Expression Characteristics Of P38MAPK And Its Role In Regulating Apoptosis Under High Temperature Stress In Sinonovacula Constricta

The razor clam(Sinonovacula constricta)is one of the economically important cultured shellfish in China,mainly distributed in low and medium tide areas along the coast.In recent years,global warming and rising temperatures may weaken the defense mechanisms of marine animals.As a typical mudflat shellfish,S.constricta could show corresponding stress for high temperature.The mechanism of apoptosis induced by high temperature involves the mitogen-activated protein kinase p38(p38MAPK)pathway.Few studies have been conducted to address the role of p38 MAPK in shellfish.In this study,we analyzed the oxidative stress response,apoptosis and tissue damage in gill and hepatopancreas tissues of razor clams under high temperature stress.The sequences of the apoptotic gene p38 MAPK from razor clam were identified and its expression characteristics were analyzed by relevant molecular biology techniques.RNAi technology was used to study the effect of inhibition of p38 MAPK gene on pathway genes and tissue apoptosis under high temperature stress.1.The effects of high temperature stress at 28°C,30°C,32°C and 34°C on the ROS content and the degree of tissue apoptosis and damage in razor clams were investigated.The content of reactive oxygen species(ROS)in gill and hepatopancreas tissues was measured using ELISA kits.The results showed that the ROS content significantly increased,and all of them were highly significant at 6 h under high temperature.The highest ROS content was found at 34°C.Apoptosis in gill and hepatopancreas tissues was observed by TUNEL staining after 72 h of high temperature.It was found that only sporadic fluorescence signals showed in gill and hepatopancreas tissues at 28℃.With the increase of temperature,the green fluorescence signals gradually increased and the degree of apoptosis gradually increased at 30℃,32℃ and 34℃,and the degree of apoptosis was the greatest at 34℃.Morphological tissue observation was performed on gills and hepatopancreas.The results of the study showed that the structure of the gill tissue was intact at 28°C.The gill lumen in the gill tissue gradually became larger as the temperature increased,and the gill lumen was largest at 34°C.The morphology of the hepatopancreas was intact at 28°C.The morphology of the lumen of the hepatic tubules began to be extruded at 30°C,and the morphology of the basement membrane was incomplete.The degree of tissue damage was most severe at 34°C.The results of the study indicated that high temperature stimulated oxidative stress in razor clams,induced tissue apoptosis and caused tissue damage.The organism begins to respond to high temperatures at 30°C,with the strongest response at 34°C.2.One p38 MAPK sequence was obtained from the razor clam.The ORF was 1080 bp encoding 359 amino acids.The predicted molecular weight(MW)was 41.54 k Da,and the theoretical isoelectric point(p I)was 5.39.The p38 MAPK was a non-transmembrane protein with no peptide signal.Amino acid composition analysis showed that the protein had 16 serine phosphorylation sites(S),14 threonine phosphorylation sites(T),and 6 tyrosine phosphorylation sites(Y).Tertiary structure prediction revealed that the protein was comprised of 40% alpha helix,15% beta strand,4% TM helix,and 13% disordered.The relative expression of p38 MAPK in different tissues was detected by q RT-PCR.The p38 MAPK was expressed in all tested tissues.In addition,the p38 MAPK was elevated to different degrees in each tissue under 34℃ for24 h.The expression of p38 MAPK in gill and hepatopancreas tissues was significantly increased,reaching 2.81 and 8.83 times as the control group,respectively(P < 0.01).After high temperature stress,the transcript expression of p38 MAPK in gill tissue increased significantly at 12 h,24 h and 72 h.The expression was generally increased in hepatopancreas tissues,and peaked at 28℃ for 24 h,30℃,32℃ for 6 h and 34℃ for 12 h,respectively.3.RNA interference technology was used to further investigate the regulatory role of p38 MAPK gene under high temperature stress.By screening the effective interference strand and the effective interference concentration of p38 MAPK gene,dsRNA-a was identified as the most suitable interference strand for this experiment,in which the interference concentration was 500 ng/μl and the interference rate was 77%.The dsRNA injection technique was used to carry out in vivo interference experiments and high temperature stress experiments.The results showed that the downstream apoptosis suppressor gene Bcl-2 increased and the pro-apoptosis gene Bax decreased highly significantly in the gill and hepatopancreas tissues.The ratio of Bcl-2 and Bax increased.In addition,ROS content was highly significantly decreased.TUNEL staining was used to detect apoptosis before and after interference.It was found that the apoptotic signals in gill and hepatopancreas tissues were reduced to a lesser extent after p38 MAPK interference.The results showed that inhibition of p38 MAPK gene can reduce ROS content and apoptosis degree by activating p38MAPK-Bcl-2/Bax dependent apoptosis pathway.