Study On The Cloning And Expression Of MyoD Gene In Grass Carp (Ctenopharyngodon Idella)

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.