近江牡蛎热休克蛋白70基因对三丁基锡的响应

Studies on Transcriptional Expression Pattern of Heat Shock Protein 70 Genes of Jinjiang Oyster Crassostrea hongkongensis Exposed to Tributyltin

为研究TBT（Tributyltin,三丁基锡）在双壳贝类HSP70s（热休克蛋白70）分子的高表达响应,以近江牡蛎（Crassostrea hongkongensis）为材料,用不同质量浓度（0、10-2、10-1、100、101、102、103ngL）的TBT对近江牡蛎处理不同时间（12、24、48、96、192 h）后,采用实时荧光定量PCR检测其3种器官（消化腺、外套膜和鳃）中HSP70和HSC70基因mRNA的表达水平.结果显示：10-1~102ngL的TBT能诱导近江牡蛎HSP70和HSC70基因mRNA水平显著高于对照组（P〈0.05）,其表达水平均为先升后降,在96 h达到峰值;103ngL的TBT也能诱导近江牡蛎HSP70和HSC70基因显著高表达（P〈0.05）,但随着处理时间延长,mRNA表达出现抑制现象.能诱导HSP70和HSC70基因表达水平显著高于对照（P〈0.05）的TBT临界浓度均在10-2~10-1ngL范围内,可见2种基因对TBT的响应灵敏度高且相同,反应强度也没有显著差异（P〉0.05）.3种器官对TBT的响应灵敏度也相同,但在消化腺和鳃中的响应强度都极显著（P〈0.01）高于外套膜.研究表明,近江牡蛎HSP70和HSC70基因对极低质量浓度的TBT就有高表达响应,可以作为TBT污染的早期预警指标;近江牡蛎较适宜的指示器官是消化腺和鳃,其次是外套膜.

The 70 kDa heat shock proteins contain a constitutive type （ heat shock cognate 70, HSC70） and an inducible type （ heat shock protein 70, HSP70） encoded by different genes in marine bivalves. The two genes can respond extensively and sensitively to various marine pollutants. However, the response characteristics of the two genes to tributyhin （TBT） , the most harmful marine organotin pollutant, are unknown so far. In the present research, 315 individuals of Jinjiang oysters （Crassostrea hongkongensis） with an average shell length of （ 10 ± 1 ） cm were randomly divided into 7 groups （45 oysters/group） , and were treated with TBT seawater at 0 （control） , 10-2, 10-1, 10°, 101^ 102^ and 103 ng/L, respectively, then randomly sampled （5 oysters/sampling time） at 12, 24, 48, 96, and 192 h. The digestive gland, gills and mantle were taken and rapidly frozen in liquid nitrogen overnight, and then stored at - 80 ℃. Fluorescent real-time quantitative RT-PCR was employed to detect the expression levels of HSP70 and HSC70 genes of Jinjiang oysters treated at various TBT concentrations for different times. The results were as follows： the relative HSP70 and HSCTO mRNA expression levels were significantly （P 〈0. 05） higher than those of the oysters in the control. The levels increased at first, peaked at 96 h, and gradually dropped with increment of treatment time at TBT concentrations ranging 10-1 ng/L through 102 ng/L. They were also significantly higher at first, but greatly or very significantly lower （P 〈 0.01 ） than those in the control at 103 ng/L. Furthermore, the two genes were significantly induced to transcribe mRNA by the same threshold of TBT concentration range （10-2^-10- 1^ ng/L） in the digestive gland, gills and mantle of the Jinjiang oysters. This suggested that the two genes in the three organs had the same high sensitivity of response to TBT, and have potential to be used as an early warning biomarker of TBT pollution. Moreover, the mRNA levels of the two genes in the digestive gland and gills were very significantly higher （P 〈 0. 01 ） than those in the mantle, so the digestive gland and gills of Jinjiang oyster were the most suitable indicator organs for early warning of marine environmental pollution, but the mantle was also available.