家蚕Met1基因的表达与组织定位分析
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摘要
选择家蚕基因组中与果蝇Dm Met(Methoprene tolerant protein)同源的基因Bm Met1进行研究。预测Dm Met和Bm Met1的结构域均是一个b HLH-PAS(basic helix-loop-helix Per-ARNT-SIM)型转录因子,具有典型的DNA结合结构域。在原核系统表达获得了Bm Met1蛋白并制备了抗体。qRT-PCR和Western blotting检测结果显示,无论是Bm Met1基因mRNA的转录还是Bm Met1蛋白的表达,在家蚕5龄中后期和吐丝期的翅原基组织中都呈现较高水平,而在蛹期呈现较低的水平。通过免疫组织化学方法分析Bm Met1的组织定位,结果显示该蛋白质在5龄第6天和吐丝期家蚕胸节表皮、脂肪体及翅原基等组织中都有较高水平的表达,在蛹期上述部位和组织的表达水平较低。分别将蜕皮激素(20E)和保幼激素类似物(Methoprene)按照2μg/头的剂量注射到5龄第3天幼虫第2~3胸节间,qRT-PCR检测幼虫翅原基组织中的Bm Met1受到Methoprene的诱导上调表达,且在处理2 h后表达水平最高。上述结果暗示Bm Met1可能参与了保幼激素对家蚕翅原基生长分化的调控。
Bm Met1,the homologous gene of fruit fly Dm Met( Methoprene tolerant protein) in silkworm( Bombyx mori),was chosen to analyze its sequence structure. Structural domain prediction for Dm Met and Bm Met1 showed that both of them are b HLH-PAS( basic helix-loop-helix Per-ARNT-SIM) transcription factors,which contain the typical DNAbinding structural domain. The recombinant Bm Met1 protein was expressed in prokaryotic system and the relevant antibody was prepared. Real-time fluorescence quantitative PCR( qRT-PCR) result of Bm Met1 gene and Western blotting suggested that both mRNA transcription and Bm Met1 protein expression were at high levels in wing disc during the middle and late stages of the 5th larval instar and during the wandering stage,but were at low levels during the pupa stage. The result of immunohistochemical localization showed that Bm Met1 had high expression level in larval epidermis,fat body and wing disc at day 6 of the 5th instar and during the wandering stage,while it was expressed at low level during pupa stage. After ecdysone( 20E) and juvenile hormone analog( Methoprene) was injected respectively into the 2nd to 3rd inter-thoracic segment of day 3 silkworm larvae of the 5th instar at 2 μg/larva dosage,qRT-PCR as-say showed that the expression of Bm Met1 was induced by Methoprene and exhibited the highest up-regulation at2 h post injection. These results suggest that Bm Met1 is involved in the regulation of silkworm wing disc growth and differentiation triggered by juvenile hormone.
Bm Met1,the homologous gene of fruit fly Dm Met( Methoprene tolerant protein) in silkworm( Bombyx mori),was chosen to analyze its sequence structure. Structural domain prediction for Dm Met and Bm Met1 showed that both of them are b HLH-PAS( basic helix-loop-helix Per-ARNT-SIM) transcription factors,which contain the typical DNAbinding structural domain. The recombinant Bm Met1 protein was expressed in prokaryotic system and the relevant antibody was prepared. Real-time fluorescence quantitative PCR( qRT-PCR) result of Bm Met1 gene and Western blotting suggested that both mRNA transcription and Bm Met1 protein expression were at high levels in wing disc during the middle and late stages of the 5th larval instar and during the wandering stage,but were at low levels during the pupa stage. The result of immunohistochemical localization showed that Bm Met1 had high expression level in larval epidermis,fat body and wing disc at day 6 of the 5th instar and during the wandering stage,while it was expressed at low level during pupa stage. After ecdysone( 20E) and juvenile hormone analog( Methoprene) was injected respectively into the 2nd to 3rd inter-thoracic segment of day 3 silkworm larvae of the 5th instar at 2 μg/larva dosage,qRT-PCR as-say showed that the expression of Bm Met1 was induced by Methoprene and exhibited the highest up-regulation at2 h post injection. These results suggest that Bm Met1 is involved in the regulation of silkworm wing disc growth and differentiation triggered by juvenile hormone.
