乙酰化修饰对马氏珠母贝植核免疫的作用

Lysine acetylation in the allograft-induced stress response of Pinctada fucata martensii

摘要:

目的 马氏珠母贝植核育珠技术会导致受体贝发生免疫排斥甚至死亡，严重影响了珍珠的质量和品质。本研究通过分析提高蛋白质乙酰化修饰水平对马氏珠母贝植核免疫的影响，从而探究乙酰化修饰在其植核后的作用。方法 通过注射赖氨酸去乙酰化酶抑制剂 (lysine deacetylase inhibitor，KDI)混合物，并采用荧光定量PCR (qRT-PCR)、蛋白印迹 (western blot，WB)、抗氧化酶活性检测以及养殖指标等多水平综合分析蛋白质乙酰化修饰水平对马氏珠母贝植核免疫的影响。结果 注射KDI混合物后，首先检测HDAC 6/10 (10006221)、Sir2家族成员 (10031932和10020349)这3个去乙酰转移酶基因的表达水平，发现均在不同时间出现显著下调，鳃组织总蛋白乙酰化水平在72 h显著提高，表明注射药物后提高了乙酰化水平；其次使用qRT-PCR检测免疫相关因子的时序表达，结果显示，IRAK1、IL-17、NF-κB和CASP 2基因的表达在12 h都出现显著上调，IκK在48 h显著上调，而TRAF 3从48 h后则出现显著下调，表明经KDI刺激后受体贝机体细胞免疫增强；抗氧化酶GSH-Px、POD和CAT酶活性在12 h和24 h表现出显著上调的趋势，表明受体贝机体体液免疫增强；最后，结合养殖数据分析，发现60 d的存活率和留珠率显著提高。结论 乙酰化修饰参与了马氏珠母贝的植核免疫反应，KDI可以作为免疫调节剂调节马氏珠母贝的植核免疫应答，为深入探究乙酰化修饰的生物学功能和调控机制奠定基础，为合理控制珍珠贝免疫反应提供理论支撑。

Abstract:

The pearl oyster, Pinctada fucata martensii, can experience transplant rejection or even host death following allografting. This study investigated the impact of increased protein acetylation on the immune response of P. fucata martensii after implantation, aiming to elucidate the role of acetylation modifications in this process. A mixture of broad-spectrum lysine deacetylase inhibitors (KDI) was injected into P. fucata martensii to assess the effects of increased protein acetylation using multiple analytical methods, including quantitative real-time PCR (qRT-PCR), Western blot (WB), antioxidant enzyme activity assay, and culture indicators. Following KDI injection, the relative expression of HDAC 6/10 (10006221), and two mumbers of the Sir2 family (10031932 and 10020349) were significantly down-regulated at different time points. Meanwhile, the acetylation level of total proteins in the gills was significantly increased at 72 h, indicating enhanced protein acetylation in gill tissue. Additionally, the expression of IRAK1, IL-17, NF-κB, and CASP2 were significantly up-regulated at 12 h, while IκK was significantly up-regulated at 48 h. In contrast, TRAF3 expression was significantly down-regulated from 48 h onward, suggesting that KDI stimulation enhanced cellular immunity in recipient oysters. Furthermore, antioxidant enzymes such as GSH-Px, POD, and CAT were significantly up-regulated at 12 and 24 h post-KDI treatment, indicating enhanced humoral immunity. The retention and survival rates of oysters were significantly higher at 60 d post-KDI treatment compared to the control group. These results suggested that acetylation modifications play a key role in allograft-induced stress in P. fucata martensii, and KDI may serve as immunomodulator to regulate the implantation process. In summary, this study provided an important basis for exploring the immune response and adaptive mechanisms associated with acetylation modifications in P. fucata martensii during implantation, and offered theoretical support for the rational control of the immune response in P. fucata martensii.