Gene Clone,Expression Analysis Of Lv-JIP4 In Litopenaeus Vannamei And Its Function In Response To Low-temperature Stress

C-Jun NH2-terminal kinase（JNK）-interacting proteins 4（JIP4）is an important scaffold protein that can participate in the mitogen-activated protein kinases（MAPKs）signalling pathway in response to various external environmental stimuli.However,the characteristics and functions of JIP4 gene are currently still not well understood in crustaceans.In the present study,a novel JIP4 gene was identified in Litopenaeus vannamei and designated Lv-JIP4.The full-length Lv-JIP4 c DNA was 3,923 bp in length and contained a 170 bp 5’-untranslated region（UTR）,a 72 bp3’-UTR,and a 3,681 bp open reading frame.The Lv-JIP4 protein contained three functional domains,including the Jnk-Sap K＿ap＿N,JIP＿LZII and WD40 domains.Lv-JIP4 m RNA was broadly distributed in all studied tissues（muscle,gill,brain,heart,nerve,stomach,eye stalk,hepatopancreas and intestine）,with the highest level in muscle.The in situ hybridization results showed that the Lv-JIP4 was scattered in muscle fibres and near the sarcolemma.Green fluorescent protein subcellular localization experiments showed that Lv-JIP4 protein was distributed in the cytoplasm of He La cells,but not in the nucleus.The transcript levels of Lv-JIP4 in muscle were found to be significantly increased by low temperature,low p H,high salinity,poly（I:C）,and LPS challenges,indicating that it might play a crucial role in responding to adverse stresses.The transcript levels of Lv-JIP4,Lv-p38a^MAPK and Lv-p38b^MAPK in muscle were significantly upregulated after challenge with low temperature,suggesting that Lv-JIP4 may be the positive regulator of Lv-p38a^MAPK and Lv-p38b^MAPK under low temperature stress conditions.In addition,after knockdown of Lv-JIP4 expression,the protein levels of total Lv-p38^MAPKand phospho-Lv-p38^MAPK in the experimental ds RNA group were attenuated compared with those in the control group after 48 hours（h）,60 h and 72 h of low-temperature stress,indicating that it might activate and phosphorylate the Lv-p38^MAPK protein in response to cold stress.Here,we elucidated the molecular characteristics and the potential role of Lv-JIP4 and further refined the signal transduction mechanism of L.vannamei in response to low-temperature stress.