A Preliminary Study On Insulin/IGF Signaling(IIS) Pathway Related Genes And Their Effects On Growth Of Litopenaeus Vannamei

Insulin signaling(IIS)pathway plays an important role in the growth,development,metabolism,reproduction and longevity of the different organism.By analyzing the genomics and transcriptomics data from Litopenaeus vannamei,we screened a series of IIS-related genes,such as IGFBP,IR,ALS,GSK3 and Fox O sequences in vivo.These results indicated that a signaling pathway which is similar to the IIS of vertebrates exist in shrimp.This signaling pathway could regulate the growth,development,metabolism and survival in shrimp,as well as that of in vertebrates.Due to the large differences in physiological function between crustaceans and vertebrates and the lack of model organisms,it is difficult to research the composition and functional mechanism of IIS in crustaceans.L.vannamei is one of the most important mariculture species,its growth traits become the most compelling characteristics.In this study,we analyzed the structural evolution and expression characteristics of IIS pathway related genes in L.vannamei by using bioinformatics methods,and verified their growth functions in vivo experiment.The results of this study provide a theoretical basis for elucidating the effects of IIS pathway related genes on the growth of L.vannamei and breeding superior varieties,and also provide new clues for further research on the crustacean IIS pathway.The thesis mainly research on the following two aspects:1.The structure and expression analysis of IIS pathway related genes.Some sequences annotated as IGFBP,IR,ALS,GSK3 and Fox O are screened by using bioinformatics technology from the genome and transcriptome of L.vannamei.Then,we analyse these sequences’ structure and phylogeny,and identify the accuracy of annotated sequence according to the conserved domain and the position of sequences related to other species on the phylogenetic tree.Analysing conserved amino acid sites and motif by using multi-sequence alignment to analyse the function of these sequences.According to the expression profiles of different early development stages,different molting stages and different tissues,we screen the most appropriate sequence to carry out ds RNA interference expression in vivo.Under the preliminary screening,we verify 3 IGFBPs,9 IRs,13 ALSs,1 GSK,and 1 Fox O complete gene sequences,respectively.Sequence structure analyse showed that Lv IGFBPs consisted of three domains : from N-terminal to C-terminal is IB(insulin growth factor binding)domain,Kazal-type serine protease inhibitor(Kazal)domain and immunoglobulin C2(IGc2)domain in turn;Lv IRs belong to tyrosine kinase receptors superfamily,they contain L1-Cys-L2 domain,three fibronectin Ⅲ domain(FN3),and tyrosine kinase catalysis(Tyr Kc)domain;Lv ALSs include 10-20 LRR domains;Lv GSK3 belongs to Ser/Thr kinase,and it contains a highly conserved Ser/Thr protein kinase catalysis(S＿TKc)domain;Lv Fox O has a conserved N-terminal Forkhead DNA-binding(FH)domain and a C-terminal Transactivation domain(TAD).Gene expression profiles analysis showed that Lv IGFBP1 exhibit high expression in most tissues and different developmental stages,while Lv IGFBP2 and Lv IGFBP3 are only slightly expressed in hemocytes;the expression level of Lv IR2 peak at P1 stage,and it is highly expressed in most tissues,except for the gonads;Lv ALS8 is began expressing from zygote to P1 stage,and it is highly expression in hepatopancreas,muscles,hemocytes and ovary;Lv GSK3 exhibit high expression in different early developmental stages,most adult tissues and pre-molting(D3)stage;Lv Fox O is highly expressed in most tissues,especially in muscles.2.The ds RNA interference experiment of IIS-related genes.This thesis aims to research the effect of IIS pathway related genes on shrimp growth.We use adult shrimp as the experimental material,so we choose the gene which highly expressed in P1 stage and muscles(Lv IGFBP1 、Lv IR2 、Lv ALS8、Lv GSK3 and Lv Fox O)to conduct ds RNA interference experiment.Each individual was injected with optimal dosage.The experiment lasted for two weeks.We measured the weight and length traits of the samples,and detected the interference efficiency by using real-time quantitative PCR.Data analysis indicated that after two weeks ds RNA interference experiment,the increment of body weight in the ds Lv IGFBP1 group was about 60% of the control groups,which was significantly lower than two control groups;the increment of body weight in the ds Lv IR2 group was 76% of control groups,which was slightly lower than that of control groups;in contrast,the increment of body weight in the ds Lv ALS8 group was 119% of control groups,which was slightly higher than that of control groups;the RNA interference of Lv GSK3 resulted in a significantly smaller increment of body weight than that of control groups,and affect the expression of molting related genes;the Lv Fox O RNAi resulted in a high fatality,the fatality rates of the two interferences were 75% and 50%respectively,which were significantly higher than those of the control groups.The final results suggested that Lv IGFBP,Lv GSK3 and Lv IR have a positive effect on growth to some extend;Lv ALS could inhibit the growth of shrimp;and Lv Fox O is essential for shrimp’s survival.In this thesis,we elucidated the structure of five IIS pathway related genes(IGFBP,IR,ALS,GSK3,Fox O)in L.vannamei and their effects on the growth of L.vannamei,which provided the data basis for the research of related genes in crustaceans in the future,and also provided some theoretical basis for molecular breeding.