Study On The Muscle Nutritional Composion Analysis And The Cloning, Expression Of GH Gene In Carassius Auratus Gibelio Var

Carassius auratus gibelio var originated in Qi-river of Henan Province. It is very famous for its many advantages such as rapid growth, delicious flavor, good effectiveness and so on. As the first name it has formally been listed in the"List of the protection of wild animals in Henan Province"by Henan Province People's Government. In order to boom market and enrich people's lives, it is increasingly urgent to develop aquaculture in ponds. It has become an important subject for the research of the biological characteristics, the law of growth and so on. But the growth and development of animals were regulated by many factors including gene, environment, nutrition and endocrine. In endocrine system, growth hormone (GH) takes up a centerlly regulative place. GH is a very important protein hormone which is secreted by the anterior pituitary cells and plays many important roles on animals, such as regulating somatic growth and stimulating appetite, affecting various metabolic activities and improving the food transformation rate, etc. Although GH has so many functions, it is limited in the application and functional researches because the natural growth hormone content is very low and difficult to purify. Along with the development of modern biotechnology, studies have shown that the recombinant growth hormone has the same biological activity as the natural growth hormone. It becomes possible that the recombinant growth hormone is applied to research and production.Therefore, in this research, we acted Carassius auratus gibelio var as the research object. We preliminarily evaluated its quality and nutritional value through the rate of muscle determination and the muscle biochemical composition analysis, in order to provide a theoretical basis for the research of Carassius auratus gibelio var nutritional physiology. The methods of RT-PCR and RACE were used to clone the GH cDNA full-length sequence of Carassius auratus gibelio var from the pituitary. Then the amino acids and nucleotides sequence of GH were analyzed, and Prokaryotic and eukaryotic expression vector (pQE30-GH and pPIC9K-GH) were constructed to express the GH recombined protein, in order to lay a solid foundation for the next growth hormone detection of biological activity, the study of transgenic fish and the development of a safe, efficient, new-type gene product regulating fish growth. The results showed that: 1) Analysis of nutritional composition and evaluation of nutritional Quality in muscle of Carassius auratus gibelio varCarassius auratus gibelio var is the fish that its rate of muscle content is higher (65.72%). Its moisture, protein, fat content and ash were 75.56%, 19.39%, 2.96% and 1.17% respectively. Total content of 17 amino acids was 18.22%. Total content of 7 essential amino acids was 7.37%. Essential amino acids concent (40.45%) and the ratio of essential amino acids and non-essential amino acids (0.68) are in line with the standards about 40 percent and above 0.60 respectively required by WHO/FAO. Essential amino acid content is higher than the baby minimum requirement of the WHO/FAO score pattern. Particularly lysine is abundant, coincidence to egg protein score pattern standards and 30.0% higher than the WHO/FAO score pattern. It can be considered that Carassius auratus gibelio var is a nutrient-rich quality fish.2) GH cDNA of Carassius auratus gibelio var was cloned firstly.The GH cDNA full-length of Carassius auratus gibelio var was about 1191 bp,consisted of a open reading frame about 633 bp ,5'and 3'untranslated regions about 55 bp and 503 bp respectively. 5'untranslated region included one highly conserved Kozak sequence in Eukaryotes, 3'untranslated region included the signal sequence ATTAAA that has the founction of shearing and processing poly(A) tail from the mRNA precursor in Eukaryotes. It encoded 210 amino acids including one signal peptide about 22 amino acids locating in its N-termina1 and one mature peptide about 188 amino acids after analyzing by SignalP3.0. The molecular weight of GH protein was 23.78 ku. There were 4 Cysteines in mature peptide. The two disulfide bonds formed by the 4 Cysteines played an important role to the normal folding of GH and maintaining the space structures, which could make GH an effective physiological function.3) The relationship between GH gene structure and evolution of vertebrate was analyzed and discovered.After comparing nucleotide and amino acid sequence of Carassius auratus gibelio var GH with those of other animals, the results showed that:①There are three very conservative regions in GH family, to the serial number of Carassius auratus gibelio var GH amino acids as the standard, from No. 28 to No. 44, No. 70 to No. 111, No. 180 to No. 210. The highly conservative region from No. 180 to No. 210, containing 10 non-polar amino acids (including three cysteines), was the molecules hydrophobic region, which had relations to the molecular structure and stability. In addition, except for the signal peptide, there were 30 amino acids the same in the animals compared, the 30 amino acid residues could be considered to take very important action to the GH biological activity and conformational space.②Mature GH evolved from a common ancestor. Along with the process of biological evolution, GH also evolved into different GH molecules; the GH molecules of Carassius auratus gibelio var and carps differd earlier, then catfish, salmon trouts later, amphibians, birds and mammals the latest.4) The prokaryon expression vector pQE30-GH was constructed and the recombined protein was expressed in E.coli.Carassius auratus gibelio var GH gene was successfully inserted in the prokaryotic expression vector pQE30, which successfully constructed a prokaryotic expression vector pQE30-GH. The expression vector structure and sequence were correct proved by sequencing. The GH recombined protein, the molecular weight is about 24 ku, was expressed after induced by IPTG, However, the output was only 6.6% of all bacteria protein.5) The eukaryon expression vector pPIC9K-GH was constructed, and the recombined protein was expressed in GS115.The pPIC9K-GH eukaryon expression vector was successfully constructed, and the pPIC9K-GH vector was transmited into GS115 by electric conversion, and the GH recombined protein was successfully expressed induced by methanol. Experimental results of the optimum expression conditions showed that the expression level of recombined protein was closely related to methanol concentration, pH of mediumand and inducing time, and the optimum expression conditions were 0.5～0.75% methanol, pH7.0～7.5 and 2-day inducing time.