| It is for the first time in the world to obtain a bisexual fertile population of allotetraploid crucian carps, which have been used to produce triploid crucian carps with obvious advantages in growth rate. The fast growth rate of the triploids was considered to be related to their sterility. It is known that the growth of fish is achieved by protein synthesis of the organism. The protein metabolism directly influences fish growth. The extrinsic protein in the fish body first degraded into peptides and dissociated amino acids. Under the effect of peptidase, the peptides would further degrade into di/tripeptides and dissociated amino acids. The transport carrier in fish intestinal canal is responsible for the absorption of di/tripeptides and amino acids. The metabolism of amino acid happens in the intestine canal or liver. In this study, three key genes that are involved in the protein metabolism of different ploidy cyprinids were studied with their expression patterns being discussed. The major results in this paper were presented as follows:1.Aminopeptidase N (aminopeptidase N, APN) is a protein digestive enzymes, and it plays an important role in protein digestion.We cloned a part-length cDNA of APN in the red crucian carp, triploid and allotetraploid.The results showed that the different ploidy cyprinids APN sequence is relatively conserved, and the phylogenetic tree showed that the red crucian carp, triploid and allotetraploid were closest relationship. By real-time quantitative PCR, we found that the intestinal distribution of APN is most abundant in triploid.The APN gene has a maternal genetic trait and can digest protein of the yolk sac in early embryonic development of different ploidy cyprinids.However, the expression of triploid APN is higher in the most stages of early embryonic development, which indicates the efficiency of protein digestion is higher than that of red crucian carp, and therefore triploid has apparent protein digestion advantage of the protein metabolism.2. Peptide transporter1(PepTl) localized at the brush-border membranes of intestinal epithelial cells plays plays a key role in the absorption of di/tripeptides from the diet,and it belongs to the proton-dependent oligopeptide transporter (POT) family.We cloned a full-length cDNA of PepTl in the red crucian carp, triploid and allotetraploid.The results showed that the different ploidy cyprinids PepTl sequence is relatively conserved, and the phylogenetic tree showed that the red crucian carp, triploid and allotetraploid were closest relationship.By real-time quantitative PCR, we found that the distribution of the fore-intestinal PepTl is most abundant in triploid, and its expression gradually reduced along the longitudinal intestinal,which shows that small peptides are mainly absorbed by the intestinal.The PepTl gene has a maternal genetic trait, and that can transport di/tripeptides of protein digestion of the yolk sac in early embryonic development of different ploidy cyprinids.However, the expression of triploid PepTl is higher in the most stages of early embryonic development, which indicates the efficiency of the absorption of di/tripeptides is higher than that of red crucian carp, and therefore triploid has apparent advantage of the absorption of protein.3. Glutamate dehydrogenase (GDH) is an important branch-point enzyme between carbon and nitrogen metabolism, and it plays a very important role in the amino acid metabolism. We cloned a full-length cDNA of GDH in the red crucian carp, triploid and allotetraploid.The results showed that the different ploidy cyprinid GDH sequence is relatively conserved, and the phylogenetic tree showed that the red crucian carp, triploid and allotetraploid were closest relationship. By real-time quantitative PCR, we found that the GDH widely distributed in all tissues,and the expression of the GDH mRNA in triploid liver tissue was higher than that of red crucian carp during the breeding and non-breeding season.The GDH gene has a maternal genetic trait.However, the expression of triploid GDH is higher in the most stages of early embryonic development, which indicates the efficiency of the amino acid metabolism is higher than that of red crucian carp, and therefore triploid has apparent advantage of the amino acid metabolism.4. This chapter studied the growth performance, Serum biochemical parameters, and protein metabolism-related gene expression changes in different ploidy cyprinids. Each ploidy fish was a treatment,and each treatment was three replicates.All fishs were about59.0g and fed30days.The results of growth experiment showed that weight gain rate of triploid is2.13times that of the red crucian carp,which provides the reference data of the triploid growth advantage.The concentration of serum urea nitrogen and thyroid hormone (T3, T4) in the triploid is lower that of the red crucian carp,and the concentration of serum aspartate and aminotransferase are higher that of the red crucian carp.Triploid adult intestinal APN, PepTl and liver GDH gene expression was significantly increased.Therefor,the triploid has a protein metabolic advantage.5. Effect of protein levels and sorts on triploid crucian carp APN, PepTl, and GDH gene expression were studied. Preparation of semi-purified diets of the five kinds of protein levels, and two kinds of protein sorts, each feed of a deal with each treatment repeated fed the same number of initial weight of about50g incubator crucian carp for feeding40days.The results of growth experiment showed that the protein level and type significantly effected on the triploid intestinal APN,GDH, PepTl gene expression. There is a correlation between the expression of triploid APN gene and intestinal location and protein concentration. Protein levels effects mainly on expression of the GDH in the triploidy crucian carp intestinal, and its expression was significantly upregulated with protein leve increased.Lower protein and higher protein could increase the expression of the triploid intestinal PepTl.Fish meal compared with soybean meal, it could increase the expression of the triploid intestinalGDH,PepT1and APN.6. The effect of sodium butyrate to the growth characteristics, the serum biochemical parameters and the expression of APN, GDH, and PepTl genes in the intestinal canal of triploid crucian carps were studied. Sodium butyrate with five different dosages were added into the basal diet of triploid crucian carps for feeding49days. After that the growth characteristics, the serum biochemical parameters and the expression abundant of APN, GDH, and PepTl genes in the intestine canal were detected. It was reveled that with the increasing in the dosage of sodium butyrate, the certain growth rate of triploid crucian carps in different test groups first increased and then decreased. The test group of triploid crucian carps with0.25g/kg sodium butyrate in the basal diet possessed the highest growth rate of21.12%and the lowest feed coefficient of2.03. The test group with0.50g/kg sodium butyrate had the most serum total protein, which was2.47times to that of the control group. The activities of the glutamic oxaloacetic transaminase (AST) and the glutamic pyruvic transaminase (ALT) were higher than that of the control. However, the difference in the ureanitrogen concentration between the test and control groups was not significant (P>0.05). The additive of sodium butyrate could significantly improve the expression of PepTl mRNA in the intestine of triploid crucian carps with dose dependent manner. The regulation effect of sodium butyrate to the intestinal APN expression was first down regulation then up regulation, the different that to the expression abundant of GDH.7.The effect of microbial ecological agent to the growth characteristics and the expression of APN, GDH, and PepTl genes in the intestine of triploid crucian carps were studied. The dosages of microbial ecological agent in the basal diet of triploid crucian carps were0.0g/kg, 0.1g/kg and0.2g/kg, respectively. After56days of feeding, the growth rate and expression abundant of APN, GDH, and PepTl genes in the intestinal canal of triploid crucian carps were detected. It was proved that the triploid crucian carps of0.1g/kg group possessed the highest weight rate and the growth rate, which was1.47and1.22times to that of the control and was extra significantly higher (p<0.01). However, the test group of triploid crucian carps with the largest dosage0.2g/kg possessed1.41and1.18times of add-on rate and the certain growth rate to that of the control with a decreasing trend. The probiotics could significantly regulate the expression of the triploid intestina GDî–’APN and PepTl. There is a correlation between the expression of these genes and intestinal location and protein concentration.The results of these studies may clarify the mechanism of action of the feed additive probiotics, and which can provide a theoretical reference for improving the feed formulations in triploid.
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