胞质型磷脂酶A2基因克隆及其表达量随大黄鱼仔稚鱼生长发育的变化

Cytosolic Phospholipase A_2 Gene: Cloning and Expression Level Change with Growth Development of Large Yellow Croaker( Larmichthys crocea) Larvae

利用同源克隆技术和cDNA末端快速扩增(RACE)技术克隆大黄鱼胞质型磷脂酶A2(cPLA2)基因的cDNA全长。此外,应用实时定量PCR法检测不同日龄大黄鱼仔稚鱼cPLA2基因的表达量。序列分析结果表明:cPLA2基因全长2 550 bp(GenBank登录号:KF006240.1),其中5'端非编码区176 bp,开放阅读框2 169 bp,3'端非编码区205 bp,共编码723个氨基酸。系统进化树分析结果表明:克隆所得大黄鱼cPLA2基因与其他鱼类的cPLA2基因亲缘关系较近,与哺乳动物的亲缘关系较远。定量检测结果表明:大黄鱼仔稚鱼cPLA2基因的表达量随日龄的增加为先显著升高(1、3、7日龄vs.15日龄,P<0.05),在15日龄时达到最高值,随后显著下降(15日龄vs.19日龄,P<0.05),之后趋于平稳。由此可知,大黄鱼从仔稚鱼到幼鱼早期的变形过程中,磷脂分解代谢的关键酶cPLA2基因的表达量呈现有规律的变化,这可能对机体保持体内磷脂的动态平衡、维护细胞膜的流动性具有重要的意义。大黄鱼仔稚鱼cPLA2基因表达量的变化趋势在一定程度上可以衡量大黄鱼消化系统的发育情况。

In this present study, the full length of cytosolic phospholipase A2（ cPLA2 ） cDNA from large yellow croaker （ Larmichthys crocea） was obtained by homology-based cloning and rapid amplification of cDNA ends （RACE） techniques. And, the expression level of cPLA2 gene of large yellow croaker larvae at different days of age was measured by real-time quantitative PCR method. Sequence analysis results showed that the full- length of cPLA2 cDNA （GenBank accession No. ： KF006240.1 ） was 2 660 bp, which consists of 2 169 bp in size with an open-reading frame （ORF） encoding 723 amino acids, a 176 bp 5＇-untranslated region （5＇UTR） and a 205 bp 3＇-untranslated region （3＇UTR）. Phylogenetic tree analysis results showed that the cPLA2 gene of large yellow croaker had a closer relationship with other fish species, but had a distant relationship with mammalian. Quantitative determination results showed that with the days of age of large yellow croaker increasing, the expression level of cPLA2 gene was significantly increased at first （ 1, 3 and 7 days of age vs. 15 days of age, P〈0.05） and the 15 days of age had the highest value, then significantly decreased （15 days of age vs. 19 days of age, P〈0.05）, maintained stable until the end of the experiment. In conclusion, the key enzyme of phospholipid catabolism--cPLA2 gene expression level show a regular variation during the deformation process from larvae to juvenile of large yellow croaker, which may has an important significance for keeping the dynamic equilibrium of phospholipids and maintaining the fluidity of membrane. The change trend of cPLA2 gene expression level of large yellow croaker larvae can estimate the development of digestive system of large yellow croaker in a certain degree.