Gene Cloning of SCD-1a and SCD-1b in Donghai No.1 Large Yellow Croaker (Larimichthys crocea) and Their Responses to Cold Stress
LI Ming-Yun1,*, MIAO Liang1, CHEN Ying-Ying1, LI Chong1, ZHANG Li-Ning2
1 State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, China; 2 Zhejiang Mariculture Research Institute, Wenzhou 325005, China
Abstract Large yellow croaker (Larimichthys crocea) is a kind of warm-temperature fish. Low temperature in winter can cause large death of cultured L. crocea, so it is necessary to carry out researches on how to deal with low temperature stress. In order to study the sequence characteristics of stearoyl coenzyme A desaturase 1 (SCD-1) gene of Donghai No.1 large yellow croaker and their responses to cold stress, the full length of SCD-1a (GenBank No. MT325796) and SCD-1b (GenBank No. MT325797) cDNA were cloned by rapid amplification of cDNA ends (RACE) technique, and expression level under chronic and acute cold stress were analysed by qRT-PCR. The full length of SCD-1a cDNA was 3 095 bp which contained a 1 014 bp ORF and encoded 337 amino acid. The full length of SCD-1b cDNA was 2 636 bp which contained a 1 008 bp ORF and encoded 335 amino acid. SCD-1a and SCD-1b both contained 3 conserved histidine rich regions, and the sequence similarity was 72.2%. The predicted tertiary protein structure of SCD-1a and SCD-1b were highly similar and both contained 4 transmembrane helical structure. In the phylogenetic tree based on amino acids, the SCD of teleost divided into 2 clusters, the SCD-1a and SCD-1b of L. crocea located in 2 clusters separately, indicating that they were different subtypes. Under normal growth temperature (18 ℃), the expression levels of SCD-1a and SCD-1b were both high in liver and lower in other tissues. During chronic-cold stress (decreasing slowly from 12 ℃ to 6 ℃ by the method of cooling at the rate of 0.5 ℃/12 h and continuing to cool down after maintaining for 12 h), the expression of SCD-1a significantly increased in gill, skill, muscle, intestine and brain, and the expression of SCD-1b significantly increased in skill, muscle, intestine, brain and heart, nevertheless SCD-1a expression decreased significantly in liver. During acute cold stress (from 12 to 8 ℃ immediately), the expression level of SCD-1b significantly increased in all tissues, and SCD-1a expression significantly increased in all tissues except skill. The results of this study have certain reference significance for understanding the mechanism of response and adaptation of L. crocea to low temperature stress.
LI Ming-Yun,MIAO Liang,CHEN Ying-Ying, et al. Gene Cloning of SCD-1a and SCD-1b in Donghai No.1 Large Yellow Croaker (Larimichthys crocea) and Their Responses to Cold Stress[J]. 农业生物技术学报, 2020, 28(10): 1820-1829.
[1] 常玉梅, 徐丽华, 李世国, 等. 2012. 鲤耐低温候选基因CcSCD全长cDNA的克隆及功能预测[J]. 水产学杂志, 25(3): 1-6. (Chang Y, Xu L, Li S, et al.2012. Isolation and characterization of full length cDNA sequence of stearoyl-CoA desaturase gene responding to low temperature from common carp (Cyprinus carpio)[J]. Chinese Journal of Fisheries, 25(3): 1-6.) [2] 冀德伟, 李明云, 王天柱, 等. 2009. 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, 28(1): 1-4. (Ji D W, Li M Y, Wang T Z, et al.2009. Effects of low temperature stress periods on serum biochemical indexes in large yellow croaker Pseudosciaena crocea[J]. Fisheries Science, 28(1): 1-4.) [3] 李明云, 冀德伟, 吴海庆, 等. 2010a. 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, 18(2): 323-328. (Li M Y, Ji D W, Wu H Q, et al.2010a. 2-DE analysis in liver of Pseudosciaena crocea during low temperature stress[J]. Journal of Agricultural Biotechnology, 18(2): 323-328.) [4] 李明云, 苗亮, 陈莹莹, 等. 2018. 