摘要
花鲈(Lateolabrax maculatus)作为我国重要的海产经济鱼类,在养殖过程中经常受到高温胁迫的影响,严重制约花鲈养殖产业的发展。已有研究表明,热休克蛋白5(HSPA5)作为关键的应激反应蛋白,在细胞热响应过程中发挥重要功能。为深入研究花鲈HSPA5在高温胁迫下的分子响应机制,本研究首先克隆了花鲈hspa5基因,并采用实时荧光定量PCR(qPCR)技术,分析了在30℃高温条件下不同处理时间对花鲈肝脏中hspa5基因表达的影响。结果显示,随着处理时间的延长,hspa5基因的表达量逐渐上升,并在处理12 h时达到最高水平,随后呈下降趋势,表明hspa5在花鲈肝脏组织应对高温胁迫时具有显著的响应。对正常条件下的花鲈肝脏组织HE染色,初步观察了组织特征和细胞状态。原位杂交结果显示,hspa5 mRNA主要定位于肝细胞胞质中。综上所述,本研究通过qPCR、HE染色和原位杂交技术,揭示了hspa5基因在花鲈肝脏组织响应高温胁迫的表达模式。研究结果不仅有助于深入理解hspa5在花鲈响应高温胁迫时的功能机制,还为花鲈的养殖管理和耐高温性状选育提供了一定的理论依据。
The Chinese spotted sea bass(Lateolabrax maculatus)inhabits the coastal regions of China,Japan,and Korea and ranges from the southern border of Vietnam to the western coast of the Korean Peninsula.This species holds considerable economic significance in China and is highly valued for its nutritional content and taste.Temperature is a crucial environmental factor influencing the biological functions of aquatic animals.Global warming and the widespread practice of intensive aquaculture have rendered high-temperature stress a major challenge for L.maculatus farming.Heat shock proteins(HSPs)are essential in cellular responses to thermal stress.Among them,HSPA5(also known as GRP78 or Bip)is a member of the HSP70 family that plays an essential role in mitigating endoplasmic reticulum stress induced by various stressors,including temperature fluctuations,oxidative stress,and nutrient deprivation.In this study,we elucidated the molecular response mechanism of L.maculatus to high-temperature stress and focused on the role of HSPA5 in liver tissue.We cloned the hspa5 gene from the L.maculatus,performed amino acid sequence and evolutionary analyses,and investigated its expression and localization in liver tissues to clarify its role in heat stress response.To further investigate the properties and functions of HSPA5,we cloned the open reading frame(ORF)of hspa5 using the L.maculatus reference genome and conducted bioinformatic analyses.The ORF of hspa5 was 1965 bp in length and encoded a 654-amino-acid protein.The first 1–16 amino acids formed a signal peptide.The protein was predicted to possess a molecular mass of 72.23 kDa,an isoelectric point of 4.95,and an instability coefficient of 31.01.It was characterized as a stable,hydrophilic protein without transmembrane structural domains.Sequence comparison and conserved domain analysis revealed that L.maculatus HSPA5 exhibited higher homology with bony fish than with mammals or birds and possessed a conserved domain characteristic of the HSP70 superfamily.Structural modeling indicated that the secondary structure of HSPA5 was predominantly composed ofα-helices,accompanied by some irregular coiling.To investigate the response mechanism of hspa5 in L.maculatus under high-temperature stress,we analyzed its expression in liver tissue using real-time quantitative polymerase chain reaction(qPCR)under a 30℃heat stress condition.The fish were acclimated in an environment with temperatures ranging from 15 to 20℃,salinity of 28–30,pH levels of 7.5–8.0,dissolved oxygen>5.0 mg/L,and ammonia nitrogen levels<1.0 mg/L for 2 weeks.The expression of hspa5 mRNA was measured at 0,6,12,24,and 72 h after heat stress exposure.The results showed a gradual increase in hspa5 expression over time,peaking at 12 h,followed by a decline.This indicated the crucial role of hspa5 in the hepatic response to high-temperature stress in L.maculatus.To determine the cellular localization of hspa5 in response to heat stress,we performed in situ hybridization and hematoxylin-eosin(HE)staining of liver tissue sections.In situ hybridization results demonstrated that hspa5 mRNA was predominantly localized in the cytoplasm of hepatocytes and confirmed the active involvement of HSPA5 in the hepatic response to thermal stress.HE staining revealed characteristic liver tissue morphology,including distinct features(e.g.,the central vein and hepatocytes),under both normal and high-temperature conditions.These findings suggest that hepatocytes are likely the primary cell type that responds to high-temperature stress and serves a protective function.Under heat stress,hepatocytes may enhance their cellular tolerance to heat injury by upregulating hspa5 expression.This increased expression may aid in alleviating liver damage induced by high-temperature stress and promote liver tissue repair.In summary,this study elucidated the expression pattern of the hspa5 gene in L.maculatus liver tissue under high-temperature stress using real-time qPCR,HE staining,and in situ hybridization.The findings enhance our understanding of the functional role of hspa5 in heat stress responses and offer a theoretical basis for the management,selection,and breeding of high-temperature-tolerant L.maculatus strains.
作者
刘小强
冯春晖
温海深
李昀
姜天宇
齐鑫
LIU Xiaoqiang;FENG Chunhui;WEN Haishen;LI Yun;JIANG Tianyu;QI Xin(Key Laboratory of Mariculture,Ministry of Education,Ocean University of China,Qingdao 266003,China)
出处
《渔业科学进展》
北大核心
2025年第4期118-128,共11页
Progress in Fishery Sciences
基金
国家重点研发计划“海洋农业与淡水渔业科技创新”重点专项“重要水产养殖生物种质资源挖掘和创新利用”项目课题三“海水鱼种质资源体系化保存与创制”(2022YFD24005103)资助。
关键词
花鲈
高温胁迫
热休克蛋白5
荧光定量PCR
原位杂交
Lateolabrax maculatus
Heat stress
hspa5
Quantitative real-time PCR
in situ hybridization
