Identification of an Alpha-Tubulin Gene from the Chinese Mitten Crab Eriocheir sinensis: Expression Profiles under Immune Challenge and during Larval Development

Abstract

In this study, an alpha-tubulin gene (EsTUBA, GenBank: KJ509188) was isolated and identified from the Chinese mitten crab Eriocheir sinensis. The EsTUBA mRNA expression patterns under larval brachyurization development and immune challenge stress by Vibrio parahaemolyticus injection in adult crabs were evaluated. EsTUBA cDNA was 1,695 base pairs (bp) with an open-reading frame of 1,356 bp encoding 451 amino acids (49.95 kDa) and containing GTPase domain and C-terminal Tubulin domain. Sequence alignment showed that EsTUBA shared 95-98% identity and exactly similar structural features with its counterparts reported in other animals. Phylogenetic analysis indicated that EsTUBA clustered into one group together with Tubulins from other crustaceans. Sequence alignment, structure comparison and bioinformatic analyses revealed that EsTUBA is member of the Tubulin family. EsTUBA was fluctuant expressed in all tested larval development stages and various organs, and the levels of EsTUBA mRNA expressed in hepatopancreas, gill and intestine, are significantly expression fluctuated and induced after bacteria V. parahaemolyticus injection, while in heart, is almost consistently expressed at 0, 6, 12, 24, 36 and 48 h post-injection compared to control. The molecular cloning of EsTUBA gene is important for further study on the function of this gene.

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“…Two primer sets were designed to perform real-time PCR using TOYOBO SYBR ® Green Realtime PCR Master Mix (TOYOBO, Japan) in Funglyn FTC-3000 (Funglyn Biotech Inc, Canada). β-actin was used as internal standard as it was proved previously to be housekeeping gene during various stimulus (Li et al, 2015). Negative controls missing cDNA template were included in this experiment.…”