Gene Cloning And Expression Of Aminopeptidase N In Ctenopharyngodon Idellus

Protein is an important part of aquatic animal nutrition,and aminopeptidase N plays an important role in the digestion of protein and the metabolic balance.In this study, aminopeptidase N cDNA's full-length sequences has been cloned,and its structure and functions has been analyzed.The changes of APN gene expression in the grass carp development process and the APN gene expression differences of the different tissues, and the changes of APN gene expression of the circadian in the intestinal have been studied by a real-time PCR technology.In this study,also,the different protein level and protein source influencing on the APN gene expression in the foregut has been studied.Therefore, this study could lay the foundation for the reserch of the relationship between gene function and structure of aminopeptidase N and revealing the molecular mechanism of protein digestion and metabolism in aquatic animals.The major results are as follows:1.The means of RT-PCR,RACE techniques were used to clone the APN full-length cDNA gene sequence. The bioinformatic analysis of APN gene was carried out.The results showed that:the full length of cDNA sequence of APN had3258nucleotides, including27nucleotides at5'UTR and552nucleotides at3'UTR; its open reading frame had2679nucleotides encoding a892-amino-acid peptide;the deduced amino acid sequences of APN gene from C. idellus displayed the highest similarity with Danio rerio (75.4%), but varied to other animals from61.2%to58.8%;the encoded protein molecular weight was predicted at100.61ku with p1at5.18; phylogenetic analysis showed that the sequence of APN gene was clustered with D. rerio as its closest neighbor, which shared a sequence similarity of81.5%, and had lower similarity with other animals from60.2%to54.3%;the APN protein had no signal peptide and one helix trans-membrane region, but its amino acid sequence of the region demonstrated a low homology relationship to other vertebrates.2.The mRNA abundance of APN gene was assayed using real-time PCR.The results showed that:during embryonic development, the APN mRNA expression is relatively low,while the APN gene was expressed at highest level in the gastrula stage;during post-embryonic,the mRNA abundance of APN gene, in general, showed a consistent increasing trend from hatching stage to7d post-hatch; and.the APN gene expression changes with some range of fluctuation during14d post-hatch to35d post-hatch,but lower than he7d post-hatch, in general; the APN gene was differentially expressed at different tissues with a gradient from higher to lower among the tissues of fore-intestine, hind-intestine, liver, mid-intestine, kidney, muscle, spleen and heart, respectivelty.The effects of the circadian rhythms on APN expression of C. Idellus showed that there was a time-dependent pattern at higher rhythm during06:00-18:00and lower rhythm during18:00-06:00.3.The effects of the feeds containing different protein sources on the APN gene expression in the foregut showed that there were no significant differences on the APN mRNA expression in the foregut between the FM group and SM Group at7d; there were significant differences between the FM group and SM Group from14to56d(P<0.05);and the APN mRNA expression of the SM Group was lower than that of the FM group. The effects of the feeds containing different protein levels on the APN gene expression in the foregut showed that, in general, there was a consistent increasing trend from the22.3%CP to27.3%CP group; and there was a consistent downward trend from the27.3%CP to42.1%CP group at different time periods. The results indicated that the APN mRNA expression of Ctenopharyngodon idella correlates tightly to the developmental stage, and showed tissue variability and circadian rhythms.The APN mRNA expression of Ctenopharyngodon idella had adaptive reaction to protein nutrition.