Studies On The Effect Of Blue Light For Conidia Discharge Of Pandora Neoaphidis And Its Related Receptor Protein Genes

Pandora neoaphidis?Entomophthorales?is an obligated aphid pathogenic fungi of Entomophthoralean fungi,that causes epizootics in aphid populations by means of active discharge of condia to control aphid effectively.Some studies indicate that blue light can significantly increase the number of ejections of fungal spores,and the characteristic is inseparable from the photoreceptors that sense various light information.However,the lack of photoreceptor study in P.neoaphidis has led to great limitations on the molecular mechanism theory research of its popular ecological adaptability and its biological control application potential.In this study,we studied the effects of blue light on spore-discharging capability by comparing the distance of spore ejection and the speed of spore ejection in the presence or absence of blue light irradiation.Meanwhile,the micro-projectile data of conidia were obtained by high-speed microscopy and used to mathematically model the ejection path using MATLAB software.Subsequently,the prokaryotic expression system was used to express and purify the blue receptor protein.Based on the amino acid sequence,the interaction between the two proteins were predicted by computer simulation.Finally,the effects of light on the spore ejection and the biological properties of light-associated blue receptor protein were illuminated.The results were as followed.1.Study on the effect of blue light on the discharge of P.neoaphidis conidiaBy measuring the discharge distance of P.neoaphidis spores under blue light irradiation and dark environment,it was found that the discharge distance of P.neoaphidis spores under blue light irradiation was higher than that under dark condition,both in vertical distance and horizontal distance.After comparing and analyzing the resultant velocity of spore discharge under two different conditions,it was found that the resultant velocity of spore under blue light irradiation was significantly higher than that under dark conditions,and it was further verified the positive effects of blue light on the ability of discharge in P.neoaphidis spores.In addition,the fitting curves of the discharge path of conidia under the two treatment conditions were obtained respectively,y=-0.0325x²+0.207x+0.3132 for blue light exposure treatment,and y=-0.0775x²+0.5316 x-0.062 for dark conditions.2.Dynamic observation and path prediction of the P.neoaphidis conidiaUsing high-speed microscopy,spore-discharging video of P.neoaphidis was successfully captured,and the speed was calculated using the high-speed camera video processing software i-SPEED Suit.When the spores were in suitable condition and in a high humidity environment,the ejection speed can reach 2.07 m/s.When the environmental humidity and temperature of the spores were not suitable,the ejection speed decreases.At the initial stage of ejection,the speed at 0.1365 m/s,the speed gradually slowed down to 1.052×10^-3m/s due to the viscosity of the air.In addition,based on the known spore physical parameters?ejection angle,mass,size,etc.?,the spore ejection path was fitted by using MATLAB software,and the fitting curve was y=-9199.3x²+0.8624x+0.00008?R²=0.8941?.3.Prokaryotic expression of blue light receptor proteins PnWC-1 and PnWC-2The full-length sequence of the pnwc-2 cDNA was obtained by transcriptome data and RACE cloning technology,and the preliminary bioinformatics analysis of the gene was carried out.It was found that the protein domain of the gene has greater similarity with other species.However,P.neoaphidis was classified as a separate branch in the amino acid homology comparison.Subsequently,the PnWC-1 protein was expressed in vitro using the pnwc-1 gene obtained previously,and the recombinant plasmid pET-wc-1 with pET-28a as a vector and the recombinant plasmid pCold-wc-1 with pCold-TF as vector were constructed.The expression results showed that the target protein was inclusion body of expression of pET-wc-1.However,using pCold-wc-1 recombinant protein expression,the target protein was soluble protein.In addition,the recombinant vector pCold-wc-2 was constructed and the recombinant protein PnWC-2 was obtained by low temperature induction,and the target protein was soluble protein.4.Simulation analysis of interaction between blue light receptor protein PnWC-1 and PnWC-2According to the amino acid sequences of pnwc-1 and pnwc-2,it was predicted that the random coils in the secondary structure was most,followed by?-helix,the?-sheet ratio was low;using the amino acid sequence of the two proteins,the?G?binding free energy?in the PPA-Pred prediction interaction process was-11.73kcal/mol,and the dissociation constant Kd was 2.51e-09 M.SWISS-MODEL was used to predict the tertiary structure of the two proteins,and the two tertiary structures were used as models to perform molecular docking simulation.The interaction area between the two proteins was 1072.7?²,and the free energy generated by this method was used.?ⁱG was-12.2 kcal/mol,and during the interaction,it was found that five amino acid sites formed hydrogen bonds.