A Study On Light Chain Sequence Of IgM In Five Species Of Salmonidae

Fish is an organism which evolves the immune globulin first,great changes was reflected in the evolution boundary of fish immunoglobulin gene sequence,and the origin of salmon fishes is the special geographical period,so the study of salmon fishes immunoglobulin gene sequence do a great favor to the ancient geography,ancient ecology,climate change,and so many other aspects such as the evolution system of the vertebrates.Therefor,this text deteced the immunoglobulin light chain sequence of four kinds of salmon fish such as Oncorhynchus mykiss aguabonita(USA), Oncorhynchus masou masou,Hucho taimen Pallas,Salvelinus leucomaenisf pluvius by molecular biology,bio-informatics and so on,and do sequence similarity comparison and functional analysis with the rainbow trout(Oncorhynchus mykiss Walbaum).First of all,obtain the total RNA from spleen of Hucho fish the major immune organs,and useRT-PCR technique to obtain its immunoglobulin(immunoglobulin,Ig) light chain cDNA clones, detected the immunoglobulin light chain sequence with 51 randomly selected positive clone colonies.Obtain 46 sequences which was complete and non-repetitive,the results comparing with BLUAT internet showed that it's similarity with the sequence X68517.1 reported on GenBank is as high as 90%;by DNAstar software analyze the amino acid sequence,the sequences automatically cluster into two clusters,the amino acid sequence homology is more than 75%in each cluster, while the amino acid sequence similarity is 40.9%in the boundary,infered that the Ig which come from this experiment should be from two different family.From structural domain variability analysis of the nucleotide sequences,we can see that in the Hucho fish Ig LV the part of framework (FR) is relatively conservative,has a smaller mutation rate;the part of complementarity determining regions(CDRs) has a higher mutation rate,especially CDR3 area.There are different link methods in the CV link locus of Hucho fish immunoglobulin light chain,so the diversity of Hucho fish immunoglobulin light chain is more abundant.Secondly,amplify and detect the immunoglobulin(IgM) light chain nucleotide sequence which come from four kinds of salmon fishes:Oncorhynchus mykiss aguabonita,Oncorhynchus masou masou,Hucho taimen Pallas,Salvelinus leucomaenisf pluvius with a pair of highly specific primers,then analyze the genetic information contained in them and compare with the rainbow trout(Oncorhynchus mykiss Walbaum) homologous nucleotide sequences(from NCBI) which had been published,at the same time carried out the evolution and inter-relationship analysis between the five kinds of salmon fishes.Nucleotide homology comparison revealed that:Salvelinus leucomaenisf pluvius and Oncorhynchus masou masou,Oncorhynchus mykiss aguabonita and rainbow trout constituted two various branches which ware opposite to Hucho taimen Pallas.Nei's D_A genetic distance analysis showed:Salvelinus leucomaenisf pluvius had the closest genetic distance with Oncorhynchus masou masou while the farthest genetic distance with the rainbow trout.By the amino acid sequence comparison in the five kinds of salmon fishes immunoglobulin light chain variable region we can see that Salvelinus leucomaenisf pluvius had the highest sequence similarity in contrast to other species;the highest and lowest similarity appeared in the CDR1 and CDR3 in the similarity of CDR District while appeared in the FR4 and FR3 in the similarity of FR District.The results of this experiment not only reflected the physical and chemical properties of the Hucho fish immunoglobulin,but also identified that there were plenty of immunoglobulin types even in the same individual,perhaps it is because the rearrangement of primary RNA transcription complex of the immunoglobulin took place in the editing process,or it may be because the B lymphocytes have taken place differentiation in the process of transfer to the different organizations,that is,a high degree of somatic cell mutation leading to immunoglobulin diversity.By the research in the immunoglobulin Physical and Chemical Properties of 5 kinds of salmon fishes we saw that,their Physical and Chemical Properties can not be exactly same even if it was the same kind of fish,it may be caused by their different growth-environmental and geographical distribution;in Comparison of its nucleotide sequence we found that besides Oncorhynchus mykiss aguabonita and rainbow trout which had the highest basyl similarity,the other three kinds of fishes had larger basyl diversity,but the amino acids of the five kinds of salmon fishes all had high similarity in the protein polypeptide chain which could indicate that although the basyl had idiovariations in evolution of salmon fishes,but there were not obviously diversify on the amino acid sequence of the merger protein polypeptide chain and physico-chemical properties,so the nucleotide sequence of immunoglobulin reflected the link of the classifications place of the salmon fishes to be researched and the species evolution time.