Molecular Biological Studies on Functional Properties of Fish Myosin with Reference to Carp Fast Skeletal Muscle

以体重0.5 ~ 0.8kg的鲤鱼鲤鱼（Cyprinus carpio）为研究对象，在10或30℃环境下驯化至少5周，并利用其背侧快速骨骼肌分离肌球蛋白重链C端三分之一的轻肌球蛋白（LMM）。SDS-PAGE微序列分析显示，10个驯化的鲤鱼的LMM成分中含有一个n端氨基酸序列，与30个驯化的鲤鱼不同。编码LMM n端区域的DNA片段通过PCR或逆转录-PCR扩增，表明两个驯化基团进一步含有多个取代的氨基酸。以10和30个氯丙酸驯化的鲤鱼快速骨骼肌cDNA文库为探针，分离到编码LMM的cDNA克隆。所有克隆至少覆盖了LMM的全部长度。鉴定出在10个和30个驯化的鱼中主要表达的cdna类型，以及在所有驯化温度下都存在的中间类型。对驯化于10、20和30℃的鲤鱼3′非翻译区DNA片段的探针进行Northern blot分析，进一步证实了驯化温度特异性同种异构体的存在。此外，我们还发现三种异构体的mRNA水平以驯化温度依赖的方式发生改变。结果表明，10 ~ 30种氨基酸之间的氨基酸序列一致性为95.6%，10 ~ 30种之间的氨基酸序列一致性为97.8%。与等温品种相比，30 - c驯化型与等温品种的相似度高于10 - c驯化型。以上结果表明，30 - C型的LMM比10 - C型的LMM更耐热。

Carp, Cyprinus carpio (0.5-0.8kg in body weight), were acclimated to either 10 or 30ﾟC for a minimum of 5 weeks, and their dorsal fast skeletal muscles were used for isolation of light meromyosin (LMM), C-terminal one third of myosin heavy chain. The microsequence analysis after SDS-PAGE revealed that the LMM component from the 10ﾟC-acclimated carp contained an N-terminal amino acid sequence different from that of the 30ﾟC-acclimated carp. DNA fragments encoding an N-terminal region of LMM were amplified by PCR or reverse transcription-PCR,demonstrating that the two acclimated groups further contained several amino acids substituted.cDNA clones encoding LMM were isolated from cDNA libraries constructed from fast skeletal muscles of carp acclimated to 10 and 30ﾟC,using the above-mentioned DNA as a probe. All clones covered at least the full length of LMM.cDNAs were identified which were the predominant types expressed in 10ﾟC-and 30ﾟC-acclimated fish, as well as an intermediate type present at all acclimation temperature. Northern blot analysis using probes ofthree kinds of DNA fragments from the 3'untranslated region of carp acclimated to 10,20 and 30ﾟC further confirmed the presence of acclimation temperature-specific isoforms. In addition, it was found that mRNA levels of three isoforms were altered in an acclimation temperature-dependent manner. The deduced amino acid sequence identity of LMM isoforms were 95.6% between the 10 and 30ﾟC types and 97.8% between the 10ﾟC and intermediate types. When these were compared with those of homoiotherms, the 30ﾟC-acclimated type was more similar to those of homoiotherms than was the 10ﾟC-acclimated type. These results suggest that the 30ﾟC type of LMM is more thermostable than that of 10ﾟC type.