| Trichinosis is a worldwide epidemic food-borne zoonotic nematodosis,which not only brings huge economic losses to animal husbandry,but also poses a great threat to human health.The infection is usually caused by the host’s ingestion of the flesh containing the infected larvae.The larvae will cause a variety of tissue and organ damage in the host,and even lead to the death of the host in serious cases.Meat infected with Trichinella spiralis(T.spiralis)is often treated by freezing.However,T.spiralis has a certain tolerance to low-temperature,which will affect the effect of harmless meat treatment to a certain extent.In this study,the trehalose and trehalase of T.spiralis(Ts TRE)under low-temperature stress were studied to explore the role of trehalose and Ts TRE in the mechanism of resistance to low-temperature.In this study,real-time quantitative PCR(q PCR)primers were synthesized according to the Ts TRE gene sequence published in Gen Bank,the survival rate of muscle larvae and the transcriptional level of Ts TRE was detected at 42℃,37℃,25℃,4℃,0℃ and-10℃ to explore the effect of different temperature stress on T.spiralis muscle larvae(ML)and screen out the optimal stress temperature.The results showed that under the same culture time,the survival rate of ML was the highest at 37℃,reaching 98.33%.It decreased gradually with the decrease of culture temperature or the increase of culture time,indicating that both low-temperature and high-temperature were not conducive to the survival of ML in vitro.Under the same culture time,low-temperature stress induced a significant decrease in transcription level of Ts TRE,and the most significant decrease was observed at 4℃(P<0.01).It is inferred that T.spiralis ML can participate in the process of resistance to low-temperature by inhibiting the expression of Ts TRE.And 4℃ can be used as the optimal stress temperature for subsequent experiments.In this study,in order to further verify the relationship between low-temperature stress and Ts TRE and trehalose,transcription and protein levels of Ts TRE at 4℃ were detected by q PCR and Western blot,Ts TRE activity was measured by 3,5-dinitrosalicylic acid assay and trehalose content of the ML was detected by the anthrone colorimetric method.The results showed that 4°C could significantly reduce transcription,protein expression levels and Ts TRE activity,which reached its lowest at 1 h and decreased by about 64.96%,68.89% and 36.98% compared with 37℃control group(P<0.01),and with increasing time of low-temperature stress,Ts TRE transcription and protein levels showed a "W" trend.Ts TRE activity changed little and was always lower than that in 37℃ control group.The change trend of trehalose content was opposite to Ts TRE expression,showing an "M" trend and peaked at 3 h(P<0.01),about 1.6 times that of the 37℃control group.The above results further proved that Ts TRE responded to low-temperature more quickly.The down-regulation of Ts TRE expression level and inhibition of Ts TRE activity further promote the accumulation of trehalose.Ts TRE and trehalose coordinate to keep the body in balance of the T.spiralis ML.The immunofluorescence results showed that Ts TRE is primarily found in the ML’s cuticle,midgut,hindgut,and reproductive primordia.The green fluorescence signal of the low-temperature treatment group was significantly lower when compared to the control group(P<0.01).These results indicate that trehalose was not synthesized in large amounts by T.spiralis muscle larvae in a good growth environment,and trehalase was mainly responsible for decomposing trehalose to provide nutrients for T.spiralis muscle larvae.However,when it was in a stress environment,the trehalase was inhibited and trehalose was rapidly accumulated to play its role in resisting low-temperature stress,which was consistent with the previous results.After the trehalase inhibitor validamycin(J),trehalose(H)and Ts TRE polyclonal antiserum(X)were co-cultured with T.spiralis at 37℃,based on the results of Ts TRE transcription level and Ts TRE enzyme activity,the optimal concentration of the three preparations was 10 m M,10 m M and 2 mg/m L,respectively,and the optimal action time was 24 h.After co-culture with T.spiralis ML under the best conditions of the three preparations,they were subjected to low-temperature at4℃ for 1 h.37℃ was used as the control group(CK),and 4℃ was used as the low-temperature treatment group(L).Under low-temperature stress,the effect of different preparations on the transcription level of Ts TRE gene,protein expression level and trehalose content of T.spiralis ML were also detected.The results showed that compared with the L group,the Ts TRE transcription level and protein expression of the J+L group,H+L group were significantly increased.The Ts TRE transcription level and protein expression of the X+L group were significantly down-regulated compared with that in L group..The trehalose content of T.spiralis ML in J+L group,H+L group and X+L group were significantly higher than that of the L group.Among them,the H+L group had the most significant effect,which was about 1.5 times of the L group(P<0.01).These findings suggest that,validamycin increased the content of trehalose by inhibiting the activity of Ts TRE,and further causing the elevated expression of the Ts TRE;The addition of trehalose directly increased the content of trehalose in ML,and then increased the expression of Ts TRE;the Ts TRE polyclonal antiserum reacted with Ts TRE antigen and antibody to neutralize some Ts TRE,and weakened the ability of the substrate(trehalose of T.spiralis ML),resulting in an increase in trehalose content in T.spiralis ML.In conclusion,trehalose in T.spiralis ML is mainly involved in its resistance to low-temperature,and the mechanism is to inhibit the expression and activity of Ts TRE,and make the T.spiralis can accumulate large amounts of trehalose to protect itself and reduce the damage caused by low-temperature.After T.spiralis ML were placed in 10 m M validamycin,10 m M trehalose and 2 mg/m L Ts TRE polyclonal antiserum,the ML were cultured at 37℃ for 24 h,and then were moved to 4℃for 1 h,3 h,6 h,9 h and 12 h.The survival rate,SOD activity,MDA content and GSH content of ML were detected to explore the regulation of Ts TRE on the antioxidant capacity of T.spiralis ML under different agents.The results showed that during the same culture time,compared with the L group,the survival rate of ML in the J+L group,H+L group and X+L group were significantly increased,and the H+L group had the most significant increase.Low-temperature could significantly increase the SOD activity,MDA content and GSH content of ML;Compared with the L group,the J+L group,H+L group and X+L group can cause the increase of SOD activity and GSH content,and slow down the accumulation of MDA,and with the increase of low-temperature stress time,the effects of H+L group were the most significant.The results showed that Ts TRE is involved in the regulation of increasing trehalose content in ML and favors its survival at low-temperatures.Low-temperature could lead to accumulation of ROS in ML,resulting in membrane lipid peroxidation,and the accumulation of MDA causing oxidative damage.Antioxidant enzymes such as SOD and antioxidant substances such as GSH could play their roles to reduce the oxidative damage.Under the action of Ts TRE,the high concentration of trehalose in T.spiralis can further increase the activity of key enzymes in its antioxidant system and the content of antioxidant substances to improve its antioxidant capacity.In summary,this study demonstrated that low-temperature stress inhibited the expression and activity of Ts TRE,and Ts TRE was involved in low-temperature resistance by increasing the trehalose content in T.spiralis ML.The high concentration of trehalose in T.spiralis ML can improve the antioxidant capacity of T.spiralis by protecting the activities of related enzymes,and reduce the oxidative damage caused by low-temperature.This study provides a basic theoretical basis for further exploring the molecular mechanism of low-temperature resistance of T.spiralis. |
