牦牛肌红蛋白的基因克隆测序、分离纯化、含量及其与乳酸脱氢酶和苹果酸脱氢酶活力的关系

为探索牦牛适应高原缺氧环境的分子机制,测定了麦洼牦牛心肌和骨骼肌中肌红蛋白（Mb）含量及乳酸脱氢酶（LDH）、苹果酸脱氢酶（MDH）活力,并对牦牛Mb进行了分离纯化和基因克隆测序。牦牛心肌和骨骼肌中Mb含量显著高于水牛和黄牛（P〈0.01）;牦牛、黄牛和水牛心肌MDH/LDH活力比均显著高于骨骼肌;牦牛心肌MDH/LDH活力比显著高于水牛和黄牛,但在不同牛种骨骼肌之间无显著差异。各组织Mb含量与MDH/LDH活力比呈显著正相关。试验用RT-PCR方法从牦牛心肌中克隆了Mb基因,与普通牛相比,核苷酸序列同源性为99.5%,氨基酸序列完全相同;用盐析、CM-Sephadex阳离子交换层析、Sephadex G-50凝胶层析等方法分离纯化了牦牛Mb,SDS-PAGE显示其分子量约为17 ku。

Yak Myoglobin:Gene Cloning and Sequencing, Purification, Contents and Their Relation to Activities of Lactate Dehydrogenase and Malate Dehydrogenase

To investigate the molecular mechanism for yak to adapt the hypoxia environment of plateau,experiments were carried out to measure myoglobin concentration,activities of lactate dehydrogenase(LDH) and malate dehydrogenase(MDH) in heart and skeletal muscles of yak.Also,yak myoglobin was purified and its gene was cloned and sequenced.Yak heart muscle and skeletal muscle contained significantly higher concentration of myoglobin than buffalo and yellow cattles.Heart tissues had significantly higher content of myoglobin and MDH/LDH ratio than skeletal muscles(P<0.01) in yak,yellow cattle and buffalo.Heart MDH/LDH ratio in yak was significantly higher than those of buffalo and yellow cattle,however,skeletal muscle MDH/LDH ratio showed no difference among the species tested.Significant positive relationship between myoglobin content and MDH/LDH ratio was observed in heart and skeletal muscles.The full-length coding sequence of yak myoglobin was cloned by RT-PCR using total RNA extracted from heart muscle of Maiwa yak.The amplified yak myoglobin gene shares 99.5% homology with that of bovine and the same amino acid sequence.Myoglobin was purified from yak heart muscle by salting-out,CM-Sephadex cation exchange chromatography,and Sephadex G-50 gel chromatography.The molecular weight of purified yak myoglobin was approximately 17 ku as shown on SDS-gel.