德国小蠊Akirin基因的克隆及表达分析

Cloning and Expression Analysis of Akirin Gene from Blattella germanica

Akirin基因是新近发现的在果蝇NF-κB依赖的Imd信号通路调控中不可或缺的转录因子。在除果蝇以外的昆虫中,Akirin基因是否参与Imd通路的调控,亦或对NF-κB依赖的Toll通路也有调控作用还未有报道。本研究旨在初步研究德国小蠊Blattella germanica中Akirin基因的基本特征、在不同组织中的表达模式及注射法RNAi实验对Akirin基因沉默效果,以期为Akirin基因功能的研究奠定基础。通过与已报道的其它昆虫的Akirin基因进行同源比对,利用RACE（rapid amplification of c DNA ends）技术,成功从德国小蠊中克隆到1个Akirin基因,该基因全长864 bp,开放阅读框（ORF）大小为543 bp,编码180个氨基酸。蛋白序列比对及进化树结果显示,Akirin基因有非常保守的核定位信号序列,且与内华达古白蚁Zootermopsis nevadensis的亲缘关系最近,同源性为86%。荧光定量PCR的结果表明Akirin基因在德国小蠊检测的4个组织中均有表达,但表达水平有差异,在血淋巴的表达水平最高,其次为脂肪体。通过注射法RNAi实验成功抑制了Akirin基因的表达水平,并且RNAi的效果随时间的延长而更强,在注射72 h后,Akirin基因m RNA的表达水平下降了90%。上述研究为Akirin基因的功能及德国小蠊防治研究提供了科学依据。

Akirin gene is a recently discovered transcription factor,which is indispensible in the regulation of Imd signaling pathway that is required for the NF-κB in Drosophila melanogaster.However,the function of Akirin in the regulation of Imd or Toll signaling pathways in other insects was still poorly understood.This study was aimed to investigate the basic characteristics of Akirin gene in Blattella germanica,the expression patterns in different tissues and the silencing effect of RNAi injection on Akirin gene,so as to lay a foundation for the study of Akirin gene function.A Akirin gene of B.germanica（Bg Akirin） was cloned by homologous comparison with other reported Akirin genes and RACE（rapid amplification of c DNA ends） technique.The full-length of Akirin was 864 bp and the open reading frame（ORF） was 543 bp,encoding 180 amino acids.Protein sequence alignment and the results of phylogenetic tree indicated that the Bg Akirin had a very conserved nuclear localization signal sequence.And Bg Akirin shared 86% identity with the known Akirin（Zootermopsis nevadensis）.The results of real-time quantitative PCR revealed that the expression of Akirin gene was detected in four tissues,but with different expression levels among different tissues.Bg Akirin was mainly expressed in the hemolymph and fat body.The expression level of Akirin gene was inhibited successfully by injection of RNAi,and the effect of RNAi increased with time.The m RNA expression level of RNAi gene decreased by 90% after 72 hours.This study might provide scientific reference for the function of Akirin gene and the control of B.germanica.