Functional study of kuruma shrimp immune-related genes

在这项研究中，我们利用dsRNA干扰来阐明和表征这些基因在体内的功能。转谷氨酰胺酶（Tgase）和凝血蛋白（CP）是虾血凝级联反应的关键分子。我们利用双链RNA干扰（dsRNAi）技术在体内验证了转谷氨酰胺酶和凝血蛋白在虾血液凝固系统中的功能参与，以及它们对抗微生物肽和酚氧化酶原系统相关基因mRNA表达的影响。注射1 μg和10 μg的Tgase-和CP- dsrna后，1 d时，Tgase mRNA在鳃、心脏、血细胞、肝胰腺、肠和淋巴器官的表达均受到抑制，而CP基因仅在鳃和心脏组织中受到抑制。然而，在注射后第7天，通过mRNA表达、凝血和蛋白数据可以观察到两种基因和剂量的全身基因沉默。白斑病毒和Vibr . More . io penaecida攻毒试验揭示了Tgase和CP在对虾免疫系统中的关键作用。在Tgase和CP沉默后，抗微生物肽（甲壳碱和溶菌酶）和参与酚氧化酶原系统的基因（酚氧化酶原和〓-2巨球蛋白）受到抑制，这表明虾的血液凝固和体内的生物防御之间存在关联。这一发现提示了螯合动物和甲壳类动物中参与酚氧化酶原系统的抗菌肽和基因激活的可能的替代机制。综上所述，本研究1)提供了血液凝固和抗微生物肽与酚氧化酶原系统相关基因的关联证据，从而提出了这些分子激活的另一种机制；2)基因沉默效力的研究结果将为dsRNA干扰在对虾疾病和病原体控制和管理中的应用提供预防措施。这项研究有助于更好地了解虾免疫系统的基本生物学。少

In this study, we elucidated and characterized the function of these genes in vivo using dsRNA interference. Transglutaminase (Tgase) and clotting protein (CP) are critical molecules involved in the blood coagulation cascade of shrimp. We demonstrate in vivo the functional involvement of transglutaminase and clotting protein in the shrimp blood coagulation system using double stranded RNA interference (dsRNAi) and their influence in the mRNA expression of antimicrobial peptides and genes involved in the prophenoloxidase system. Expression of Tgase mRNA was inhibited in gills, heart, hemocyte, hepatopancreas, intestine and lymphoid organ while the CP gene was suppressed only in gills and heart tissues on day-1 post injection with 1 μg and 10 μg of Tgase- and CP-dsRNA, respectively. However, at day-7 post injection, systemic gene silencing was observed for both genes and dosages as shown by mRNA expression, blood coagulation and protein data. Challenge test with white spot virus and Vibr … More io penaecida revealed the critical function of Tgase and CP in the immune system of shrimp. Suppression of antimicrobial peptides (crustine and lysozyme) and genes involved in the prophenoloxidase system (prophenoloxidase and 〓-2 macroglobulin) following Tgase and CP silencing demonstrate an association between blood coagulation and humoral biodefenses in shrimp. This finding suggests a possible alternative mechanism on the activation of antimicrobial peptides and genes involved in the prophenoloxidase system in chelicerates and crustaceans. In summary, this study 1)provided evidence on the association of blood coagulation and anti-microbial peptides and genes involved in the prophenoloxidase system and thus, presents an alternative mechanism on the activation of these molecules 2) gene silencing potency results will serve as a precaution on the application of dsRNA interference in the control and management of shrimp diseases and pathogens. This study contributed to the better understanding of the basic biology of the shrimp immune system. Less