Expression analysis of the inducible nitric oxide synthase(iNOS)gene of the Pelodiscus sinensis
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维普
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一氧化氮(NO)参与生物体内多种重要的生理病理活动,可增强机体的免疫功能,而诱导型一氧化氮合酶(iNOS)是一氧化氮合成过程中的关键限速酶.为了探明iNOS在中华鳖(Pelodiscus sinensis)先天免疫中的作用,研究了iNOS基因在中华鳖组织的分布及外源刺激物对其基因和蛋白表达的影响.荧光定量PCR结果显示,iNOS基因在中华鳖的心脏、肝脏、肾脏、脾脏、肌肉、小肠和大肠中均有表达,其中在大肠中表达量最高,其次是小肠、肾脏和脾脏.在受脂多糖(lipopolysaccharides,LPS)刺激后,肝、脾、肾、肌肉和小肠iNOS mRNA的表达显著升高.免疫组化实验结果显示LPS刺激后 12 h、24 h iNOS蛋白在小肠、脾脏表达水平显著高于生理盐水对照组.研究结果表明iNOS可能参与了中华鳖的机体免疫反应.
Considered one of China's commercially important aquatic species of China,the Chinese softshell turtle(Pelodiscus sinensis)contributes a high portion of the total aquatic products sold in the country.P.sinensis is directly exposed to the aquatic environment,and because the water is very nutrient-rich,it is vulnerable to in-fection by opportunistic pathogens.Nitric oxide(NO)participates in various important physiological and patho-logical activities in organisms.It can enhance the immune function of the body,and inducible nitric oxide syn-thase(iNOS)is a key rate-limiting enzyme in the process of nitric oxide synthesis.In some organisms,it has been demonstrated that treatment with lipopolysaccharides(LPS),a gram-negative bacteria cell product,can lead to a pronounced increase in iNOS expression.The study investigated the distribution of iNOS genes in P.sinensis tissues and the effects of exogenous stimuli on their gene and protein expression to investigate the role of iNOS in the innate immunity of P.sinensis.Quantitative real-time PCR(qRT-PCR)was used to examine the distribution of iNOS in various turtle tissues,including the muscle,spleen,liver,heart,kidney,and small and large intestines.The results of qPCR showed that the iNOS gene was expressed in the heart,liver,kidney,spleen,muscle,and small and large intestines.And the highest expression level was in the large intestine,fol-lowed by the small intestine,kidney,and spleen.P.sinensis were equally distributed into two groups.The chal-lenge group was chosen to receive an injection of LPS,while the control group was given of saline injections.Five turtles from each group were dissected to collect the liver,small intestine,spleen,kidney,and muscle at various intervals(0,3,6,12,24,and 48 hours).After LPS stimulation,iNOS mRNA expression was sig-nificantly elevated in the liver,spleen,kidney,muscle,and small intestine.The results of IHC showed that the expression levels of iNOS protein in the small intestine and spleen were significantly higher than that in the saline-treated group at 12 and 24 hours after LPS stimulation.In conclusion,LPS can cause a significant rise in iNOS in P.sinensis at the mRNA and protein levels.In this situation,iNOS might be connected to numerous im-munological reactions in various organs.However,more research will be required to completely understand the immunological roles of iNOS in P.sinensis.The research indicates that iNOS may be involved in the immune re-sponse of P.sinensis.It will help to understand the process by which P.sinensis acquires immunity against various pathogens.
维普
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