The Physiological Regulation Of Acipenser Baerii Vitellogenesis And Annotation And Analysis Of Acipenser Sinensis ESTs

There are 27 existent species of sturgeons in the world. Sturgeon is one of the most ancient groups of actinopterygian fishes, which belongs to the order of Acipenseriformes, and the family of Acipenseridae, Acipenser. Chinese sturgeon, Acipenser sinensis, is an anadromous species that only spawns in the Yangtze River of China. Siberian sturgeon, Acipenser baerii, mostly presents in all of the major Siberian river basin from Ob River in the west to Kolyma River in the east. It becomes one of the most popular aquaculture fishes in china currently. The sturgeon eggs are the main ingredient for caviar. The development situation of sturgeon eggs are directly influence the caviar quality and the rate of return on investment and create higher profit of sturgeon breeding enterprise.Vitellogenesis is the process of yolk formation via nutrients being deposited into the oocyte. The yolk constitutes the main storage compartment in egg including amino acid, lipid, Ca2+, etc. And yolk also is the major internal food supply on which most embryos rely. Therefore, the vitellogenesis is crucial importance for animal reproductive development. Therefore, the study on physiological regulation of vitellogenesis will increase the understanding of sturgeon growth, sexual development, reproduction etc, and will also provide the theoretical foundation for sturgeon protection and cultivation.Over the past few decades, bioinformatics technology developed rapidly, which produced a tremendous amount of information related to molecular biology, and have became an useful tool to refine the important molecular information about biological growth, development and reproduction etc. Therefore, we analysis the sturgeon EST library to obtain more useful genomic information and provide insight into the molecular mechanisms of the biological process behind Chinese sturgeon.This study includes three parts as follow: 1. Study of Siberian sturgeon Acipenser baerii vitellogenesis, hormone regulation and lipid metabolismThis section with Siberian sturgeon as the research object, we observed the morphology during development, determined the blood steroid, lipid, lipase, total protein, Ca2+ and Pi level in different ovary developmental stage.Our results shown that serum E2, T, total protein (TP), very low density lipoprotein (VLDL), triglycerides (TG), Ca2+ and Pi concentrations were increased since stageⅡ(early vitellogenesis) to IV (late vitellogenesis), and then decreased till stageⅥ. Serum cholesterol (TC) and low-density lipoprotein (LDL) amounts were gradually increasing during ovary development. LPL activity in ovary increased during vitellogenesis with a higher level in stageⅣ. The result indicated that, in response to circulating E2, Vtg (Ca2+ and Pi concentrations can represent the serum Vtg and phospholipid level) and lipid (e.g. VLDL and TG) were induced synthesis, and relevant enzymes expression and activity were regulated (e.g. ovary LPL activity). E2 have reached the peak till stageⅣ, which induced the protein and lipid in a large amounts synthesis and with a highest concentration in blood stream. In this stage, oocytes greatly increase in size, most oocyte proteins and lipids are derived from blood Vtg and VLDL. During late vitellogenesis to spawning stage, the oocyte undergoing the final mature and almost having finished accumulating nutrition. The E2 synthesis greatly decreased, and so did the blood protein, lipid and ovary LPL activity.The variations of blood lipid concentration were also regulated by blood lipase. Serum LPL and HL activity were gradually increasing and insignificantly higher in stageⅤ, and then decreased. We also found that two serum lipases activity positively correlated with serum VLDL, TG and TC, and negatively correlated with high-density lipoprotein (HDL). The variation tendency of serum LPL, HL activity and lipids concentration, and the close correlationship between the two lipases and lipids during ovarian development suggested that LPL and HL participate in the regulation of blood lipid metabolism. The LPL and HL also play important role in TC, LDL and HDL metabolism during ovary development. 