【作者】 王立新；

【导师】 陈宏； 罗建仁；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 MyoD基因是生肌调节因子MRFs家族的主要成员之一,是脊椎动物胚胎期肌肉发育的主导调控基因之一,对骨骼肌的形成和分化起主要作用,MyoD缺失可导致成肌细胞的增殖和分化无法进行。而MSTN(myostatin,肌肉生长抑制素)基因属于TGF-β(transforming growth factor beta,转化生长因子)超家族,其功能是抑制成肌细胞的过度增殖,是肌肉发育的负调控基因。现代肌肉发育生物学的研究表明,影响脊椎动物肌肉生长和肌肉品质的肌纤维的数量在出生前就已决定,出生后肌肉的生长主要靠肌纤维长度和周径的增大,而肌纤维的增粗又会影响肌肉的嫩度和肉用品质。同时获得高的肌肉生长(净肉率)和优良的肉质品质是肉用动物生产一直追求但却无法实现的理想目标。MyoD及其相关基因的发现为解决这一难题提供了可能,但对于该基因的研究目前还主要集中在基因的结构功能、组织表达、多态性以及肌肉瘤的医学诊断上,而在肉用动物生产中的研究还未受到重视。本研究以我国池塘主要养殖品种草鱼为研究对象,旨在通过对草鱼MyoD基因克隆、重组表达以及在不同发育阶段表达规律的研究,为鱼类肌肉品质改良提供理论依据,也为鱼类高效养殖探索新的途径,该项研究对于我国这样一个水产养殖大国来说无疑具有很高的社会和经济价值。 为此本研究用RT—PCR和RACE技术从受精后22h(水温约为24℃)的草鱼肌节期胚胎总RNA中扩增获得了草鱼MyoD全长cDNA序列,并对核苷酸和氨基酸序列进行了分析,构建了草鱼MyoD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;与此同时,本研究还对MyoD基因在草鱼不同发育阶段的表达规律及其与负调控基因MSTN之间的表达关系进行了初步研究,得到如下结果: 1.首次克隆并获得了草鱼MyoD全长cDNA序列。 草鱼MyoD cDNA序列全长为1597bp,其中编码蛋白的开放阅读框位于186～1011bp,长825bp,共编码275个氨基酸;编码的蛋白的分子量为30.87kD。蛋白质结构分析表明草鱼MyoD基因具有该家族基因典型的碱性螺旋环螺旋(basic helix-loop-helix,bHLH)结构域,其中编码肽链的第1-84个氨基酸为草鱼MyoD基因的Basic区,第98～142个氨基酸为草鱼MyoD基因的HLH结构域;SignalP 3.0和Tmpred(prediction oftransmembrane regions and orientation)在线结构分析也发现,该肽链没有信号肽和跨膜区段,这和该基因只在胚胎期骨骼肌细胞中表达的组织特异性相符。 2.分析并揭示了MyoD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MyoD基因的核苷酸序列和氨基酸序列深入分析发现:①MyoD基因的保守性较高,且氨基酸肽链的长度随动物由低等到高等有逐渐加长更多还原

【Abstract】 MyoD is a member of myogenic regulation factors family, playing an important role in the process of vertebrate embryo muscle development. Since its main function is to control the forming and differentiation of skeleton muscle, the myoblast of vertebrate can’t proliferate and differentiate without MyoD. Myostatin, a negative regulation gene for muscle development, belongs to the TGF-β supper family, its function was to depress the myoblast proliferate overly. The muscle developmental biological research has showed that the number of myofibres was decided prenatally, and no increasing on postnatal, so increasing the myobibres length and diameter was the way that skeleton muscle grew. However, increasing the diameter of myofibres would affect the muscle quality. To improve the quantity and quality of muscle synchronously is the perfect result we want, but it is impossible to achieve this goal with feeding approach. Now it may become the truth because of the discovery of MyoD and MRFs family which offers a new way to control and regulate the vertebrate muscle development. At present, all the research focus on the MyoD gene structure, expression pattern, Polymorphism and diagnosis rhabdomyosarcoma in physic, but how to control the muscle in domestic animals by the MyoD has not been reported. So the Grass carp (Ctenopharyngodon idella), one role fish species cultured in ponds was chosen to be object in this research. The purpose of our research is to study on the expression pattern and molecule mechanism of the muscle development in grass carp by cloning and expressing the grass carp MyoD, and explore a new way for improving the quantity and quality of grass carp. It is meaningfull and valuable for China with developed fishery.In this research, RT-PCR and RACE were used to clone the grass carp MyoD cDNA sequence, from the embryo at 22 h post-fertilization. Then the amino acids and nucleotides sequence of grass MyoD were analyzed, and expression vectors (pBV220-MyoD-D and pPICZαA-MyoD) were constructed to express the MyoD recombined protein. At the same time, the expression pattern of MyoD and Myostatin also has been explored in different stage of grass carp. The result showed that:1. MyoD cDNA of the grass carp was cloned firstly.The full-length MyoD cDNA sequence of Grass carp was 1597 bp, with 825 bp open read frame, locating at 186-1011 bp, coding 275 amino acids, including a bHLH domain which composed of basic domain (1th to 84th amino acids) and HLH domain (98th to 141th amino acids). The molecular weight of MyoD protein was 30.87 kD;in which there wasn’t signal peptide after analyzing by SignalP3.0 and Tmpred. So it wasn’t excretion protein and accorded with its speciality that it expressed only in the embryo muscle.2. The relationship between MyoD gene structure and evolution of vertebrate was analyzedand discovered.After comparing nucleotide and amino acid sequence of grass carp MyoD with those of other animals, the results showed that: ? the MyoD is high conservative, and the length of MyoD peptide increase from 226 amino acids(Branchiotoma belcheri) to 319 amino acids {Homo sapiens) with the animal evolving from low vertebrate to high vertebrate, even among the fish there was huge difference. And the homology of nucleotide and amino acid are accorded with relativeship among the animals;(2) The bHLH domain of vertebrate MyoD was higher conservative than other section, there were two amino acid difference between the low vertebrate and high vertebrate. However, the composition of amino acid and length of basic domain are quite different.3. The prokaryon expression vector pBV220-MyoD-S was constructed and the recombined protein was expressed in E.coli.The pBV220-MyoD-S expression vector was reconstructed by reconstructed PCR, and the structure was proved corrected by sequencing. The MyoD recombined protein, the molecular weight is about 34kD, was expressed induced at 42 °C. However, the output was only 10.8% of all bacteria protein.4. The eukaryon expression vector pPICZaA-MyoD was constructed, and the recombined protein was expressed in GS115.The pPICZaA-MyoD eukaryon expression vector was constructed in E.coli, and the pPICZaA-MyoD vector was transmited into GS115 by LiCl. And the MyoD recombined protein was expressed in pH7.5-8.0 BMGY medium, after inducing 2day with 0.5% methanol, the ouput was about 250mg/L. The result showed that the expression level of recombined protein was closely related to pH of medium.5. The relationship between MyoD gene (the positive regulation gene for muscle development) and Mystatin gene (the negative regulation gene for muscle development) was explored firstly in grass carp.The total RNA from grass carp in different developing stage was extracted to detect the MyoD and myostatin mRNA level using RT-PCR. The results showed that the MyoD mRNA was transcripted from 16 h post-fertilization to 30days post-hatch, and there were two peaks of MyoD transcription, one appeared at 16h post-fertilization, another appeared at 20d post-hatch. However, the myostatin was different, it could not be detected before lyear post-hatch, while faint expression could be detected after lyear post-hatch. Once the myostatin gene was expressed, the expresson of MyoD gene would be stoped.更多还原