Cloning, charaterization, and expression of heat inducible genes relating to stress condition of black tiger shrimp, Penaeus monodon

Abstract

Stress tolerance was determined in heat induced P. monodon exposed to pathogenic and environmental stresses. Shrimp initially induced by heat (35 ºC, 2 h) were subjected to WSSV challenge and ammonia exposure. The results showed that the survival rates of heat induced shrimp exposed to WSSV and ammonia were 15 and 20 %, respectively, higher than that of un-induced shrimp, indicating that stress tolerance to WSSV and ammonia can be enhanced by heat acclimation. Consequently, expression patterns of P. monodon genes including aquaporin (PmAQP1), glucosamine-6-phosphate deaminase (PmGluN6P-deaminase), and C- type lectin domain containing protein (PmCLP) were determined. Full length cDNA sequences of PmAQP1 (786 bp ORF encoding 261 amino acids), PmGluN6P-deamiase (948 bp ORF encoding 315 amino acids), and PmCLP (678 bp ORF encoding 225 amino acids) were firstly identified and characterized. Expression patterns of these 3 genes in various tissues of juvenile P. monodon were investigated using RT-PCR and quantitative analyses of the expression levels of the target genes were determined. The constitutive levels of Aquaporin (PmAQP1) were detected in hepatopancrease, gill, ovaries, testes, intestine, stomach and heart. The level in gill appeared to be greater than others tested tissues. The expression of PmAQP1 was not detected in haemocyte, epidermis, lymphoid organs and muscle. The result of PmAQP1 expression in shrimp exposed to ammonia stress indicated that the up-regulation of PmAQP1 was detected in heat induced shrimp much sooner than that of un-induced shrimp, indicating that the expression of PmAQP1 might be involved in the enhancement of ammonia stress tolerance in the shrimp. Expression of PmGluN6P-deaminase was also determined in ammonia exposed shrimp. The result revealed no significant difference of the expression level was detected between heat-induced and un-induced shrimp, indicating no relation in PmGluN6P-deaminase expression and ammonia stress tolerance. The involvement of PmCLP in WSSV tolerance was determined. The up-regulation of PmCLP in heat induced shrimp after exposed to WSSV for 72 h indicated the potential functional role of PmCLP in pathogenic stress tolerance of P. monodon. In addition, recombinant PmAQP1 was conducted and successfully produced using pET17b System. The recombinant protein will be useful for antibody construction and further used as biological tool for determining health status of the shrimp.