Cloning Of Key Genes For Yolk Deposition Of Chinese Sturgeon (Acipenser Sinensis) And The Research On Their Responses To Different Dietary Lipid Levels

Chinese sturgeon?Acipenser sinensis?is large reproductive migratory species,which is native to Yangtze River of China.Besides,It is also a rare and endangered species,so artificial propagation for the species protection of Chinese sturgeon has already been developed in China for many years.However,the sexual maturation in controlled breeding is limited to be obtained as it is hard for Chinese sturgeon to develop from stage II to stage III,which is a key stage of vitellogenic deposition.Thus,in order to protect this species better,it is very essential to investigate themolecular mechanism of vitellogenic deposition and hydrolysis as well as nutrition regulation to the poor sexual maturity.In this article,some key genes about ovary development were firstly and systematically isolated and characterized from Chinese sturgeon?Acipenser sinensis?.In addition,it also explored the effects of dietary lipid levels on ovary development of Chinese sturgeon according to their ovary development status and gene expressions analysis after one-year feeding trial.The main results are shown as follows:1. Identification,m RNA expression profiling and characterization of vitellogenic deposition and hydrolysis related genes in ovary from Chinese sturgeonThe full-length c DNA sequences of vitellogenic deposition and hydrolysis related key genes?vtgr,atp6v1c1,atp6v1h,ctsb,ctsd and ctsl?were firstly and systematically isolated and characterized from Chinese sturgeon Acipenser sinensis.Their complete c DNA sequences contained 3640 bp,1638 bp,1751 bp,1317 bp,1483 bp and 1438bp,respectively.And their c DNAs consisted of an ORF of 2568 bp,1152 bp,1434 bp,1020 bp,1191 bp and 1020 bp,respectively and encoded amino acid sequences of 855,383,477,339,396 and 339 aa,respectively.Alignment of predicted polypeptide sequences indicated that amino acid sequences of Chinese sturgeon Vtgr,Atp6v1c1,Atp6v1h,Ctsb,Ctsd and Ctsl were similar to those from other vertebrates,exhibiting76.4-85.7%,93.2-97.1%,93-97.7%,75.9-83.2%,66.4-92.7%and 71.5-87.9%amino acid sequence similarity and 70.4-81.5%,88-93.5%,89.2-94.8%,68.5-78.2%,58.9-86.1%and 63.2-80.5%amino acid sequence identities,respectively.The domain analysis showed that the protein sequence of Chinese sturgeon VTGR possesses eight cysteine-rich Ligand-binding domains?LBD?,five YWTD?-propeller domains,three epidermal growth factor-like domains?EGF 1,2 and 3?,a transmembrane domain?TM?and a cytoplasmic domain?TM?and a cytoplasmic domain?CD?.The protein sequence of Chinese sturgeon ATP6V1C1 contained a similar architecture with mammals,including seven casein kinase II phosphorylation sites,five N-myristoylation sites,four protein kinase C phosphorylation sites,two tyrosine kinase phosphorylation sites,one amidation site,two O-glycosylation sites and one membrane helix.The protein sequence of Chinese sturgeon ATP6V1H possessed five repeats and twenty seven?-helices.Similar with the mammals,Chinese sturgeon CTSB,CTSD and CTSL all possessed a signal peptide.The protein sequence of Chinese sturgeon CTSB contained a similar architecture with mammals,including three conservative active sites(C¹⁰⁸,H²⁷⁸,and N²⁹⁸),a oxyanion hole(Q¹⁰²)of cysteine proteinase and a conserved fragment of occluding loop in the mature peptide sequence,which formed the typical domain architecture of cysteine proteinase.Multiple alignment of the Chinese sturgeon CTSD sequence with those from other species indicated that the two N-glycosylation sites and two active sites of the deduced protein were well conserved.The propeptide,and aspartyl protease domain were found in Chinese sturgeon and other vertebrates.The alignment also indicated the characteristic ERWNIN motif in the proregion of cathepsin L was conserved in vertebrates including Chinese sturgeon.The m RNAs of these genes mentioned above were highly expressed in ovary of stage?and stage?and they also expressed in other various tissues at different levels.According to the amino acid sequence similarity and homology,domain and phylogenetic analysis,Chinese sturgeon and spotted gar had the closet relationship.All in all,these genes were relatively conservative in evolution.These results will be beneficial for us to elucidate the molecular basis of Chinese sturgeon ovary development.2. A series of effects of dietary lipid levels on ovary development of Chinese sturgeon broodstockA one-year feeding trial was conducted to determine the effects of dietary lipid level on growth,gonad development and vitellogenesis-related gene expressions exploring the mechanism of lipid on ovary development.Three isonitrogenous experimental diets were compounded with different lipid levels?10%,14%and 18%,respectively?.Results of the feeding trial showed that the ovary developed as lipid levels,even two Chinese sturgeon in the 18%group was in stage?and the thickness of ovary area in longitudinal views?d?and the ovary area proportion?Po?of the whole reproductive organs in itransversal views all increased as lipid levels.Besides,there was significant collection between ovary development and the lipid levels in dietary by chi-squared test.In addition,dietary lipid promoted ovary development and elevated the expressions of vtgr,atp6v1c1,atp6v1h,ctsb and ctsd in the ovary in a dose-dependent manner.These results indicated that those up-regulated genes might play essential roles in vitellogenic deposition and hydrolysis in response to dietary lipid.This study did not only explore the molecular characterization and function of vitellogenic deposition and hydrolysis related genes,which would be beneficial for us to elucidate the molecular basis of Chinese sturgeon ovary development but also provide novel insights on the nutrition of broodstock.