Molecular cloning and mRNA expression analysis of carp embryonic, slow and cardiac myosin heavy chain isoforms
Molecular cloning and mRNA expression analysis of carp embryonic, slow and cardiac myosin heavy chain isoforms
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DOI：
10.1242/jeb.01978
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发表时间：
2006-01
影响因子：
2.8
通讯作者：
Yoshiaki Nihei;Atsushi Kobiyama;D. Ikeda;Yosuke Ono;S. Ohara;N. Cole;I. Johnston;S. Watabe
中科院分区：
文献类型：
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作者：
Yoshiaki Nihei;Atsushi Kobiyama;D. Ikeda;Yosuke Ono;S. Ohara;N. Cole;I. Johnston;S. Watabe
从鲤鱼（Cyprinus carpio L.）MYHemb 1、MYHemb 2和MYHemb 3。从适应10°C（MYHS 10）和30°C（MYHS 30）的成年鲤鱼的慢肌中也分离MYH DNA克隆。系统发育分析表明，MYHemb 1和MYHemb 2属于快速骨骼肌MYH进化枝。相比之下，MYHemb 3的序列与成人慢肌亚型MYHS 10和MYHS 30相似。通过北方印迹分析首次在受精后61 h的胚胎中检测到MYHemb 1和MYHemb 2转录本。在心跳阶段，表达高峰出现在1个月大的青少年。当MYHemb 2检测不到时，MYHemb 1在7个月大的青少年中继续以低水平表达。MYHemb 3转录物与MYHemb 1转录物出现的时期几乎相同（61 h.p.f.），这些基因表现出相似的表达模式。整体原位杂交分析显示MYHemb 1和MYHemb 2的转录本在肌节的内部表达，而MYHemb 3则在肌节的表面部分表达。MYHS 10和MYHS 30 mRNA在孵化时首次检测到。在成体阶段，慢肌MYH mRNA的表达依赖于驯化温度。MYHS 10 mRNA在10和20°C的驯化温度下表达，但在30°C下不表达。相反，MYHS 30 mRNA在所有驯化温度下均强烈表达。主要MYH成绩单中发现在成人慢肌和胚胎在孵化时表达在成人快肌在某些驯化温度，但不是其他。从10° C驯化的成鱼（MYHcard）的心肌中分离MYH DNA克隆。在61 h. p.f.首次检测到MYH卡mRNA，但仅在成人心肌中观察到强信号。因此，本研究揭示了一个复杂的模式MYH基因的表达与发育阶段，肌肉类型和驯化温度。到目前为止，没有一个骨骼肌MYH在幼年晚期强烈表达，表明MYH II基因家族的进一步发育调控成员仍有待发现。
SUMMARY Three embryonic class II myosin heavy chains (MYHs) were cloned from the common carp (Cyprinus carpio L.), MYHemb1, MYHemb2 and MYHemb3. MYH DNA clones were also isolated from the slow muscle of adult carp acclimated to 10°C (MYHS10) and 30°C (MYHS30). Phylogenetic analysis demonstrated that MYHemb1 and MYHemb2 belonged to the fast skeletal muscle MYH clade. By contrast, the sequence of MYHemb3 was similar to the adult slow muscle isoforms, MYHS10 and MYHS30. MYHemb1 and MYHemb2 transcripts were first detected by northern blot analysis in embryos 61 h post-fertilization (h.p.f.) at the heartbeat stage, with peak expression occurring in 1-month-old juveniles. MYHemb1 continued to be expressed at low levels in 7-month-old juveniles when MYHemb2 was not detectable. MYHemb3 transcripts appeared at almost the same stage as MYHemb1 transcripts did (61 h.p.f.), and these genes showed a similar pattern of expression. Whole mount in situ hybridization analysis revealed that the transcripts of MYHemb1 and MYHemb2 were expressed in the inner part of myotome, whereas MYHemb3 was expressed in the superficial compartment. MYHS10 and MYHS30 mRNAs were first detected at hatching. In adult stages, the expression of slow muscle MYH mRNAs was dependent on acclimation temperature. MYHS10 mRNA was expressed at an acclimation temperature of 10 and 20°C, but not at 30°C. In contrast, MYHS30 mRNA was strongly expressed at all acclimation temperatures. The predominant MYH transcripts found in adult slow muscle and in embryos at hatching were expressed in adult fast muscle at some acclimation temperatures but not others. A MYH DNA clone was isolated from the cardiac muscle of 10°C-acclimated adult fish (MYHcard). MYHcard mRNA was first detected at 61 h.p.f., but strong signals were only observed in the adult myocardium. The present study has therefore revealed a complex pattern of expression of MYH genes in relation to developmental stage, muscle type and acclimation temperature. None of the skeletal muscle MYHs identified so far was strongly expressed during the late juvenile stage, indicating further developmentally regulated members of the MYH II gene family remain to be discovered.