The Mechanism Of Several Bt Proteins Toxic To Scarabaeoidea Larvae

The larvae of white grubs living in soil can cause great damage to the roots of crops,meanwhile adults can feed on aerial crop structures.In China,white grubs are currently endangering large areas of crops,causing significant reduction of output and great economic losses.White grubs are difficult to control because of its soil living habits,the damage to the crops are much more seriously.So,it need new strategy to control these insect pests.In previous work,Bacillus thuringiensis strain HBF-18 has been confirmed toxic to H.oblita and H.parallela larvae,and containing three toxin genes,vip1Ad1,vip2Ag1 and cry8-like gene.The Vip1 Ad1 and Vip2Ag1 protein have confirmed act as binary toxin that have high toxicity against both H.oblita and H.parallela larvae,and its toxicity significant higher than Cry8-like protein.For the requirements of pests control in the field,we research on the mechanism of Vip1Ad1/Vip2Ag1 and Cry8-like protein with the aim of evaluating its industrialization prospect.The main results as follows:1.We investigated the methods of extraction and purification of Vip1 Ad1 and Vip2Ag1.The results showed that Vip1 Ad1 and Vip2Ag1 are secretory proteins,and it is inefficiency to extract these two proteins using ammonium sulfate precipitation method.But we have obtained purified Vip1 Ad1 and Vip2Ag1 from fermentation broth after treated with crossflow filtration system and then purified by anion-exchange and cation-exchange,respectively.What's more,we investigated the activation process of these two proteins.The results indicated that Vip1 Ad1(approximately 90 kDa)digested with Trypsin resulted in a protease-resistant core fragment of approximately 70 kDa(Vip1Ad1-T),but Vip2Ag1 could not be digested by both Trypsin and Chymotrypsin.The purified Vip1Ad1 and Vip2Ag1 mixture have high activity against H.parallela larvae with the LCso of 2.33 ?g/g soil,but not for the individual Vip1 Ad1 and Vip2Ag1.The purified Vip1Ad1 and Vip2Ag1 mixture have no activity to Colaphellus bowvingi,Anoplophora glabripennis,Plutella xyllostella and Nilaparvata lugens.The results of Histopathological effects of ViplAd1/Vip2Ag1 binary toxin on H.parallela larvae midgut revealed that brush border membrane was greatly destructed in the midgut from H.parallela larvae that fed with a mixture of Vip1Ad1 and Vip2Ag1 proteins(molar ratio 1:1),but not for individual ViplAdl and Vip2Ag1.The results are consistent with the fact that the biological activities of Vip1 Ad1 and Vip2Agl binary toxin are elicited by the combination of the two component.2.The protoxin of Vip1 Ad1 and Vip2Ag1 could not binding each other in solution;And,the Vip1Ad1,either protoxin or trypsinized,bound to BBMV from H.oblita,whereas the Vip2Ag1 alone did not;The binding of Vip2Ag1 was determined only when the BBMV were reacted with trypsinized Vip1Ad1,but not when they were reacted with Vip1Ad1 protoxin;In particular,the Vip2Ag1 bound to Vip1Ad1 oligomers-BBMV complex;The ViplAdl oligomers could generated both in solution and on the cell surface after Vip1Ad1 monomers bound with BBMV,but in either scenario,the Vip1Ad1 oligomers were generated only after proteolysis of Vip1Ad1 protoxin.The results of kinetic study with Vip1Ad1 and BBMV from H.oblita revealed that Vip1Ad1 bound specifically to binding sites on the BBMV,which not shared with Cry8-like.In addition,we identified Vip1Ad1-binding proteins in the midgut of H.oblita larvae.The results demonstrated that the Vip1Ad1 binding protein is not cholesterol and not GPI-anchored protein.Theses results provides important basic data for the further study of mode of action of Vip1Ad1/Vip2Ag1 against H.oblita larvae.3.The results of binding competition assays of fluorescence-labeled Cry8-like with BBMV in H.oblita larvae demonstrated,for the first time,that Cry8-like bound specifically to binding sites on the BBMV that shared with Cry 8Ga.Our work provides important basic data,and promotes the understanding of insecticidal mechanism of Cry8 proteins against H.oblita larvae.4.Furthermore,Cry8-like-binding proteins in midgut from H.oblita larvae were identified by pull-down assays,and by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The results showed that five proteins were identified as Cry8-like-binding.They are serine protease,transferrin-like,uncharacterized protein LOC658236 of Tribolium castaneum,ATPase catalytic subunit,and actin.These identified Cry8-like-binding proteins were different with those previously confirmed as receptors for Cry 1A proteins in lepidopteran insect species such as aminopeptidase,alkaline phosphatase and cadherin.