一个二化性家蚕醛酮还原酶基因的克隆及序列与表达分析（英文）
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摘要
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序列克隆进表达载体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 c DNA 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 c DNA 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 AKR2 E subfamily of AKR2 family,and the amino acid sequence showed over 30%identity with that of AKR2 E proteins. Therefore,this protein was named as AKR2 E-like. The ORF segment of akr2 e-like gene was cloned into the plasmid p ET-28 a( +) 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 ark2 e-like gene was developmentally regulated and showed a constant increase in the early stage of pupation in diapause-destined group. The highest level of akr2 e-like mRNA expression was found on the 4 th day after pupation in diapause-destined pupae. While in the non-diapause-destined group,expression of akr2 e-like gene had no obviously fluctuation after pupation. These results supported the hypothesis that AKR2 E-like protein plays a key role in the diapause preperation of bivoltine silkworm.
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 c DNA 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 c DNA 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 AKR2 E subfamily of AKR2 family,and the amino acid sequence showed over 30%identity with that of AKR2 E proteins. Therefore,this protein was named as AKR2 E-like. The ORF segment of akr2 e-like gene was cloned into the plasmid p ET-28 a( +) 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 ark2 e-like gene was developmentally regulated and showed a constant increase in the early stage of pupation in diapause-destined group. The highest level of akr2 e-like mRNA expression was found on the 4 th day after pupation in diapause-destined pupae. While in the non-diapause-destined group,expression of akr2 e-like gene had no obviously fluctuation after pupation. These results supported the hypothesis that AKR2 E-like protein plays a key role in the diapause preperation of bivoltine silkworm.
引文
[1] PENNING T M.The aldo-keto reductases (AKRs):overview[J].Chem Biol Interact,2015,234:236-246
[2] 舒楠,陈子珺.醛酮还原酶的研究进展[J].药物生物技术,2017,24(2):85-89
[3] BARSKI O A,TIPPARAJU S M,BHATNAGAR A.The aldo-keto reductase superfamily and its role in drug metabolism and detoxification[J].Drug Metab Rev,2008,40(4):553-624
[4] DANKS H V.Seasonal adaptations in Arctic insects[J].Integr Com Biol,2004,44(2):85-94
[5] EGI Y,AKITOMO S,FUJII T,et al.Silkworm strains that can be clearly destined towards either embryonic diapause or direct development by adjusting a single ambient parameter during the preceding generation[J].Entomol Sci,2014,17(4):396-399
[6] YAMAMOTO K,WILSON D K.Identification,characterization,and crystal structure of an aldo-keto reductase (AKR2E4) from the silkworm Bombyx mori[J].Arch Biochem Biophys,2013,538(2):156-163
[7] HAHN D A,DENLINGER D L.Energetics of insect diapause[J].Annu Rev Entomol,2011,56:103-121
[8] COLEMAN R A,LEE D P.Enzymes of triacylglycerol synthesis and their regulation[J].Prog Lipid Res,2004,43(2):134-176
[9] ZHAO L,JONES W.Expression of heat shock protein genes in insect stress responses[J].Invertebr Survival J,2012,9(1):93-101
[10] PETRASH J M,MURTHY B S,YOUNG M,et al.Functional genomic studies of aldo-keto reductases[J].Chem Biol Interact,2001,130-132(1/3):673-683
[2] 舒楠,陈子珺.醛酮还原酶的研究进展[J].药物生物技术,2017,24(2):85-89
[3] BARSKI O A,TIPPARAJU S M,BHATNAGAR A.The aldo-keto reductase superfamily and its role in drug metabolism and detoxification[J].Drug Metab Rev,2008,40(4):553-624
[4] DANKS H V.Seasonal adaptations in Arctic insects[J].Integr Com Biol,2004,44(2):85-94
[5] EGI Y,AKITOMO S,FUJII T,et al.Silkworm strains that can be clearly destined towards either embryonic diapause or direct development by adjusting a single ambient parameter during the preceding generation[J].Entomol Sci,2014,17(4):396-399
[6] YAMAMOTO K,WILSON D K.Identification,characterization,and crystal structure of an aldo-keto reductase (AKR2E4) from the silkworm Bombyx mori[J].Arch Biochem Biophys,2013,538(2):156-163
[7] HAHN D A,DENLINGER D L.Energetics of insect diapause[J].Annu Rev Entomol,2011,56:103-121
[8] COLEMAN R A,LEE D P.Enzymes of triacylglycerol synthesis and their regulation[J].Prog Lipid Res,2004,43(2):134-176
[9] ZHAO L,JONES W.Expression of heat shock protein genes in insect stress responses[J].Invertebr Survival J,2012,9(1):93-101
[10] PETRASH J M,MURTHY B S,YOUNG M,et al.Functional genomic studies of aldo-keto reductases[J].Chem Biol Interact,2001,130-132(1/3):673-683
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