刘杰^1,2, 聂鸿涛^1,2, 姜坤银^1,2, 王政兴^1,2, 孙晓彤^1,2, 霍忠明^1,2, 闫喜武^1,2
1.大连海洋大学 水产与生命学院, 大连 116023;2.大连海洋大学 辽宁省贝类良种繁育工程技术研究中心大连 116023

摘要:
为了解TYR基因与蛤仔免疫的关系，本实验利用荧光定量PCR技术研究了菲律宾蛤仔五个群体（白蛤、白斑马蛤、斑马蛤、养殖和野生群体）的鳃组织和肝胰腺组织在LPS胁迫下TYR基因在不同时间（0 h、3 h、12 h、24 h、48 h）的表达特性。结果表明，在LPS注射后鳃组织中TYR6基因表达水平在白蛤和白斑马蛤3 h、6 h、12 h，野生蛤仔3 h，斑马蛤3 h、6 h，养殖群体6 h、12 h显著上调（P<0.05），3h野生蛤仔和斑马蛤达到峰值，6 h白蛤、白斑马蛤、养殖群体达到峰值（P<0.05）；在肝胰腺中，养殖群体和白斑马蛤6 h，白蛤6 h、24 h，野生群体24 h，斑马蛤3 h显著上调，3 h野生蛤仔和斑马蛤达到峰值，6 h养殖群体、白蛤、白斑马达到峰值（P<0.05）；鳃组织中TYR10基因表达水平在白蛤、野生群体和白斑马蛤3 h，养殖群体3 h、6 h，斑马蛤3 h、12 h显著上调（P<0.05），肝胰腺组织中TYR10基因表达水平在白蛤3 h，野生群体3 h、6 h、12 h，斑马蛤、养殖群体6 h显著上调（P<0.05），推测TYR基因在五个菲律宾蛤仔群体的鳃和肝胰腺中参与了免疫应答。通过对TYR基因的氨基酸序列进行二级结构分析和系统发育树分析，找到两个铜离子结合位点和6个组氨酸残基，并发现TYR6基因和长牡蛎同源性最高，为39.43%，TYR10基因和大珠母贝同源性最高，为51.04%。本文首次探讨在LPS胁迫下菲律宾蛤仔TYR基因的表达特性，为进一步探究TYR基因与菲律宾蛤仔免疫应答机制提供参考。
关键词:菲律宾蛤仔(Ruditapes philippinarum)酪氨酸酶基因(Tryosinase,TYR)基因表达荧光定量PCR
DOI：10.11759/hykx20190715004
分类号:S917.4
基金项目:国家重点研发计划“蓝色粮仓科技创新”重点专项（2018YFD0901400）；国家贝类产业技术体系建设专项（CARS-48）；辽宁省教育厅科研项目（500917201013）；辽宁“百千万人才工程”项目；大连市青年科技之星项目（2016RQ065）

TYR gene expression in gills and hepatopancreas of five populations of Ruditapes philippinarum under LPS Stress
LIU Jie^1,2, NIE Hong-tao^1,2, JIANG Kun-yin^1,2, WANG Zheng-xing^1,2, SUN Xiao-tong^1,2, HUO Zhong-ming^1,2, YAN Xi-wu^1,2
1.College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China;2.Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province, Dalian Ocean University, Dalian 116023, China
Abstract:
Manila clam (Ruditapes philippinarum) is the most commercially utilized mollusk worldwide. It is characterized by a robust immune system. In mollusks, TYR genes play important roles in shell pigmentation, melanin synthesis, and immune system. The present study investigated the relationship between TYR gene and immunity of Manila clam. Five populations of Manila clam (white clam, white zebra clam, zebra clam, cultured, and wild population clam) were exposed to lipopolysaccharide (LPS) stress, and TYR expression in gills and hepatopancreas was studied using quantitative fluorescence polymerase chain reaction at different time points (0h, 3h, 12h, 24h, and 48h). Post LPS injection, TYR6 expression in gills of wild clams and white zebra clams significantly increased at 3h, 6h and 12h, for wild clams at 3h, zebra clams at 3h and 6h, and cultured clams at 6h and 12h (P<0.05). Maximum TYR6 expression was observed at 3h in wild clams and cultured white clams and at 6h in white zebra clams (P<0.05). For hepatopancreas, the expression of TYR6 significantly increased in white zebra clams and cultured clams at 6h, white clams at 6h and 24h, wild clams at 24h, and zebra clams at 3h post LPS exposure. This expression peaked at 3h in wild and zebra clams, and at 6h in cultured clams, white clams, and white zebra clams (P<0.05). TYR10 expression in gills of white clams, white zebra clams, and wild clams significantly increased after LPS injection at 3h, in cultured clams at 3h and 6h, and zebra clams at 3h and 12h (P<0.05). For hepatopancreas, the expression of TYR10 significantly increased in white clam at 3h, wild population at 3h, 6h, 12h, zebra clams and cultured clams at 6h after LPS injection (P<0.05). The gills and hepatopancreas of clams are known to participate in immune response. Further, secondary structure analysis and phylogenetic tree analysis of the amino acid sequence of TYR gene revealed two copper ion binding sites and six histidine residues. TYR6 and TYR10 genes showed highest homology of 39.43%, and 51.04%, respectively, with Crassostrea oyster. Thus, induction of stress in Manila clams was accompanied by the expression of both TYR6 and TYR10 genes suggesting a possible role of TYR genes in the immune response of clams, which should be further explored to understand the underlying mechanism. To our knowledge, this is the first report on TYR gene expression analysis in clams under LPS stress.
Key words:Ruditapes philippinarumTryosinaseGene expressionqRT-PCR





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