Characterization and functional analysis of MjChi4 from the kuruma shrimp Marsupenaeus japonicus under the environmental stresses low pH and high ammonia

Abstract

Chitinase is widely present among crustaceans and is involved in digestion, growth, moulting and viral immune defence; its adaptive regulatory mechanism under environmental stresses remains unclear. To explore the adaptive regulation of crustacean chitinase under environmental stresses, we conducted a preliminary study on the cloning and anti-stress functions of the chitinase 4 gene (MjChi4) of Marsupenaeus japonicus. The full-length MjChi4 gene cDNA was obtained through the RACE cloning technique, including an open reading frame of 1842 bp. The MjChi4 protein clusters with Penaeus monodon PmChi4 and Litopenaeus vannamei PvChi4 and is expressed mainly in the hepatopancreas. During the first 24 h of low-pH stress, MjChi4 expression showed a cyclical change, first decreasing and then recovering, and the initial increase was detected at 48 h. Under high-ammonia stress, MjChi4 transcripts increased first and then decreased before 12 h, but increased again at 48 h and 96 h. Interference with MjChi4 resulted in significant reduction in expression levels of MjChi1, MjChi2, MjChi3, prophenoloxidase b, C-type lectin, antiviral protein and C-type lysozyme and significantly upregulated the gene expression levels of the transcription factor relish, which is related to the Imd signalling pathway and cytosolic MnSOD. In addition, following RNA interference of MjChi4 under low-pH and high-ammonia conditions, the survival rate of M. japonicus decreased and the time to death was significantly shortened. This study establishes that shrimp chitinase 4 functions to resist low-pH and high-ammonia stresses and provides important genetic material for rapid screening and improvement of M. japonicus germplasm through molecular-assisted breeding.