Effects Of Propamidine On Mitochondrial Respiratory Chain Complexs Of Botrytis Cinerea

Propamidine, which have novel effect on grey mould caused by Botrytis cinerea, is type of fungicide with intellectual property in China and it is safe to environment. Early research result shows that propamidine have a certain impact on the metabolism and may inhibit the energy metabolism of B. cinerea. From the view point of biology and biochemistry it is speculated that may inhibit the expression of a subunit gene of mitochondrial complexâ…¢, which causes the inhibition of fungal growing. In order to further define the mechanism of propamidine on B. cinerea, alternative pathway, activity of complexes on propamidine- sensitive strain and propamidine-resistance strain, molecular biology is adopted in this research and results are obtained as listed below:SHAM shows no significant effect on mycelium growing of B. cinerea. Obtained by the analysis, the presence of SHAM possesses no significant impact on electron transport chain (ETC) and the inhibition of alternative oxidase (AOX) possesses no significant impact on ETC too. According to the calculation by Yun-peisun formula, propamidine and SHAM possesses significant synergistic effect on B. cinerea. The synergistic reasons may be concluded that the presence of propamidine may inhibit the oxidation of ubiquinone, which casues ETC can't function properly and AOX gene is induced to activate the alternative pathway. Howerver, because of the presence of the AOX inhibitor, SHAM, alternative pathway can't function properly too, which causes the electron can't be transported to O2 and finally oxidative phosphorylation can't function properly. As it shows that propamidine may inhibit the complex directly or indirectly, which blocked the oxidation of ubiquinone and ETC can't function properly, affect the synthesis of ATP.Complexes activity test result in vitro shows that different concentration of propamidine possesses no significant effect on the activity of four types of complexes. Besides, propamidine possesses no significant effect on propamidine-resistance strain of complexâ…¢, which can be speculated that propamidine is not simply combining with a point on complexâ…¢to affect the normal ETC function. Complexes activity test result in vivo shows that after the treatment of propamidine-sensitive strain mycelium by propamidine 1 day, the activity of complexâ… ,â…¡,â…£increase with the concentration higher and the activity of complexâ…¢shows a negative correlation with the propamidine concentration. After the treatment of propamidine-sensitive strain mycelium by propamidine 3 day, all four complexes activity show negative correlation with the propamidine concentration. Besides, after the treatment of propamidine-resistance strain mycelium by different concentration of propamidine 1 day and 3 day, each concentration group of complexâ…¢activity possesses no significant different with CK group in same time, which shows that propamidine-resistance strain mitochondrial complexâ…¢is affected little comparing with propamidine-sensitive strain. According to the test of complexes activity in vitro and in vivo, speculating that the resistance of B. cinerea to propamidine may be caused by the decrease sensitivity of complexâ…¢to propamidine. Above all, mitochondrial complexâ…¢may be one of the most important action site of propamidine.cDNA-AFLP technology is adopted and 37 abnormal or up-regulated expression TDFs are tested by 64 kinds of primer combinations in B.cinerea treated by propamidine. Furthermore 5 of 37 TDFs with typlical changes are recycled, cloned and sequencing analyzed and the result shows that 4 of 5 TDFs match with the known homologous sequences. TDF4 and TDF5 possesses high homology with cytochrome P450 alkane hydroxylase; TDF1 possesses high homology with mitochondrial phosphate carrier protein in B. cinerea; TDF2 possesses high homology with a fragment of cDNA library in T4 strain of B. cinerea, unkown function; TDF3 possesses no homology with the known sequences in NCBI and pending further research. For TDF1, up-regulated expression of mitochondrial phosphate carrier protein gene may be caused by the block of electron transport in ETC, which blocks the formation of the proton gradient between two sides of inner membrane of mitochondrial, feedback signal of ATP synthesis can't be progress is send to the whole organism. So, propamidine may block the electron transport in ETC and causes the proton gradient between two sides of inner membrane of mitochondrial can't be formed, which block the synthesis of ATP by F1-F0-ATP synthase lacking of proton motive force. For TDF4 and TDF5, the up-regulated expression of cytochrome P450 alkane hydroxylase gene may be the result of B. cinerea possesses a stress adaption to propamidine.Above all, propamidine may affect the transcript, translation, peptide extension or peptide folding, which may cause composition or conformation changes of complexâ…¢, blocks the oxidation of ubiquinone and the proton gradient between two sides of mitochondrial inner membrane that causes blocking of ATP synthesis by lacking of protonmotive force and finally causes death of mycelium because of lacking of energy.