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| Cloning of the Trypsin Gene in Larimichthys polyactis and Its Response to High Temperature Stress and Pseudomonas plecoglossicida Infection |
| LIU Hao-Wen1, LIU Feng2,*, ZHANG Tian-Le2, LI Qian3, LIU Si-Fang1, ZHU Jia-Jie4, LOU Bao2,*, YU Xiao-Ping1 |
1 College of Life Sciences, China Jiliang University, Hangzhou 310018, China;
2 Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences / Zhejiang Key Laboratory of Coastal Biological Germplasm Resources Conservation and Utilization, Hangzhou 310021, China;
3 National Engineering Research Center for Marine Aquaculture, Zhejiang Ocean University, Zhoushan 316022, China;
4 School of Marine Sciences, Ningbo University, Ningbo 315832, China |
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Abstract The frequent occurrence of summer heatwaves and visceral white-nodules disease caused by Pseudomonas plecoglossicida has emerged as critical constraints on the sustainable development of small yellow croaker (Larimichthys polyactis) aquaculture. Notably, trypsin, a key enzyme in amino acid metabolism, may play a pivotal role in mediating the physiological responses of small yellow croaker to both thermal stress and bacterial infection. To investigate the response characteristics of the trypsin gene in L. polyactis under high temperature stress and P. plecoglossicida infection, the CDS sequence of the trypsin gene (Lp_try) in L. polyactis was cloned in this study. The sequence of Lp_try spaned 744 bp, encoding 247 amino acids and containing the conserved Tryp_SPc domain. Expression analysis revealed that the Lp_try gene showed pan-tissue expression characteristics, but it showed a specific high expression pattern in the digestive system (intestine, liver) and circulatory system (heart). The expression characteristics of Lp_try gene in the liver of L. polyactis subjected to 32 ℃ high temperature stress and P. plecoglossicida infection were analyzed. The results showed that high temperature stress initially induced a significant increase of Lp_try expression (P<0.05), followed by a swift decreased with prolonged exposure. Additionally, during P. plecoglossicida infection, Lp_try expression exhibited a dynamic trend of decrease-increase-decrease over time, suggesting its regulatory role in the processes of L. polyactis in response to both high temperature stress and bacterial infection. This study provides an important foundation for further understanding of the physiological regulatory mechanisms in fish in response to high temperature stress and pathogen infection.
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Received: 21 August 2024
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Corresponding Authors:
*lengfeng0210@126.com; loubao6577@163.com
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[1] 陈亮, 梁旭方, 王琳, 等. 2009. 鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析[J]. 中国生物化学与分子生物学报, 25(12): 1115-1123.
(Chen L, Liang X F, Wang L, et al.2009. Cloning and sequence analysis of trypsin, amylase and pepsinogen genes in Siniperca chuatsi[J]. Chinese Journal of Biochemistry and Molecular Biology, 25(12): 1115-1123.)
[2] 陈睿毅, 楼宝, 詹炜, 等. 2016. 小黄鱼亲鱼驯养及苗种繁育[J]. 科学养鱼, (01): 42-43.
(Chen R Y, Lou B, Zhan W, et al. 2016. Domestication and seedling breeding of small yellow croaker[J]. Scientific Fish Farming, (01): 42-43.)
[3] 陈文波, 李卫国, 张珍, 等. 2013. 斑马鱼3种胰蛋白酶原基因的克隆、序列分析及组织表达[J]. 中山大学学报(自然科学版), 52(01): 111-117.
(Chen W B, Li W G, Zhang Z, et al.2013. Molecular cloning, sequence analysis and tissue expression of three forms trypsinogens in zebrafish, Danio rerio[J]. Journal of Sun Yat-sen University (Natural Science), 52(01): 111-117.)
[4] 储天琪, 刘峰, 陈红林, 等. 2022. 小黄鱼肝脏中hsp90b1和hspb1对温度胁迫的响应[J]. 农业生物技术学报, 30(03): 528-538.
(Chu T Q, Liu F, Chen H L, et al.2022. Response of hsp90b1 and hspb1 to temperature stress in the liver of Larimichthys polyactis[J]. Journal of Agricultural Biotechnology, 30(03): 528-538.)
[5] 崔晓莹. 2019. 大黄鱼感染变形假单胞菌带菌量测定及转录组分析[D]. 硕士学位论文, 集美大学, 导师: 王志勇, pp. 9-51.
