| In China,the sea cucumber Apostichopus japonicus(Slenka)is one of the most important aquaculture species in coastal regions with high nutritional value and economic value.However,diseases,such as skin ulceration disease,have posed a serious threat to the sea cucumber of all ages during the past decade.Therefore,it has become the focus and hot-spot of the current sea cucumber industry to research disease resistance mechanism and effective prevention and treatment deeply.In this study,A.japonicus was used as the research object.The cDNA of TRX-like protein gene from A.japonicus(denoted as AjTRX)was cloned by RACE technology.Spatial and temporal expression of AjTRX gene were analyzed by quantitative real-time PCR,and the sea cucumber were challenged by Vibrio splendidus.In addition,recombinant protein of AjTRX gene was expressed and purified by technology of genetic engineering,and its activity also was verified.Finally,the relationship between reactive oxygen species(ROS)and AjTRX gene was figured out using RNA interference.The result are listed as below:1.The cDNA sequence of AjTRX gene was firstly identified by transcriptome sequencing and RACE approaches.The full-length cDNA of AjTRX gene was of 1870 bp,consisting of a 5’-UTR of 101 bp,a long 3’-UTR of 887 bp and a 882 bp open reading frame(ORF)encoding a 293 amino acids.The predicted molecular mass and the theoretical PI of the deduced amino acids of AjTRX gene were 32.3 kDa and 5.52,respectively.Phylogenetic trees showed that AjTRX had a closer evolution relationship with TRX from Strongylocentrotus purpuratus.AjTRX gene was found to be ubiquitously expressed in all examined tissues including longitudinal muscle,coelomocytes,tube feet,intestine,respiratory tree and body wall indicating a general role in physiological processes.Temporal expression pattern of AjTRX gene in coelomocytes showed that AjTRX reached two peak expression levels at 8 h and 48 h after Vibrio splendidus challenge with 8.6 and 9.3-fold increase compared to their control groups,respectively(P< 0.01).These results indicate that AjTRX gene is involved in the immune response of A.japonicus to V.splendens infection.2.Thioredoxin(Trx)is an crucial member of the redox family.In the present study,the results of SDS-polyacrylamide gel electrophoresis(PAGE)showed that we successfully acquired the over-expressed recombinant protein of AjTRX gene(rAJTRX)in Escherichia coli.What’s more,The rAJTRX displayed obvious antioxidant activity in a dose-dependent manner,and the higher reducing activity was detected in 20 mM experimental group.These results indicate that rAJTRX may participate in the anti-oxidative reaction of the sea cucumber and show strong reducibility.3.To better understand the biological functions of AjTRX gene,we conducted the RNA interference experiment in coelomocytes.AjTRX silencing could increase intracellular ROS levels by 1.86-fold and 1.62-fold compared to each control group in vitro and in vivo,respectively.These results suggested that AjTRX gene can negatively regulate the production of ROS.All these results observed in this study strongly suggested that AjTRX can act as an antioxidant and play a very important role in the physiological processes of the sea cucumber.At the same time,AjTRX gene also participates in the process of immune response A.japonicus to V.splendens infection and is likely to negatively regulate the production of ROS during the process.These results can provide a reference for elucidating the biological function and innate immunity mechanism of AjTRX gene. |
