Functional analysis of a tyrosinase gene involved in early larval shell biogenesis in Crassostrea angulata and its response to ocean acidification@en

The formation of the primary shell is a vital process in marine bivalves. Ocean acidification largely influences shell formation. It has been reported that enzymes involved in phenol oxidation, such as tyrosinase and phenoloxidases, participate in the formation of the periostracum. In the present study, we cloned a tyrosinase gene from Crassostrea angulata named Ca-tyrA1, and its potential function in early larval shell biogenesis was investigated. The Ca-tyrA1 gene has a full-length cDNA of 2430 bp in size, with an open reading frame of 1896 bp in size, which encodes a 631-amino acid protein that includes a 24-amino acid putative signal peptide. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that Ca-tyrA1 transcription mainly occurs at the trochophore stage, and the Ca-tyrA1 mRNA levels in the 3000 ppm treatment group were significantly upregulated in the early D-veliger larvae. WMISH and electron scanning microscopy analyses showed that the expression of Ca-tyrA1 occurs at the gastrula stage, thereby sustaining the early D-veliger larvae, and the shape of its signal is saddle-like, similar to that observed under an electron scanning microscope. Furthermore, the RNA interference has shown that the treatment group has a higher deformity rate than that of the control, thereby indicating that Ca-tyrA1 participates in the biogenesis of the primary shell. In conclusion, and our results indicate that Ca-tyrA1 plays a vital role in the formation of the larval shell and participates in the response to larval shell damages in Crassostrea angulata that were induced by ocean acidification.初级贝壳形成是海洋双壳类生物的关键生命过程之一。海洋酸化可对贝壳形成过程产生显著影响。已有研究表明，参与酚氧化反应的酶类，如酪氨酸酶（tyrosinase）与酚氧化酶（phenoloxidases），可参与贝壳角质层的形成过程。本研究从葡萄牙牡蛎（Crassostrea angulata）中克隆得到一个酪氨酸酶基因，命名为Ca-tyrA1，并对其在幼虫早期贝壳生物发生过程中的潜在功能进行了探究。Ca-tyrA1基因的全长cDNA序列长度为2430 bp，包含一段长度为1896 bp的开放阅读框，可编码一个含631个氨基酸的蛋白质，其中包含一段由24个氨基酸残基组成的推定信号肽。定量反转录聚合酶链反应（qRT-PCR）分析结果显示，Ca-tyrA1的转录主要发生在担轮幼虫期；在3000 ppm处理组的早期D形面盘幼虫中，Ca-tyrA1的mRNA表达水平显著上调。整体原位杂交（whole-mount in situ hybridization, WMISH）与扫描电子显微镜分析结果显示，Ca-tyrA1的表达始于原肠胚期，并持续至早期D形面盘幼虫阶段；其杂交信号呈鞍形，与扫描电子显微镜下观察到的结构特征相符。此外，RNA干扰（RNA interference, RNAi）实验结果显示，处理组的畸形率显著高于对照组，由此表明Ca-tyrA1参与了初级贝壳的生物发生过程。综上，本研究结果表明，Ca-tyrA1在幼虫贝壳形成过程中发挥关键作用，并参与了海洋酸化诱导的葡萄牙牡蛎幼虫贝壳损伤的应激响应过程。