斜纹夜蛾视黄酸结合蛋白(Slcrabp)基因的克隆、表达及功能

摘要

视黄酸结合蛋白(Cellular retinoic acid binding protein,CRABP)属于胞内脂质结合蛋白超基因家族,参与了许多生理活动,如细胞分化、组织重建和信号转导等,但其在昆虫中肠的作用尚不明确.研究从斜纹夜蛾Spodoptera litura中肠基因表达序列标签(EST)文库中克隆获得一个编码Slcrabp的全长cDNA,该cDNA由396个核苷酸组成,编码132个氨基酸.预测CRABP蛋白质的空间结构与脂肪酸结合蛋白非常相似,含一个由10个反平行的β折叠和2个α螺旋形成的配体结合中心结构域.SlCRABP基因具有4个外显子,与脊椎动物crabp基因类似.RT-PCR检测表明,在转录水平上,Slcrabp在6龄幼虫中肠的各个时期均有较高表达.Western blot分析结果显示,SlCRABP蛋白分布广泛,在中肠、脂肪体、精巢等组织上大量表达.在中肠,其表达峰值出现在预蛹期.利用荧光标记物质8-苯胺基-1-萘磺酸(1,8-ANS)分析了重组SlCRABP蛋白与不同底物的亲和力,发现SlCRABP与不饱和长链脂肪酸有较高结合活性,如花生四烯酸钠、亚麻酸、油酸钠和油酸,但与视黄酸、视黄醇的结合力却很弱或几乎不结合,暗示斜纹夜蛾SlCRABP功能与同家族脂肪酸结合蛋白(FABP)性质相似.对6龄幼虫进行饥饿处理,结果显示经过经24 h和48 h饥饿处理后,虫体中肠SlCRABP蛋白质的表达量有显著上升,暗示SlCRABP可能参与了斜纹夜蛾体内脂类的转运过程.%Cellular retinoic acid binding protein (CRABP) belongs to a superfamily of intracellular lipid binding proteins.CRABP plays an important role in a wide range of physiological processes,including cell differentiation,tissue reconstruction and signal transduction,but its role in the insect midgut remains to be investigated.A full-length cDNA clone named Slcrabp was identified from an Expressed Sequence Tag (EST) library of the midgut of Spodoptera litura.Slcrabp cDNA is consisted of 396 nucleotides and encodes a polypeptide of 132 amino acids.The predicated spatial structure of SlCRABP is similar to that of a typical fatty acid binding protein of 10-stranded β-barrel folding and 2 α helixes,which forms a cavity where lipophilic ligand resides.Based on the genomic data of S.litura,Slcrabp gene contains 4 exons,which is similar to the vertebrate crabp genes.RT-PCR showed that Slcrabp kept high expression in the midgut throughout all six larval stages.Western blotting analysis results revealed that SlCRABP protein had abundant expression in the midgut,fat body,and testis.In the midgut SlCRABP attained the highest expression level at the prepupal stage.Binding affinity of SlCRABP was assessed by displacement assay of a fluorescent marker,1-anilinonaphthalene-8-sulfonic acid (1,8-ANS).The results showed that SlCRABP had higher binding affinity with unsaturated long chain fatty acids,including arachidonic acid,sodium acid,linolenic acid,sodium oleate and oleic acid.However,SlCRABP almost did not bind with retinoic acid or retinol.These results indicate that Slcrabp may play a role as a fatty acid binding protein.Larvae at the sixth instar stage were starved for 24 h and 48 h,respectively,and the expression of SlCRABP protein was detected.The results indicated the significant increase in the SlCRABP level in the midgut.This suggested that starvation induced the expression of SlCRABP,which may play a role in the transport of fatty acids in the midgut of S.litura.