ENU-induced Mutagenesis In Grass Carp(Ctenopharyngodon Idellus) And Cloning And Function Analysis Of Duplicated Fgfrl1 And Igfbp-5 Genes

The grass carp(Ctenopharyngodon idellus) is the largest single amount of aqucultural fish species in China, it has a big size with a long cycle of sexual maturity. Traditional way of breeding needs higher cost and longer time. The genome of grass carp still exists quite a number of main effect genes which function is unknown. Study their function has important theoretical significance and application value for economic properties such as reproduction, growth, development and tolerance of grass carp.Chemical mutagenesis has been widely applied in breeding of crops and vegetables, Because of the characteristic of the vitro fertilization of fish, we can soak the fertilized egg at different times to obtain mutant. The using of the Alkylating agent is the main method. The recent study shows, Valuable biological function mutants can obtain from model organisms by ENU- induced mutagenesis. The fertilized eggs of grass carp were treated with a range of ENU concentrations for 15 min, the deformity rate was 20.3%~78.5%, the hatchability was 86.3%~17.2%, the results indicated that the proportion of embryos with morphological abnormalities at segmentation increased with increasing ENU dose. We treated the fertilized eggs with 0.5mmol ENU, the deformity rate of embryos at segmentation was 50%, and the ENU-traeted F1 population showed difference in growth during the first year. Analysis of DNA from 20 F1 ENU-treated individuals for mutations in partial coding regions of mstn-1、mstn-2、fst-1、fst-2 loci revealed that ENU-treated can induced single nucleotide mutation. The average mutation rate at the examined loci was 0.13%. ENU-treatment of fertilized eggs can efficiently induce point mutations in grass carp, which has a potentially useful way for fish breeding.FGFRL1, fibroblast growth factor receptor like 1, belongs to the member of the FGFR family. Fibroblast growth factor receptor like 1 is a novel FGF receptor lacks the intracellular tyrosine kinase domain. Studies shows that it play an important role in cell proliferation, musculoskeletal system development. In the present study, we duplicated the c DNA of fgfrl1 genes from grass carp by RACE. The size of the fgfrl1 a c DNA was 3,405 bp with a 1,461 bp open reading frame(ORF) which can encod a 486 amino acid residues. The size of the fgfrl1 b c DNA was 2,666 bp with a 1458-bp ORF which can encoded a 485 amino acid residues. Both Fgfrl1 a and-1b contain three Ig-like domains, a transmembrane domain, two conserved tyrosine residues and a peculiar histidine-rich intracellular domain. They included six conservative cysteine residues. They shared high identities of 96% and 91% with zebrafish orthologs, respectively, the amino acid sequence identity between grass carp Fgfrl1a/-1b and human FGFRL1 was relatively low(66% and 59% separately), but it is only 56% between Fgfrl1 a and-1b. The result of q RT-PCR shows that both fgfrl1 a and-1b could be detected in the zygotes, it increased gradually during somitogenesis, then reduced. In adult fish, both fgfrl1 a and-1b expressed in all tissues, but the tissues they expressed in were different.The fgfrl1 a transcript was abundantly expressed in the heart, brain and muscle, while the fgfrl1 b m RNA showed obvious expression in the eyes, muscle and gill. Using in situ hybridization, fgfrl1 a transcripts were detected in the notochord and somite, Meanwhile, fgfrl1 b was transcribed mainly in the endoderm and lens. Juvenile grass carp were fasted for 6 days and then refeeded,the result shows that both fgfrl1 a and-1b m RNAs were significantly(p<0.05) up-regulated in the muscle and brain.the highest level appeared in 6-day of fasting, then it restored to the control level following re-feeding. Both fgfrl1 a and-1b m RNAs were down-regulated after recombinant h GH treatments, and the reduction degrees were positively related to the injecting doses. Our study lay the foundation to explore the gene function of fgfrl1 s and molecular breeding on grass carp.Insulin-like growth factor binding protein(IGFBP-5) is a secreted protein that binds to IGF and modulates IGF actions. IGFBP-5 is the most conserved member of the IGFBP family. Here, we report the spatial expression of IGFBP5 genes in adult fish. The igfbp-5a m RNA expression in liver was the highest, followed by the brain, higher expression in the kidney and eyes, and igfbp-5b m RNA expression was relatively high in muscle, liver, kidney, brain and eyes, other tissues showed lowexpression. Using in situ hybridization, igfbp-5a transcripts were detected in the somite and brain, meanwhile, igfbp-5b was transcribed mainly in notochord, somites and brain. Juvenile grass carp were fasted for 6 days and then refeeded, the result shows that igfbp-5a and igfbp-5b m RNA expression showed different tends in different tissues, in the liver and muscle with an obviously increased and then recovered, in the brain is decreased at first then increased to nomal levels. After the injection of exogenous h GH, igfbp-5a and igfbp-5b m RNA increased in muscle, but decreased in brain. These results indicate that igfbp-5s have special function or signaling pathways in different tissues.