| Bacillus thuringiensis(Bt),as an environmental friendly biological pesticide without any toxicity to humans and animals,is wildly used in the world.During its sporulation,the insecticidal crystal proteins(ICPs)were being produced,which have specific insecticidal activity.However,there are some problems in the use of Bt,such as protein degradation,short duration,insect resistance,and so on.Therefore,molecular biology technology was used to improve Bt strains genetically,providing a technical way to solve the problems in application and production of Bt biocides.Based on the previous whole genome sequencing,transcriptomics and proteomics analysis of Bt 4.0718,two genes,dpaB encoding pyridicarboxylic acid synthase B subunit and tig encoding trigger factor,were screened,which may be related to the formation of spores and crystals.The dpaB gene and tig gene were knocked out by homing endonuclease I-Sce I mediated gene deletion method to study related biological functions.The dipicolinic acid synthetase subunit B encoded by gene is a key enzyme in the biosynthetic pathway of dipicolinic acid(DPA).The knockout vector was constructed in plasmid p RP1028,which was introduced into the recipient strain Bt 4.0718 by the triparental conjugation transfer technology,after which the dpaB gene knock-out strain Bt-?dpaB and the knock-in strain Bt-dpaB were constructed.The changes in bacterial surface morphology caused by gene deletion were studied.The results showed that compared with the original strain,the stable period of Bt-?dpaB was prolonged,mother cell lysis delayed,the formation of spore cells and crystal proteins was inhibited in the fermentation medium,and the number of active spores decreased,but it can produce mature spores in GYS medium,changing the glucose concentration in the GYS medium showed that the formation of spores was significantly inhibited.The crystal protein yield and insecticidal toxicity of the knock-out strain were much lower than those of the original strains,while the phenotype of knock-in strain was partially recovered.qRT-PCR analysis was performed on genes related to sporulation and crystal proteins formation,such as spo,cry and sig regulatory genes,and the results showed that the related genes were down-regulated to varying degrees,which further revealed the effect of dpaB gene on the formation of spore and crystal proteins.DpaB protein was successfully expressed in E.coli and and its related functional studies were verified.The trigger factor(TF,encoded by tig gene),as a molecular chaperone,participates in protein export to maintain the newly synthesized protein in an open conformation,whose function is similar to the peptidyl prolyl cis-trans isomerase.Knockout the tig gene to obtain the gene knockout strain Bt-?tig,and construct the supplementary strain Bt-tig.It was found that compared with the original strain,Bt-?tig did not produce spores and insecticidal crystal proteins.In addition,the maximum biomass of Bt-?tig was reduced,and the production and insecticidal virulence of the crystal proteins against Helicoverpa armigera were significantly weakened,while the phenotype of Bt-tig was partially restored.qRT-PCR analysis showed that genes and regulators related to sporulation and crystal protein formation were down-regulated.The expression of TF protein in E.coli provides a new theoretical basis for studying the association between tig gene and crystal protein formation,which has important scientific significance and application value.Besides,in our previous study,it was detected by Pull-down and Co-IP experiments that Vip3 A could interact with the 65 k Da specific band in the midgut of Helicoverpa armigera,it was identified as alkaline phosphatase(ALP)by mass spectrometry.By constructing p ET28a-vip3 A and p ET28a-ALP expression vector,successfully expression,separation and purification of ALP and Vip3 A proteins,SPR was used to further verify the effect of Vip3 A on the midgut receptor ALP of Helicoverpa armigera. |
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