The Action Of Borrelidin Inhibit To Phytophthora Infestans Threonyl-tRNA Synthetase

Borrelidin is18-membered ring macrolide antibiotic produced by fermentation of Streptomyces rochei. Its structure includes a novel nitrile, which is unique and rare in nature. Borrelidin is possessed of anticancer activity, antiangiogenic activity, antimalarial activity and antimicrobial activity, which play a role by specifically inhibiting the activity of the threonyl-tRNA synthetase (ThrRS). However the mechanism of the reaction between Borrelidin and ThrRS is still unclear and further research is required.This study gets the IC50of borrelidin to Phytophthora infestans(P.infestans) by in vitro inhibition of borrelidin on Phytophthora infestans(P.infestans). We observe that compensation of the threonine (Thr) weaken the inhibition of borrelidin on P. infestans. We study the expression of ThrRS in E. coli system by designing primers with Nde Ⅰ and HindⅢ restriction sites for the amplification of the ThrRS sequence of P.infestans. And we conduct the determination of the in vivo enzyme activity function of borrelidin on ThrRS of P.infestans, The main results are as follows:(1) We achieved that IC50values of borrelidin on P. infestans was0.0528μg/ml by in vitro physiological tests;(2) We extracted the genome of the P.infestans with the method of salting out. designed primers with restriction sites according to P.infestans ThrRS gene sequences, cloned ThrRS sequences, then sequenced and blasted the obtained sequence. The blast result reveals that the sequence homology between the cloned gene and ThrRS sequence of T-130strain is as high as100%, which demonstrates the cloned segment is the threonyl-tRNA synthetase (ThrRS) gene of P.infestans;(3) We ligated the cloned gene into the NdeⅠ and HindⅢ had E.coli expression vector pET-32a (+) which had been cut with Ndel and HindⅢ, and transformed it into E.coli BL21(DE3), and expressed the protein in the forms of fusion. We achieved the best expression conditions by optimizing the conditions including the concentration of IPTG, inducing time, rotating speed and rotating time. The results indicated that the yield of fusion protein reached tiptop when we induced it with0.5mM IPTG at16℃,160rpm for16h.The SDS-PAGE analysis showed that we get the expected expression stripe near83.8Kda. The concentration of the ThrRS was2.23mg/mL after nickel column purification and dialysis for24h;(4)We made the purified protein react with ATP and Thr, measured the activity of enzyme by analyzing the decrement of Thr with liquid chromatography, analyzed the reaction between borrelidin with purified proteins, ATP and Thr, and measured the inhibition of Borrelidin on threonyl-tRNA synthetase of P.infestans by detecting the change of amount of Thr with liquid chromatography.In summary, the study of this paper provides a theoretical basis for exploring the direction of studying the new bio-pesticide and supplies information for the study of the mechanism of action between borrelidin and ThrRS.