家蚕微孢子虫核糖-5-磷酸异构酶A基因的克隆及表达特征分析
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摘要
核糖-5-磷酸异构酶A(ribose 5-phosphate isomerase A,RpiA)是多种生物中普遍存在的一种高度保守的蛋白酶,在磷酸戊糖途径(PPP)中起着核心作用,参与原核生物与真核生物核糖-5-磷酸(R5P)与核酮糖-5-磷酸(Ru5P)之间的可逆异构化反应及植物二氧化碳固定的卡尔文循环。为探索RpiA在家蚕微孢子虫侵染家蚕后能量代谢与物质合成过程中所发挥的作用,通过PCR扩增得到NbRpiA基因的编码区。该开放阅读框全长345 bp,编码114个氨基酸,预测蛋白质的分子质量约为13.145 kD,等电点为7.72,未发现明显已知功能结构域。实时荧光定量PCR检测发现,NbRpiA基因在家蚕微孢子虫感染家蚕后7 d内均有表达:从感染后2 h起,NbRpiA基因的表达水平呈缓慢上升的趋势,第2天达到最高,随后表达水平逐渐降低,从第4天开始表达水平大幅降低,至第7天降至最低。初步推测NbRpiA可能在家蚕微孢子虫孢子发芽及增殖阶段发挥作用。研究结果为了解NbRpiA在家蚕微孢子虫能量代谢与物质合成中的作用提供了初步线索。
Ribose 5-phosphate isomerase A( RpiA) is a highly conserved protease widely found in many organisms. It plays a central role in the pentose phosphate pathway(PPP),and participates in the reversible isomerization reactions between ribose-5-phosphate( R5 P) and ribulose-5-phosphate( Ru5 P) of prokaryote and eukaryote,as well as carbon dioxide fixation via Calvin cycle of plant. In order to study the role of RpiA on energy metabolism and substance synthesis in the process of silkworm infected by Nosema bombycis,NbRpi A gene was amplified by PCR. It contained an open reading frame(ORF) of 345 bp in length and encoded 114 amino acids. The molecular mass of the predicted protein was approximately 13. 145 k D,the isoelectric point was 7. 72,and there was no obvious characterized functional domain. qRT-PCR detection showed that NbRpi A gene was expressed in silkworm mid gut within 7 days after being infected by N. bombycis.The expression level of NbRpi A gene gradually increased from 2 h post infection,reached the highest on the 2 nd day,and then gradually decreased slowly. The expression level of NbRpi A gene decreased sharply from the 4 th day and droped to the bottom on the 7 th day. It was presumed that NbRpiA may play an important role in the germination and proliferation stages of N. bombycis in silkworm. This study will lay foundation for further understanding on the role of NbRpiA in energy metabolism and substance synthesis of N.bombycis during infection.
Ribose 5-phosphate isomerase A( RpiA) is a highly conserved protease widely found in many organisms. It plays a central role in the pentose phosphate pathway(PPP),and participates in the reversible isomerization reactions between ribose-5-phosphate( R5 P) and ribulose-5-phosphate( Ru5 P) of prokaryote and eukaryote,as well as carbon dioxide fixation via Calvin cycle of plant. In order to study the role of RpiA on energy metabolism and substance synthesis in the process of silkworm infected by Nosema bombycis,NbRpi A gene was amplified by PCR. It contained an open reading frame(ORF) of 345 bp in length and encoded 114 amino acids. The molecular mass of the predicted protein was approximately 13. 145 k D,the isoelectric point was 7. 72,and there was no obvious characterized functional domain. qRT-PCR detection showed that NbRpi A gene was expressed in silkworm mid gut within 7 days after being infected by N. bombycis.The expression level of NbRpi A gene gradually increased from 2 h post infection,reached the highest on the 2 nd day,and then gradually decreased slowly. The expression level of NbRpi A gene decreased sharply from the 4 th day and droped to the bottom on the 7 th day. It was presumed that NbRpiA may play an important role in the germination and proliferation stages of N. bombycis in silkworm. This study will lay foundation for further understanding on the role of NbRpiA in energy metabolism and substance synthesis of N.bombycis during infection.
