Regulation of tyrosine hydroxylase (TH) on physiological and immunological responses in Litopenaeus vannamei

• Main Authors: Ratchaneegorn Mapanao, 馬彩妮
• Other Authors: Winton Cheng
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/9chmsv

博士 === 國立屏東科技大學 === 熱帶農業暨國際合作系 === 105 === Tyrosine hydroxylase belongs to the biopterin-dependent aromatic amino acid hydroxylase enzyme family, is the rate-limiting step in synthesis of catecholamines (CAs) that are required for physiological and immune process in invertebrates and vertebrates under stressful conditions. The aim of this study is to clone the full length cDNA of TH gene in the pacific whiteleg shrimp, Litopenaeus vannamei (abbreviated LvTH) by 3´ and 5´ rapid amplification of cDNA ends (RACE) methods, investigate the LvTH gene expression in various tissues under hypothermal stress as well as the expression of LvTH and TH activity subjected to hypothermal stress and Vibrio alginolyticus challenges. In addition, profile the change in the distribution of LvTH transcripts in haemocytes and tissues in L. vannamei were examined. TH double-stranded (ds) RNA (LvTH-dsRNA) injected L. vannamei was used to examine specifically and effectively suppressed in haemocytes and tissues of shrimp. The RT-PCR and real-time RT-PCR as well as in situ hybridization were used in this study. Furthermore, the relationship between LvTH to catecholamine synthesis, immune response and immune related gene expression were evaluated by using LvTH-dsRNA. In present study, the full-length cDNA of TH gene was cloned from the brain of L. vannamei consists of 1699 bp (GenBank accession no: KU379701). It contained an open reading frame (ORF) of 1500 bp, 54 bp of the 5´-untranslated region (UTR), and 145 bp of the 3´-UTR, including a stop codon (TGA) and a poly A tail. The ORF is predicted to encode a protein of 500 amino acid (aa) with a predicted molecular mass of 57.38 kDa and a theoretical isoelectric point (pl) of 5.95. In addition, containing a short alpha helix domain, a catalytic core, a regulatory domain, a phosphorylation site and two potential N-linked glycosylation sites as presented in vertebrate and insect THs without acidic region and signal peptide cleavage sites at the amino-terminal, exhibited a similarity of 60.0-61.2% and 45.0-47.0% to that of invertebrate and vertebrate THs respectively. LvTH expression was abundant in gill and haemocytes. The expression of LvTH was significantly increased in haemocytes and brain within 30-120 min and 15-30 min respectively, after L.vannamei challenged with Vibrio alginolyticus at 105 cfu shrimp-1. Shrimp exposed to hypothermal stress at 18 °C significantly increased LvTH gene expression in haemocytes and brain within 30-60 and 15-60 min, respectively. The TH activity and haemolymph glucose level (haemocyte-free) significantly increased in pathogen challenged shrimp at 120 min and 60 min, and in hypothermal stressed shrimp at 30-60 min and 30 min, respectively. Using in situ hybridization, the TH positive hybridization signatures were observed in the nervous tissues (brain and circumesophageal connective) and haemocyte (hyaline cells). Shrimp received LvTH-dsRNA at 5 μg/shrimp after 3 days post injection showed significantly decreased LvTH gene expression in nervous system and haemocytes (hyaline cells) determined by real-time PCR as well as TH activity in plasma, and meanwhile, the weaken signals were detectable with in situ hybridization. The depletion of LvTH by using LvTH-dsRNA injected L. vannamei, revealed a significant increase in total haemocyte count (THC), granular cells; semigarnular cells; respiratory bursts (RBs release of superoxide anion); superoxide dismutase (SOD) activity; phagocytic activity and clearance efficiency; and the expression of lipopolysaccharide and β-1,3-glucan-bingding protein (LGBP) and peroxinectin (PE), SOD, crustin, and lysozyme genes. In addition, the reduction of TH gene expression and activity was accompanied by a decline of phenoloxidase (PO) activity per granulocyte, lower glucose and lactate levels and significantly low expression of dopamine-β dydroxilase (DBH) and crustacean hyperglycemic hormone (CHH) expression. The survival ratio of LvTH-silenced shrimp was significantly higher than that of shrimp injected with diethyl pyrocarbonate-water and nontargeting dsRNA when challenged with Vibrio alginolyticus. In conclusion, LvTH is a neural TH enzyme appears to be involved in the physiological and immune response of pacific whiteleg shrimp, L. vannamei suffering stressful stimulation which might be involved in the immune-neuroendocrine network. TH expression may happen in immune haemocytes as mobile-immune-brain during stress stimuli. The depletion of LvTH can enhance disease resistance in shrimp by upregulation specific immune parameters and downregulating the level of carbohydrate metabolites.