近江牡蛎HSC70基因对溶藻弧菌感染的反应

mRNA expression of heat-shock cognate protein 70 gene(HSC70) in the oyster(Crassostrea hongkongensis) in response to Vibrio algi-nolyticus challenge

人工注射溶藻弧菌(Vibrio alginolyticus)感染近江牡蛎(Crassostrea hongkongensis),通过实时荧光定量RT-PCR方法检测了近江牡蛎6种不同组织器官(外套膜、鳃、消化腺、闭壳肌、心脏和血细胞)中HSC70基因表达量的变化;同时采用平板活菌计数法测定了近江牡蛎不同组织器官中溶藻弧菌菌量的变化。结果显示,人工注射感染溶藻弧菌后,近江牡蛎6种组织器官中HSC70基因表达量均显著性升高(P<0.05),且随时间变化均呈现出先升高后恢复至对照水平的趋势;其中鳃组织中HSC70基因分别在第6小时和第72小时出现2次显著性高表达,且在第72小时的表达量高于第6小时(P<0.05)。在多数组织器官中,HSC70基因出现显著高表达后便急剧下降至对照水平,而在消化腺中高表达持续的时间长达18 h(第6 24小时)。在注射感染溶藻弧菌后第6小时,血细胞中HSC70基因的表达量达到峰值,接近于对照水平的15倍;而在其他组织器官中,该基因表达峰值仅为对照水平的2.5倍左右。人工注射感染溶藻弧菌后3 h,3种组织(消化腺、闭壳肌和血淋巴)中均能检测到溶藻弧菌的积累,其中闭壳肌中含菌量最高,6 h时闭壳肌中含菌量急剧下降,随后又增加并维持在一定水平直至72 h;消化腺中菌量随着时间推移而增加,到达峰值后又逐步下降;血淋巴中,溶藻弧菌含量整体上随时间不断增加,到48 h稳定在较高水平。各组织中HSC70表达量与溶藻弧菌含量之间存在一定的关联性,尤其在溶藻弧菌攻击的靶器官(消化腺)中最为明显。由此可见,病原菌感染可以诱导近江牡蛎HSC70基因高表达,并且HSC70可能参与了机体抗病原菌感染的相关作用,这为进一步研究近江牡蛎HSC70基因在抗病免疫中的作用机制奠定了基础。

We developed a fluorescent real-time quantitative RT-PCR method to evaluate the pattern of HSC70 mRNA expression in response to the bacterial infection in the oyster （Crassostrea hongkongensis）. We injected oysters with live Vibrio alginolyticus, and measured HSCTO gene expression in six tissues （mantle, gills, digestive gland, muscle, heart and hemocytes）. We measured the accumulation and distribution of V. alginolyticus using the plate counting method. HSC70 mRNA expression was significantly up-regulated in all the six tissues following injection with V. alginolyticus, and the expression was time-dependent. HSCTO gene expression increased signifi- cantly at 6 and 72 h in the gill. Similarly, levels peaked at 6 h in the digestive gland then remained high until 24 h, after which they decreased rapidly to control levels. HSC70 mRNA expression was -10 fold higher in the haemo- cytes than the control after 3 h, and the expression evels peaked at 6 h （-15 fold higher than the control）. The ex- pression of HSC70 was -2.5 fold higher in the remaining tissues than in the control. The bacterial concentration was higher in the muscle than that in other tissues （digestive gland and hemolymph） at 3 h after the oysters were injected with V. alginolyticus, though levels had declined by 6 h. The accumulation of bacteria in the muscle in- creased at 12 h and remained high up to 72 h. The change in V. alginolyticus concentration was time-dependent in the digestive gland whereas the bacteria increased with time and peaked at 48 h in the hemolymph. We observed a positive correlation between HSC70 mRNA expression and the concentration of bacteria, most obvious in the di- gestive gland. Our results suggest that bacterial challenge induces the up-regulation of HSC70 mRNA expression and suggests that HSC70 plays an important role in protecting the oyster against the bacterial pathogen.