Gene expression patterns in response to pathogen challenge and interaction with hemolin suggest that the Yippee protein of Antheraea pernyi is involved in the innate immune response.
Yippee was first identified as a protein that physically interacts with the Hemolin protein of Hyalophora cecropia. In this study, we identified a gene with a 366bp open reading frame (ORF) that encodes a 121 amino acid protein containing a conserved Yippee domain. We named this gene Ap-Yippee (Yippee gene from Antheraea pernyi), and investigated the role of the protein in the host immune response. A recombinant Ap-Yippee protein was expressed in Escherichia coli cells, and polyclonal antibodies were produced against the recombinant protein. Real-time PCR and a Western blot analysis revealed that Ap-Yippee is expressed in the hemocytes, Malpighian tubules, midgut, silk gland, epidermis, and fat bodies of A. pernyi, with the highest expression level observed in Malpighian tubules. The fifth instar larvae of A. pernyi were challenged by injecting them with nucleopolyhedrovirus (AP-NPV), the Gram-negative bacterium E. coli, the Gram-positive bacterium Micrococcus luteus, or the entomopathogenic fungus, Beauveria bassiana. These challenges with diverse pathogens resulted in differential expression patterns of the protein. A knockdown of the Ap-Yippee gene by small interfering RNA (siRNA) transfection had a significant influence on the expression of the hemolin in the pupae which was confirmed by qRT-PCR and Western blot. Furthermore, a possible protein-protein interaction between Ap-Yippee and Hemolin was explored by Far-Western blotting. Therefore, our data suggest that the Ap-Yippee protein is involved in a pathway that regulates the immune response of insects.
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
Yu
Y
Sun
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
Lishang
L
Dai
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
Yuxuan
Y
Sun
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
Lei
L
Wang
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
Cen
C
Qian
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
Guoqing
G
Wei
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China. Electronic address: zhubaojian@ahau.edu.cn.
Bao-Jian
BJ
Zhu
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China. Electronic address: clliu@ahau.edu.cn.
Chao-Liang
CL
Liu
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000276:immunology; D007113:Immunity, Innate / Q000276:immunology*; D007136:Immunoglobulins / Q000276:immunology / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D009036:Moths / Q000235:genetics* / Q000276:immunology*; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D059467:Transcriptome
D002352:Carrier Proteins; D007136:Immunoglobulins; D019476:Insect Proteins; C066846:hemolin
Antheraea pernyi; Ap-Yippee; Expression pattern; Far-Western blotting; Immune response; RNAi