2. Vitellogenin gene cloning and serum Vtg protein detection during vitellogenesis in Siberian sturgeonIn this study, we cloned vitellogenin (Vtg) cDNA from Siberian sturgeon liver using three pair primers. The cDNA encodes the mature Vtg protein are include three fragments, LvⅠ, Pv and LvⅡ, and with Vitellogenin domain, VWFD domain and serine-rich domain domain. Siberian sturgeon The cDNA encodes the mature Vtg (AbVtg) protein which has share the highest similarity 97.6% with White sturgeon Vtg, and low similarity with other teleost fish. Results of the alignment of the deduced amino acids and validation of the tree topology according with the consanguinity relationships between different species, showing that teleost fish Aa-type Vtg and Ab-type Vtg clustered together, and then cluster with the teleost untyped-Vtg (these teleost havn’t developed to Aa, Ab or C type, which include sturgeon), amphibians, avians, cyclostome fish, and teleost C-type Vtg. The species mentioned above are all oviparous vertebrates, were cluster with all the invertebrate Vtg. The phylogenetic result reflects the evolutionary relationship of taxa to a certain extent.We found Vtg mRNA expression was induced in Siberian sturgeon liver under E2-treatment. The Vtg mRNA expression levels enhanced with increasing E2 dose. The result proved E2 inductive influence on Vtg, and also suggest Vtg mRNA expression levels can reflect the ovary development situation. But this method needs killing the fish for their livers, which were limited in application. Using the Vtg antibody to detect the Vtg expression levels in blood is the better way to solve this problem.Therefore, according to the sequence of Vtg gene, we constructed three prokaryotic expression plasmids (partial LvⅠ, Pv and partial LvⅡ) based on pET23 vector. The recombinant vector was transformed into BL21 (DE3). We optimized the expression condition. The LvⅠexpression recombinant protein (LvIE) has been purified and concentrated, then used to immunize the New Zealand rabbit for antiserum produce. The purified LvIE antibody can specifically recognize the vitellogenin of Siberian sturgeon. We analyzed the Vtg content in different ovary stage, the result shown that the serum Vtg content increased since stageⅡtoⅣ, and then decreased in stage V. This variation is similar with serum Ca2+ and Pi, suggesting that Vtg synthesized in response to circulating E2, provide continuous protein to accumulate into oocytes untill final maturation. Our study provides a basis to establish a more sensitive quantitative detection method of Vtg. Therefore, using the Vtg antibody to directly detect Vtg protein in serum of Siberian sturgeon can more convenient and efficient to estimate the ovary status during development.3. Annotation and analysis of expressed sequence tags (EST) from Chinese sturgeon Acipenser sinensisIn order to advance the molecular research on Chinese sturgeon reproduction, ontogenetic development, we were seeking the keywords ’Acipenser sinensis’ for genomic information in the NCBI expressed sequence tag (EST database). There are 3,384 identified cDNA sequences have been found as Chinese sturgeon pituitary ESTs, and have been assembled into 861 unigenes. Blast analysis revealed 301 unigenes shared high similarity with genes in the public databases, and these were classified into three groups:202 known genes,81 putative genes and 8 unknown genes. The remainder (560 genes) had no significant match to any protein sequence. Further,255 unigenes and 333 unmatched unigenes were annotated with Gene Ontology (GO), which could be classified into cellular component, molecular function, and biological process. Among the known genes, the hormone genes (POMC A, POMC B, GtH alphaⅠsubunit, GtH alphaⅡsubunit and GH) were present in this library. Comparison of the Chinese sturgeon proteins (GH, GtH alpha subunit and POMC) to other species showed higher levels of homology among sturgeon species. To investigate the potential function of genes, we performed semi-quantitative RT-PCR of four hormone related genes (CPH, PPIB, STMN3 and 7B2) and four novel genes (contig 192,177,170 and 168) using different tissues from Chinese sturgeon. These results represent an advance in the rapidly growing collection of genomic resources available on A. sinensis, which can be used for future investigation in a number of different fundamental and applied areas.