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

Cloning and Expression Characteristics of Ribose-5-phosphate Isomerase A Gene of Nosema bombycis

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 kD,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.