Study On The Effects Of Mutations On The Structure And Function Of Bombyx Mori Type 1 Acetylcholinestrase Gene

Acetylcholinestrase (AChE, EC 3.1.1.7) is one of important serine hydrolases, which has function of catalyze neurotransmitter ACh to produce acetic acid and choline during nerve conduction, and finally terminates nerve impulses and maintains normal nerve conduction. AChE is mainly located at junction between neuron and neuromuscular, and inhibit delivery of nervous impulse from nervous to muscle cell. AChE is a target of nerve poison, organophosphates (OPs) and carbamates (CBs) pesticides. Pesticides bind to serine at AChE’s active center and inhibit degradation of ACh by occupy catalysis site of enzymes. Persistent binding of ACh and ACh receptor(AChR) will lead to continuous activation of muscle cell, which may cause insect convulsion or death. Also, AChE is related to neurodevelopment, nerve cell differentiation, nerve cell migration and synapse formation; participate in the proliferation and differentiation of hematopoietic cell and involved in formation of megakaryocyte; associated with tumor proliferation and cell apoptosis.Increased expression, increased membrane localization, and mutations at specific bases are main reasons causing pesticide resistance of AChE. There are two types of AChE in silkworm (AChE1 and AChE2), and AChE1 is sensitive to pesticides. AChE1 gene (acel) has been mutated to study influences of changes on structure, activity and anti-pesticide sensitivity by specific mutation (A286S, G312A, L537L). Wild type acel (wacel) and mutant ace1 (mace1) were expressed in E. coli and purified for enzyme activity measurement and inhibition dynamics analysis.3D structures of wAChEl and mAChEl were also obtained for further research of relationship between Bombyx mori AChE structure and function, wacel and macel were transfected into BmN cells, and extracted protein was exposed to phoxim and physostigmine to further study the enzyme activity and inhibiting effect. Construction the silkworm gene targeting vector (pSK-LoxpFRTA3GFP-IEneo-A3tk) and successfully konckout acel. mace1 was then transformed to silkworm. The main results are as follows:1 Influences of mutations on function of AChEl using prokaryotic system.A prokaryotic system was used in this study to investigate influences of mutations on AChE1 function. After expression, there are Bombyx mori wild type AChE1 (wAChE1) and mutant AChE1 (mAChE1). Active AChE1 proteins were obtained after refolding and purification, there were no significant differences of activity among those two. After incubation with 10-6 M physostigmine and 10-3 mg/mL phoxim, the remaining enzyme activity of mAChE1 was 4.42% and 8.86% higher than that of wAChEl’s, respectively. These results indicated that mutations may reduce sensitivity of AChEl to physostigmine and phoxim.2 Influences of mutations on structure of AChEl.The secondary structure and 3D structure of wAChE1 and mAChE1 have been predicted and analysis to study the influences of mutations on AChE1 structure. It resulted that mutations have changed the secondary and 3D structure and changed spatial configuration of active center of AChE1. Side chain of mutated Ser286 and Ala312 have extended toward central part of S285 with distances 2.80 A and 3.68 A respectively. This may influence binding of physostigmine and phoxim to serine residue at active center, and which may lead to sensitivity reduction.3 Construction of wacel and macel transgenic cells.In order to investigate their expression character in cell lines, wacel and macel were transfected into BmN cells. The expression of wacel and macel in the cells were confirmed by western blot and their expression levels were 21.51 folds and 21.69 folds of the endogenous acel level, respectively. Furthermore, the activities of AChE in wacel and mace1 transgenic cells were 10.62% and 20.21% higher than control cells respectively. Physostigmine and phoxim treatments led to 7.47% and 17.41% higher remaining activity of AChE in macel transgenic cells than that of wacel transgenic cells, respectively. The results showed that acel transgene can significantly improve ace1 expression, and ace1 mutation at a specific site can reduce the sensitivity of AChE inhibitors.4 Construction and application of Bombyx mori universal gene targeting vector.A universal knock-out gene target vector is built to achieve simple gene knock-out, conditional gene knock-out and out-in gene target in silkworm. In construction, pBluescript Ⅱ SK+ was used as backbone and insert an Actin3 drived GFP, and IE drived neo flanked by two FRT, GFP and neo were then flanked by two LoxP. For convenient gene knockin, an attP was added to the vector, an Actin3 drived HSV-tk was added as a negative selection marker.This constructor were applied in gene acel knock-out in BmN cell in silkworm. Two segments DNA sequence of acel were cloned into the gene targeting vector as upstream and downstream exchange arms, after transfected into BmN, green fluorescence was detected, and cells resistant to G418. Indicate the expression of GFP and neo. After identification by PCR, acel was knockout. Indicate the vector can be used for Bombyx mori gene target.5 Preparation of macel transgenic silkworm.Sperm-mediated gene transfer method is used in this study, and normal incubation and breeding lay eggs until pupation is denoted as GO generation under inducing of mixture of pIZT-macel with lipidosome and transformed in to Bombyx mori. Stereo fluorescence microscope was used to screen the positive pupas, eclosion, mated and spawn, denoted as G1 generation, and as so on. G2 generation moth genome DNA were extracted, and GFP and transformed macel were detected after PCR, which indicate successful preparation of macel transgenic silkworm.Influences of mutations on structure and function of AChE1 has been firstly revealed in this study, and mutations on three key position may change structure of AChE1 and reduce the sensitivity against inhibition. Bombyx mori universal gene targeting vector is constructed, and acel is knock-outed using this vector to have macel transported into silkworm. This study revealed the sensitivity of mutants of three reduced AChEl to physostigmine and phoxim, and offered new understanding of relationship between AChE1 mutations and pesticide resistance, also attributed to operation of gene target with silkworm and provides a new material for the cultivation of new pesticide-resistant varieties of Bombyx mori.