大黄鱼MIPS基因克隆及低温胁迫下的表达分析[J]. 农业生物技术学报, 26(2): 292-301. (Li M-Y, Miao L, Chen Y-Y, et al.2018. Cloning of MIPS gene in large yellow croaker (Larimichthys crocea) and the expression analysis under cold treatments[J]. Journal of Agriculture Biotechnology, 26(2): 292-301.) [5] 李明云, 吴玉珍, 冀德伟, 等. 2010b. 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, 41(3): 348-351. (Li M Y, Wu Y Z, Ji D W, et al.2010b. 2-DE analysis of liver of Pseudosciaena crocea after low temperature breeding[J]. Oceanologia et Limnologia Sinica, 41(3): 348-351.) [6] 刘浩. 2015. 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]. 硕士学位论文, 浙江海洋学院, 导师: 吴常文, pp. 22-28. (Liu H.2015. 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]. Thesis for M.S., Zhejiang Ocean University, Supervisor: Wu C, pp. 22-28.) [7] 刘家富. 2013. 大黄鱼养殖与生物学[M]. 厦门: 厦门大学出版社, pp.1-102. (Liu J.2013. Culture and Biology of Large Yellow Croaker[M]. Xiamen: Xiamen University Press, pp.1-102.) [8] 刘丽丽, 朱华, 闰艳春, 等. 2018. 鱼类耐低温耐受机制与功能基因研究进展[J]. 生物技术通报, 34(2): 1-6. (Liu L, Zhu Z, Yan Y, et al.2018. Research progress of cold tolerance mechanism and functional genes in fish[J]. Biotechnology Bulletin, 34(2): 1-6.) [9] 苗亮, 李明云, 陈炯, 等. 2014. 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, 22(10): 1314-1320. (Miao L, Li M Y, Chen J, et al.2014. Breeding of fast growth and low temperature tolerance of new variety Donghai No. 1 large yellow croaker (Pseudosciaena crocea)[J]. Journal of Agricultural Biotechnology, 22(10): 1314-1320.) [10] 苗亮, 李明云, 陈莹莹, 等. 2017. 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, 41(4): 481-489. (Miao L, Li M Y, Chen Y Y, et al. 2017. 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, 41(4): 481-489.) [11] 徐丽华, 常玉梅, 刘春雷, 等. 2011. 鲤脑组织低温差异表达候选基因的筛选[J]. 遗传, 33(3): 262-269. (Xu L H, Chang Y M, Liu C L, et al.2011. Screening cold-acclimation differential expression candidate genes in the brain of common carp (Cyprinus carpio)[J]. Hereditas (Beijing), 33(3): 262-269.) [12] 张春丹, 胡玉珍, 苗亮, 等. 2014. 网箱养殖大黄鱼越冬期间脂肪酸的相对含量变化[J]. 海洋学研究, 32(2): 80-84. (Zhang C, Hu Y, Miao L, et al.2014. Changes of fatty acid composition and content in Pseudosciaena crocea from net cage during the winter[J]. Journal of Marine Sciences, 32(2): 80-84.) [13] 张晓丽, 胡玉珍, 李明云, 等. 2013. 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, 27(11): 27-34. (Zhang X L, Hu Y Z, Li M Y, et al.2013. Changes in antioxidant level and serum enzyme activity of farmed large yellow croaker during natural water cooling in winter[J]. Marine Sciences, 37(11): 27-34.) [14] Bai Y, McCoy J G, Levin E J H, et al.2015. X-ray structure of a mammalian stearoyl-CoA desaturase[J]. Nature, 524: 252-256. [15] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J]. Methods, 25(4): 402-408. [16] Paton C M, Ntambi J M.2009. Biochemical and physiological function of stearoyl-CoA desaturase[J]. American Journal of Physiology-Endocrinology Metabolism, 297: E28-E37. [17] Polley S D, Tiku P E, Trueman R T, et al.2003. Differential expression of cold- and diet-specific genesencoding two carp liver D9-acyl-CoA desaturase isoforms[J]. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 284(1): 41-50. [18] Tamura K, Dudley J, Nei M, et al.2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular Biology and Evolution, 24: 1596-1599. [19] Xu H, Zhang D L, Yu D H, et al.2015. Molecular cloning andexpression analysis of scd1 gene from large yellow croaker Larimichthys crocea under cold stress[J]. Gene, 568(1): 100-108.
