文章摘要
梁威,杨铁柱,沈和定,许国绿,顾冰宁,薛宝宝.瘤背石磺钙/钙调蛋白依赖性蛋白激酶Ⅳ(CaMKⅣ)基因的克隆及组织表达[J].水产学报,2019,43(11):2336~2346
瘤背石磺钙/钙调蛋白依赖性蛋白激酶Ⅳ(CaMKⅣ)基因的克隆及组织表达
Cloning and tissue expression analysis of the calcium/calmodulin dependent protein kinase Ⅳ gene (CaMKⅣ) in Onchidium struma
投稿时间:2018-09-25  修订日期:2018-10-25
DOI:10.11964/jfc.20180911460
中文关键词: 瘤背石磺  CaMKⅣ  基因克隆  实时荧光定量PCR  组织表达分析
英文关键词: Onchidium struma  CaMKⅣ  gene clone  qRT-PCR  tissue expression
基金项目:国家自然科学基金(41276157);上海高校水产学一流学科建设项目
作者单位E-mail
梁威 上海海洋大学, 省部共建水产种质资源发掘与利用教育部重点实验室, 水产科学国家级实验教学示范中心, 海洋动物系统分类与进化上海高校重点实验室, 上海 201306  
杨铁柱 上海海洋大学, 省部共建水产种质资源发掘与利用教育部重点实验室, 水产科学国家级实验教学示范中心, 海洋动物系统分类与进化上海高校重点实验室, 上海 201306  
沈和定 上海海洋大学, 省部共建水产种质资源发掘与利用教育部重点实验室, 水产科学国家级实验教学示范中心, 海洋动物系统分类与进化上海高校重点实验室, 上海 201306 hdshen@shou.edu.cn 
许国绿 上海海洋大学, 省部共建水产种质资源发掘与利用教育部重点实验室, 水产科学国家级实验教学示范中心, 海洋动物系统分类与进化上海高校重点实验室, 上海 201306  
顾冰宁 上海海洋大学, 省部共建水产种质资源发掘与利用教育部重点实验室, 水产科学国家级实验教学示范中心, 海洋动物系统分类与进化上海高校重点实验室, 上海 201306  
薛宝宝 上海海洋大学, 省部共建水产种质资源发掘与利用教育部重点实验室, 水产科学国家级实验教学示范中心, 海洋动物系统分类与进化上海高校重点实验室, 上海 201306  
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中文摘要:
      钙/钙调蛋白依赖性蛋白激酶Ⅳ (CaMKⅣ)是一种广泛存在于真核生物中的调控因子,在生物体内具有多种生理功能。为了探究瘤背石磺中CaMKⅣ的作用和分子机制,为进一步阐述CaMKⅣ的生理功能提供科学的理论依据。本实验以瘤背石磺神经节为实验材料,利用RACE-PCR技术克隆得到CaMKⅣ基因的cDNA序列,并进行生物信息学分析和qRT-PCR实验。结果显示,瘤背石磺CaMKⅣ基因的cDNA全长1 603 bp,开放阅读框为1 032 bp,5'非编码区315 bp,3'非编码区256 bp,共编码343个氨基酸;预测该基因编码的多肽链原子数量是5 490,分子量约为3.87 ku,理论等电点6.12,分子式为C1741H2768N452O514S15,N端信号肽由1~29个氨基酸组成。氨基酸序列比对及系统进化树结果显示,瘤背石磺CaMKⅣ基因与加州海兔、光滑双脐螺CaMKⅣ基因的亲缘关系最为接近,其系统进化分析与传统的形态学分类相吻合。荧光定量PCR结果显示,CaMKⅣ基因在各个组织均有表达,其中在腹足的相对表达量最高,其次是背部皮肤、口器,而在神经节组织、蛋白腺、肠、肝胰腺等脏器组织的相对表达量较低,初步推测,该基因在瘤背石磺的神经调节中发挥重要作用,或为生物机体感知外界环境变量因素的分子基础。本实验为今后进一步了解瘤背石磺神经结构生理功能的调节以及与CaMKⅣ基因功能研究奠定理论支撑,也为探究海洋动物由海洋向陆地进化过程中对环境的适应机制提供参考。
英文摘要:
      Calcium/calmodulin-dependent protein kinase Ⅳ (CaMKⅣ) which has multiple physiological functions in organisms, is a regulatory factor widely found in eukaryotes. To investigate the role and molecular mechanism of CaMKⅣ in Onchidium struma in the process of evolution from sea to land, and further explain the physiological function of CaMKⅣ to provide a scientific theoretical basis, a series of experiments have been carried out. In this study, the cDNA sequence of CaMKⅣ gene was cloned by RACE-PCR using ganglion of O. struma, and bioinformatics analysis and qRT-PCR experiment were performed. The results suggested that the full-length cDNA of CaMKⅣ gene consists of 1 603 base pairs (bp), 315 bp 5' non-coding region (UTR), 256 bp 3' non-coding region (UTR) and 1 032 bp open reading frame (ORF) which encode a total of 343 amino acids. The atom of the polypeptide chain encoded by this gene was predicted. The CaMKⅣ protein was predicted to contain 5 490 atoms, the molecular weight is about 3.87 ku, the theoretical isoelectric point is 6.12, the molecular formula is C1 741H2 768N452O514S15, and the N-terminal signal peptide is composed of 1-29 amino acids. The amino acid sequence alignment and phylogenetic tree results suggest that the CaMKⅣ gene was the closest to the CaMKⅣ gene of Aplysia californica and Biomphalaria glabrata, and its phylogenetic analysis was consistent with the traditional morphological classification. The results of qRT-PCR indicated that the CaMKⅣ gene was expressed in all tissues, and the relative expression in the pleopod was the highest, followed by the dorsal skin and mouthparts, and the expression level in the ganglion, albumen gland, intestine, hepatopancreas and other organs were low. It was preliminarily speculated that the gene played an important role in the ganglion of O. struma, or it was the molecular basis for the biological body to perceive external environmental variables. The results of this experiment will provide a theoretical support for further understanding of the regulation of the physiological functions of nervous structure and the function of CaMKⅣ gene, and also provide a reference for exploring the environmental adaptation mechanism of marine animals in the process of evolution from ocean to land.
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