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基于转录组测序对翘嘴鳜(Siniperca chuatsi) 2种肌球蛋白重链基因的克隆与分析
陈之航,董浚键,孙成飞,田园园,叶 星
作者单位
陈之航 中国水产科学研究院珠江水产研究所 广州 510380上海海洋大学水产与生命技术学院 上海 201306 
董浚键 中国水产科学研究院珠江水产研究所 广州 510380 
孙成飞 中国水产科学研究院珠江水产研究所 广州 510380 
田园园 中国水产科学研究院珠江水产研究所 广州 510380 
叶 星 中国水产科学研究院珠江水产研究所 广州 510380上海海洋大学水产与生命技术学院 上海 201306 
摘要:
肌球蛋白重链(Myosin heavy chain,MYH)是骨骼肌粗肌丝的重要组成单位,其表达量高低影响肌纤维的组成和肌肉生长。为了解其在翘嘴鳜(Siniperca chuatsi)早期生长过程中的作用,本研究从前期生长差异显著的2组(快长组和慢长组)翘嘴鳜幼鱼转录组差异表达Unigene中筛选出2个MYH基因,RACE (Rapid amplification of cDNA ends)克隆到其全长cDNA(MYH-7a,MYH-7b)。MYH-7a全长为6071 bp,开放阅读框为5820 bp;MYH-7b全长为5896 bp,开放阅读框为5745 bp。序列分析显示,2个MYH均有Loop1、Loop2环、ATP结合位点等关键结构域;进化树聚类分析显示,MYH-7a与MYH-7b均属于慢肌球蛋白。实时荧光定量PCR验证发现,其在快长组样本中表达量显著高于慢长组,与转录组测序结果一致;检测其在翘嘴鳜心肌、红白肌和皮肤等14种组织的表达水平,结果显示,MYH-7a主要在心肌中表达,而MYH-7b主要在红肌中表达;在胚胎发育不同阶段,二者随着胚胎发育的进行,表达量不断增加;在幼鱼早期生长过程(孵化出膜后15 dph、30 dph和60 dph)的翘嘴鳜白肌中,在15 dph和30 dph快长组的表达量显著高于慢长组,而到60 dph时快长组的表达量均显著低于慢长组。翘嘴鳜MYH-7a、MYH-7b在快长组与慢长组鱼中的差异表达提示它们在翘嘴鳜胚胎及其早期生长发育过程中发挥重要作用。
关键词:  翘嘴鳜  转录组测序  肌球蛋白重链  cDNA克隆  表达分析
DOI:10.11758/yykxjz.20160414001
分类号:
基金项目:国家引智项目(国家外专局)(20140326016)、广东省海洋与渔业科技推广(A201601A06)、广东省科技计划项目(2015A020209034)和广州市科技计划项目(2014J4100087)共同资助
cDNA Cloning and Analyses of Two Myosin Heavy Chain Isoforms of Mandarin Fish (Siniperca chuatsi) Based on Transcriptome Sequencing
CHEN Zhihang,DONG Junjian,UN Chengfei,TIAN Yuanyuan,YE Xing
Abstract:
Myosin heavy chain (MYH) is the main component of the skeletal muscle thick filament. The expression levels of MYHs have significant influences on the formation of thick filaments and muscle development. To understand the roles of MYHs on growth during the early development of mandarin fish (Siniperca chuatsi), the differentially expressed unigenes from the transcriptome sequencing data of the fast and slow growing fish were analyzed, and two highly expressed unigenes of MYH were screened. The full length cDNAs of these two MYH-7s, MYH-7a and MYH-7b were obtained by RACE method; they were 6070 bp and 5896 bp in length, with coding regions of 5820 bp and 5745 bp, respectively. Sequencing analysis showed that both MYH-7s contain the conserved regions such as Loop1 and Loop2, and ATP binding sites. The evolutionary tree clustering analysis showed that both MYH-7s genes belong to the slow muscle type MYH. The expression patterns of the two MYH-7s in different tissues were detected by qRT-PCR. The highest expression of MYH-7a was found in heart muscle while MYH-7b was highly expressed in red muscle. The expression levels of both MYH-7a and MYH-7b increased gradually in accordance with the embryonic development stages. The expression levels of the MYH-7s in the fast growing group were significantly higher than in the slow growing groups in 15, 30-day post hatching (dph), while both genes showed lower expression levels in fast growing group than in slow growing group in 60 dph. The different expression levels in tissues and early developmental stages of these two genes in the fast and slow growing groups suggested that they play important roles in the embryonic and early development of S. chuatsi.
Key words:  Mandarin fish (Siniperca chuatsi)  Transcriptome sequencing  Myosin heavy chain  cDNA cloning  Expression analysis