| Marsupenaeus japonicus is one of the most valuable shrimps in the coastal area of China.As two important factors of environmental stress,low salinity and high nitrite nitrogen are closely related to the growth,survival and immune regulation of shrimp.Study on the molecular mechanism of M.japonicus under low salinity and high nitrite stress,which can provide an important theoretical basis for the healthy culture of M.japonicus.In this study,we analyzed the transcriptome of M.japonicus under low salinity and high nitrite nitrogen stress,and coloned a trehalose 6-phosphate synthetase(TPS)gene which was closely related to stress resistance,and studied its stress resistance and immune function by RNA interference technology.The main results are as follows:(1)Transcriptome analysis of M.japonicus under low salinity stress: The hepatopancreas of M.japonicus under low-salinity stress of 8 salinity for 6,12,24,48 and 96 hours and the control groups were analyzed by transcriptome.We obtained 89042532–105130044 Clean Reads with data volume of 6.68G-7.88 G and Q30 of 93.34-94.53%.48807 unigenes and 1746 bp N50 were obtained after Trinity assembly.Compared with the control group,811,589,1095,745 and 875 differential genes(DEGs)were obtained.We annotated DEGs to obtain pathways and genes related to osmoregulation,metabolism and immunity.The expression of 9 randomly selected differential genes(Tau D?Sar DH?CLEC?GNMT?BHMT?MAT2??PPP2C??TAUT ? AGMAT)was verified by q PCR,and the expression was consistent with the trend of RNA-seq.(2)Transcriptome analysis of M.japonicus under high nitrite stress: The hepatopancreas of M.japonicus under high nitrite stress of 80mg/L for 6,12,24,48 and 96 h and the control groups were sequenced.A total of 920785608 Clean Reads were obtained from 10 libraries,the data volume was 6.48G-7.34 G,and Q30 was more than 93.07%.46308 unigenes and 1833 bp N50 were obtained by Trinity software.Compared with the control group,593,606,1089,497 and 988 DEGs were obtained.We annotated the DEGs to obtain the pathways and genes related to immunity and metabolism.The expression of 9 randomly selected differential genes(DPD?ABCH?Pro POb?ACADL?CYP2J?PAT4?BHMT?CLEC ? PEPCK)was verified by q PCR,and the expression was consistent with the trend of RNA-seq.(3)Cloning and expression of TPS gene of M.japonicus: The total length of Mj TPS Gene c DNA of M.japonicus is 3308 bp,encoding 844 amino acids,with a molecular weight of 95.82 ku and the PI of 6.45.It has two conserved functional domains: Glyco?transf?20 domain and Trehalose?PPase domain.The phylogenetic tree showed that the Mj TPS and crustacean were one.The results of q PCR showed that the expression level of Mj TPS was the highest in hepatopancreas.The expression level of Mj TPS gene was significantly upregulated and down-regulated in 6-96 h under low salinity stress,but up-regulated and upregulated in 6-96 h under high nitrite stress.(4)Studies on the function of Mj TPS gene interference: The expression of TPS gene decreased significantly in 3-48 h,and the content of downstream trehalose also decreased significantly.The results showed that compared with the control group,the mortality of M.japonicus was significantly increased,the expression level of Mj TPS gene and the synthesis of downstream products were significantly reduced,and the expression levels of PMO25,ERP,CD,HSP90,HSP70,HSP60,HMC and CLEC2 were significantly changed,and they participated in the immune response of M.japonicus by promoting or inhibiting.Among them,CD and ERP belong to lysosomal pathway.It is speculated that when TPS gene is silenced,M.japonicus participates in immune response by inhibiting lysosomal pathway.In addition,we found that the silencing of Mj TPS gene would affect the expression of three molting related genes: CHI1,ERP and CHS,and speculated that TPS gene might be related to molting. |
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