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醛酮还原酶(aldo-keto reductase,AKR)超级家族成员以还原型烟酰胺腺嘌呤二核苷酸磷酸作为其辅酶,将醛酮类化合物还原成相应的醇类,其作用底物范围广泛,包括糖类、脂肪醛和甾体类激素等,在许多重要生物学反应中发挥关键作用.以二化性家蚕品系秋丰为材料,利用RT-PCR分别从滞育命运组和非滞育命运组克隆出AKR蛋白的cDNA序列.AKR蛋白序列由343个氨基酸组成,与黑腹果蝇和人的AKR分别有43％和38％的序列一致性.基于蛋白质序列构建的系统发育树显示,家蚕AKR蛋白属于AKR2E亚家族,其氨基酸序列与该家族成员序列一致性超过30％,故命名为AKR2E-like.将akr2e-like基因的cDNA序列克隆进表达栽体pET-28a(+)中进行原核重组表达,SDS-PAGE电泳和Western blot分析显示重组蛋白被成功表达.qRT-PCR分析表明,ark2e-like基因表达受发育环境调节:在滞育诱导条件下,该基因在家蚕化蛹早期的表达量持续增加,在化蛹后第4天mRNA丰度达到最高值;而在非滞育诱导条件下,化蛹后ark2e-like基因表达量并无明显波动.上述结果表明,ark2e-like基因有可能在家蚕二化性品系的滞育准备期起关键作用.“,”Aldo-keto reductase (AKR) super family comprises proteins that catalyze the NADPH-dependent reduction of ketones and aldehydes,and converses aldehyde to alcohols.They have a wide variety of substrates,including sugars,fatty aldehyde and steroid hormones,which play a key role in some important biological reactions.In this study,the cDNA encoding AKR protein was isolated from bivoltine silkworm strain Qiufeng in both diapause-destined group and non-diapause-destined group using RT-PCR technology.The cDNA encoding a 343 amino acids protein showed 43％ and 38％ identity with AKRs from Drosophila melanogaster and Homo sapiens,respectively.Phylogenetic analysis suggested that this protein belonged to AKR2E subfamily of AKR2 family,and the amino acid sequence showed over 30％ identity with that of AKR2E proteins.Therefore,this protein was named as AKR2E-like.The ORF segment of akr2e-like gene was cloned into the plasmid pET-28a(+) to construct a recombinant expression plasmid.SDS-PAGE and Western blot results revealed that the His-tagged fusion protein was successfully expressed.Moreover,qRT-PCR analysis demonstrated that the expression of ark2e-like gene was developmentally regulated and showed a constant increase in the early stage of pupation in diapause-destined group.The highest level of akr2e-like mRNA expression was found on the 4th day after pupation in diapause-destined pupae.While in the non-diapause-destined group,expression of akr2e-like gene had no obviously fluctuation after pupation.These results supported the hypothesis that AKR2E-like protein plays a key role in the diapause preperation of bivoltine silkworm.