An ShK-domain serine protease of Eriocheir sinensis regulates the PO activity to resist Spiroplasma eriocheiris infection
An ShK-domain serine protease of Eriocheir sinensis regulates the PO activity to resist Spiroplasma eriocheiris infection
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中华绒螯蟹ShK结构域丝氨酸蛋白酶调控PO活性抵抗绒毛螺原体感染
DOI：
10.1016/j.fsi.2020.06.046
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Qingguo Meng
中科院分区：
文献类型：
--
作者：
Xiaohui Cao;Yinyue Lu;Jiyun Li;Xiaoli Xia;Qi Gao;Wei Gu;Wen Wang;Qingguo Meng
从中华绒螯蟹（Eriocheir sinensis）中发现了一种新的丝氨酸蛋白酶（EsShK-SP）。EsShK-SP cDNA全长1927 bp，包含一个1260 bp的开放阅读框，编码420个氨基酸，包括一个信号肽、两个ShK结构域和Tryp-SPC结构域。实时荧光定量PCR结果显示EsShK-SP主要在血细胞、鳃、肠和神经中表达，在心脏、肌肉和肝胰腺中表达较弱。感染后1 ~ 9 d EsShK-SP表达显著上调。将Tryp-SPC结构域与pGEX-4 T-1载体连接，进行原核表达，获得重组蛋白rSPC。当rSPC和S.绒螯菌刺激E.中华绒螯蟹PO活性显著上调。亚细胞定位结果表明，重组EsShK-SP主要定位于果蝇S2细胞的胞质中。绝对实时荧光定量PCR和激光共聚焦扫描显微镜结果均表明，EsShK-SP在S2细胞中过表达可降低S.绒螯蟹同时，EsShK-SP的过表达也能提高S2细胞的PO活性和细胞活力。EsShK-SP双链RNA干扰后48 ~ 96 h，PO原的表达水平和PO活性均显著下降。利用RNAi技术敲低EsShK-SP基因，使S. EsShK-SP沉默组在S.绒螯菌感染同时，EsShK-SP-dsRNA组的蟹存活率下降。以上结果表明EsShK-SP在E. sinensisagainstS.通过proPO系统的调节，
A novel serine protease contains two ShK-domain was found from the Chinese mitten crabEriocheir sinensis(EsShK-SP). The full-length EsShK-SP cDNA is 1927 bp and contains a 1260-bp open reading frame encoding a protein of 420 amino acids, including a signal peptide, two ShK domain, and Tryp-SPC domain. Quantitative real-time PCR showed thatEsShK-SPwas expressed mainly in the hemocytes, gills, intestine, and nerve, but weakly in heart, muscle, and hepatopancreas. After infected with Spiroplasmaeriocheiris, the expression ofEsShK-SPwas significantly up-regulated from 1 d to 9 d. The Tryp-SPC domain was ligated with pGEX-4T-1 vector and prokaryotic expressed to obtain recombinant protein rSPC. When rSPC andS. eriocheirisstimulated the hemocytes ofE. sinensis, the PO activity was significantly up-regulated. The subcellular localization revealed that recombinant EsShK-SP was mainly located in the cytoplasm of Drosophila S2 cells. Both absolute real-time PCR and confocal laser scanning microscope results showed that over-expression of EsShK-SP in S2 cells could decrease the copy number ofS. eriocheiris. Meanwhile, the over-expression of EsShK-SP also increased the PO activity and cell viability of S2 cells. After EsShK-SP RNA interference using dsRNA, the expression levels of proPO and activity of PO decreased significantly from 48 h to 96 h. The knockdown of EsShK-SP by RNAi resulted in the copy number ofS. eriocheirisin the EsShK-SP silenced group was significantly increased compared to the control groups duringS. eriocheirisinfection. Meanwhile, the survival rate of crabs decreased in the EsShK-SP-dsRNA group. The above results indicated that EsShK-SP plays an important immune role duringE. sinensisagainstS. eriocheiristhrough regulation of the proPO system.