【作者】 高春生；

【导师】 范光丽； 王艳玲；

【作者基本信息】 西北农林科技大学 ， 基础兽医学， 2008， 博士

【摘要】 淇河鲫(Carassius auratus gibelio var)原产于河南省淇河,以生长快、味道美和效益高等优点而久负盛名,河南省人民政府已正式行文将其列入《河南省重点保护野生动物名录》并以第一名予以公布。为了繁荣市场,丰富人民生活,发展池塘养殖已变得日益迫切,对淇河鲫的生物学特性、生长变化规律等方面的研究已成为重要的课题。而动物的生长和发育受多种因素的调节和控制,包括遗传,营养,环境和内分泌等。在内分泌系统中,生长激素(GH)占据着中心调节作用的位置,它是由脑垂体间叶细胞分泌的一种多肽激素,具有调节生长发育,参与体内代谢调节,增进食欲,提高食物转化率等作用,然而垂体中GH含量极低,分离纯化比较困难,使得GH生理功能的进一步研究和应用受到限制。随着现代生物技术的发展,已有研究表明基因重组的GH与天然的GH一样具有生物活性,基因重组GH应用于研究和生产已经成为可能。因此,我们以淇河鲫作为研究对象,通过含肉率测定、肌肉生化成分分析,对其品质、营养价值作出初步评定,为淇河鲫营养生理的研究提供理论基础;用RT-PCR和RACE技术从淇河鲫垂体总RNA中扩增获得了淇河鲫GH全长cDNA序列,并对核苷酸和氨基酸序列进行了分析,构建了淇河鲫GH基因的原核和真核表达载体,获得了相应的重组基因工程菌,并对基因工程菌进行了诱导表达研究,为下一步开展淇河鲫GH的生物活性检测、转基因鱼研究以及研制开发成为安全、高效的新型调节鱼类生长的基因制剂奠定坚实的基础。主要结果如下:1)淇河鲫肌肉营养成分分析及营养价值评定淇河鲫是含肉率(65.72%)较高的鱼类;肌肉水分、蛋白质、脂肪、灰分含量分别为75.56%、19.39%、2.96%、1.17%;氨基酸分析表明,淇河鲫肌肉氨基酸总量(18.22%)、必需氨基酸量(7.37%)均较高,必需氨基酸占氨基酸总量的百分比(40.45%)和必需氨基酸与非必需氨基酸的比值(0.68)均符合WHO/FAO提出的40%左右和在0.60以上的要求,而且必需氨基酸含量高于婴儿需要量为底限的WHO/FAO评分模式;尤其是赖氨酸含量较丰富,达到鸡蛋蛋白质评分模式标准,较WHO/FAO评分模式高出30.0%,可以认为淇河鲫是一种营养丰富的优质鱼。2)克隆并获得了淇河鲫GH全长cDNA序列淇河鲫GH cDNA序列全长1191 bp,其中含Poly(A) 14 bp, 5’端非编码区长55 bp (含有在真核生物中高度保守的Kozak序列),阅读框长633 bp,3’端非编码区长503bp(含真核生物mRNA前体3’剪切和加poly(A)尾的信号ATTAAA序列),共编码210个氨基酸;编码的蛋白的分子量为23.78 ku。蛋白质结构分析表明淇河鲫GH信号肽为前22个氨基酸,成熟肽为188个氨基酸,成熟肽中有四个半胱氨酸(C),形成两个二硫键,对GH的正常折叠、维持空间结构以发挥有效的生理功能有着重要作用。3)分析并揭示了淇河鲫GH基因结构和脊椎动物进化的关系通过对不同分类地位脊椎动物GH基因的核苷酸序列和氨基酸序列深入分析发现:①GH家族有三个非常保守的区域,以淇河鲫GH氨基酸序号为标准,第28位到第44位、第70位到第111位、第180位到第210位;特别是从第180位到第210位这个区域高度保守,含有的非极性氨基酸达10个(其中3个半胱氨酸),为分子的疏水区,与GH分子的结构及稳定性有关;此外,除去信号肽部分,有30个氨基酸在所比较的动物中完全相同,可以认为这30个氨基酸残基对GH专一性、生物活性及空间构象有重要的作用。②成熟的GH是从一个共同的祖先进化而来,随着生物的进化过程,GH趋异进化成不同物种的GH分子;淇河鲫和鲤形目鱼类GH分歧较早,鲶形目鱼类的GH分歧次之,鲑鳟鱼类的GH分歧较晚,两栖类、禽类和哺乳类动物GH分歧最晚。4)构建了淇河鲫GH基因的原核表达载体并获得了重组蛋白本研究在原核表达载体pQE30上成功插入了淇河鲫GH基因,成功构建获得了pQE30-GH原核表达载体,通过测序验证构建的表达载体结构和序列正确无误。该载体经IPTG诱导后,SDS-PAGE电泳检测在约24 ku处获得了表达产物,经薄层扫描分析重组蛋白表达量较低,占菌体蛋白的6.6%。5)构建了淇河鲫GH基因毕赤酵母真核表达载体pPIC9K-GH,并获得了基因工程菌和重组蛋白。构建了淇河鲫GH基因真核表达载体pPIC9K-GH,该载体经电转化获得了整合有淇河鲫GH基因的重组毕赤酵母基因工程菌,重组菌经甲醇诱导后,成功获得了重组蛋白;表达条件优化试验结果表明,培养液的pH值、甲醇诱导浓度、诱导时间均对重组蛋白的表达有较大影响,其中诱导重组菌表达的适宜甲醇浓度为0.5～0.75%,适宜在弱偏碱性的培养液中表达,表达的最适pH值为7.0～7.5;诱导2天即达到最大表达量。更多还原

【Abstract】 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.更多还原