| RNase1 is one of the most important pancreatic secretion digestive enzymes,and is belong to RNase A superfamily.Gene duplication of RNase1 in leaf monkeys is closely related to its leaf-eating diet,and RNase1 becomes one of the hottest gene in learning feeding habits.Cyprinidae fish are important economic fish in China with wide feeding habits.In this study,RNase1 gene contained complete CDS of 8 cyprinidae fishes were cloned.The mRNA expression levels were determined by real-time quantitative PCR.In addition,the protein expression levels of different tissues were analyzed by western blot.(1)The protein coding region(CDS)of RNase1 was amplified using Genome walking technology.Bioinformatics analysis showed: The length of amplified RNase1 of Cluter alburnus was 940 bp,the CDS was 447 bp coding 149 amino acids,the predicted molecular weight was 17.49 KDa,and the theoretical isoelectric point(PI)was 9.10;The length of amplified RNase1 of Xenocypris davidi was 1401 bp,the CDS was 456 bp coding 152 amino acids,the predicted molecular weight was 17.49 KDa,and the theoretical PI was 9.10;The length of amplified RNase1 of Aristichthys nobilis was 2014 bp,the CDS was 456 bp coding 152 amino acids,the predicted molecular weight was17.33 KDa,and the theoretical PI was 8.53;The length of amplified RNase1 of Hypophthalmichthys molitriac was 2756 bp,the CDS was 456 bp coding 152 amino acids,the predicted molecular weight was 16.80 KDa,and the theoretical PI was 9.05;The length of amplified RNase1 of Ctenopharyngodon idellus was 1033 bp,the CDS was 456 bp coding 152 amino acids,the predicted molecular weight was 16.80 KDa,and the theoretical PI was 9.05;The length of amplified RNase1 of Megalobrama amblycephala was 856 bp,the CDS was 456 bp coding 152 amino acids,the predicted molecular weight was 17.32 KDa,and the theoretical PI was 9.11;The length of amplified RNase1 of Elopichthys bambusa was 1439 bp,the CDS was 456 bp coding 152 amino acids,the predicted molecular weight was 17.40 KDa,and the theoretical PI was 9.11;The length of amplified RNase1 of Cluter alburnus was 1643 bp,the CDS was 456 bp coding 152 amino acids,the predicted molecular weight was 17.26 KDa,and the theoretical PI was8.94.BLAST analysis showed the similarity of the 8 amino acids sequences was between65.7% to 99.2%.The length of signal peptide sequence was about 24 amino acids.All these sequences have the “CKXXNTF” signature motif,ribonuclease activity required catalytic triad(His12–Lys41–His119)and conserved cysteine residues.The NJ treeshowed M.Amblycephala RNase1 and C.Alburnus RNase1 has the closest relation,E.bambusa RNase1 and X.Davidi RNase1 has the closest relation,A.Nobilis RNase1 and H.Molitriax RNase1 has the closest relation,and they have the largest relation with medaka RNase1.(2)?-actin was used as the reference gene in real-time RCR.The expression level of RNase1 in muscle was most in A.nobilis,least in M.amblycephala.The expression levels of RNase1 in gonads,heart,liver,spleen,kidney were significantly different in H.molitrix and A.nobilis compared with other fishes(P<0.05).The expression level of RNase1 in gills,brain,intestine were significantly different in A.nobilis compared with other fishes(P<0.05).The expression level of RNase1 in blood was significantly different in C.alburnus compared with other fishes(P<0.05).The expression level in intestine was highest in all fishes except E.bambusa.The expression levels in blood in H.molitrix and A.nobilis were higher than that in other fishes.(3)We prepared the antibody of M.amblycephala RNase1 using rabbit as the immune animal,and research the expression levels of protein in different tissues of M.amblycephala,C.carpio,C.alburnus and A.nobilis.The result of western blot showed RNase1 was only expressed in liver and intestine in 4 fishes.In M.amblycephala,C.carpio and C.alburnus,the protein expression level in intestine is higher than the expression level in liver.In A.nobilis,the protein expression level in liver is higher than the expression level in intestine. |
PDF Full Text Request