Cloning and expression analysis of PP2A-B55 gene in Metapenaeus affinis
As an important economic shrimp,Metapenaeus affinis has high nutritional value.However,due to the fact that their broodstock mainly rely on marine fishing,the amount of wild broodstock resources has been decreasing year by year.The realization of fully artificial cultivation of M.affinis broodstock can effectively alleviate the pressure,and research on the ovarian development of the broodstock regulatory mechanism is of great significance.Reversible protein phosphorylation is an important regulatory mechanism in eukaryotic organisms,mainly involving protein kinases and protein phosphatases.PP2A,as one of the main cell cycle regulatory enzymes,is involved in regulating almost every stage of the cell cycle.In order to understand the role of protein phosphatase 2A(PP2A)in the process of cell cycle regulation in organisms,the PP2A-B55 gene of M.affinis was cloned by RACE and its role in the process of growth and development was preliminarily explored using qRT-PCR.The full-length cDNA sequence of MaPP2A-B55 gene was obtained by RACE amplification,totaling 2 100 bp,of which the ORF was 1 332 bp,encoding 443 amino acids,with a predicted molecular weight of 51.229 kDa and a theoretical isoelectric point of 6.04.The transmembrane structure,signal peptide,and corresponding glycosylation sites were not predicted in the MaPP2A-B55 amino acid sequence,but 52 phosphorylation sites were detected.Multiple alignments showed that the amino acid sequence consistency of MaPP2A-B55 was the highest among Litopenaeus vannamei and Penaeus monodon,with a consistency of 99.55％,followed by Scylla paramamosain.The phylogenetic tree showed close phylogenetic relationships with L.vannamei,P.monodon,and Marsupenaeus japonicus,and later clustered with other crustaceans.The quantitative results indicated that among the detected tissues MaPP2A-B55 had the highest expression level in muscle and ovarian tissue,followed by gill.Afterwards,the expression level of MaPP2A-B55 was detected at different stages of ovarian development,and the results showed significant differences.It increased from stage Ⅰ to stage Ⅱ,then decreased and remained stable,and then significantly increased at stage Ⅴ(P＜0.05),and suddenly decreased at stage Ⅵ.At the same time,the expression pattern of MaPP2A-B55 was also detected at various stages of larval development,with high expression at the oosperm stage during larval development and then decreasing.It reached its lowest point at the nauplius stage Ⅵ,and then began to rise and remain stable at the zoea stage Ⅰ.These research results indicate that PP2A-B55 gene may be involved in the ovarian maturation and larval development process of M.affinis.
Metapenaeus affinisPP2A-B55cloningexpressionovarian developmentlarval development
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