Cloning And Expression Of Gene Encoding Inhibitor Of Apoptosis Protein2from Nilaparvata Lugens

The brown planthopper (Nilaparvata lugens) is one of the major pests of rice in China and many Asian countries. Traditional management of BPH (brown planthopper) is primarily spraying chemical pesiticides, which usually led to development resistance of BPH to these chemicals and resulted in serious pollution of environment. Our previous work showed that during the normal development of BPH larvae, apoptosis was observed in the midgut tissues. In this work, we focus on cloning and expression analyses of BPH apoptosis-related genes, which will provide some reference data to understand the molecular mechanism of midgut apoptosis in BPH.There were no reports of cloning and expression of IAPs homologous genes in BPH. In this work, reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were employed to clone an inhibitor of apoptosis protein2gene (LAP2). The full-length of LAP2cDNA is3250bp, including375bp of5’untranslation region (UTR),907bp of3’UTR and1968bp of open reading frame (ORF). The ORF codes for a putative protein with655amino acids in length. The molecular weight and isoelectric point of the predicted peptide is73.1kD and4.89, respectively. BPH IAP2contains "BIR1","BIR2","BIR3" and "RING" domains found in all other insect IAP2s. Blast analysis showed that BPH IAP2was highly homologous to LAP2in Bombyx mori, Harpegnathos saltator, Acromyrmex echinatior with over30%amino acid identity. Secondly, Differential expressions of IAP2in Midgut, fat bodies and carcass of Nilaparvata lugens were analyzed using Real-time quantitative PCR with β-actin as a reference gene. The results indicated that the expression levels of IAP2in carcass was10times higher than that in fat bodies and33times higher than that in Midgut. In the meantime,BPH LAP2was cloned into the prokaryotic expression vector pET23a to result a expression vector pET23a-IAP2. The vector was transformed and expressed in E. coli BL21strain. The transfromants were induced by IPTG. The cells were collected and lysated. The recombinant protein was separated on SDS-PAGE and Western blot hybridization. The result showed that the hybridization signal is consistent with the theoretical molecular mass of the protein, and the expression level was high.This work laid a foundation for further study on the function of IAP2. In addition, it provided some reference data for investigation of other IAPs’ roles in the apoptosis pathway. More importantly, this work offered some clues for developing novel management of BPH. For instance, new drugs can be designed to inhibit these genes or proteins, to induce apoptosis of BPH. Thus controlling BPH can be achieved by disable its normal physiological functions.