三疣梭子蟹(Portunus trituberculatus)FABP基因克隆及其在卵巢发育过程中的表达特征研究

CLONING AND EXPRESSION ANALYSIS OF FATTY ACID-BINDING PROTEIN IN OVARIAN DEVELOPMENT OF PORTUNUS TRITUBERCULATUS

脂肪酸结合蛋白(fatty acid-binding protein,FABP)是胞内脂质结合蛋白超家族成员,在长链脂肪酸的摄取、转运及代谢调节中发挥重要作用。本研究采用RACE技术克隆获得三疣梭子蟹(Portunus trituberculatus)脂肪酸结合蛋白基因(简称Pt FABP)c DNA全长,并采用实时荧光定量PCR技术测定了FABP基因在三疣梭子蟹不同组织和不同发育阶段卵巢中的表达情况,探讨了Pt FABP在卵巢发育过程中的作用。Pt FABP基因全长893bp,包括289bp的5’UTR,405bp的ORF和199bp的3’UTR。该基因编码长度为134个氨基酸的多肽,其理论p I为5.92,分子量为15.13k Da。Pt FABP氨基酸序列与中华绒螯蟹同源性最高(72%),且有一个Lipocalin结构域。q PCR的结果表明:FABP在三疣梭子蟹所有组织中均有表达,在卵巢中表达量最高,肝胰腺、眼柄中表达量中等,心脏、鳃、肌肉和血淋巴细胞中的表达较低。在卵巢发育过程中Pt FABP的表达量呈现先上升后下降的趋势,卵巢发育至近成熟期(Ⅳ期)时达到最高值,而在成熟排卵后不断降低。这与三疣梭子蟹卵巢发育伴随着脂类大量积累相关。因此上述研究结果表明,FABP可能参与调控三疣梭子蟹卵巢的发育,并且为后期胚胎的发育储存能量。

As being the members of intracellular lipid-binding protein superfamily, fatty acid-binding proteins （FABPs） play an important role in absorption, transportation, and metabolic regulation for long-chain fatty acids. To understand the function of these FABPs during the ovarian development of Portunus trituberculatus, we applied reversed transcript PCR （RT-PCR） and rapid amplification of cDNA ends （RACE） techniques to clone the FABP （coded as PtFABP）, and detected its gene expression by quantitative real-time PCR. The full-length of FABP is 893bp, including a 289bp 5＇-untranslated region （UTR）, a 199bp 3＇-UTR and a 405bp opening reading frame （ORF） encoding 134 amino acid protein whose isoelectric point （pI） is 5.92 and molecular mass is 15.13kDa. The deduced amino acid sequence of FABP exhibited the highest identity with that of Eriocheir sinensis （72%）. FABP mRNA was detected in the hemocyte, hepatopancreas, muscle, gill, eyestalk, heart and ovary. As the result of real-time quantitative PCR （qPCR） shows, the PtFABP was expressed in all the tested tissues, of which the highest in ovary, intermediate in hepatopancreas and eyestalk, lowest in heart, gill, muscle and hemocyte. Moreover, the expression of the PtFABP mRNA in the ovarian development stages was increased first and then decreased, it appeared at stage 1V （nearly-mature）, and then decreased after ovulation. We believe that this phenomenon was resulted from the substantial accumulation of lipids during ovarian development stage in order to maintain normal growth state after ovulation. Therefore, the PtFABP is important in regulating the ovarian development and storing energy for embryo development of P. trituberculatus.