碳酸盐碱度胁迫下凡纳滨对虾基因的差异表达

以广盐性养殖的凡纳滨对虾(Litopenaeus vannamei)为研究对象,采用抑制消减杂交(suppression subtractive hybridization,SSH)和定量PCR的方法,研究其在高碳酸盐碱度胁迫下转录组水平的基因表达差异,以期从基因组水平研究对虾对高碳酸盐碱度胁迫的适应机制。结果表明,以高碳酸盐碱度(20 mmol/L)胁迫第4天凡纳滨对虾鳃组织和低碱度(2 mmol/L)对照组鳃组织为材料,通过斑点杂交筛选,发现鳃组织中有158个克隆子表达上调,291个克隆子表达下调。挑选150个高表达差异的克隆子进行测序,获得100个序列,其中50个得到成功注释。经过GO分析,这些注释的差异基因主要分为8大类群,其中碳酸酐酶基因(CA)、Na+-K+-ATPase基因(NKA-α)等与离子调控相关的基因表达量上调,而溶菌酶基因等与先天免疫相关的基因表达量下调,这些结果表明高碳酸盐碱度胁迫下,凡纳滨对虾以增加离子调控的方式进行酸碱平衡的调控,同时其免疫功能受到抑制。此外,还对CA和NKA-α两个基因在鳃和触角腺中的时空表达规律进行了研究,发现高碳酸盐碱度胁迫9 d过程中,两个基因在鳃组织中的表达均呈现先高表达后回落的现象,而在触角腺中NKA-α基因则一直维持较高表达水平,说明CA和NKA-α基因在凡纳滨对虾高碳酸盐碱度适应离子调控中起着关键作用,同时还发现除了鳃组织之外,触角腺同样参与了调控。本研究从转录水平初步筛选了高碳酸盐碱度胁迫下凡纳滨对虾的表达差异基因,探索了凡纳滨对虾的耐盐碱机制,对培育适于盐碱地水产养殖的优良品种有着重要的意义。

Gene expression profiles of Litopenaeus vannamei in response to carbonate alkalinity stress

Saline-alkali water-bodies are common in China.Alkalinity stress is considered to be one of the primary stressors for shrimp in saline-alkali water.Thus,an improved understanding of the molecular response to alkalinity stress is critical for advancing the sustainability of shrimp culture.Our objective was to evaluate the effect of car-bonate alkalinity on global gene expression in Litopenaeus vannamei,a species of shrimp that is cultured throughout the world.We constructed two subtractive cDNA libraries from the gills of shrimp that were exposed to either 20 mmol/L alkalinity water or control water for 4 days.Dot blot expression analysis revealed that 158 clones were up-regulated and 291 clones were down-regulated following exposure to alkalinity stress.These clones were subsequently sequenced and up to 100 genes were identified from the forward and reverse libraries,of which 50 were well annotated.These differentially expressed genes were divided into a number of biological gene ontology groups related to catalytic activity,cell,structural molecule activity,binding,transporter activity,repro-duction,enzyme regulator activity,and cellular process.Ion transportation genes,such as carbonic anhydrase(CA) and Na+-K+-ATPase(NKA-α),were up-regulated while immune response genes(e.g.,lysozyme) were down-regulated.We evaluated expression of two differentially expressed genes(CA and NKA-α) in the gills and antennal gland of shrimp prior to exposure and following exposure to 20 mmol/L carbonate alkalinity water for 1–9 d.Exposure to carbonate alkalinity resulted in an increase in CA mRNA and NKA-α mRNA expression in the gills and antennal gland.The majority of the increase occurred on day 1.Our results suggest that expression of carbonic anhydrase and Na+-K+-ATPase genes plays an important role in the response to alkalinity stress in L.vannamei,particularly in the gill and antennal gland.To our knowledge,this is the first study to use shrimp SSH cDNA libraries to detect global gene expression alterations in response to alkalinity stress.Alkalinity stress stimulated ion regulated processes and slowed down the gene expression related to immune system and reproduc-tion in L.vannamei.The alkalinity-regulated genes characterized in the present study may be convenient begin-ning points to study the molecular basis of alkalinity adaptation.The physiological role of these genes in envi-ronmental adaptation remains to be explored.Understanding how alkalinity triggers regulation of gene expression deserves further attention.