引文
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[19] 钱永华,鲁兴萌,金伟,等.家蚕微孢子虫(Nosema bombycis)向家蚕BmN细胞接种与增殖的观察[J].蚕业科学,2003,29(3):260-264
[20] HE X,FU Z,LI M,et al.Nosema bombycis (Microsporidia) suppresses apoptosis in BmN cells (Bombyx mori)[J].Acta Biochim Biophys Sin (Shanghai),2015,47(9):696-702
[2] HE X,HE X,LIU H,et al.Proteomic analysis of BmN cells (Bombyx mori) in response to infection with Nosema bombycis[J].Acta Biochim Biophys Sin (Shanghai),2014,46(11):982-990
[3] LIU H,CHEN B,HU S,et al.Quantitative proteomic analysis of germination of Nosema bombycis spores under extremely alkaline conditions[J/OL].Front Microbiol,2016,7:1459[2018-05-01].https://www.ncbi.nlm.nih.gov/pubmed/27708628.DOI:10.3389/fmicb.2016.01459
[4] 武文琦,丛旭珍,殷爱红,等.变异链球菌核糖-5-磷酸异构酶A的表达、纯化与鉴定[J].首都医科大学学报,2012(2):227-232
[5] WAMELINK M M,GRUNING N M,JANSEN E E,et al.The difference between rare and exceptionally rare:molecular characterization of ribose 5-phosphate isomerase deficiency[J].J Mol Med (Berl),2010,88(9):931-939
[6] HOLMES M A,BUCKNER F S,VAN VOORHIS W C,et al.Structure of ribose 5-phosphate isomerase from Plasmodium falciparum[J].Acta Crystallogr,F,2006,62(5):427-431
[7] GONTERO B,CáRDENAS M L,RICARD J.A functional five-enzyme complex of chloroplasts involved in the Calvin cycle[J].Eur J Biochem,1988,173(2):437-443
[8] ESSENBERG M K,COOPER R A.Two ribose-5-phosphate isomerases from Escherichia coli K12:partial characterisation of the enzymes and consideration of their possible physiological roles[J].Eur J Biochem,1975,55(2):323-332
[9] LOBLEY C M,ALLER P,DOUANGAMATH A,et al.Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118[J].Acta Crystallogr,F,2012,68(12):1427-1433
[10] CAPRILES P V,BAPTISTA L P,GUEDES I A,et al.Structural modeling and docking studies of ribose 5-phosphate isomerase from Leishmania major and Homo sapiens:a comparative analysis for Leishmaniasis treatment[J].J Mol Graph Model,2015,55:134-147
[11] ZHANG R G,ANDERSSON C E,SKARINA T,et al.The 2.2? resolution structure of RpiB/AlsB from Escherichia coli illustrates a new approach to the ribose-5-phosphate isomerase reaction[J].J Mol Biol,2003,332(5):1083-1094
[12] STERN A L,BURGOS E,SALMON L,et al.Ribose 5-phosphate isomerase type B from Trypanosoma cruzi:kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism[J].Biochem J,2007,401(1):279-285
[13] LI Z,WANG Y,WANG L,et al.Molecular and biochemical responses in the midgut of the silkworm,Bombyx mori,infected with Nosema bombycis[J/OL].Parasit Vectors,2018,11(1):147[2018-05-01].https://www.ncbi.nlm.nih.gov/pubmed/29510742.DOI:10.1186/s13071-018-2755-2
[14] VAN DER GIEZEN M,TOVAR J,CLARK C G.Mitochondrion-derived organelles in protists and fungi[J].Int Rev Cytol,2005,244:175-225
[15] KAUR P K,DINESH N,SOUMYA N,et al.Identification and characterization of a novel ribose 5-phosphate isomerase B from Leishmania donovani[J].Biochem Biophys Res Commun,2012,421(1):51-56
[16] WOODRUFF W W Ⅲ,WOLFENDEN R.Inhibition of ribose-5-phosphate isomerase by 4-phosphoerythronate[J].J Biol Chem,1979,254(13):5866-5867
[17] PARK K E,ANDERSON L E.Appearance of three chloroplast isoenzymes in dark-grown pea plants and pea seeds[J].Plant Physiol,1973,51(2):259-262
[18] DE SINATTI V V C,BAPTISTA L P R,ALVES-FERREIRA M,et al.In silico identification of inhibitors of ribose 5-phosphate isomerase from Trypanosoma cruzi using ligand and structure based approaches[J].J Mol Graph Model,2017,77:168-180
[19] 钱永华,鲁兴萌,金伟,等.家蚕微孢子虫(Nosema bombycis)向家蚕BmN细胞接种与增殖的观察[J].蚕业科学,2003,29(3):260-264
[20] HE X,FU Z,LI M,et al.Nosema bombycis (Microsporidia) suppresses apoptosis in BmN cells (Bombyx mori)[J].Acta Biochim Biophys Sin (Shanghai),2015,47(9):696-702