Cloning of amylase gene in Macrobrachium rosenbergii and its expression regulation

【Objective】Macrobrachium rosenbergii amylase gene(AMY) was cloned, biological information analysis and expression regulation were studied, to provide theoretical foundation for further understanding biological function of AMY gene in the growth and development of M. rosenbergii.【Method】The coding area sequence(CDS) of AMY gene in M. rosenbergii was cloned by PCR. Bioinformatic analysis of AMY sequencing was performed using ExPASy ProtParam, ExPASy ProtScale, SignalP-5.0, MegAlign and Lasergene v8.0 softwares. Real-time fluorescence quantitative PCR(RTPCR) was used to detect the expression of AMY gene in different tissues(including abdominal ganglion, stomach, heart, gill, gonad, hepatopancreas, muscle and intestine), and further to explore its expression in the muscle of individuals from fast-growing families(FG) and slow-growing families(SG).【Result】The length of CDS of AMY gene was 2121 bp, encoding 706 amino acids residues. The molecular weight of AMY protein was 76.87 kD, and the theoretical isoelectric point (pI) was 4.63, belonging to an unstable hydrophilic protein, which contained two typical amylase domains. The secondary structure of AMY protein was consisted of α-helix, extended strand and random coil, accounting for 13.88%, 21.39% and 64.73%, respectively. The homology of amino acid sequence of AMY gene in M. rosenbergii was the highest(62.6%) and lower(48.9%) compared with the amino acid sequences of Procambarus clarkii and Pinctada maxima, respectively. Phylogenetic tree constructed based on the amino acid sequence similarity of AMY genes howed that M. rosenbergii had the closest genetic relationship with P. clarki, but had the far genetic relationship with Eriocheir sinensis. AMY gene was widely expressed in the different tissues, but with differences between sexes and tissue specificity. The details were as follows:it had the extremely higher expression in the intestines and gills of female individuals than that of male individuals (P<0.01, the same below), it had the significantly higher expression in gill of female individuals than that of male individuals(P<0.05), but the expression in the hepatopancreas of female individuals was lower than that of male individuals. In addition, the expression level of AMY gene in muscle of in FG was extremely higher than that in SG.【Conclusion】AMY gene plays an important role in growth and development of M. rosenbergii. It is widely expressed in different tissues of M. rosenbergii, but with sex specificity and tissue specificity.