Carp Cyprinus Carpio IECs GLS And TOR Gene CDNA Clone And Gln To IECs Protein Synthesis Influence And Mechanism Research

In this study,using research model of suspension primary carp intestinal epithelial cells (IECs) by established in this study,combining with isotope tracer technique,molecular cloning, bioinformatics and fluorescence quantitative PCR method,the gene eDNA sequence of target of rapamyein(TOR),which is a signal regulation molecular of protein synthesis in intestinal epithelial cells(IECs),and glutaminase(GLS) were cloned,GLS and TOR gene expression and the GLS activity in the carp intestinal tract different section,further the influence of L-glutarnine(Gin) on carp intestines different section IECs ability of protein synthesis and mechanism were studied.The results are as follows:1.Established the research model of carp suspension primary IECs.The results showed that IECs isolated from carp intestine were round under light microscopy,the polarization characteristics can be seen clearly.A high proportion of IECs,representing the total number of cells,95%,cell viability,93%.And the extraction DNA from isolation IECs is purity,the extraction total RNA structure is complete.2.The carp GLS eDNA was cloned.This sequence contains from start eodon 1 to the terminal termination codon 1788bp opening reading frame,coded includes 595 amino acid residue GLS protein precursor,molecular weight 65.96kDa.The nucleotide sequence with the zebrafish homology is 89.9%,with the original chicken homology is 69.4%,with mouse K-and the L-GLS homology respectively is 70.2%and 65.2%,with rat K-and the L-GLS homology respectively is 70.4%and 65.9%,with human K-and the L-GLS homology respectively is 69.8%and 64.3%.The protein structure analysis indicated that carp GLS amino acid sequence includes two very conservative regions,respectively GLS protein family region from 171 to 457 amino acid residue and anchor protein repetitive sequence from 484 to 570 amino acid residues.118,355,409,438 and 502 serine residue phosphorylation position spot,205 threonine residue phosphorylation position spot and 393,511 tyrosine residue phosphorylation position spot of GLS are highly conservative and important to the GLS function regulation.The GLS protein includes 18 cysteine residues,and has highly conservative 10 cysteine residues,which locates at GLS protein family region and anchor protein repetitive sequence,relates with GLS protein molecular structure,stable and subcellular localization. 3.The carp TOR cDNA was cloned.This sequence contains from start codon 1 to the terminal termination codon 7548bp opening reading frame,coded includes 2515 amino acid residue GLS protein,molecular weight 286.03kDa.The nucleotide sequence with the ,-'ebrafish homology is 92.0%,with the original chicken homology is 78.0%,with the platypus, mouse and rat homology respectively is 74.5,78.4 and 78.5%,and with human homology is 78.6%.The protein structure analysis indicated that Carp TOR protein includes 5 structure region.Between amino acid residues 1-348 of the HEAT repeat,between 349-2515 amino acid residue of PIKKs protein family's domain.The PIKKs protein family's domain includes between 1365-1948 amino acid residue is FAT.domain,between 1985-2078 amino acid residues is the FRB domain(FKBP 12-RAP complex binding domains),between 2119-2397 amino acid residues is kinase catalytic region,between 2485-2515 amino acid residue is FATC domain,which domains are very conservative and play vital role to the TOR protein's spatial conformation and the biological activity.The N-terminal 93I-1039 amino acid residue sequence includes the endoplasmic reticulum and the golgiosome localization sequence, which is very important to TOR protein located in the endoplasmic reticulum and Golgi apparatus membrane. 4.Alanine aminotransferase(AIaAT),aspartate aminotransferase(AspAT),glutamate dehydrogenase(GDH),malate dehydrogenase(MDH) and lactate dehydrogenase(LDH) activities in carp different intestinal section have been determined.The results showed that the AIaAT activity in 5 intestinal section is significant higher than other various intestinal section (P＜0.05),2 intestinal section enzyme activity is significant higher than 1-2,4 and 6-7 intestinal sections(P＜0.05),2,4 sections activity is significant higher than 1,6-7 sections(P＜0.05),1,6-7 section activity is not significant difference(P＞0.05).The AspAT activity in 2 section is significant higher than other various sections(P＜0.05),3-5 sections is significant higher than 1,6-7 sections(P＜0.05),1,6-7sections is not significant difference(P＞0.05).The GDH activity in 3 section is signifcantly higher than other various intestines sections(P＜0.05),2, 4-6 sections is not significant difference(P＞0.05),but significant higher than 1 and 7 section (P＜0.05),1 and 7 section is not significant difference(P＞0.05).The MDH activity in 3 scection is significant higher than other various section(P＞0.05),2 section is significantyl higher than 1,4-7 sections(P＞0.05),4 section is significant higher than 1,5-7 sections (P＞0.05),1 and 5 section is not significant difference(P＞0.05),but is significant higher than 6-7 sections(P＞0.05),6-7 section is not significant difference(P＞0.05).The LDH activity in 2 scection is significantly higher than other various sections(P＞0.05),4 section is significantyl higher than 1,3,5-7 sections(P＞0.05),3 section is significantly higher than 1, 5-7 sections(P＞0.05),1 and 5-7 section is not significant