Preparation And Study On Anti-TMV Activity Of Quercetin Nanoliposomes

Plant viruses that are intracellular parasitic pathogens,can cause the serious loss of crops production,and are more difficult to control.Besides planting anti-virus varieties,the application of anti-viral agents is currently a major option for controlling the plant viral diseases.To cope with the the common mixed infection of viruses,the development of new antiviral agents has become a current research hotspot.Quercetin is a good bioactive molecule effective in antivirus.This paper prepared the quercetin-loaded nanoparticle combining nanotechnology and conducts systematic research for the development of new plant antiviral agents.In this paper,quercetin nanoliposomes was successful done by the film-ultrasonic technique.L-?-phosphatidylcholine and cholesterol as raw materials,Tween 80 as a surfactant,and quatemized chitosan(HACC)as a stabilizer.The morphology,particle size,PDI,Zeta potential,and encapsulation efficiency were determined.The structural changes of liposome before and after modification were studied by FTIR and TEM.Meanwhile,through formula optimization,the optimal concentration of HACC was determined.To investigate the stability of nanoliposomes,the effects of pH,temperature and storage time on the release rate of quercetin was studied.The results showed that :(1)the load quercetin system was quercetin nanoliposomes,the system volume was 100 mL,the initial dose of quercetin was 10 mg,the mass ratio of quercetin,L-?-phosphatidylcholine and cholesterol was 1:10:2,and the nanoliposomes had good quality.(2)The encapsulation rate of H-TQ-NPs obtained by optimized prescription was 88%,the average particle size was 300 nm,the Zeta potential was +38.5 mV,and the PDI index was 0.31.(3)The optimal concentration of HACC is 0.3g/L,which is helpful to improve the stability of nanoliposomes.(4)The weakly acidic(6.0)and low temperature(4°C)are the advantage of keeping the stability of quercetin nanoliposomes within 20 days.In order to determine the antiviral activity of quercetin nanoliposomes(Q-NPs),this study was conducted based on the N.benthamiana-TMV model,regarding relative accumulation of TMV CP mRNA as an indicator,and the application of 0.1 mM/L quercetin spraying twice at 12 h intervals can effectively inhibit the infection and replication of TMV.Western Blot and qRT-PCR detection showed that the inhibitory effect of Q-NPs on TMV was 10% and 42% higher than the same concentration of free quercetin at the gene and protein levels.The aqueous solution of Q-NPs was safe for N.benthamiana and Samsun NN,and no phytotoxicity and leaf malformation were found after spraying.To elucidate the antiviral mechanism of quercetin,based on the NCBI database,this study selected six genes with different subcellular localizations in the Hsp70 family of N.benthamiana as the research object,NbHsp70er-1(located in the endoplasmic reticulum),NbHsp70cp-1(located in the chloroplast),NbHsp70,NbHsp70c-A,NbHsp70c-B,and NbHsp70c-C(located in the cytoplasm).The effect of Q-NPs on the expression of 6 genes was studied by qRT-PCR.Through abiotic stress,it was found that the relative expression of NbHsp70cp-1 and NbHsp70c-A were up-regulated in response to heat stress,and the relative expression of other genes did not change significantly;In addition,pre-treatment with Q-NPs can reduce the up-regulation of NbHsp70cp-1 and suppress its response to heat stress.Biological stress(TMV infection)test results showed that NbHsp70er-1 and NbHsp70c-A were up-regulated during TMV replication,and the relative expression levels of other genes did not change;Q-NPs were used after TMV inoculation,the relative expression of NbHsp70er-1 and NbHsp70c-A was down-regulated,and the replication of TMV treated with Q-NPs had a lower amount.The results show that Q-NPs treatment can down-regulate the expression of NbHsp70cp-1,NbHsp70er-1 and NbHsp70c-A,and the expression of Hsp70 in different organelles is beneficial to multiple virus replication,so quercetin nanoliposomes can inhibit the corresponding Hsp70 expression to prevent multiple viral diseases.The experiment investigated the possible mechanism of Q-NPs preventing TMV-Q-NPs can inhibited the expression of NbHsp70er-1 and NbHsp70c-A induced by TMV replication,thereby reducing the accumulation of TMV in the early stage of the disease.