卵形鲳鲹MyoG基因克隆及其胚胎组织表达分析

Gene cloning of MyoG in Trachinotus ovatus and its embryonic tissues analysis

【目的】明确卵形鲳鲹胚胎发育中肌细胞生成素基因(MyoG)的表达规律,为阐明MyoG在卵形鲳鲹胚胎生长发育中的作用机制打下基础。【方法】采用RT-PCR扩增卵形鲳鲹MyoG基因全长序列,通过ProtParam、GOR IV、SWISS-MODEL、TMHMM、SignalP、PSORT及NetNGlyc 1.0等在线软件对其进行生物信息学分析;运用实时荧光定量PCR检测MyoG基因在卵形鲳鲹不同胚胎发育时期(受精卵、卵裂期、囊胚期、原肠胚期、胚胎形成期、眼囊期、耳囊期、心脏跳动期、晶体出现期和孵化期)的表达情况。【结果】从卵形鲳鲹胚胎组织中克隆获得的MyoG基因全长为1379 bp,编码233个氨基酸残基,其蛋白分子量为26059.19 Da,理论等电点(pI)为8.72;不稳定指数为75.23,即MyoG蛋白稳定性较差;总平均疏水指数(GRAVY)为-0.764,属于亲水性蛋白。卵形鲳鲹MyoG蛋白无跨膜结构,无信号肽序列,在第49位氨基酸处存在1个潜在的糖基化位点;卵形鲳鲹MyoG蛋白主要存在于细胞质和微体中,不属于分泌蛋白,其二级结构中以无规则卷曲占比最高(59.23%),其次为α-螺旋(21.46%)和延伸链(19.31%)。MyoG基因相对表达量在卵形鲳鲹胚胎发育过程中呈明显的先升高后降低变化趋势。其中,在胚胎发育的前期(受精卵至胚体形成期)MyoG基因相对表达量极低,但从眼囊期开始其相对表达量迅速升高,至心脏跳动期达最高值;眼囊期、耳囊期和心脏跳动期3个时期的MyoG基因相对表达量均极显著高于胚胎发育前5个时期(P<0.01,下同);从晶体出现期开始,MyoG基因相对表达量极显著降低。【结论】MyoG基因除了在卵形鲳鲹眼囊期、耳囊期和心脏跳动期的分化形成过程中发挥重要作用外,在卵形鲳鲹胚胎器官分化形成后仍发挥着重要作用。

【Objective】To investigate the expression pattern of myogenin gene(MyoG)during embryonic development of Trachinotus ovatus,and lay the foundation for elucidating the mechanism of MyoG in the embryonic development of T.ovatus.【Method】RT-PCR was used to amplify the full-length sequence of the MyoG gene of T.ovatus,and the bioinformatics analysis was carried out with online software such as ProtParam,GOR IV,SWISS-MODEL,TMHMM,SignalP,PSORT and NetNGlyc 1.0.Real-time fluorescence quantitative PCR was used to detect the expression of MyoG mRNA in different embryonic development stages(zygote,cleavage stage,blastula stage,gastrula stage,embryo formed stage,optic vesicle stage,otocyst vesicle stage,heart pulsation stage,formation of eye lens and hatching stage)in T.ovatus.【Result】The MyoG gene cloned from embryonic tissues of T.ovatus was 1379 bp in length,encoding 233 amino acid residues,its protein molecular weight was 26059.19 Da,theoretical isoelectric point(pI)was 8.72.The instability index was 75.23.Which indicated that MyoG protein was not stable.The total average hydrophobic index(GRAVY)was-0.764,indicating that it belonged to hydrophilic protein.MyoG protein had no transmembrane structure,no signal peptide sequence,and a potential glycosylation site at amino acid 49.The MyoG protein mainly existed in the cytoplasm and the microbody(peroxisome),not belonging to asecretory protein.In the secondary structure,random coil accounted for the highest proportion(59.23%),followed byα-helix(21.46%)and extended strand(19.31%).The relative expression trend of MyoG gene was firstly increased and then decreased during the embryonic development of T.ovatus.Among them,the relative expression level of MyoG gene was extremely low in the early stage of embryonic development(from the zygote to the embryo formed stage),but the relative expression level of MyoG increased rapidly from the optic vesicle stage,and reached the highest value in the heart pulsation stage.The relative expression of MyoG gene in the three phases of optic vesicle stage,otocyst vesicle stage and heart pulsation stage were extremely significantly higher than that in the first five phases of embryonic development(P<0.01,the same below).From the phase of formation of eye lens,the relative expression of MyoG gene was extremely significantly reduced.【Conclusion】The MyoG gene not only plays an important role in the differentiation and formation of the optic vesicle stage,otocyst vesicle stage and heart pulsation stage of the T.ovatus,but also plays an important role after the embryonic organ differentiation of T.ovatus.