| Kuruma shrimp Marsupenaeus japonicus (Bate,1888) is an important commercial and maricultured species in the coastal areas of China. There are two morphologically similar varieties in M. japonicus which are characterized by diagnostic color banding patterns on the carapace, and the two varieties have different geographical distributions and exist high genetic differentiation. As a major digestive enzyme for carbohydrates, alpha-amylase (1,4-alpha-D-glucan-4-glucanohydrolase, EC 3.2.1.1) plays an important role in the food digestion and nutrition metabolism of shrimp. In this study, we cloned the alpha-amylase genes (MjAmy-1 and MjAmy-2) of the two varieties, analyzed their molecular structures and compared the differences of the two genes. Moreover, we examined the real-time quantitative expression and protein levels of MjAmy-1 in various tissues in order to gain more insight into its function in food digestion, and tested the expression patterns of MjAmy-1 during larval development to better understand the development of a-amylase synthetic ability and nutritional utilization in the larval kuruma shrimp. The results are as follows:(1) The newly identified alpha-amylase gene of variety I was designated as MjAmy-1, and its cDNA sequence was 1651 bp in length with an ORF of 1539 bp encoding a polypeptide of 512 amino acids which contained a predicted signal peptide of 17 amino acids. The calculated molecular weight (MW) of the deduced protein was 57.10KDa, and the theoretical pI was 4.99. The DNA sequence of MjAmy-1 was 3387bp and had an complex exon-intron organization which was composed of eleven exons interrupted by ten introns. Multiple sequence alignment of MjAmy-1 amino acid sequence with the corresponding sequences of other species showed high identities, among which similarity to that of Litopenaeus vannamei reached 93.16%. In addition, phylogenetic analysis indicated that it shared the closest phylogenetic relationship with the published sequence of L. vannamei. Furthermore, the deduced protein of MjAmy-1 contained the typical domains and conserved motifs commonly present in a-amylases from other species. Given these, we could comfirm that the newly cloned MjAmy-1 belonged to the a-amylase family.(2) A quantitative real-time PCR was performed to quantify the relative mRNA expression pattern of MjAmy-1, the results showed that MjAmy-1 was abundantly expressed in hepatopancreas. Quantification of a-amylase protein levels in different tissues suggested that a-amylase existed ubiquitously in all the examined tissues with the highest amount in hepatopancreas followed by intestine and stomach. Moderate levels of a-amylase was detected in muscle and heart, and a lower concentration in eyestalks and gills. Preferential expression of both mRNA and a-amylase protein was observed in hepatopancreas, indicating that MjAmy-1 was mainly synthetized and secreted by the hepatopancreas and it was possibly involved in the digestive function of this organ and related to the process of metabolism.(3) The expression pattern of MjAmy-1 during larval development from eggs to postlarva was analyzed by qRT-PCR. The mRNA expression of MjAmy-1 was almost undetectable as early as the stage of eggs, while it was present in slight amounts at nauplius. After first feeding at zoea 1, the transcriptional level increased dramatically and peaked at mysis 1, then dropped severely at mysis 2 and decreased slowly until postlarva 2. Thereafter, the expression quantity of MjAmy-1 again increased progressively and remained at high levels from postlarva 3 until end of the research. It was suggested that the pattern and level of a-amylase expression in M. japonicus during larval development was coincident with ontogenetic and behavioral changes.(4) The newly identified alpha-amylase gene of variety II was named MjAmy-2, it contained an ORF of 1539 bp encoding a polypeptide of 512 amino acids which contained a predicted signal peptide of 17 amino acids. The calculated molecular weight of the deduced protein was 57.04KDa, and the theoretical pI was 4.99. The DNA sequence of MjAmy-2 was 3382bp and was composed of eleven exons and ten introns. Homologous analysis showed that MjAmy-2 shared 98.83% identity with MjAmy-1, and its identites with the corresponding sequences of other species was 52-93%. The deduced protein of MjAmy-2 showed highly conservation with all animal a-amylases on sequences of catalytic residues, chloride binding sites, cysteines and calcium binding sites, and it also contained the typical domain A and domain C, all of these suggested that MjAmy-2 was a member of α-amylase family.(5) There are significant differences between the nucleotide sequences especially the intron sequences of MjAmy-1 and MjAmy-2, and six amino acid residues changes were also found in the peptide sequences of MjAmy-1 and MjAmy-2. Moreover, some variations were also observed in the secondary structures of the two genes. Analysis of the genetic diversities between the a-amylase genes of the two varieties suggested that variety I seemed to be higher polymorphic. The genetic differentiation indices Fst and gene flow values Mm of the two populations confirmed that the two varieties were genetically distinct. In the present study, we also found ten SNPs in the a-amylase genes which could distinguish the two varieties. |
PDF Full Text Request