高原鼠兔组织乳酸脱氢酶同工酶的特异性
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摘要
高原鼠兔(Ochotona curzoniae)是青藏高原高海拔地区特有的物种,为了研究其对高原环境的适应机制,用聚丙烯酰胺凝胶电泳观察心、肝、肺、肾、脑、骨骼肌和睾丸组织乳酸脱氢酶(Lactate dehydrogenase,LDH)同工酶谱,我们发现,在繁殖季节,高原鼠兔LDH同工酶谱在各组织中都表现出六条带,与睾丸和精子中的同工酶谱完全一样。而平原SD大鼠的体细胞组织中只有五条带,睾丸中却有六条带,其中从点样孔往下第三条为LDH-C4。我们推测,Ldh-c基因不仅在高原鼠兔精子中表达,而且在体细胞同样表达。为证明这一假设的可靠性,我们克隆了Ldh-c全长序列,分析了Ldh-c在体细胞中是否表达,以及海拔变化对睾丸中Ldh-c基因表达的影响。用聚丙烯酰胺凝胶电泳观察心、肝、肺、肾、脑、骨骼肌和睾丸LDH同工酶谱;用分子及生物信息学方法测定并分析高原鼠兔Ldh-c的cDNA序列;用Real-time PCR比较高原鼠兔睾丸内Ldh-c在不同海拔mRNA水平的表达。结果:同工酶谱显示,高原鼠兔各组织中均表现出位置一致的6条带。高原鼠兔Ldh-c基因全长1 624bp,包括106bp的5′端非编码区,519bp的3′端非编码区和一个999bp的编码区。开放式阅读框架起始于107位核苷酸起始密码子ATG,终止于1 105位核苷酸TAA终止密码子。高原鼠兔Ldh-c基因的编码区序列与牛,人,大鼠,小鼠分别有84.98,86.09,76.38和76.88%的同源性。玉树鼠兔睾丸中的Ldh-c在mRNA水平上的表达显著低于海北鼠兔(P<0.05),同时在体细胞组织中并未发现Ldh-c基因表达。
以上结果表明,高原鼠兔Ldh-c基因并不在体细胞组织中表达。体细胞组织和睾丸中表达一致的六条带仍有待进一步研究。尽管高原鼠兔是一种对高原环境高耐受性的动物,低氧对其生理仍有一定的影响。我们推测,随着海拔的升高,高原鼠兔Ldh-c基因的表达受抑制,抑制精子获能,从而对其生殖造成影响。
Plateau pika (Ochotona curzoniae) is native to the Qinghai-Tibet plateau. To study their adaptive mechanisms, the expression patterns of lactate dehydrogenase (LDH) isoenzymes spectrum in their tissues were measured through polyacrylamide gel electrophoresis (PAGE). Our results indicated that, in mating season, six bands of LDH isoenzymes were showed in tissues of plateau pikas, the expression patterns were identical to that in testes and spermatozoa. It was showed that five bands of LDH isoenzymes in tissues of Sprague-Dawley rats, but six bands in testes. The third band in LDH isoenzymes spectrums in testes of Sprague-Dawley rats was LDH-C4. We speculated that, Ldh-c gene was not only expressed in testes of plateau pika, but also in somatic cells. To prove the inference, we cloned the full-length sequence of Ldh-c, determined whether Ldh-c was expressed in somatic cells, and explored the effects of elevation on the Ldh-c expression of plateau pika. The expression patterns of lactate dehydrogenase (LDH) isoenzymes spectrum were measured through polyacrylamide gel electrophoresis (PAGE); The Ldh-c gene was sequenced and analyzed by molecular and bioinformatics approach; the mRNA expression levels in different altitudes (3200 and 4266 m) were detected by real-time PCR.
The results showed that six bands of LDH isoenzymes were showed in tissues of plateau pikas .The full-length pika Ldh-c cDNA was 1624 bp with a 999 bp open-reading frame encoding a peptide of 332 amino acids, a 106 bp 5' untranslated region (UTR), and a 519 bp 3' UTR. The coding sequence of pika Ldh-c cDNA and LDH-C subunit shared 77-86% and 74-88% homology to that of rat, mouse, cow and human, respectively. The mRNA expression level in 3200 m was obviously higher than that in 4266 m, while in somatic cells Ldh-c gene was not found.
In conclusion, these results suggested that the plateau pika Ldh-c gene is not expressed in somatic cells. Six bands of LDH isoenzymes in all tissues Needs for further research. Although plateau pika is a high hypoxia tolerate mammal with high ration of oxygen utilization to cope with plateau hypoxia, hypoxia has some effects on the fertility of plateau pika. We speculated that, with the elevation increasing, the plateau pika Ldh-c gene expression was inhibited, which affected their reproduction.
以上结果表明,高原鼠兔Ldh-c基因并不在体细胞组织中表达。体细胞组织和睾丸中表达一致的六条带仍有待进一步研究。尽管高原鼠兔是一种对高原环境高耐受性的动物,低氧对其生理仍有一定的影响。我们推测,随着海拔的升高,高原鼠兔Ldh-c基因的表达受抑制,抑制精子获能,从而对其生殖造成影响。
Plateau pika (Ochotona curzoniae) is native to the Qinghai-Tibet plateau. To study their adaptive mechanisms, the expression patterns of lactate dehydrogenase (LDH) isoenzymes spectrum in their tissues were measured through polyacrylamide gel electrophoresis (PAGE). Our results indicated that, in mating season, six bands of LDH isoenzymes were showed in tissues of plateau pikas, the expression patterns were identical to that in testes and spermatozoa. It was showed that five bands of LDH isoenzymes in tissues of Sprague-Dawley rats, but six bands in testes. The third band in LDH isoenzymes spectrums in testes of Sprague-Dawley rats was LDH-C4. We speculated that, Ldh-c gene was not only expressed in testes of plateau pika, but also in somatic cells. To prove the inference, we cloned the full-length sequence of Ldh-c, determined whether Ldh-c was expressed in somatic cells, and explored the effects of elevation on the Ldh-c expression of plateau pika. The expression patterns of lactate dehydrogenase (LDH) isoenzymes spectrum were measured through polyacrylamide gel electrophoresis (PAGE); The Ldh-c gene was sequenced and analyzed by molecular and bioinformatics approach; the mRNA expression levels in different altitudes (3200 and 4266 m) were detected by real-time PCR.
The results showed that six bands of LDH isoenzymes were showed in tissues of plateau pikas .The full-length pika Ldh-c cDNA was 1624 bp with a 999 bp open-reading frame encoding a peptide of 332 amino acids, a 106 bp 5' untranslated region (UTR), and a 519 bp 3' UTR. The coding sequence of pika Ldh-c cDNA and LDH-C subunit shared 77-86% and 74-88% homology to that of rat, mouse, cow and human, respectively. The mRNA expression level in 3200 m was obviously higher than that in 4266 m, while in somatic cells Ldh-c gene was not found.
In conclusion, these results suggested that the plateau pika Ldh-c gene is not expressed in somatic cells. Six bands of LDH isoenzymes in all tissues Needs for further research. Although plateau pika is a high hypoxia tolerate mammal with high ration of oxygen utilization to cope with plateau hypoxia, hypoxia has some effects on the fertility of plateau pika. We speculated that, with the elevation increasing, the plateau pika Ldh-c gene expression was inhibited, which affected their reproduction.
引文
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