difference(P＞0.05).The result indicated that AlaAT,AspAT,GDH,MDH and LDH activities are higher in carp midintestine (2-5 intestines sections).5.The GLS activity and gene expression in carp different intestines section was studied. The results showed that GLS activity in carp intestine 3-4 sections is significantly higher than 1-2 sections and 6～7 sections(P＜0.05),5 section is significantly higher than 1,6-7 sections (P＜0.05);2 section is significantly higher than 1,7 sections(P＜0.05);6 section is significantly higher than 1,7 sections(P＜0.05);1 section is significantly higher than 7 sections(P＜0.05).The GLS the mRNA expression quantity in 3-4 intestinal sections is significantly higher than 7 sections,other various sections is not signifcant difference.The result indicated that the GLS activity and the mRNA expression quantity in the carp midintestine(2-5 sections) are higher.6.The TOR gene expression in different intestinal section of different body weight carp was studied.The results showed that 1 section,18.1g group TOR the mRNA relative expression quantity is significantly lower than other weight groups(P＜0.05),between 18.1g and 31.1g group as well as between 40.6,58.0,and 73.9g groups were no significant differences(P＞0.05);2-5 sections and 6-7 sections,18.1g and 31.1g group TOR mRNA relative expression level was significantly lower than the other weight group(P＜0.05),18.1g and 31.1g group as well as between 40.6,58.0,and 73.9g groups were no significant differences(P＞0.05).40.6,58.0,73.9g group relative TOR mRNA expression in different section was no significant difference(P＞0.05),but 18.1g group,1 section was significantly higher than 6-7 sections,31.1g group 1 section was significantly higher than 2-5 sections and 6-7 sections(P＜0.05).The result indicated that with weight increasing,TOR the mRNA expression quantity in carp intestinal tract increased,simultaneously the fore and midgut expression quantity is higher in the young age stage(before 30g).7.The protein synthesis ability in different section IECs of the carp intestinal tract and the influence of Gin on IECs synthesis ability of the different intestines section were studied. The results showed that 2-3 sections is significantly higher than other section(P＜0.05),but not significant difference each other(P＞0.05);1 section is significantly higher than 4-7 sections(P＜0.05);4 section is significantly higher than 5-7 sections(P＜0.05),5-7 sections is not significant difference(P＞0.05).IECs protein synthesis rate of 1-3 sections,1mg/L Gin group is significantly higher than 0mg/L Gin group(P＜0.05),4-7 sections,lmg/L and 0mg/L Gln group is not significantly difference(P＞0.05).The result indicated IECs protein synthesis ability of the carp fore and midgut(1-3 section) is higher than hindgut(5-7 sections);Gin promoted fore and midgut(1-3 section) IECs protein synthesis ability,but has on influence on hindgut(5-7 sections).8.Studied the influence of TOR inhibitor RAP,GLS inhibitor DON and Gin on IECs protein synthesis ability.The results showed that protein synthesis rate of 1mg/L Gin group was significantly higher than 0mg/L Gin group(P＜0.05),lmg/L Gln+RAP,DON or RAP+DON group were significantly lower than 1mg/L Gln group(P＜0.05),higher than 0mg/L Gin group(P＜0.05).1mg/L Gln+DON group was significantly higher than 1mg/L GIn+RAP and 1mg/L Gin RAP+DON Group.1mg/L Gln+RAP group was significantly higher than 1mg/L Gln+RAP+DON Group.The results indicated that GLS and TOR signaling molecules are involved in regulation of Gin improving the ability of fish IECs protein synthesis.9.The influence of Gin on the GLS gene expression of carp IECs was studied.The results showed that 30-120min,1mg/LGln group GLS mRNA relative expression was significantly higher than 0mg/LGln group(P＜0.05).1mg/LGln group,120min GLS mRNA relative expression was significantly lower than 70min(P＜0.05),but no significant differences with 30min(P＞0.05);0mg/LGln group,30-120min,GLS mRNA relative expression difference was not significant(P＞0.05).The results indicated that Gln can influence GLS gene expression of carp IECs.10.The influence of Gln on the TOR gene expression of carp IECs was studied.The results showed that 1mg/L Gin group IECs TOR mRNA expression was significantly higher than 0mg/L Gin group(P＜0.05),Gln 1mg/L+RAP group was significantly lower than 1mg/L Gin group(P＜0.05),but no significant difference with 0mg/L Gln(P＞0.05),Gln 1mg/L+DON group was no significant difference with 1mg/L Gin group(P＞0.05),but significantly higher than Gin 1mg/L+RAP group and 0mg/L Gln group(P＜0.05).15min group IECs TOR mRNA relative expression was significantly higher than 5min group(P＜0.05);30min group was significantly higher than 5min group(P＜0.05),but was no significant difference with 15min group(P＞0.05).The results indicated that Gln through TOR signaling molecules regulating TOR gene expression,increase TOR mRNA expression levels. GLS did not take part in Gln on the regulation of TOR gene expression process of carp IECs.In summary,TOR,this is a signal regulation molecular of protein synthesis in IECs, esists in carp intestine.With the weight gain,TOR mRNA expression in the intestine gradually increase,the fore and midgut expression quantity is higher in the young age stage (before 30g).The carp fore and midgut IECs protein synthesis ability is higher,Gln promoted fore and midgut IECs protein synthesis ability.Gln improves IECs protein synthesis ability to receive TOR and the GLS regulation.