鳜twist2序列特征、时空表达模式及靶向miRNAs预测分析  

Sequence characterization,spatiotemporal expression patterns and predictive analysis of miRNAs targeting twist2 in Siniperca chuatsi

作　　者：程聪益 朱鑫[2] 曾维 宾琴 王玮 褚武英[2] 张建社[2] CHENG Congyi;ZHU Xin;ZENG Wei;BIN Qin;WANG Wei;CHU Wuying;ZHANG Jianshe(State Key Laboratory of Developmental Biology of Freshwater Fish,College of Life Sciences,Hunan Normal University,Changsha 410081,China;Hunan Provincial Key Laboratory of Aquatic Animal Nutrition and Quality Control,College of Biological and Chemical Engineering,Changsha University,Changsha 410022,China)

机构地区：[1]湖南师范大学生命科学学院省部共建淡水鱼类发育生物学国家重点实验室,湖南长沙410081 [2]长沙学院生物与化学工程学院水生动物营养与品质调控湖南省重点实验室,湖南长沙410022

出　　处：《中国水产科学》2024年第2期144-154,共11页Journal of Fishery Sciences of China

基　　金：国家自然科学基金国际合作重点项目(31820103016);国家自然科学基金区域创新发展联合重点项目(U21A20263);国家自然科学基金青年基金项目(32002370)。

摘　　要：Twist2在调控动物发育过程发挥了重要作用。为解析鳜(Siniperca chuatsi) twist2基因的表达模式以及参与肌肉发育调节的功能和机制,本研究从鳜基因组数据库获得twist2基因序列,采用生物信息学方法对Twist2蛋白特征和同源进化进行了分析。鳜twist2基因所编码的蛋白由162个氨基酸组成,具有一个保守功能域bHLH结构域。基于氨基酸序列的多序列比对结果显示,Twist2蛋白在脊椎动物中具有较高的相似性;采用RT-qPCR技术分析了鳜twist2基因的时空表达规律,发现twist2基因在不同发育阶段存在表达差异,原肠期之前表达水平较低,从神经胚期开始显著上升,尾芽期表达最高;在鳜组织中的表达情况显示,该基因在脾脏中表达量较高,其次是脑和红肌。采用整胚原位杂交技术检测其早期胚胎不同发育阶段的twist2基因定位,发现其主要在神经胚层和体节中特异性表达。采用Targetscan和RNAhybrid工具对可能靶向鳜twist2基因的miRNAs进行预测,结果发现miR-30a、miR-30b、miR-30e-5p和miR-204可能作用于twist2 3'UTR,提示twist2是miR-30a、miR-30b、miR-30e-5p和miR-204的潜在靶基因。鳜短期饥饿胁迫实验显示,在饥饿3 d时twist2的表达与上述miRNAs的表达呈明显的负相关,表明twist2与上述4个miRNAs存在潜在的调控关系。因此,本研究结果将有助于从分子水平了解twist2基因的序列特征、时空表达规律以及其调控肌肉生长发育的潜在功能,为鱼类发育生物学以及健康养殖提供理论依据。Twist2(Twist family transcription Factor 2)is a member of the basic Helix-Loop-Helix(bHLH)family that plays an important role in the regulation of animal development.However,the expression patterns and regulatory roles of twist2 genes in fishes are poorly understood.This study aimed to understand the expression pattern of the twist2 gene and its functions in the regulation of muscle development in Siniperca chuatsi.The twist2 gene sequence was obtained from the transcriptome database of S.chuatsi and Twist2 protein characterization and homology were analyzed using bioinformatic methods. The protein encoded by the twist2gene of S. chuatsi consists of 162 amino acids with a conserved functional bHLH structural domain and the results of multiple sequence comparison based on the amino acid sequences showed that the Twist2 protein has a high similarity in vertebrates. The spatiotemporal expression pattern of the twist2 gene of S. chuatsi was analyzed by RT-qPCR and the expression pattern of the twist2 gene was found to be differentially expressed at different developmental stages. The expression level was low before the mid-gastrula stage, increased significantly from the neurula stage, reached its highest level in the tail-bud stage, and was widely expressed in S. chuatsi tissues;its expression was highest in the spleen, followed by the brain and red muscle. Whole embryo in situ hybridization was used to detect the localization of twist2 at different developmental stages in early embryos and it was found to be specifically expressed mainly in the neuroectoderm and somites. Targetscan and RNAhybrid tools were used to predict miRNAs that might target the mandarin twist2 gene. miR-30a, miR-30b, miR-30e-5p, and miR-204 were found to potentially act on the twist2 3' UTR, suggesting that twist2 is a potential target gene for miR-30a, miR-30b, miR-30e-5p, and miR-204. The short-term starvation stress experiment in S. chuatsi showed that the expression of twist2 after three days of starvation was significantly negatively cor

关 键 词：鳜 twist2 时空表达 MIRNA 短期饥饿 

分 类 号：S917[农业科学—水产科学]