Quantification Of HSC70 Gene MRNA In Crassostrea Hongkongensis Treated With Trichlorfon

Estuarine and coastal waters of China not only serve as prime habitat for asignificant fraction of commercial and recreational marine fisheries, but also receiveindustrial and municipal wastes from a rapidly expanding coastal economy. Molecularbiomarkers, due to their sensitivities in early warning to indicate biological damagesfollowing the exposure with low concentrations of the pollutants, have been shown tobe very sensitive indicators of environmental pollution.Heat shock proteins (HSPs) are a family of highly conserved proteins and aredetected in all cells, prokaryotic and eukaryotic. Hsps are especially useful biomarkersbecause their induction is much more sensitive to stress than traditional indices. TheHSP70 family, the most widely studied of the HSPs, is an ideal candidate forbiomarkers of environmental pollution.Crassostrea hongkongensis (Lam and Morton, 2003) is one of the mostimportant and best-known oysters in China. They are widespread about Chinesecoastal waters and extensively cultivated in estuarine areas of southern China seas. Itis an ideal biomarker to monitor environmental conditions of Chinese coastal waters.HSC70 gene expression in C. hongkongensis exposure to trichlorfor wasinvestigated by Real time quantitative PCR.For selecting reliable reference gene, the expression levels of three commonlyused housekeeping genes 13-actin, glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and 18S rRNA, were examined in C. hongkongensis. The tissues analysedby quantitative real-time PCR were gill, mantle, digestive gland and adductor. Theexperiments were performed to investigate the transcriptional stability within andbetween tissues at 3hours, 7days and 20 days exposure to 0.5 mg/l trichlorfor. Theresults were as follows:β-actin gene expressed stably in all tissue samples of theoyster in the experiments. Therefore, it can be used for normalization of real-timePCR results for gene expression studies in a wide variety of samples in C.hongkongensis. However, GAPDH gene stably expressed just in gill and digestivegland, and 18S rRNA gene just in the digestive gland. They can be used as internalcontrols only in these tissues.The effect of Trichlorfor on the HSC70 was investigated in four different tissuesof Crassostrea hongkongensis. Oysters were exposed to 0.5, 0.2, 0.05, 0.01, 0.001,0.0001mg/l Trichlorfor for 3 hours, 7 days and 20 days. The tissues analysed were gill,mantle, digestive gland and adductor. Exposure to Trichlorfor produced statistically significant changes in the expressiori of HSC70. But the expression of HSC70 showedtissue-dependence. In the gill, mantle and digestive gland, trichlorfor significantlyinduced HSC70 expression that reached maximum levels, decreasing thereafter.However, in the adductor, HSC70 expression significantly decreased compared withcontrol, and recovered when exposure time prolonged. In our study, trichlorforexposure concentration 0.5mg/l can't induce HSC70 significant expression in the gill,mantle and digestive gland, but it can induce HSC70 significantly lower expressionthan that of control in the adductor. In the mantle, the threshold HSC70 inductionconcentration of trichlorfor is estimated at 0.001-0.0001mg/l, and 0.01-0.001mg/l in thedigestive gland and adductor, respectively. In the gill, trichlorfor exposureconcentration as low as 0.0001mg/l can induce HSC70 significantly higher expressionthan that of control. In the gill, HSC70 response is most sensitive to Trichlorfor. Sogills are a readily available tissue for studying biomarkers and can be useful inindicating pollutants such as Trichlorfor.This study indicated that the HSC70 was asensitive indicator of exposure to Trichlorfor.