The Function And Mechanism Of Aj-catalase In The Regulation Of Intestinal Flora Homeostasis In Sea Cucumber

The sea cucumber is an economically aquaculture species in the coastal area of China.In recent years,due to the intensive farming of sea cucumber,the farm environment is populated which caused the serious diseases.Therefore,it is important to understand the immune defense mechanism of sea cucumber.It knows that the intestinal tissue plays an important role not only in absorbing nutrients,but also in the process of defense against pathogenic bacteria.And catalase is an important antioxidant enzyme in the body.It demonstrated that the catalase played an important role in the maintenance of body intestinal homeostasis redox balance in intestinal immune responses.For these reasons,we will study the regulation of intestinal immunity by Apostichopus japonicas catalase(Aj-CAT)and its related mechanisms.This study found that Aj-CAT showed obvious abnormal expression in four tissues(intestine,respiratory tree,body wall,antennae)at the transcriptional level.The results showed that Aj-CAT participates in the immune function of sea cucumber.Because the intestine is an important immune organ of the body,the experiment will mainly study the important role of Aj-CAT in intestinal immunity of sea cucumber.Firstly,the model of oral injection of pathogenic bacteria was established,and FITC was used to label Vibrio splendidus to determine the distribution and retention time of pathogenic bacteria in intestine.The results showed that Vibrio splendidus distributed in the intestine of sea cucumber within 3hours after oral injection,and its retention time was short.After 6 hours,the intestinal Vibrio splendidus disappeared almost completely.Subsequently,the expression of Aj-CAT in the intestinal tract of sea cucumber was detected.The results showed that Aj-CAT showed significant up-regulation at both the transcriptional level and the enzyme activity level under the stimulation of Vibrio splendidus.Secondly,Aj-CAT was silenced in vivo using RNAi technology.It was found that the survival rate of sea cucumber under the stimulation of pathogenic bacteria was significantly reduced when Aj-CAT was silenced.It demonstrated that the immune function of Aj-CAT in sea cucumber,and play an important role in intestinal immune responses of sea cucumber.The experiment continued to explore the regulatory mechanism of Aj-CAT on intestinal immunity of sea cucumber.The experiment used 16 s RNA sequencing to detect changes in the intestinal flora of the Aj-CAT gene silenced group compared with the control groups injected of the DEPC or de RNA-GFP.It was found that after the Aj-CAT gene was silenced,the number and structure of the intestinal flora changed significantly.At the same time,after Aj-CAT was silenced,the pathogenic bacteria in the intestine increased significantly at the generic level.The experiment continued to detect the retention of the sea cucumber in the normal and Aj-CAT silent sea cucumber after exogenous injection of Vibrio splendidus and Pseudomonas.The result indicated that the abundance of these two pathogenic bacteria in the intestine increased significantly after Aj-CAT was silenced.The above results indicated that Aj-CAT played an important role in the steady-state balance of intestinal flora of sea cucumber.Because Aj-CAT can regulate the ROS level of the organism,this study further explored the regulatory role of Aj-CAT in the intestinal ROS of sea cucumber.After Aj-CAT was silenced,tissue slices showed that the folds in intestinal layer of sea cucumber were significantly reduced,compared with the normal sea cucumbers,and the ROS levels in the intestines increased significantly.Therefore,it found that Aj-CAT can affect the intestinal structure and the homeostasis of intestinal flora by regulating the level of ROS in intestinal tract.Because intestinal flora plays an important role in the process of intestinal immune defense,which destroys the status of intestinal flora,the intestinal immune mechanism of sea cucumber is affected.Therefore,the results of this experiment show that Aj-CAT can regulate the level of ROS in intestinal tract of sea cucumber Changing the structure of intestinal tract and the homeostasis of bacterial flora of sea cucumber eventually participates in the regulation of intestinal immunity of sea cucumber.