(Cui X Y.2019. Determination of the pathogen load after Pseudomonas plecoglossicida infection and transcriptomic analysis in Larimichthys crocea[D]. Thesis for M.S., Jimei University, Supervisor: Wang Z Y, pp. 9-51.)
[6] 杜翠红, 苏文金, 卢宝驹, 等. 2011. 鳜鱼(Siniperca chuatsi)幽门垂中两种胰蛋白酶的cDNA克隆及其生物信息学分析[J]. 海洋与湖沼, 42(02): 221-228.
(Du C H, Su W J, Lu B J, et al.2011. Cloning and bioinformatic analysis of two trypsin genes from mandarin fish Siniperca chuatsi[J]. Oceanologia et Limnologia Sinica, 42(02): 221-228.)
[7] 冯文荣. 2011. 文昌鱼胰蛋白酶基因的鉴定、表达和功能研究[D]. 硕士学位论文, 中国海洋大学, 导师: 张士璀, pp. 19-71.
(Feng W R.2011. Characterization, expression and functional analysis of trypsin gene in amphioxus Branchiostoma japonicum[D]. Thesis for M.S., Ocean University of China, Supervisor: Zhang S G, pp. 19-71.)
[8] 何雨, 刘峰, 张天乐, 等. 2024. 高温胁迫对小黄鱼肝脏组织结构和细胞凋亡的影响[J]. 浙江农业学报, 36(01): 58-66.
(He Y, Liu F, Zhang T L, et al.2024. Histological study on the effect of high temperature stress on liver tissue in Larimichthys polyactis[J]. Acta Agriculture Zhejiangensis, 36(01): 58-66.)
[9] 胡永乐, 梁旭方, 王琳, 等. 2010. 斜带石斑鱼胰蛋白酶原和淀粉酶全长cDNA的克隆与序列分析[J]. 热带海洋学报, 29(05): 125-131.
(Hu Y L, Liang X F, Wang L, et al.2010. Molecular cloning and sequence analysis of pancreatic trypsinogen and amylase from orange-spotted grouper (Epinephelus coioides)[J]. Journal of Tropical Oceanography, 29(05): 125-131.)
[10] 黎军胜, 李建林, 吴婷婷. 2006. 罗非鱼胰蛋白酶Ⅰ mRNA克隆与分析[J]. 水生生物学报, 30: 209-213.
(Li J S, Li J L, Wu T T.2006. Cloning and analysis of trypsinⅠmRNA in tilapia[J]. Acta Hydrobiologica Sinca, 30: 209-213.)
[11] 凌统. 2007. 中华倒刺鲃胰蛋白酶的cDNA克隆与序列分析[D]. 硕士学位论文, 西南大学, 导师: 李英文, pp. 9-27.
(Ling T.2007. Cloning and sequence analysis of trypsin cDNA in Spinibarbus chinensis[D]. Thesis for M.S., Southwest University, Supervisor: Li Y W, pp. 9-27.)
[12] 刘峰, 刘阳阳, 楼宝, 等. 2016. 温度对小黄鱼体内抗氧化酶及消化酶活性的影响[J]. 海洋学报, 38(12): 76-85.
(Liu F, Liu Y Y, Lou B, et al.2016. Effect of water temperature on antioxidant and digestive enzymes activities in Larimichthys polyactis[J]. Haiyang Xuebao, 38(12): 76-85.)
[13] 邱杨玉, 郑磊, 毛芝娟, 等. 2012. 大黄鱼(Larimichthys crocea)内脏白点病的病原分离和组织病理学观察[J]. 微生物学通报, 39(03): 361-370.
(Qiu Y Y, Zheng L, Mao Z J, et al.2012. Isolation and identification of the causative agent and histopathology observation of white-spots disease in internal organs of Larimichthys crocea[J]. Microbiology China, 39(03): 361-370.)
[14] 饶秋华, 刘洋, 黎露, 等. 2022. 大黄鱼内脏白点病的病原分离鉴定及致病性[J]. 福建农林大学学报(自然科学版), 51(06): 800-808.
(Rao Q H, Liu Y, Li L, et al.2022. Isolation, identification and pathogenicity of the pathogen of visceral white spot disease of Larimichthys crocea[J]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 51(06): 800-808.)
[15] 杨莉, 欧都, 齐亚银, 等. 2015. 寒冷应激对新疆阿勒泰羊脂肪酸合成酶mRNA表达的影响[J]. 江苏农业科学, 43(10): 28-30.
