at 6 ℃ the expression were 99.89 and 110.17 fold higher than before cold stress. And there were no significant changes in intestine, kidney, liver and spleen (P＞0.05). During acute cold stress (from 12 ℃ to 8 ℃ immediately and keeping 8 ℃ for 4 h), the expression level of MIPS were significantly up-regulated (P＜0.05) in all the detected tissues except intestine. The 2 biggest expression changes were appeared in brain and muscle, which were 54.53 and 32.89 fold higher than before cold stress. The expression changes in both chronic and acute cold treatments indicated MIPS participates in the response to cold stress and has possible relationship to cold adaptability of L. crocea, brain and muscle are the 2 most important tissues in the response to cold stress. These results provide reference for researching the function of MIPS gene and the mechanism of response to cold stress, as well as breeding low-temperature tolerant cultivars of L. crocea.
参考文献:[1] 刘家富, 韩坤煌. 我国大黄鱼产业的发展现状与对策[J]. 福建水产, 2011, 33(5): 4-8. Liu J F, Han K H. Current development situation and countermeasure of large yellow crocker industry in China[J]. Journal of Fujian Fisheries, 2011, 33(5): 4-8 (in Chinese). [2] 徐镇, 江锦坡, 陈寅儿. 不同品系大黄鱼致死低温的研究[J]. 宁波大学学报(理工版), 2006, 19(4): 462-464. Xu Z, Jiang J P, Chen Y E. Study on low lethal temperature of different strains of Pseudosciaena crocea[J]. Journal of Ningbo University (NSEE), 2006, 19(4): 462-464 (in Chinese). [3] 苗亮, 李明云, 陈炯, 等. 快长、耐低温大黄鱼新品种东海1号的选育[J]. 农业生物技术学报, 2014, 22(10): 1314-1320. Miao L, Li M Y, Chen J, et al. Breeding of fast growth and low temperature tolerance of new variety Donghai No. 1 large yellow croaker (Pseudosciaena crocea)[J]. Journal of Agricultural Biotechnology, 2014, 22(10): 1314-1320 (in Chinese).[4] 徐丽华, 常玉梅, 刘春雷, 等. 鲤脑组织低温差异表达候选基因的筛选[J]. 遗传, 2011, 33(3): 262-269.Xu L H, Chang Y M, Liu C L, et el .Screening cold-acclimation differential expression candidate genes in the brain of common carp (Cyprinus carpio) [J].Hereditas (Beijing) ,2011, 33(3): 262―269[5] Xu H, Zhang D L, Yu D H, et al. Molecular cloning and expression analysis of scd1 gene from large yellow croaker Larimichthys crocea under cold stress[J]. Gene, 2015, 568(1): 100-108. [6] 刘浩. 大黄鱼硬脂酰辅酶A去饱和酶-1(SCD-1)基因的克隆与鉴定及在低温条件下对膜流动性影响[D]. 舟山: 浙江海洋学院, 2015. Liu H. Molecular clone, identification of Stearoyl-CoA desaturase 1 (SCD1) gene in large yellow croaker (Pseudosciaena crocea) and effect of SCD1 on membrane fluidity under cold stress[D]. Zhoushan: Zhejiang Ocean University, 2015 (in Chinese). [7] 苗亮，李明云*，陈莹莹,等. 大黄鱼冷诱导结合蛋白（CIRP）基因cDNA克隆及低温胁迫对其时空表达的影响?[J].水产学报,2017,41(4):481-489.MIAO Liang, LI Mingyun, CHEN Yingying,et el. Cloning of cold inducible RNA-binding protein (CIRP) gene in Larimichthys crocea and the expression analysis under cold treatments?[J]. Journal of Fisheries of China, 2017,41(4):481-489.[8] Majumder N D, Ram T, Sharma A C. Cytological and morphological variation in hybrid swarms and introgressed population of interspecific hybrids (Oryzarufipogon Griff . x Oryza sativa L. ) and its impact on evolation of intermediate types[J]. Euphytica, 1997,94:295-302.[9]宋颖琦, 杨谦. 拟南芥肌醇-1-磷酸合酶类蛋白基因的克隆表达[J]. 哈尔滨工业大学学报, 2005,37(12):1641-1643.Song Y Q, Yang A Q . Cloning and expression of the cDna ofmyo - inositol 1 phosphatesynthase - like protein gene from Arab idopsis tha liana[J]. Journal of harbin institute of technology ,2005,37(12):1641-1643[10] 冀德伟, 李明云, 王天柱, 等. 