家蚕核转录因子NF-κB对不同细胞系中免疫相关基因表达的调控分析

NF-κB transcription factors regulate the expression of immunity-related genes in different cell lines of silkworm(Bombyx mori)

昆虫Toll和免疫缺陷(immune deficiency,IMD)信号通路在进化上非常保守,主要通过核转录因子κB(nuclear factor-kappa B,NF-κB)调节抗菌肽等免疫相关基因的表达。然而,关于家蚕NF-κB转录因子Rels和Relish对抗菌肽等免疫相关基因表达调控的差异目前尚未见系统报道。本研究克隆了家蚕BmRelA、BmRelB和BmRelish1基因,并构建了它们的真核细胞过表达载体。将重组载体分别转染家蚕BmE和BmN细胞后,利用实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)检测了抗菌肽及免疫相关基因的表达变化。结果表明,家蚕抗菌肽基因Defensin2和Gloverin2的表达主要受Relish1调控,Moricin的表达主要受RelA和RelB调控,其他抗菌肽的表达则受二者共同调控。此外,家蚕肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)、β-1,3-葡聚糖识别蛋白(β-1,3-glucan recognition proteins,βGRPs)、溶菌酶(lysozymes,Lys)、类溶菌酶蛋白(lysozyme and lysozyme-like proteins,LLPs)、一氧化氮合酶(nitric oxide synthase,NOS)等蛋白的编码基因均响应Rels和Relish的诱导,在不同细胞系中不同程度上调表达,提示这些分子的表达也受到Toll或IMD通路的调节。相比于果蝇Toll和IMD信号通路转录因子对抗菌肽基因表达调控具有特异性,本研究发现家蚕Rels和Relishl对抗菌肽基因表达的调控模式更为复杂,为进一步解析昆虫Toll和IMD通路的效应机制和反馈机制提供了实验依据。

The Toll and immune deficiency(IMD)signaling pathways in insects are highly conserved in evolution and regulate the expression of antimicrobial peptides(AMPs)and other immune-related genes mainly through nuclear factor-kappa B(NF-κB)transcription factors.However,the differences of NF-κB transcription factors Rels and Relish in the expression regulation of AMPs and other immune-related genes in silkworm(Bombyx mori)have not been systematically reported.In this study,the BmRelA,BmRelB and BmRelish1 genes were cloned and their eukaryotic cell overexpression vectors were constructed.After the recombinant vectors were transfected into BmE and BmN cells,the expression of AMPs and immune-related genes was detected by real-time fluorescence quantitative PCR.The results showed that the expression of AMP genes Defensin2 and Gloverin2 was mainly regulated by Relish,the expression of Moricin was mainly regulated by RelA and RelB,and the expression of other AMP genes was jointly regulated by both.In addition,the expression levels of peptidoglycan recognition proteins(PGRPs),β-1,3-glucan recognition proteins(βGRPs),lysozymes(Lys)and lysozyme-like proteins(LLPs),and nitric oxide synthase(NOS)were up-regulated to varying degrees in different cell lines in response to RelA,RelB and Relish1,suggesting that the expression of these immune-molecules was also regulated by Toll or IMD pathways in silkworm.Compared with the regulatory specificity of transcription factors in Drosophila Toll and IMD signaling pathways on the expression of AMPs,this study found that the regulatory patterns of Rels and Relish1 on the expression of AMPs in silkworm are more complex,which provides an experimental basis for further analysis of the effect mechanism and feedback mechanism of Toll and IMD pathways in insects.