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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。
Vibrio alginolyticus was infected with Crassostrea hongkongensis by real-time fluorescence quantitative RT-PCR. The contents of 6 different tissues and organs (mantle, gill, digestive gland, Blood cells), and the changes of the amount of Vibrio alginolyticus in different tissues and organs of Oyster oyster were determined by plate viable count method. The results showed that the expression of HSC70 gene in six tissues and organs of Oyster oyster was significantly increased after artificial inoculation with V. alginolyticus (P <0.05), and showed the tendency of first increasing and then recovering to the control level The HSC70 gene in gill tissue showed two significant high expression at 6 hours and 72 hours, respectively, and the expression level of HSC70 in gill tissue was higher than that of 6th hour (P <0.05) at 72 hours. In most tissues and organs, HSC70 gene was dramatically down-regulated to the control level after it was significantly overexpressed, while up-regulated in the digestive gland for up to 18 h (6th hour 24 hours). The expression level of HSC70 gene in blood cells peaked at 6 hours after infection with V. alginolyticus, which was close to 15 times of the control level. In other tissues and organs, the peak value of HSC70 gene expression was only 2.5 times of the control level. Vibrio alginolyticus accumulation could be detected in 3 kinds of tissues (digestive gland, adductor muscle and hemolymph) 3 h after infection with Vibrio alginolyticus, Clostridium in a sharp decline in bacteria, and then increased and maintained at a certain level until 72 h; digestive gland bacteria increased over time, reaching a peak and then gradually decreased; hemolymph, Vibrio alginolyticus content Overall, it increased with time and remained stable at 48 h. There was a certain correlation between the expression level of HSC70 and the content of Vibrio alginolyticus in all tissues, especially in the target organ (digestive gland) attacked by V. alginolyticus. Thus, pathogen infection can induce overexpression of HSC70 gene, and HSC70 may be involved in the role of the body against pathogenic bacteria infection, which laid the foundation for further study on the mechanism of HSC70 gene in disease resistance and immunity of oyster oyster.