Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, College of Animal Science and Technology, College of Veterinary Medicine, Zhejiang Agriculture and Forestry University, Hangzhou, 311300, China.
Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, College of Animal Science and Technology, College of Veterinary Medicine, Zhejiang Agriculture and Forestry University, Hangzhou, 311300, China.
Fish Shellfish Immunol. 2020 Apr;99:392-402. doi: 10.1016/j.fsi.2020.02.043. Epub 2020 Feb 19.
This study investigated the function of Troponin T (TnT) in the mud crab, Scylla paramamosain. The 1952 bp cDNA sequence of TnT was cloned from S. paramamosain using rapid amplification of cDNA ends (RACE) PCR. The quantitative real-time PCR analysis showed that TnT was highly expressed in the muscle and heart of S. paramamosain. Challenging with white spot syndrome virus (WSSV) or Vibrio alginolyticus (VA), two common pathogens that infect mud crabs, enhanced the expression of TnT in S. paramamosain. Knockdown of TnT using TnT-dsRNA led to up-regulating the expression of immune-related genes, such as c-type-lectin, toll-like-receptor, crustin antimicrobial peptide and prophenoloxidase. The cumulative mortality of WSSV- and VA-infected crabs was significantly increased following TnT knockdown. After WSSV or VA infection, TnT knockdown caused a significant reduction in phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC), indicating a regulatory role of TnT in the innate immune response of S. paramamosain to pathogens. Apoptosis of hemocytes was higher in crabs treated with TnT-dsRNA compared with control crabs treated with phosphate-buffered saline. Knockdown of TnT increased apoptosis of hemocytes following VA infection, but reduced hemocyte apoptosis following WSSV infection. In summary, TnT may enhance the immune response of S. paramamosain to WSSV infection by regulating apoptosis, THC, PO activity and SOD activity. And TnT may play a positive role in the immune response against VA infection by regulating apoptosis, THC, SOD activity and PO activity.
本研究调查了肌钙蛋白 T（TnT）在锯缘青蟹（Scylla paramamosain）中的功能。通过快速扩增 cDNA 末端（RACE）PCR 从锯缘青蟹中克隆出 1952bp 的 TnT cDNA 序列。定量实时 PCR 分析显示，TnT 在锯缘青蟹的肌肉和心脏中高度表达。用白斑综合征病毒（WSSV）或溶藻弧菌（VA）两种常见的感染锯缘青蟹的病原体刺激后，TnT 在锯缘青蟹中的表达增强。用 TnT-dsRNA 敲低 TnT 导致免疫相关基因如 c 型凝集素、Toll 样受体、抗菌肽 crustin 和酚氧化酶原的表达上调。在 TnT 敲低后，WSSV 和 VA 感染的蟹的累积死亡率显著增加。在 WSSV 或 VA 感染后，TnT 敲低导致酚氧化酶（PO）活性、超氧化物歧化酶（SOD）活性和总血细胞计数（THC）显著降低，表明 TnT 在锯缘青蟹先天免疫反应中对病原体具有调节作用。与用磷酸盐缓冲液处理的对照蟹相比，用 TnT-dsRNA 处理的蟹的血细胞凋亡更高。在 VA 感染后，TnT 敲低增加了血细胞凋亡，但在 WSSV 感染后降低了血细胞凋亡。总之，TnT 可能通过调节凋亡、THC、PO 活性和 SOD 活性来增强锯缘青蟹对 WSSV 感染的免疫反应。并且 TnT 可能通过调节凋亡、THC、SOD 活性和 PO 活性在对 VA 感染的免疫反应中发挥积极作用。