(Yang L, Ou D, Qi Y Y, et al.2015. Effect of cold stress on mRNA expression of fatty acid synthase in Xinjiang Altay sheep[J]. Jiangsu Agricultural Sciences, 43(10): 28-30.
[16] 郑凯迪, 冯波, 李云, 等. 2012. 胭脂鱼胰蛋白酶cDNA克隆以及不同蛋白含量日粮和饥饿对mRNA表达和酶活力的影响[J]. 水生生物学报, 36(01): 9-17.
(Zhen K D, Feng B, Li Y, et al.2012. Molecular cloning of trypsin and the effect of dietary activity in juvenile Chinese sucker (Myxocyprinus asiaticus Bleeker)[J]. Acta Hydrobiologica Sinca, 36(01): 9-17.)
[17] Aguilar A, Mattos H, Carnicero B, et al.2022. Metabolomic profiling reveals changes in amino acid and energy metabolism pathways in liver, intestine and brain of zebrafish exposed to different thermal conditions[J]. Frontiers in Marine Science, 9: 835379.
[18] Andersen S M, Rune W, Marit E.2016. Functional amino acids in fish nutrition, health and welfare[J]. Frontiers in Bioscience (Elite edition), 8(1): 143-169.
[19] Bagath M, Krishnan G, Devaraj C, et al.2019. The impact of heat stress on the immune system in dairy cattle: A review[J]. Research in Veterinary Science, 126: 94-102.
[20] Bai Y L, Qu A, Liu Y, et al.2022. Integrative analysis of GWAS and transcriptome reveals p53 signaling pathway mediates resistance to visceral white-nodules disease in large yellow croaker[J]. Fish & Shellfish Immunology, 130: 350-358.
[21] Belhadj I S, Taha N, Abdeljelil G, et al.2014. Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage. A review[J]. International Journal of Hyperthermia, 30(7): 513-523.
[22] Besson M, Vandeputte M, Arendonk J V, et al., 2016. Influence of water temperature on the economic value of growth rate in fish farming: The case of sea bass (Dicentrarchus labrax) cage farming in the Mediterranean[J]. Aquaculture, 462: 47-55.
[23] Chen L H, Zeng W H, Rong Y Z, et al.2022. Nutritional composition and textural quality of wild-caught and cage-cultured small yellow croaker (Larimichthys polyactis)[J]. Journal of Food Composition and Analysis, 107(10):104370.
[24] Cheng T, Chen J D, Dong X D, et al.2023. Protective effect of steroidal saponins on heat stress in the liver of largemouth bass (Micropterus salmoides) revealed by metabolomic analysis[J]. Aquaculture Reports, 33: 2352-5134.
[25] Douglas S E, Gallant J W.1998. Isolation of cDNAs for trypsinogen from the winter flounder, Pleuronectes americanus[J]. Marine Biotechnology, 6: 214-219.
[26] Gudmundsdóttir A, Gudmundsdóttir E, Oskarsson S J, et al.1993. Isolation and characterization of cDNAs from Atlantic cod encoding two different forms of trypsinogen[J]. Eurpean Journal of Biochemistry, 217: 1091-1097.
[27] Hegyi E, Sahin-Tóth M.2017. Genetic risk in chronic pancreatitis: The trypsin-dependent pathway[J]. Digestive Diseases and Sciences, 62(7): 1692-1701.
[28] Holecek M.2015. Ammonia and amino acid profiles in liver cirrhosis: Effects of variables leading to hepatic encephalopathy[J]. Nutrition, 31(1): 14-20.
[29] Jia Z, Wang M, Zhang H, et al.2018. Identification of a clip domain serine proteinase involved in immune defense in Chinese mitten crab (Eriocheir sinensis)[J]. Fish and Shellfish Immunology, 74: 332-340.
[30] Jiang J, Shi D, Zhou X Q, et al.2015. In vitro and in vivo protective effect of arginine against lipopolysaccharide induced inflammatory response in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian)[J]. Fish and Shellfish Immunology, 42(2): 457-464.
[31] Klomklao S, Benjakul S, Visessanguan W, et al.2006. Trypsins from yellowfin tuna (Thunnus albacores) spleen: Purification and characterization[J]. Comparative Biochemistry and Physiology, Part B, 144(1): 47-56.
[32] Koshikawa N, Hasegawa S, Nagashima Y, et al.1998. Expression of trypsin by epithelial cells of various tissues, leukocytes, and neurons in human and mouse[J]. The American Journal of Pathology, 153(3), 937-944.
