Seawater carbonate chemistry and gene expression stability of Hong Kong oyster (Crassostrea hongkongensis)@en

The increase of CO2 by anthropogenic activities leads to a decrease of pH in the ocean surface due to ocean acidification (OA) process. Generally, OA not only reduces the rate of calcification in marine environments but also affects various physiological activities, especially in calcifiers, including edible oysters. Quantitative real-time PCR (qRT-PCR) is often used to detect gene expression in response to OA, which relies on the stability of internal control. However, the appropriate internal controls for OA experiments remain scarce especially in the marine calcifiers. Hence, this study developed internal controls for qRT-PCR assays using the Hong Kong oyster (Crassostrea hongkongensis) as a model to reveal gene expression profile during development under OA. In this study, 17 housekeeping genes were selected as the possible candidate of the internal controls. After a comprehensive interpretation from the multiple algorithms and software, GAPDH paired with RL23 is recommended for the normalization for planktonic larvae and benthic juveniles, but beyond that, TUBB and EF2 are recommended for post-metamorphic stage. Moreover, GAPDH and EF2 were suitable for various pH treatments, and TUBB, RL35 and RL23 could be the alternatives for OA experiments. These results are instrumental for the selection of internal control in Crassostrea hongkongensis during the development, and shed light on other molecular OA experiments in marine invertebrates for reference.人为活动引发的二氧化碳浓度升高，会通过海洋酸化（Ocean Acidification, OA）过程导致海洋表层海水pH值下降。通常而言，海洋酸化不仅会降低海洋环境中的钙化速率，还会干扰多种生理活动，尤其对包括食用牡蛎在内的钙化生物产生负面影响。实时定量聚合酶链反应（Quantitative real-time PCR, qRT-PCR）常被用于检测响应海洋酸化的基因表达水平，而该技术的应用高度依赖内参基因的稳定性。然而，适用于海洋酸化实验的内参基因仍较为匮乏，针对海洋钙化生物的相关筛选研究更是寥寥无几。因此，本研究以香港牡蛎（Crassostrea hongkongensis）为实验模型，开发适用于qRT-PCR检测的内参基因，以揭示其在海洋酸化环境下发育过程中的基因表达谱特征。本研究共选取17个持家基因作为内参基因的候选序列。经多种算法与软件综合分析评估后，本研究推荐将GAPDH与RL23配对，用于浮游幼虫和底栖稚贝阶段的基因表达标准化分析；而针对变态后阶段，则推荐采用TUBB与EF2作为内参基因组合。此外，GAPDH与EF2适用于不同pH梯度处理的实验体系，而TUBB、RL35及RL23可作为海洋酸化实验的备选内参基因。本研究结果可为香港牡蛎发育过程中的内参基因筛选提供重要参考，同时也可为海洋无脊椎动物的其他海洋酸化相关分子生物学实验提供借鉴思路。