Application Of Virus Induced PPO Gene Silencing In Lotus

Lotus(Nelumbo nucifera Gaertn.)leaf is round,whole or slightly undulate.It is mainly grown in Hubei,Jiangsu and Anhui provinces,which was listed as a food with medicinal food homology by the Ministry of Health in 2002.Lotus leaf has the function of lipid regulation that can assist to treat a variety of cardiovascular diseases,such as atherosclerosis and hypercholesterolemia,etc.It has been used in a variety of compoun d preparations and as an important raw material source of lipid-lowering Chinese patent medicines.Polyphenol oxidase(PPO)is a kind of copper-binding enzyme commonly found in plants and animals.It oxidizes phenols into quinones,which self-polymerize and combine with other substances to form brown or black pigment precipitates.They can form a physical barrier to protect plant tissues.However,the function of PPO in lotus leaves is still unknown and needs further investigation.In this study,PPO gene in lotus leaf seedlings was silenced by virus induced gene silencing(VIGS).The phenotype and physiological changes of lots were observed in order to investigate the influence of PPO and its function in lotus leaf.This study founded a method to illustrate the effects of genes on lotus leaves,and provided a theoretical basis to strengthen the resistance of lotus leaves,and to improve the food and medicinal value.The main experimental results of this study are as follows:1.Through the multi-bioinformatics method,the PPO family of lotus leaf was found to have three structural domains: Tyrsinase,PPO1＿DWL and PPO1＿KFDV,with the length of about 600 aa and the isoelectric point of 5.87-8.59.PPO in lotus leaf was divided into two clusters: PPO1 and AUS There were great differences in both structure and function betweenPPO1 and AUS.The amino acid residues contained in most of PPOs in lotus leaves were leucine,accounting for 8.9% ～ 9.2%,followed by proline and lysine.The residue bases of negative charge(aspartic acid and glutamic acid)were 76-79,and the residue bases of positive charge(arginine and lysine)were 67-82.In general,polar amino acids were more than non-polar amino acids.The PPO instability coefficients of lotus leaves were all greater than 40,the fat coefficient was 69.28 ～ 84.04,and the total average hydrophilicity was all less than 0,indicating that this family were all unstable hydrophilic lipid-binding proteins.Two active sites were found inPPO of lotus leaf,namely,the tyrosine Cu A binding region and the tyrosine Cu B binding region,and both sites were located in the tyrosine domain.According to the PPO functional domain of lotus leaves,they all have Nglycosylation sites,casein kinase Ⅱ phosphorylation sites,N-myrioylation sites,and protein kinase C phosphorylation sites,but AUS does not have amylation sites.Nnaus1 has a unique cell adhesion sequence RGD(arginine-glycine-aspartic acid).2.The recombinant plasmid pTRV2-PDS was successfully constructed,transformed into Agrobacterium GV3101 and injected into the leaves of the plant.The leaves were found to be seriously disgreen,indicating that the VIGS system was successfully established in lotus leaves.The three PPO genes of lotus leaves were combined with PTRV2 vector to build recombinant plasmid.After injecting leaves,brown spots were found on the surface of leaves of the three silencing types,and the root hairs of the roots were significantly reduced.NnPPO1 silencing group and NnAUS2 silencing roots almost had no root hairs.The q PCR results showed that the content of PPO in the silencing group was significantly lower than that in the control group,indicating that the silencing effect was better.3.In the NnPPO1 silencing group,the other three oxidation indexes except SOD were lower than those in the control group,indicating that the antioxidant capacity of lotus leaves had a downward trend after silencing NnPPO1 gene.SOD and CAT in NnAUS1 silencing group were lower than those in control group,while POD and MDA in NnAUS1 silencing group were higher than those in control group,indicating that the degree of cell membrane peroxidation was increased and the cell membrane was damaged.In the NnAUS2 silencing group,the enzyme activity indices were significantly increased,while MDA barely changed,suggesting that the content of reactive oxygen species in lotus leaves was increased and physiological activity was abnormal after NnAUS2 silencing.Histochemical staining results showed that after silencing PPO gene,the contents of superoxide anion and hydrogen peroxide in lotus leaves increased,which corresponded to the increase of protective enzyme activity.4.Scanning electron microscopy showed that compared with the frontal leaves in the control group,the NnPPO1 and NnAUS1 silencing groups became flattened,while some areas in the NnAUS2 silencing group collapsed and formed local plaques.The number and volume of papillae on the dorsal side of leaves in the silenced group were significantly smaller than that in the control group,and some cells in the silenced group even had no papillae.It shows that after PPO is silenced,the positive and negative sides of lotus leaf have changed,and self-cleaning ability and floating ability have been affected.