Cloning, Mrna Expression Analysis And Regulation Research In Vivo Of Full Length Type Collagen CDNA From â…  Grass Carp (Ctenopharyngodon Idellus)

Collagen are a class of fibroid and distinct supramolecular structural proteins associated with the extracellular matrix of connective fibrotic tissues of most vertebrates, which is secreted by the connective tissue cells and other types of cells (such as liver, lung, spleen and brain cells), its main function is the junction protein as cytoskeleton of three types fibers (microtubules, microfilaments and intermediate fibers). In muscle tissue of fishes, collagen plays an important role in maintaining the texture of fish muscle and the formation of muscle fibers as a structural protein of muscle connective tissue. the crisp, toughness and hardness of Grass carp (Ctenopharyngodon idellus) muscle increased significantly by feeding natural plant Vicia faba L. Due to this, indigene call it as " crisp grass carp " generally. Previous studies have shown that the collagen contents were significantly increased in muscle from crisp grass carp .In order to explore the collagen regulation mechanism which broad bean acts on grass carp muscle, the present study focused on collagen typeⅠ,the main component of collagen, from grass carp muscle. In the study , we cloned the full-length cDNA ofα1 andα2 genes (COL1A1, COL1A2) which are encoded collagen typeⅠof grass carp, analyzed amino acid sequence and homology of the two genes; the bioinformatics such as physico-chemical parameters of COL1A1 and COL1A2 protein peptide were analyzed by using ProtParam software on line; the expression patterns of the two genes mRNA in the 8 tissues from grass carp and crisp grass carp had been analyzed by using semi-quantitative RT-PCR technique; based on the qRT-PCR and Western blot technology, the expression of COL1A1 and COL1A2 in muscle from grass carp and crisp grass carp had been analyzed in mRNA and protein levels. The main research results include:1 Molecular cloning and analysis of full length typeⅠcollagen cDNA from grass carp (Ctenopharyngodon idellus)In this present study, total RNA was isolated from muscle of Ctenopharyngodon idellus and COL1A1 and COL1A2 cDNAs were obtained by Reversed Transcript PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The whole cDNA length of COL1A1 is 5772bp, consisting of a 4347bp open reading frame (ORF) which encodes 1449 amino acids. The COL1A2 cDNA is 4899bp in length, consisting of a 4059bp ORF and encoding 1353 amino acids. Amino acid sequence of COL1A1 and COL1A2 genes had been analyzed by using SignalP software on line, the results showed that amino acid sequence of COL1A1 and COL1A2 genes both had one 24 amino acid signal peptide. They are M1-G24 and M1-S24 respectively. Homology aligntment of two amino acid among grass carp and other species showed that grasp carp had the maximum homology of 93.90%,95.00% and 93.60%,94.40%with zebra fish and goldfish, which had a highest similarity with grass carp. Moreover, In phylogenetic tree constructed based on COL1A1 and COL1A2 amino acid sequences, the 2 genes were firstly grouped to zebrafish and goldfish, which were more closely related to each other than to COLlA1 and COLlA2 from other species.2 Bioinformatic analysis of COLlA1 and COLlA2 protein peptide from grass carp (Ctenopharyngodon idellus)The physico-chemical parameters of COL1A1 and COL1A2 protein peptide were analyzed by using ProtParam software on line, the results showed that the protein peptide molecular weight were 137.2KD and 127.4KD respectively, the Theoretical pI were 5.44 and 9.37 respectively. By using PriPred software on line, the secondary structure characterization of COLlA1 and COLlA2 protein peptide analysis showed that the two protein peptides possess 6 and 5α-helixes,12 and 8β- sheetes respectively ,others are ruleless coil regions. By using ProSite software on line, the domains,functional sites of COL1A1and COL1A2 protein peptide analysis showed that the two protein peptides possess 18 and 16 regions of triple helical repeats, 22 and 21 low complexity regions,17 and 10 function domains respectively.By using ProtScale software on line, the hydrophobicty and hydrophilicity of COLlA1 and COLlA2 protein peptide analysis showed that the Grand average of hydropathicity of two protein peptides all is -0.739, which indicate hydrophobicty of the two protein peptides is high. The instability index is computed to be 25.15 and 24.28 respectively,the function analysis of microelement banding showed that COL1A1 protein peptide possess 2 calcium-binding sites and one zinc-binding sites, COL1A2 protein peptide possess one zinc-binding sites only.3 Tissue expression and regulation research of COL1A1and COL1A2 mRNA from grass carp (Ctenopharyngodon idellus) by feeding Vicia faba LBy semi-quantitative RT-PCRmethod, COL1A1and COL1A2 gene mRNA were determined in 8 tissues( muscle, intestine, hepatopancreas, gills, skin, fins, kidney and spleen tissues ) of grass carp and crisp grass carp. The results showed that 2 genes were expressed widely in 8 tissues of grass carp and crisp grass carp. the expression content of 2 genes mRNA in gill,kidney,skin and fin were significantly higher than other tissues (p <0.05). The results of crisp grass carp tissues showed that the expression contents of COL1A1 genes mRNA in skin and fin were significantly higher than other tissues (p <0.05), at the same time, it's lower in hepatopancreas and spleen tissues (p<0.05). The expression content of COL1A2 genes mRNA in gill,skin and fins were significantly higher than other tissues (p <0.05), at the same time, it's lower in kidney and spleen tissues (p<0.05). The comparison of expression of COL1A1 and COL1A2 in different tissues of crisp grass carp and grass carp by RT-PCR showed that these tissues of crisp grass carp were higher than these in the grass carp except intestine,kidney and spleen tissues.4 Based on the quantitative real-time RT-PCR and western bloting technology, analyzed the expression content of COL1A1,COL1A2 mRNA and protein levels from grass carp and crisp grass carpThe expression patterns of the COL1A1 and COL1A2 mRNA were detected using realtime-PCR in muscle from grass carp and crisp grass carp. The results showed that the expression content of 2 genes mRNA in muscle of crisp grass carp are 2.7 and 2.1time than grass carp respectively (p<0.05). The COL1A1 and COL1A2 protein levels were detected using western blotting technology in muscle from grass carp and crisp grass carp. The results showed that the expression content of 2 genes protein in muscle of crisp grass carp were significantly higher than grass carp (p<0.05).Above all, full length typeⅠcollagen cDNA from grass carp (Ctenopharyngodon idellus) was cloned and its Bioinformatic was analyed. Furthermore, typeⅠcollagen (COL1A1 and COL1A2) widely expression in 8 tissues ( muscle, intestine, hepatopancreas, gills, skin, fins, kidney and spleen tissues ) of grass carp and crisp grass carp was observed for first time. This study discussed the expression regulation of typeⅠcollagen from grass carp under the effect of Vicia faba L primarily,and researched the expression regulation of collagen and typeⅠcollagen under the effect of Vicia faba L in gene and protein levels from grass carp ulteriorly. Undoubtedly, the results of this study provided basic data to explore the molecule regulation mechanism of collagen under the effect of Vicia faba L from grass carp muscle, which also provided the new research direction for the nutrition regulation of collagen from grass carp.