| Destruxin A（DA）is a cyclopeptide lactones produced by the entomogenous fungus,Metarhizium anisopliae as a key pathogenic factor.It has good insecticidal activity,but the molecular mechanisms is not elucidated yet.To discover its action targets,our group in previous studies identified about 100 suspected DA-binding proteins from silkworm Bm12 cells based on the principles of drug affinity response target stability（DARTS）.It is necessary to validate the interaction of DA with these proteins.Therefore,in this research,five proteins,i.e.,calreticulin（BmCRT）,ATPaseαsubunit（BmATPaseα）,dipeptidyl peptidase 3（BmDPP3）,inner nuclear membrane protein MAN1（BmMAN1）,protein disulfide isomerase A5（BmPDIA5）were selected to carry on further experiments of proteins expression and purification,and the interactions in vivo and in vitro.The main results are listed as follows:1.The vectors of five proteins:p ET-sumo-BmCRT,p DE-BmATPaseα,p ET-sumo-BmMAN1-M,p ET32-BmDPP3,p ET32-BmPDIA5 were successfully constructed and expressed in Escherichia coli.Then,the five proteins were isolated and purified（BmMAN1-M is a partial fragment,without transmembrane region）.2.Surface plasmon resonance（SPR）and Biolayer interferometry（BLI）were employed to measure the affinity between DA and above proteins in vitro.The affinity constants（KDvalue,mol/L）of DA and BmCRT,BmATPaseα,BmMAN1-M,BmDPP3 and BmPDIA5were determined by SPR as 1.98×10-4,2.56×10-4,3.6×10-3,1.81×10-4and 9.26×10-5,respectively;while the KDvalues（mol/L）of DA with BmDPP3 and BmPDIA5 evluateded by BLI as 5.87×10-5and 4.47×10-5,respectively.Obviously,although a few difference exists in SPR and BLI,the results absolutely indicated that DA respectively binds the four proteins,BmCRT,BmATPaseα,BmDPP3 and BmPDIA5 with the KDvalues of 10-4～10-5mol/L.The KDvalue of about 10-3mol/L shows the less interaction of DA and BmMAN1,however,it might be due to the incomplete sequence of BmMAN1（excluding its transmembrane region）.3.Insect two hybrid（I2H）technology was used to study the effects of DA on protein interactions in vivo.The results indicated the interactions of five proteins were reduced with a DA-dosage-dependent manner.Under the treatment of 2 mg/L,DA significantly inhibited the interactions of BmATPaseα-BmATPaseγ,BmMAN1-BmMAD3,BmDPP3-BmKEAP1,and BmCRT-BmPDIA5.While at the dosages of 0.2 mg/L and 0.02 mg/L,DA moderately inhibited the same interactions.However,DA at 0.002 mg/L did not effect on these interactions.4.ELISA was used to determine the effects of DA on the enzyme activity of BmDPP3 and BmPDIA5.The experimental results showed that DA can inhibit the enzyme activity of BmDPP3 to a certain extent,but DA has no effect on BmPDIA5.5.Based on the method of q PCR,the effects of DA on the expression of five protein genes of BmCRT,BmATPaseα,BmMAN1,BmDPP3,and BmPDIA5 in Bm12 cells of silkworm were detected.The results showed that DA induced up-regulation of the expression of BmDPP3 gene and inhibited the expression of BmCRT protein gene at a dose of 2 mg/L,.DA has no significant effect on the expression of the above five genes under lower concentration conditions.In conclusion,the experimental results support that BmCRT,BmATPaseα,BmDPP3and BmMAN1 are the DA-binding proteins.This research gives a new insight to understand the molecular mechanisms of DA against insects. |
