关岭牛FABP1FABP2基因克隆及其组织表达分析

Cloning and tissue expression analysis of Guanling cattle FABP1 and FABP2 genes

  • 摘要: 【目的】明确肝脏型脂肪酸结合蛋白(FABP1)和肠型脂肪酸结合蛋白(FABP2)基因的分子特征及其在不同年龄段和不同品种牛各组织中的表达情况,为后续研究FABP1和FABP2对牛脂肪酸的调控作用机制提供理论依据。【方法】采用RT-PCR扩增关岭牛FABP1FABP2基因编码区(CDS)序列,通过ProtParam、ProtScale、SOPMA、SWISSMODEL、TMHMM-2.0、SignalP-5.0和NetPhos-3.1等在线软件进行生物信息学分析,并以实时荧光定量PCR检测FABP1FABP2基因在3日龄关岭牛(犊牛)、24月龄关岭牛(成年牛)及24月龄杂交牛(关岭牛×利木赞牛)各组织中的表达情况。【结果】 FABP1、FABP2基因CDS序列全长分别为384和399 bp,对应编码127和132个氨基酸残基,其编码蛋白二、三级结构均以无规则卷曲和延伸链为主,无跨膜结构域和信号肽剪切位点,且属于亲水性稳定蛋白。FABP1、FABP2氨基酸序列分别包含19和18个磷酸化位点,其中苏氨酸在2个氨基酸序列中均广泛分布。FABP1、FABP2氨基酸序列同源比对及系统发育进化分析结果均显示,关岭牛与马、绵羊和山羊的亲缘关系较近,而与鸡和鸭的亲缘关系较远,与其他物种的相似性都在75.0%以上。FABP1、FABP2基因在不同年龄段和不同品种牛的各组织中均有表达,其中,FABP1基因以在肝脏和小肠中的相对表达量较高,且犊牛和杂交牛多个组织中的FABP1基因相对表达量极显著高于成年关岭牛(P<0.01,下同); FABP2基因则主要在小肠中高表达,且成年关岭牛小肠中的FABP2基因相对表达量极显著高于犊牛、显著高于杂交牛(P<0.05)。【结论】 FABP1FABP2基因在关岭牛中保守性较高,且在不同年龄阶段及不同组织中均有表达,尤其在肝脏和小肠中的表达水平较高,故推测FABP1FABP2基因协同调控关岭牛脂肪酸的合成代谢。

     

    Abstract: 【Objective】 The molecular characteristics of liver-type fatty acid binding protein(FABP1)and intestinaltype fatty acid binding protein(FABP2)genes and their expression in various tissues of cattle of different ages and breeds were clarified to provide a theoretical basis for subsequent studies on the mechanisms of the regulatory effects of FABP1 and FABP2 on bovine fatty acids.【Method】 The sequences of the coding regions(CDS)of FABP1 and FABP2 genes in Guanling cattle were amplified by RT-PCR,and bioinformatics analysis was performed by online softwares such as ProtParam, ProtScale, SOPMA, SWISS-MODEL, TMHMM-2.0, SignalP-5.0 and NetPhos-3.1, and the expression of FABP1 and FABP2 genes in various tissues of 3-day-old Guanling cattle(calves),24-month-old Guanling cattle(adults)and 24-month-old crossbred cattle(Guanling cattle×Limousin cattle)was examined by real-time fluorescence quantitative PCR.【Result】 The full-length CDS sequences of FABP1 and FABP2 genes were 384 and 399 bp,respectively,encoding 127 and 132 amino acid residues. The secondary and tertiary structures of their encoded proteins were dominated by random coil and extended chain without transmembrane structural domains and signal peptide shear sites,and they were hydrophilic stable proteins. The amino acid sequences of FABP1 and FABP2 contained 19 and 18 phosphorylation sites respectively,of which threonine was widely distributed in both amino acid sequences. Homology comparison and phylogenetic evolutionary analysis of the amino acid sequences of FABP1 and FABP2 showed that Guanling cattle were closely related to horses,sheep and goats,while they were more distantly related to chickens and ducks,and the similarities with other species were also above 75.0%. FABP1 and FABP2 genes were expressed in all tissues of cattle of different ages and breeds,among which,the relative expression of FABP1 gene was higher in liver and small intestine,and the relative expression of FABP1 gene in several tissues of calves and crossbred cattle was extremely significantly higher than that of adult Guanling cattle(P<0.01,the same below). The FABP2 gene was highly expressed mainly in the small intestine,and the relative expression of FABP2 gene in the small intestine of adult Guanling cattle was extremely significantly higher than that of calves and significantly higher than that of crossbred cattle(P<0.05).【Conclusion】FABP1 and FABP2 genes are highly conserved in Guanling cattle and are expressed in different age stages and tissues,especially in the liver and small intestine,so it is hypothesized that FABP1 and FABP2 genes synergistically regulate fatty acid anabolism in Guanling cattle.

     

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