Study On The Insecticidal Mechanism Of Novel Vip1/Vip2 Protein

Bacillus thuringiensis(Bt)insecticidal proteins,Vip1Ad1 and Vip2Ag1,are a group of binary toxins,which have good insecticidal activity against larva of Holotrichia oblita and Holotrichia parallela.However,the mechanism of their insecticidal effect is not clear.In order to explore the mechanism of action,this study firstly optimized the purification methods of Vip1Ad1 and Vip2Ag1 proteins and prepared high-purity insecticidal proteins.The interaction of Vip1Ad1,Vip2Ag1 and BBMV from H.oblita and H.parallela were further analyzed.The mechanism of Vip1Ad1 and Vip2Ag1 acting on the midgut of the H.oblita and H.parallela was revealed.The main results are as follows:1.The high efficiency purification system of Vip1Ad1 and Vip2Ag1 proteins was established.Firstly,the recovery,purity and activity of Vip1Ad1 and Vip2Ag1 proteins purified by ammonium sulfate precipitation,membrane filtration and ion exchange chromatography were compared.The recovery rate of Vip1Ad1 showed that maximum was membrane filtration(82.50%)and the minimum was ammonium sulfate precipitation(62.15%).The recovery rate of Vip2Ag1 showed that maximum was membrane filtration(79.30%)and the minimum was ammonium sulfate precipitation(40.75%).The purity of Vip1Ad1 showed that maximum was ion exchange chromatography(76.84%)and the minimum was membrane filtration(47.56%)The purity of Vip2Ag1 showed that maximum was ion exchange chromatography(97%)and the minimum was ammonium sulfate precipitation(67.26%).The activity of Vip1Ad1 showed that Vip1Ad1 protein,purified by ion exchange chromatography,bound to BBMV of H.oblita,but the Vip1Ad1 protein,purified by ammonium sulfate precipitation,was not.So,we choosen the ion exchange chromatography to purify Vip1Ad1 and Vip2Ag1 proteins,after comparing the above three methods.2.The oligomeric characteristics of the Vip1Ad1 protein were analyzed.SDS-PAGE result showed that the activated Vip1Ad1 protein consisted of monomer and oligomers in the solution.The tolerance of Vip2Ag1 to trypsin was poor and it was easy to be digested completely by trypsin.The c(s)sedimentation coefficient distribution showed that oligomers included tripolymer,tetramer,pentamer,hexamer and heptamer.3.The affinity constants of Vip1Ad1 and Vip2Ag1 proteins were analyzed.The ligand blot and isothermal titration result showed that the activated Vip1Ad1 protein and Vip2Ag1 protein could bind and form a complex in 20 mmol Tris-HCl.The ELISA result showed that the affinity constant measured was 58.90 ± 3.9nmol/L.The sedimentation coefficient of binding between Vip2Ag1 and digested Vip1Ad1 was further analyzed by ultracentrifugation and the result showed that Vip2Ag1 protein could bind to oligomers of Vip1Ad1.4.The interreaction characteristics of Vip1Ad1,Vip2Ag1 and BBMV in the midgut of H.oblita were analyzed.Firstly,The ELISA result showed that the binding constant of Vip1Ad1 protoxin to the BBMV in the midgut of H.oblita was 94.3 ±14.3 nmol/L.Secondly,the binding constant of activated Vip1Ad1 to BBMV in the midgut of H.oblita was 96.5 ±27.8 nmol/L,and the binding constant did not change when Vip2Ag1 protein was added.The 9 bands of BBMVs bind to Vip1Ad1 and were analyzed and identified by ligand blot and LC-MS/MS.It was found that leptinotarsa decemlineata actin,pediculus humanus corporis myosin-9,actias artemis mRNA for serine protease like protein,agrilus planipennis myosin-IA and cobitis choii cytoplasmic beta-actin 1 could bind to Vip1Ad1.5.The interreaction characteristics of Vip1Ad1,Vip2Ag1 and BBMV in the midgut of H.parallela were analyzed.Vip1Ad1 protoxin was digested during incubating with BBMVs of H.parallela larvae.In addition,monomer and oligomers bound to BBMVs of H.parallela larvae.Vip2Ag1 also could bind to BBMVs of H.parallela,but the amount of binding was small.When Vip1Ad1 was added,the binding amount of Vip2Ag1 was increased.