[33] Leon L R, Bouchama A.2015. Heat stroke[J]. Comprehensive Physiology, 5: 611-647.
[34] Li C W, Wang S L, Ren Q L, et al.2020. An outbreak of visceral white nodules disease caused by Pseudomonas plecoglossicida at a water temperature of 12 ℃ in cultured large yellow croaker (Larimichthys crocea) in China[J]. Journal of Fish Diseases, 43(11): 1353-1361.
[35] Li S W, Liu Y J, Li B L, et al.2022. Physiological responses to heat stress in the liver of rainbow trout (Oncorhynchus mykiss) revealed by UPLC-QTOF-MS metabolomics and biochemical assays[J]. Ecotoxicology and Environmental Safety, 242: 113949.
[36] Ling Z N, Jiang Y F, Ru J N, et al.2023. Amino acid metabolism in health and disease[J]. Signal Transduction and Targeted Therapy, 8(1): 345.
[37] Liu F, Zhang T L, He Y, et al., 2023. Integration of transcriptome and proteome analyses reveals the regulation mechanisms of Larimichthys polyactis liver exposed to heat stress[J]. Fish and Shellfish Immunology, 135: 108704.
[38] Olsvik P A, Vikeså V, Lie K K, et al.2013. Transcriptional responses to temperature and low oxygen stress in Atlantic salmon studied with next-generation sequencing technology[J]. BMC Genomics, 14(1): 817.
[39] Page M J, Di Cerae E.2008. Serine peptidases: Classification, structure and function[J]. Cellular and Molecular Life Sciences, 65(7-8): 1220-1236.
[40] Qian B, Xue L.2016. Liver transcriptome sequencing and de novo annotation of the large yellow croaker (Larimichthy crocea) under heat and cold stress[J]. Marine Genomics, 25: 95-102.
[41] Ruan G L, Li Y, Gao Z X, et al.2010. Molecular characterization of trypsinogens and development of trypsinogen gene expression and tryptic activities in grass carp (Ctenopharyngodon idellus) and topmouth culter (Culter alburnus)[J]. Comparative Biochemistry and Physiology part B, 155: 75-85.
[42] Sendler M, Lerch M M.2020. The complex role of trypsin in Pancreatitis[J]. Gastroenterology, 158(4): 822-826.
[43] Shah D, Mital K.2018. The role of trypsin: Chymotrypsin in tissue repair[J]. Advances in Therapy, 35(1): 31-42.
[44] Suzuki T, Srivastava A S, Kurokawa T.2002. cDNA cloning and phylogenetic analysis of pancreatic serine protease from Japanese flounder, Paralichthys olavaceus[J]. Comparative Biochemistry and Physiology part B, 131: 63-70.
[45] Wang B, Feng L, Jiang W D, et al.2015. Copper-induced tight junction mRNA expression changes, apoptosis and antioxidant responses via NF-κB, TOR and Nrf2 signaling molecules in the gills of fish: Preventive role of arginine[J]. Aquatic Toxicology, 158: 125-137.
[46] West N E, Qian H S, Guzik T J, et al.2001. Nitric oxide synthase (nNOS) gene transfer modifies venous bypass graft remodeling: Effects on vascular smooth muscle cell differentiation and superoxide production[J]. Circulation, 104(13): 1526-1532.
[47] Wu D D, Wang G D, Irwin D M, et al.2009. A profound role for the expansion of trypsin-like serine protease family in the evolution of hematophagy in mosquito[J]. Molecular Biology and Evolution, 26(10): 2333-2341.
[48] Xiong L, Teng J L, Botelho M G, et al.2016. Arginine metabolism in bacterial pathogenesis and cancer therapy[J]. International Journal of Molecular Sciences, 17(3): 363.
[49] Xu A L, ShangGuan J B, Li Z B, et al.2023. Effects of dietary vitamin E on the growth performance, immunity and digestion of Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂ by physiology, pathology and RNA-seq[J]. Aquaculture, 575: 739752.
[50] Zeng X, Jin S, Jiang J, et al.2017. Predict the relationship between gene and large yellow croaker's economic traits[J]. Molecules, 22(11): 1978.
[51] Zhao Y, Yan M Y, Jiang Q, et al.2021. Isoleucine improved growth performance, and intestinal immunological and physical barrier function of hybrid catfish Pelteobagrus vachelli×Leiocassis longirostris[J]. Fish and Shellfish Immunology, 109: 20-33. |
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