不同低温胁迫时间对大黄鱼血清生化指标的影响[J]. 水产科学, 2009, 28(1): 1-4. Ji D W, Li M Y, Wang T Z, et al. Effects of low temperature stress periods on serum biochemical indexes in large yellow croaker Pseudosciaena crocea[J]. Fisheries Science, 2009, 28(1): 1-4 (in Chinese). [11] 李明云, 冀德伟, 吴海庆, 等. 低温胁迫下大黄鱼肝脏蛋白质组双向电泳分析[J]. 农业生物技术学报, 2010, 18(2): 323-328. Li M Y, Ji D W, Wu H Q, et al. 2-DE analysis in liver of Pseudosciaena crocea during low temperature stress[J]. Journal of Agricultural Biotechnology, 2010, 18(2): 323-328 (in Chinese). [12] 李明云, 吴玉珍, 冀德伟, 等. 低温选择大黄鱼(Pseudosciaena crocea)的肝脏蛋白质组双向电泳分析[J]. 海洋与湖沼, 2010, 41(3): 348-351. Li M Y, Wu Y Z, Ji D W, et al. 2-DE analysis of liver of Pseudosciaena crocea after low temperature breeding[J]. Oceanologia et Limnologia Sinica, 2010, 41(3): 348-351 (in Chinese). [13] 张晓丽, 胡玉珍, 李明云, 等. 养殖大黄鱼在自然海区降温不同阶段抗氧化水平及血清酶活性的变化[J]. 海洋科学, 2013, 37(11): 27-34. Zhang X L, Hu Y Z, Li M Y, et al. Changes in antioxidant level and serum enzyme activity of farmed large yellow croaker during natural water cooling in winter[J]. Marine Sciences, 2013, 37(11): 27-34 (in Chinese). [14] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408. [15]李春艳, 宋清晓, 刘建宁, 等. 东方山羊豆肌醇-1-磷酸合酶基因的克隆与分析[J]. 家畜生态学报 2010, 31(5): 219-225.Li C Y , Song Q X, Liu J N,et el.Cloning and Analysis of Myo-Inositol-1-Phosphate Synthases in Galega Orientalis[J].Acta Ecologiae Animalis Domastici Sep .2010,31(5):219-225[16] Stein A J, Geiger J H. The crystal structure and mechanism of l-L myo-inositol-1-phosphate synthase[J]. Biol Chem. 2002,277(11):484-491.[17] Stieglitz K A, Yang H, Roberts M F. Reaching for mechanistic consensus across life kingdoms: structure and insights into catalysis of the myo-inositol-1-phosphate synthase (MIPS) from Archaeoglobus fulgidus[J]. Biochemistry. 2005,44(1):213-224.[18] Majumder A L, Johson M D, Henry S A. L-myo-in-ositol-1-phosphate synthase[J]. Bioehem Biophys Acta, 1997,13(48):245.[19] Majeem, Maitra S, Dastidar K G. A novel salt-tolerant 1-Myo-inositol-1-phosphate synthase from porteresia coarctata (Roxb.) tateoka, a halophytic wild rice: molecular cloning, bacterial over expression, characterization and functional introgression into tobacco-conferring salt tolerance phenotype[J]. BiolChem, 2004,279:285-289.[20] Kawsar H I, Ohtani K, Okumura K. Organization and transcriptional regulation of myo-inositol operon in Clostridium per, ringens[J]. FEMS Microbiol Lett, 2004,23(5l):285-289.[21] Guimin Guan, Peihua Dai, Ishaiahu Shechter. cDNA cloning and gene expression analysis of human myo-inositol-1-phosphate synthase[J]. Archives of Biochemistry and Biophysics, 2003,417:251-259. [22] 柳哲胜, 刘庆昌, 翟红. 甘薯肌醇-1-磷酸合成酶基因的克隆及序列分析[J]. 农业生物技术学报, 2006, 14(2): 219-225.Liu Z s, Liu Q c, Zhai H. Cloning and Sequence Analysis of Myo Inositol-1-phosphateSynthase Gene in Sweetpotato[J]. Journal of Agricultural Biotechnology 2006,14 (2): 219~225[23] Chun J A, Jin U H, Lee J W, et a1. Isolation and characterization of a myo-inositol-1-phosphate synthase cDNA from developing sesame (Sesamum indicum L.) seeds: functional and differential expression, and salt-induced transcription during germination[J]. Planta, 2003,216(5):874-880.[24] 徐德立. 低温胁迫对草鱼ZC-7901细胞系和淡水白鲳CBS细胞系某些细胞功能影响的研究[D]. 2002, 浙江大学.Xu D L. The study of the effects of cold stress on some cell fuction of the CtenophaYyngodonidella cell line ZC一7901 and the Colossoma brachypomum cell line CBS[D].2002, Zhejiang University Hangzhou, China
