Cloning, Identification And Expression Analysis Of Three Hormone Receptor Genes And HSP Genes In Flower Buds

Anguilla marmorata (A. marmorata) is a typical catadromy fish. It has high nutritional value and market foreground, and must be migrate to from seawater to freshwater growth. At present, the exploration of A. marmorata in osmoregulation and immune responses have been transferred from cell morphology and pathological detection level to molecular level. There are few reports about the hormone receptor gene of migratory fish in salinity adaptation studies and heat shock protein (HSP) in the immune responses. Arginine vasotocin receptors(AVTRs) and isotocin rexeptor (ITR) belong to G protein coupled receptors (GPCR). After binding with the ligand, the G protein is activated by activating the different signal transduction pathways to produce a variety of biological effects, which plays an important role in the process of osmotic adjustment. In addition,HSP is a functional protein,when the cells are stimulated by adverse factors,it can play a role in cell protection. This paper takes A. marmorata as research materials, and investigate the expression pattern of AVTRs and ITR in different salinity environment and the expression pattern of HSP after Aeromonas hydrophila (A. hydrophila) infection. The results are as follows:(1) Cloning, identification and expression analysis of AVTRs and ITR genes in A.marmorataWe cloned the full-length AVTRV1a2, AVTRV2 and ITR of A. marmorata by using rapid amplification of cDNA ends (RACE). The full length of three genes cDNA were 3049 bp, 2101 bp and 2191 bp; the open reading frame (ORF) was predicted as 1260 bp,1651 bp and 1131 bp; encoded 419, 551 bp and 376 amino acids, respectively. The length of the 5’and 3’ UTR were 237 bp and 1552 bp,158 bp and 287 bp,69 bp and 1060 bp.DNA Star software predict the molecular weight was 47.784 KD, 59.884 KD and 42.970 KD; the isoelectric point was 8.664, 8.705 and 9.292. Amino acid sequence analysis results showed that each receptor contains seven alpha helix membrane structure of G protein coupled receptor family. Through the multiple sequence comparison and phylogenetic tree analysis, we found that AVTRVla2, AVTRV2 and ITR gene highly conserved in the fish.We used fluorescence quantitative PCR technology, and found AVTRV1a2, AVTRV2 and ITR of A. marmorata gene were widely distributed in the brain, gill, intestine, liver,spleen, kidney, muscle and heart tissues. Different genes has tissue specificity. We choose three salinity environment: salinity 0 (Fresh water,FW),salinity 10 (Brackish water,BW)and 25 (Sea water, SW), brain, gill, intestine three tissue analysis. Three genes mRNA expression showed significant changes (P< 0.05). The expression of AVTRV1a2 protein in the brain was analyzed by western blot, and the expression of protein increased in the BW group and then decreased in the SW group (P< 0.05). In addition, in gills, we explore the relevance of three receptor gene and NKA, CFTR and NHE8 gene respectively. The results showed that there is a strong correlation between three receptor gene and NKAα1, NHE8,and a moderate correlation between three receptor gene and CFTR.(2) Cloning and expression analysis of AmHSP90, AmHSP70 and AmHSC70This is the first study to clone three A. marmorata heat shock protein gene(AmHSP90, AmHSP70 and AmHSC70) full-length cDNA. The full length of AmHSP90,AmHSP70 and AmHSC70 were 2527 bp, 2443 bp and 2247 bp; ORF were 2181 bp, 1932 bp and 1950 bp, encoded 643, 726 and 649 amino acids, respectively; the theoretical molecular weight were 83.6, 70.5 and 71.21 KD, the isoelectric point were 5.47, 5.44 and 5.28; 5’ and 3’ UTR were 58 bp and 288 bp,79 bp and 432 bp,79 bp and 218 bp,respectively. The amino acid sequence showed that AmHSP90 has a typical histidine kinase domain, contained five conserved HSP90 protein family signals, MEEVD signal characteristic sequence was located C terminal. AmHSP70 and AmHSC70 contained the ATP/GTP binding site, a bilateral nuclear localization signal (KK and RRLRT), three conserved domains and HSP70 family cytosolic motif EEVD classic in the 3’ end. The multiple alignments and phylogenetic analysis results showed that the three heat shock protein genes were highly conserved.Analysis the distribution of AmHSP90, AmHSP70 and AmHSC70 by qPCR technique in eight tissues, the results showed that the expression of heat shock protein in liver,intestine, heart and muscle were higher than those other tissues. A. marmorata were injected with normal saline and A. hydrophila,AmHSP90,AmHSP70 and AmHSC70 mRNA expression were changed significantly (P< 0.05) after A. hydrophila infection. The results showed that AmHSP90, AmHSP70 and AmHSC70 played an important role in the immune response in the four tissues, especially the liver tissue was more sensitive to A.hydrophila challenge.