凡纳对虾半胱氨酸天冬氨酸蛋白酶（Pvcaspase Droc）协调溶藻弧菌感染的凋亡清除

摘要(中文译文)

从溶藻弧菌感染前后凡纳滨对虾肝胰腺组织的差异表达基因中鉴定并克隆了Pvcaspase Dronc。Pvcaspase Dronc的cDNA全长为908个碱基对，编码266个氨基酸。保守结构域分析显示，Pvcaspase Dronc含有一个caspase募集结构域。多物种氨基酸比对表明，Pvcaspase Dronc与黑腹果蝇Dronc（NP_524017.1）具有81%的相似性。系统发育树结果表明，Pvcaspase Dronc与哺乳动物caspase-9亲缘关系最近。亚细胞定位分析显示，Pvcaspase Dronc在S2细胞中呈点状、均匀分布。Pvcaspase Dronc在凡纳滨对虾的12个组织中广泛分布，其中在眼柄和肝胰腺中表达水平最高。溶藻弧菌感染显著上调了肝胰腺、血细胞、肌肉和肠道中Pvcaspase Dronc的表达。通过dsRNA成功实现了对Pvcaspase Dronc的沉默，注射后24小时和48小时在血细胞、鳃和肝胰腺中均确认了显著的敲降效率。在对虾中Pvcaspase Dronc表达降低后，溶藻弧菌感染导致细菌菌落计数增加、存活率降低，表明抗菌防御能力受损。通过流式细胞术分析，在溶藻弧菌感染后0至24小时内，Pvcaspase Dronc沉默对虾的血细胞凋亡率较对照组显著降低。本研究通过体内和体外沉默或过表达，探究了Pvcaspase Dronc的分子特征和抗菌功能，为对虾免疫防御机制提供了初步认识，并为理解对虾如何应对溶藻弧菌提供了初步基础。

Abstract

Pvcaspase Dronc was identified and cloned from differentially expressed genes in the hepatopancreatic tissue of Penaeus vannamei before and after infection with Vibrio alginolyticus. The full-length cDNA of Pvcaspase Dronc comprises 908 base pairs, encoding 266 amino acids. Conserved domain analysis revealed that Pvcaspase Dronc contains a caspase recruitment domain. Multispecies amino acid alignment showed that Pvcaspase Dronc shares 81% similarity with Drosophila melanogaster Dronc (NP_524017.1). Phylogenetic tree results indicated Pvcaspase Dronc is most closely related to mammalian caspase-9. Subcellular localization analysis revealed punctate, uniform distribution of Pvcaspase Dronc throughout S2 cells. Pvcaspase Dronc exhibited widespread distribution across 12 tissues of Penaeus vannamei, with highest expression levels in the eyestalk and hepatopancreas. Vibrio alginolyticus infection significantly upregulated Pvcaspase Dronc expression in the hepatopancreas, hemocyte, muscle, and intestine. Silencing of Pvcaspase Dronc by dsRNA was successfully achieved, with significant knockdown efficiency confirmed at both 24 and 48 h post-injection in hemocyte, gill and hepatopancreas. Following reduced expression of Pvcaspase Dronc in shrimp, infection with Vibrio alginolyticus resulted in increased bacterial colony counts and reduced survival rates, indicating impaired antimicrobial defense capacity. Through flow cytometry analysis, the apoptosis rate of hemocyte in Pvcaspase Dronc silenced shrimp was significantly reduced compared to control group within 0-24 h after Vibrio alginolyticus infection. This study investigated the molecular characteristics and antibacterial function of Pvcaspase Dronc through in vivo and in vitro silencing or overexpression, providing preliminary insights into the immune defense mechanisms of shrimp and offering initial understanding of how shrimp responds to Vibrio alginolyticus.

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