长牡蛎自噬调控基因CgSVIP在病原胁迫响应中的生物标志物功能

Research on the autophagy regulatory gene CgSVIP of Crassostrea gigas as a biomarker of pathogen stress

  • 摘要:
    目的 探明长牡蛎响应病原体相关分子模式(PAMPs)和病原刺激过程中CgSVIP的表达特征,发掘CgSVIP作为病害预警生物标志物的应用方式,解析CgSVIP参与免疫应答调控的机制。
    方法 采用生物信息学、实时荧光定量PCR (RT-qPCR)和蛋白免疫印迹(Western blot)技术,解析了CgSVIP在长牡蛎病害预警中潜在生物标志物的调控机制及参与病原刺激诱导的自噬调控过程。
    结果 脂多糖(LPS)、肽聚糖(PGN)、葡聚糖(GLU)和聚肌胞苷酸Poly(I:C)刺激长牡蛎12 h后,血淋巴细胞中CgSVIP的mRNA相对表达水平均显著上升,且在转录组差异表达基因中最显著,平均FPKM值为106.938。长牡蛎受LPS刺激12、24和48 h,以及灿烂弧菌刺激3、6、12、48和72 h后血淋巴细胞中CgSVIP的mRNA绝对表达量显著上升,同时对2023年3—10月采集的养殖长牡蛎进行灿烂弧菌刺激12 h后发现,除3月采集的长牡蛎外,其他样本血淋巴细胞中CgSVIP的mRNA绝对表达量均显著升高。长牡蛎在实验室和养殖过程中受到PAMPs或病原刺激时,CgSVIP的mRNA绝对表达量为3.18~3.79 lg (拷贝数/mL),在正常状态下CgSVIP的mRNA绝对表达量为2.93~3.35 lg (拷贝数/mL),初步认定CgSVIP用于长牡蛎病害预警的mRNA绝对表达量阈值为3.35 lg (拷贝数/mL)。为解析CgSVIP参与免疫应答调控的机制,利用卡马西平(CBZ)刺激长牡蛎后,其血淋巴细胞中CgSVIP蛋白表达水平显著上升,为DMSO组的1.67倍;利用RNA干扰(RNAi)技术抑制长牡蛎CgSVIP的表达并进行LPS刺激12 h后,发现血淋巴细胞中CgSVIP及自噬相关基因CgLC3、CgBeclin1、CgAtg5和CgP62的mRNA相对表达水平均显著降低,分别降低为NC-RNAi+LPS组的53%、16%、38%、19%和40%;检测自噬相关基因CgLC3蛋白切割水平,与NC-RNAi+LPS组相比,CgSVIP-RNAi+LPS组中CgLC3Ⅱ/CgLC3Ⅰ比值显著降低,为NC-RNAi+LPS组的73%。
    结论 CgSVIP通过影响长牡蛎血淋巴细胞自噬参与抗细菌免疫应答,可以作为潜在的病害预警生物标志物。

     

    Abstract: This study aimed to explore the expression characteristics of CgSVIP in response to pathogen-associated molecular patterns (PAMPs) and pathogen stimulation in Crassostrea gigas, to discover the application of CgSVIP as a biomarker for disease early warning, and to analyze the mechanism of CgSVIP in regulating immune responses. This experiment utilized bioinformatics, quantitative real-time PCR (RT-qPCR), and Western blot to analyze the regulatory mechanism of CgSVIP as a potential biomarker in disease early warning for C. gigas and its involvement in the autophagy regulation process induced by pathogen stimulation. It was found that after stimulation with lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid Poly(I:C) for 12 h, the mRNA expression level of CgSVIP in haemocytes of C. gigas significantly increased under all four PAMPs stimulations, and the average expression difference level was the most significant among the differentially expressed genes in the transcriptome, with an average FPKM value of 106.938. Further, when C. gigas was stimulated with LPS and Vibrio splendidus, the absolute expression value of CgSVIP in haemocytes significantly increased at 12, 24 and 48 h after LPS stimulation, reaching 3.71, 3.52 and 3.55 lg copies/mL respectively; and significantly increased at 3, 6, 12, 48 and 72 h after V. splendidus stimulation, reaching 3.31, 3.12, 3.11, 3.42 and 3.26 lg copies/mL respectively. After stimulation with V. splendidus for 12 h from March to October 2023, the expression level of CgSVIP in haemocytes significantly increased except in March, reaching 3.52, 3.57, 3.45, 4.42, 3.77, 3.75 and 4.06 lg copies/mL respectively, with the highest expression in July samples. Therefore, the absolute expression value of CgSVIP was within the range of 2.93-3.35 lg copies/mL without PAMPs and pathogen stimulation, and within the range of 3.18-3.79 lg copies/mL with stimulation. It was preliminarily determined that the threshold for CgSVIP to respond to PAMPs and pathogen stress was 3.35 lg copies/mL. To analyze the mechanism of CgSVIP in regulating immune responses, the expression level of CgSVIP protein in haemocytes of C. gigas significantly increased after stimulation with carbamazepine (CBZ), reaching 1.67 fold that of the DMSO group. After inhibiting the expression of CgSVIP in C. gigas by RNAi and stimulating with LPS for 12 h, the expression levels of CgSVIP and autophagy-related genes CgLC3, CgBeclin1, CgAtg5 and CgP62 in haemocytes significantly decreased, reduced to 53%, 16%, 38%, 19% and 40% of the NC-RNAi+LPS group, respectively. The ratio of CgLC3II/CgLC3I was significantly decreased in the CgSVIP-RNAi+LPS group compared with the NC-RNAi+LPS group, reaching 0.73 fold that of the NC-RNAi+LPS group. CgSVIP can be used as a potential biomarker for disease early warning in C. gigas, and it participates in the regulation of immune responses by affecting autophagy in haemocytes and mediating antibacterial immune responses. Through changes in gene expression as a biomarker, early warning of C. gigas aquaculture can be achieved to reduce mortality and production losses caused by diseases.

     

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