| Bacillus thuringiensis(Bt)is an entomopathogenic bacterium,which mainly relies on insecticidal protein to kill pests.It is lethal to many agricultural pests such as Lepidoptera,Coleoptera,Diptera.It has been widely used in the control of agricultural pests and at present in this world it is widely used in insecticidal microorganism.The insecticidal crystal proteins of Cry protein and Cyt protein produced by BT in the stable growth phase and the Vip protein produced by Bt during the vegetative period have been widely used in the field of agricultural pest control.However,the newly discovered Sip protein showed strong insecticidal toxicity to coleoptera larvae.At present,the harm of coleoptera pests in agricultural production in China is increasing,and it has caused serious economic losses.Therefore,the in-depth study of Sip protein is of great significance to the control of Coleoptera's agricultural pests.In this study,the truncated sipa gene with signal peptide(the first 30 amino acids)was successfully cloned from strain QZL38.The coding region length of the gene was 1005 bp,.About37 k Da insecticidal protein was successfully expressed in E.coli,and the Sipa protein was purified.The purified protein of Sipa was obtained.Cry8 Ca,which also has insecticidal activity to coleoptera pests,was activated,expressed,extracted and purified.The histopathological changes of midgut of Sipa and Cry8 Ca protein were observed by electron microscope.The results showed that the microvilli were swollen and shedded,and the space between midgut cells was enlarged,and the mesenteric tissue of Sipa protein was swollen and exfoliated,and the cell space of midgut was enlarged,and the cell space of midgut was enlarged.Endoplasmic reticulum is swollen and broken,mitochondria hollow deformation and so on.These results suggest that the Sipa protein is destructive to the midgut tissue of Colaphellus bowringi Baly.,and the corresponding receptor protein is predicted.Microvilli swelling and exfoliation were also observed in the midgut of Colaphellus bowringi Baly.fed with Cry8 Ca protein,but there was no significant change in the midgut cell space and endoplasmic reticulum(ER).The results showed that Cry8 Ca protein had a slight damage to the midgut tissue of Colaphellus bowringi Baly.During feeding,it was found that the protein could inhibit the feeding and growth of Colaphellus bowringi Baly.The BBMV,of the midgut of Colaphellus bowringi Baly was extracted for feed 10 days.The Sipa protein was used to bind to BBMV in vitro.The results showed that there was a clear band about 37 k Da in the midgut of Colaphellus bowringi Baly.It was confirmed that the Sipa protein has a corresponding receptor in the midgut BBMV of Colaphellus bowringi Baly.The unlabeled Cry8 Ca protein was added to the reaction system of BBMV Sip binding protein,which was 5,10,50 and 500 fold,respectively,and the competitive binding test was carried out.The results showed that Cry8 Ca protein had no competitive effect on the binding of Sipa protein to the midgut of Colaphellus bowringi Baly,suggesting that there may be different receptors between Cry8 Ca protein and Sipa protein in the midgut BBMV of Colaphellus bowringi Baly.In order to delay the occurrence of pest resistance,it is an urgent task to find out a new Bt gene which can replace the commercial gene without cross resistance.Further isolation and identification of the receptor proteins of Sip toxin in the midgut BBMV of different species of Colaphellus bowringi Baly will help to understand the mechanism of action and resistance of Sip toxin.The proteins with different mechanisms can broaden the idea of improving insecticidal activity and alleviate insect resistance and provide theoretical guidance for directed evolutionary transformation and molecular design of Sip. |
PDF Full Text Request