Cloning And Characterization Of Some Immunity Related Genes From Haliotis Diversicolor Supertexta

Small abalone Haliotis diversicolor supertexta is the most commercially important cultured abalone in southern China. Since late 2000, outbreaks of mass mortality in cultured abalone have caused catastrophic losses to aquaculturists. Therefore, cloning and characterization of the immunity-related genes from small abalone are essential for explaining the disease-resistance mechanism and searching for the new pathway of managing disease outbreaks, which may contribute to the sustained development of abalone culture.EST analysis and SMART RACE techniques were combined together to clone immune related genes from small abalone. Three full lengths cDNA of samif (macrophage migration inhibitory factor), sadpt2 (dermatopontin 2) and saβgrp (β-1,3-glucan recognition protein), and three cDNA fragments of sapgrp (peptidoglycan recognition protein) subgroup were obtained. Real time PCR was used to analyze their expression patterns after Vibrio Parahaemolyticus infection and tributyltin (TBT) exposure. Their complete sequences were confirmed by head to toe PCR. Meanwhile, MIF genomic DNA was cloned by PCR. The main results are reported as follows:(1) The full length cDNA of samif was of 535 bp and the deduced protein was composed of 128 amino acids, with an estimated molecular mass of 14.0 kDa. The full length samif genomic DNA comprises 3237 bp, containing three exons and two introns. Real time quantitative PCR analysis revealed that samif gene is constitutively expressed in 6 selected tissues, and its expression level in hepatopancreases is higher than that in the other tissues (p <0.01). Samif expression level in the hepatopancre at 24 and 48 h after V. Parahaemolyticus injection was upregulated most significantly (p <0.01), but there was no significant change after exposure to TBT (p >0.05).(2) The full length cDNA of sadpt2 was of 584 bp, consisting of a 5'-terminal UTR of 58 bp, an open reading frame of 471 bp and a 3'-terminal UTR of 55 bp. The deduced protein was composed of 156 amino acids, with an estimated molecular mass of 18.3 kDa. Real time quantitative PCR analysis revealed that sadpt2 expression level in the hepatopancre at 24 h after V. Parahaemolyticus injection was upregulated significantly (p <0.05), which indicates that sadpt2 is an immune inducible protein and can participate in immune defence against V. Parahaemolyticus. The expression of sadpt2 was not significant difference between treatment and control after TBT exposure (p >0.05). (3) The full length cDNA of saβgrp was of 1459 bp contained an open reading frame of 987 bp that coded for a protein of 328 amino acids, with an estimated molecular mass of 36.9 kDa. Different from other reportedβGRPs which contain a signal peptide, the putative saβGRP does not contain a signal peptide belongs to non-secretary proteins. Real time quantitative PCR analysis revealed that after V. Parahaemolyticus injection, the expression level of saβgrp in hepatopancreases at 24 h was most significantly higher than that of the control group (p <0.01), while at 48 h post-injection, it was significantly higher than that of the control group (p <0.05). The expression of saβgrp was also not significant difference between treatment and control after TBT exposure (p >0.05).(4) The lengths of cDNA fragment of three sapgrp subgroups are 804 bp, 1015 bp and 728 bp respectively, showing high homology with Biomphalaria glabrata pgrp gene short form, which we inferred that the three sapgrp subgroups may be three isomers of small abalone pgrp gene short form.