中华绒螯蟹Bco1基因的表达模式及其对高pH胁迫的响应

Expression pattern and high pH stress response of the Bco1 gene in the Chinese mitten crab （Eriocheir sinensis）

摘要:

结合生物信息学、实时荧光定量PCR、原位杂交及HPLC技术研究了中华绒螯蟹（Eriocheir sinensis）Bco1基因的序列特征、时空表达模式和亚细胞定位，并探讨了高pH慢性胁迫下肝胰腺的Es-Bco1表达量、色泽及类胡萝卜素含量的变化。结果表明：（1）Es-Bco1开放阅读框（Open reading frame，ORF）全长1 614 bp，编码537个氨基酸，属于RPE65超家族，并与三疣梭子蟹（Portunus trituberculatus）BCO1高度相似；（2）Es-Bco1在多个组织中均有表达，高表达于内膜、眼柄和肝胰腺；蜕壳周期中蜕壳后期（AB期）肝胰腺Es-Bco1表达量最高；卵巢发育阶段Ⅰ~Ⅲ期的肝胰腺Es-Bco1表达量显著上升，此后显著下降；（3）原位杂交结果显示，在蜕壳AB期和卵巢发育Ⅲ期，Es-Bco1 mRNA主要定位于肝胰腺的F细胞（纤维细胞）和R细胞（吸收细胞）中；（4）幼蟹在pH 8.0（对照）、9.0和10.0条件下胁迫30 d后，肝胰腺中的Es-Bco1表达水平显著上调，其肝胰腺亮度值（L^*）、黄度值（b^*）、总类胡萝卜素及β-胡萝卜素含量显著下降。综上，中华绒螯蟹Bco1主要在内膜、眼柄和肝胰腺中表达，且在蜕壳后期和卵巢Ⅲ期肝胰腺中高表达。此外，高pH胁迫可导致肝胰腺中Es-Bco1上调，降低类胡萝卜素含量，表明Bco1在中华绒螯蟹响应高pH胁迫过程中类胡萝卜素裂解起着重要作用。

Abstract:

This article combines bioinformatics， quantitative real-time PCR， in situ hybridization， and HPLC techniques to investigate the sequence characteristics， spatiotemporal expression patterns， and subcellular localization of the Bco1 gene in the Chinese mitten crab （Eriocheir sinensis）. It also explores the variations in Es-Bco1 expression， color， and carotenoid content in the hepatopancreas under chronic stress caused by high pH levels. The findings reveal that the full-length of Es-Bco1 open reading frame （ORF） spans 1 614 bp and encodes 537 amino acids. It belongs to the RPE65 superfamily and exhibits a high similarity to BCO1 in the Portunus trituberculatus.（2） Es-Bco1 was expressed in multiple tissues， with high expression in the endoderm， eyestalks， and hepatopancreas； the expression of Es-Bco1 in the hepatopancreas was highest during the post-molt stage （stage AB）； the expression of Es-Bco1 in the hepatopancreas significantly increased during ovarian stages Ⅰ-Ⅲ and then significantly decreased. （3） In situ hybridization results showed that Es-Bco1 mRNA was mainly localized in the F cells （Fibrillar cell） and R cells （Resorptive cell） of the hepatopancreas during the AB stage and ovarian development stage Ⅲ. （4） After 30 days of exposure to pH 8.0 （control）， 9.0， and 10.0， the expression level of Es-Bco1 in the hepatopancreas of juvenile crabs significantly increased， while the lightness value （L^*）， yellowness value （b^*）， total carotenoid content， and β-carotene content in the hepatopancreas significantly decreased. In conclusion， Bco1 in E. sinensis is mainly expressed in the epidermal layer， eyestalks， and hepatopancreas， with high expression during the post-molt stage and ovarian stage Ⅲ. Furthermore， high pH stress can upregulate Es-Bco1 expression in the hepatopancreas and reduce carotenoid content， indicating that Bco1 plays an important role in carotenoid cleavage of E. sinensis in response to high pH stress.