A Primary Study On The Mechanism Of AChE Regulating Midgut Apoptosis In Nilaparvata Lugens

The brown planthopper(BPH),Nilaparvata lugens is one of the major insect pests of rice in the middle and south of China.In terms of control,the direct and effective method is using chemical pesticides.But the long-term application of chemicals has caused serious consequences,such as suriously environmental pollution,to the resurgence of BPH with drug resistance.During the last several decades,scientists tried to apply developing and planting new rice varieties with high resistant levels,but BPH has strong ability to to adapt to new varietions,which constrains the promotion and application of the new rice to some extent.Carrying out the research of BPH biology can provide clues for developing effective and environment-friendly control measures.AChE is a key enzyme in animal nerve system.Its classical function is to hydrolyse neurotransmitter-acetylcholine to control normal nerve signals transmission in animal body.One of it non-classic functions is involved in apoptosis.We should pay more attention to AChE in insects for it is the direct target enzyme of pesticides,in addition,mutation or abnormal expression of AChE may result in the insecticide-resistance of BPH.Furthermore,this enzyme is closely related to apoptosis process.Since midgut tissue is an important interface of BPH with rice,confirming that AChE is involved in midgut apoptosis and looking for some protein interacted with AChE can provide us new knowledge and some valuable information on co-evolution of BPH and rice,promote breeding novel resistant rice and developing scientific management method of BPH.In this study,we used immunohistochemistry(IHC)technology combined with TUNEL staining techniques were used to study the expression profiles,subcellular location of AChE in apoptotic cells,as well as its involvement in apoptosis.The juice extracted from the susceptible rice TN1,the resistance rice B5 and TKM-6 was used to treat midgut cells,respectively.The results showed that insect-resisted group,the apoptosis ratio and the expression level of AChE are higher in the groups treated with resistance rice than that with TN1 rice.Next,real-time fluorescent quantitative PCR was applied to verify the expression profiles of AChE in each sample groups.The data revealed a similar trends as obtained in immunohistochemical reaction.In order to discover,we used the yeast two hybrid cDNA library was constructed and used to screen new proteins interacting with AChE during apoptosis.The results may be helpful in revealing the new functions of AChE in apoptosis and demonstrating the signaling pathways in the future.Firstly,AChE cDNA was amplificated and used to construct a bait vector pGBKT7-AChE.Total RNA was extracted from the third instar nymphs of biotype I,and used as template to retro-transcribe into complementary DNA(cDNA)by SMART reverse transcription technology.Then total cDNAs were generated by LD – PCR,and then ligased cDNAs with linear carrier pGADT7-Rec and transformed into Y187 yeast competent cells to produce yeast two hybrid cDNA library.The construct pGBKT7-ACHE was used as bait to screen BPH cDNA library.At first,we screened at SD /-Leu /-Trp/X-a-Gal/AbA auxotroph medium was used to perform the first round screen,then SD/-Ade /-His /-Leu /-Trp /-X-a-Gal/Ab A auxotroph medium was employed to carry out the second round screen.The target genes were amplified from the resulting positive clones,cloned into T-A vector and sequenced.The gene sequences were analyzed and the candidate proteins interacting with AChE were determined.This study will strenthen our understanding of the interacting mechanism of BPH and rice,especially the function of AChE involved in insect apoptosis,including:(1)the midgut apoptotic level in BPH exposed to resistant rice and the relationship between midgut apoptosis and rice resistant level,which may supply some important information about the rice resistance mechanism indirectly;(2)identification of proteins interacting with AChE,and determination of their functions may provide certain reference for understanding the molecular mechanism of AChE involved in apoptosis and revealing the related signaling pathways.