Evaluation Of Anti-aphid Activity Of Bacillus Thuringiensis Cry Toxin And Elucidation Of The Mechanism

Cry toxins extracted from Bacillus thuringiensis in its sporulation stage is a important toxin protein with specific insecticidal activity against a variety of agricultural pests.cry gene has also been widely used in transgenic crops,and has become an effective way to control target insects such as Lepidoptera,Coleoptera in agricultural production.With the increasing area of these GM crops,the Hemiptera aphids,which are insensitive to Cry,occupy the living space of the original target insects and gradually upgrade from secondary pests to major pests.At present,the main method of controlling aphids is chemical control,which runs counter to the purpose of expanding GM crops to reduce the use of chemical pesticides.Therefore,it has important theoretical significance and application prospects to uncover the reason why Cry toxins exhibit low activity against aphids and to reveal the mechanism of Cry antiaphid.First,we evaluated the anti-caries activity of different Cry toxins.Different Cry toxins were expressed in E.coli: Cry1Ab1,Cry2Ab12,Cry7Ab4,Cry-related(a Cry41 homologous protein,the Cry protein with the highest anti-aphid activity to date)and SCAN2-2(Cry46 homologous protein),And constructed cry2Ab12 transgenic tobacco,cry-related transgenic tobacco,cry-related + Tma12 bivalent transgenic tobacco(Tma12 encodes a fern-derived chitinase)The toxicity levels of Cry toxin and transgenic tobacco against Myzus persicae were investigated by membrane capsular method and leaf disc method.The results showed that the Cry1Ab1 protein was not substantially toxic to Myzus persicae;SCAN2-2 shows high toxicity to Myzus persicae cry2Ab12 transgenic tobacco is less toxic to Myzus persicae;Both cry-related transgenic tobacco and cry-related + Tma12 bivalent transgenic tobacco showed higher anti-aphid activity.In addition,the winged mites that landed on the bivalent transgenic plants all died and no offspring were found to be harmful.Secondly,in this study,the binding proteins of Cry1Ab1,Cry7Ab4,Cry2Ab12,Cryrelated,Cry1 F and Vip3 Aa were isolated and identified from the whole population of Myzus persicae by using Pull-down and LC-MS/MS techniques.The results indicate that all Cry toxins have binding proteins associated with phagocytosis,which may mean that Cry proteins can enter the aphid cels by endocytosis;All Cry toxins do not have binding proteins such as cadherin(CAD)and alkaline phosphatase(ALP)(CAD and ALP are common receptors for Lepidoptera insect recognition Cry),which may be reason that Cry toxins low toxic for aphid;Cathepsin-B and ATP-dependent-6-phosphofructokinase derived from the symbiotic bacteria of Myzus persicae are Cryrelated unique binding proteins,which may be the reason for the highest Cry-related anti-spasmodic activity.Using the String database,protein interaction network prediction of the binding protein showed that the molecular process of partial Cry on aphids involved MAPK,Hippo or Wnt pathway.Finally,we examined the in vitro interaction of Cry-related with Cathepsin-B.The role of Cathepsin-B in the Cry-related anti-caries process was explored.We cloned Cathepsin-B from Myzus persicae,which expresses in E.coli.It was confirmed by immunoblot analysis that Cry-related and Cathepsin-B could interact directly in vitro,which further indicates that Cry-related is most toxic to aphids.Cathepsin-B’s interaction is achieved.We determined the enzymatic properties of Cathepsin-B and found it to be a typical cysteine protease.Cysteine protease plays an important role in apoptosis.Cry-related is an anti-cancer crystal protein that can induce apoptosis.Therefore,the interaction between Cry-related and Cathepsin-B may synergistically induce cell Malignant apoptosis.Molecular docking showed that Cry-related binding to Cathepsin-B blocked the active site of Cathepsin-B,suggesting that Cry-related may also affect the normal protein degradation of aphids,which in turn affects the growth and development of aphids.Based on this,this study proposes the Cry anti-caries mechanism hypothesis: The CAD,ALP of aphids is significantly different from other insects.Most Cry toxins are unable to bind to their common receptors,so they are not sensitive to aphids,and cryrelated can also exert anti-caries effect by inducing malignant apoptosis of aphid cells or affecting protein degradation and utilization through interaction with Cathepsin-B.Cry-related can also affect the growth and development of aphids by interacting with ATP-dependent-6-phosphofructokinase derived from commensal bacteria.The results of this study can provide a theoretical basis for how to improve the anti-aphid activity of Cry,and it is of great significance to effectively prevent and control aphids.