引文
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[20]MURAMATSU D,KINJOH T,SHINODA T,et al.The role of 20-hydroxyecdysone and juvenile hormone in pupal commitment of the epidermis of the silkworm,Bombyx mori[J].Mech Dev,2008,125(5/6):411-420
[2]RIDDFORD L M,TRUMAN J W,CHERBAS P.Ecdysone receptors and their biological actions[J].Vitam Horm,2000,60(1):1-73
[3]JONES G.Molecular mechanisms of action of juvenile hormone[J].Annu Rev Entomol,1995,40:147-169
[4]RIDDFORD L M.Juvenile hormone action:a 2007 perspective[J].J Insect Physiol,2008,54(6):895-901
[5]ASHOK M,TURNER C,WILSON T G.Insect juvenile hormone resistance gene homology with the b HLH-PAS family of transcriptional regulators[J].Proc Natl Acad Sci USA,1998,95(6):2761-2766
[6]LETUNIC I,COPLEY R R,PILS B,et al.SMART 5:domains in the context of genomes and networks[J].Nucleic Acids Res,2006,34(D1):D257-D260
[7]MASSARI M E,MURRE C.Helix-loop-helix proteins:regulators of transcription in eucaryotic organisms[J].Mol Cell Biol,2000,20(2):429-440
[8]KONOPOVA B,JINDRA M.Juvenile hormone resistance gene Methoprene-tolerant controls entry into metamorphosis in the beetle Tribolium castaneum[J].Proc Natl Acad Sci USA,2007,104(25):10488-10493
[9]PARTHASARATHY R,TAN A,PALLI S R.b HLH-PAS family transcription factor methoprene-tolerant plays a key role in JH action in preventing the premature development of adult structures during larval-pupal metamorphosis[J].Mech Dev,2008,125(7):601-616
[10]MIURA K,ODA M,MAKITA S.Characterization of the Drosophila Methoprene-tolerant gene product.Juvenile hormone binding and ligand-dependent gene regulation[J].FEBS J,2005,272(5):1169-1178
[11]GODLEWSKI J,WANG S,WILSON T G.Interaction of b HLH-PAS proteins involved in juvenile hormone reception in Drosophila[J].Biochem Biophys Res Commun,2006,342(4):1305-1311
[12]BAUMANN A,BARRY J,WANG S,et al.Paralogous genes involved in juvenile hormone action in Drosophila melanogaster[J].Genetics,2010,185(4):1327-1336
[13]CHARLES J P,IWEMA T,EPA V C,et al.Ligand-binding properties of a juvenile hormone receptor,Methoprene-tolerant[J].Proc Natl Acad Sci USA,2011,108(52):21128-21133
[14]GUO E,HE Q,LIU S,et al.MET is required for the maximal action of 20-hydroxyecdysone during Bombyx metamorphosis[J/OL].PLo S ONE,2012,7:e53256[2017-02-06].https://doi.org/10.1371/journal.pone.0053256
[15]胡启豪,刘学术,马琼,等.保幼激素和蜕皮激素对家蚕翅原及分化的影响[J].华南师范大学学报(自然科学版),2017,49(4):62-67
[16]PARTCH C L,GAEDNER K H.Coactivator recruitment:a new role for PAS domains in transcriptional regulation by the b HLH-PAS family[J].J Cell Physiol,2010,223(3):553-557
[17]杨鑫华.家蚕变态发育期间20E和JHⅢ水平及20E受体和相关转录因子的表达分析[D].广州:华南师范大学,2010
[18]KAYUKAWA T,MINAKUCHI C,NAMIKI T,et al.Transcriptional regulation of juvenile hormone-mediated induction of Kruppel homolog 1,a repressor of insect metamorphosis[J].Proc Natl Acad Sci USA,2012,109(29):11729-11734
[19]KAYUKAWA T,NAGAMINE K,ITO Y,et al.Kruppel homolog 1inhibits insect metamorphosis via direct transcriptional repression of Broad-complex,a pupal specifier gene[J].J Biol Chem,2016,291(4):1751-1762
[20]MURAMATSU D,KINJOH T,SHINODA T,et al.The role of 20-hydroxyecdysone and juvenile hormone in pupal commitment of the epidermis of the silkworm,Bombyx mori[J].Mech Dev,2008,125(5/6):411-420