柞蚕胆绿素还原酶基因ApBVRB的克隆及表达分析
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摘要
柞蚕(Antheraea pernyi)幼虫体壁颜色表现出高度多样性。胆绿素还原酶(biliverdin reductase B,BVRB)可催化血红素降解过程中产生的胆绿素还原成胆红素,参与昆虫体色形成。为明确BVRB在柞蚕幼虫体色形成中的作用,克隆柞蚕胆绿素还原酶基因ApBVRB,检测其mRNA表达水平,同时调查胆绿素还原酶的活性。ApBVRB编码205个氨基酸,预测蛋白质分子质量为50.67 kD,等电点为5.15。氨基酸序列比对表明,ApBVRB与家蚕的黄素还原酶(flavin reductase,FR)具有75%的序列相似度。qRT-PCR检测结果表明,ApBVRB在柞蚕幼虫的所有受试组织器官中均表达,除丝腺外,青、黄体色幼虫之间的相对表达量并没有显著差异。酶活性检测发现,胆绿素还原酶活性在青、黄蚕的血淋巴和丝腺中有极显著差异,推测可能由于血淋巴中该酶活性的差异而导致柞蚕幼虫表现为青、黄2种体色。
Antheraea pernyi larvae show a high degree of diversity in cuticle color. Bilirubin reductase(BVRB) can catalyze the biliverdin produced in the process of heme degradation into bilirubin, and participate in the formation of cuticle color. In order to explore the function of BVRB in the cuticle color formation of A. pernyi, ApBVRB gene of A. pernyi was isolated and the relative expression level of mRNA and enzyme activity of biliverdin reductase were investigated. Results showed that ApBVRB contains an ORF encoding a protein of 205 amino acids with predicted molecular weight of 50.67 kD and isoelectric point of 5.15. Amino acid sequence alignment showed that ApBVRB has 75% sequence identity with Bombyx mori flavin reductase(FR). qRT-PCR investigation demonstrated that ApBVRB mRNA was expressed in all tested tissues and organs, and its relative expression showed no significant difference between green and yellow larvae except that in silk gland tissue. However, there were extremely significant difference in the activity of biliverdin reductase in hemolymph and silk gland between green and yellow individuals. The results indicated that activity of bilirubin reductase in hemolymph might influence the cuticle color of A. pernyi.
Antheraea pernyi larvae show a high degree of diversity in cuticle color. Bilirubin reductase(BVRB) can catalyze the biliverdin produced in the process of heme degradation into bilirubin, and participate in the formation of cuticle color. In order to explore the function of BVRB in the cuticle color formation of A. pernyi, ApBVRB gene of A. pernyi was isolated and the relative expression level of mRNA and enzyme activity of biliverdin reductase were investigated. Results showed that ApBVRB contains an ORF encoding a protein of 205 amino acids with predicted molecular weight of 50.67 kD and isoelectric point of 5.15. Amino acid sequence alignment showed that ApBVRB has 75% sequence identity with Bombyx mori flavin reductase(FR). qRT-PCR investigation demonstrated that ApBVRB mRNA was expressed in all tested tissues and organs, and its relative expression showed no significant difference between green and yellow larvae except that in silk gland tissue. However, there were extremely significant difference in the activity of biliverdin reductase in hemolymph and silk gland between green and yellow individuals. The results indicated that activity of bilirubin reductase in hemolymph might influence the cuticle color of A. pernyi.
引文
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[2] SHIRATAKI H,FUTAHASHI R,FUJIWARA H.Species-specific coordinated gene expression and trans-regulation of larval color pattern in three swallowtail butterflies[J].Evol Dev,2010,12(3):305-314
[3] SHAMIM G,RANJAN S K,PANDEY D M,et al.Biochemistry and biosynthesis of insect pigments[J].Eur J Entomol,2014,111(2):149-164
[4] O'BRIEN L,HOSICK P A,JOHN K,et al.Biliverdin reductase isozymes in metabolism[J].Trends Endocrinol Metab,2015,26(4):212-220
[5] PEREIRA P J,MACEDO-RIBERIRO S,PARRAGA A,et al.Structure of human biliverdin Ⅸβ reductase,an early fetal bilirubin Ⅸβ producing enzyme[J].Nat Struct Biol,2001,8(3):215-220
[6] SMITH L J,BROWNE S,MULHOLLAND A J,et al.Computational and experimental studies on the catalytic mechanism of biliverdin Ⅸβ reductase[J].Biochem J,2008,411(3):475-484
[7] BLUMENTHAL S G,STUCKER T,RASMUSSEN R D,et al.Changes in bilirubins in human prenatal development[J].Biochem J,1980,186(3):693-700
[8] 张宝乐,李国勤,章鹤,等.鸭BLVRA基因cDNA克隆及其在输卵管子宫部的mRNA表达量与青壳性状的相关性分析[J].畜牧兽医学报,2010,41(9):1082-1089
[9] 王晓庆,徐廷生,雷雪芹,等.鸡血红素加氧酶-1(HO-1)和胆绿素还原酶A(BLVRA)基因表达量对蛋壳颜色的影响[J].中国农学通报,2014,30(35):64-68
[10] 苏伦安.试论柞蚕体色的演变、遗传及在生产上的作用[J].安徽农学院学报,1981(1):57-60
[11] 石生林,姜义仁,杨瑞生,等.柞蚕黄体色大茧型品种沈黄2号的选育及杂交组合的选配[J].蚕业科学,2014,40(4):660-665
[12] THOMPSON J D,GIBSON T J,PLEWNIAK F,et al.The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Res,1997,25(24):4876-4882
[13] KUMAR S,STECHER G,TAMURA K.MEGA7:Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[J].Mol Biol Evol,2016,33(7):1870-1874
[14] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2 -ΔΔCtmethod[J].Methods,2001,25(4):402-408
[15] 刘文琪,李雍龙.日本血吸虫胆绿素还原酶的测定[J].中国寄生虫学与寄生虫病杂志,2004,22(2),106-108