| For a long time,organic synthetic pesticides have been the most important means to prevent flies in breeding farms because of its high efficiency,economy and convenient usage.However,due to the long-term and large-scale usage of organic synthetic insecticides,"3R" problems(resistance,recurrence,resideue)has been caused,thus the effect of the insecticides is also difficult to be ensured,even producing the harm to human beings and polluting environmental.Biological insecticides have the characteristics for low toxicity,easy to be degradation,harmless to people and animals,and no resistance to pests,which can meet the demands of ecological development.It is one of the important measures for the development of new insecticides in the future.In recent years,the development of biological insecticides is relatively rapid and widely used,especially to Bacillus thuringiensis.the principle of insect control is the endotoxin(parasporal crystal)secreted by it has stomach toxin,leading to no intaking,and finally dies from the hunger,further the nerve disorder and cell wall rupture was followed.Bacillus thuringiensis Israeli subspecies was originally used to kill the eggs and larvae of Diptera mosquitoes.In this experiment,it was found that the endotoxins CryIVD gene of this subspecies had the strong killing effect on houseflies(especially on their eggs).Referring to the published target gene sequence from NCBI Gen Bank(accession number eu 124377.1),primer 6.0 software was used to design primers for amplification of CryIVD gene,the gene was clone into pMD19-T vector to construct the clone plasmid.Then,the enzyme digestion was carried out to identify and determine the sequence,and the homology analysis was carried out.Meanwhile,the prokaryotic expression vector pGEX-4T-Cryivd also was constructed,it was expressed by induction of IPTG,and the expression products were identified by SDS-PAGE and immunoblotting techniques.In the acute toxicity test,the recombinant expression of CryIVD was mixed into houseflies foods to let them intake,then investigated the LD50 value to evaluate its effect.The results showed that:(1)the target gene was successfully amplified and its size was about 660 bp,sequence analysis also displayed that it had the highest homology to Bacillus thuringiensis strain dab-bt2 cry4d gene,arrived at 100%,it was the closest to Bacillus thuringiensis strain dab-bt2 cry4d gene in genetic distance in phylogenetic tree.After induction,the target protein can successfully be expressed,its size is about 51kD fusion protein;In vitro toxicity test,CryIVD protein has a good killing effect on fly eggs,and its LD50 could reach 7.87 × 107cells/g,but the effect on housefly adults was lower than that of killing houseflies eggs,its LD50 was only 6.30 × 105cells/piece.The conclusion could be made that the recombinant CryIVD has a ideal killing effect on the eggs of housefly,and it maybe a potential alternative to chemical medicine. |
