Optimization Of Prokaryotic Expression Conditions Of Harpins Protein And Its Effects On Grape Growth And Downy Mildew Resistance

Grape quality and yield are severely reduced by various diseases.Downy mildew is one of the most common destructive grape diseases worldwide,particularly in regions with warm and humid climates.Downy mildew causes serious damage to young fruits,stems and particularly leaves.To date,the application of chemical pesticides is the main method to prevent and control grape downy mildew,which not only leads to environmental pollution but also increases the risk of leaving pesticide residues.Harpins protein belongs to microbial protein preparation pesticide,which has the characteristics of safety,non-toxicity,eco-friendliness and no drug resistance.In order to verify the effect of harpins protein on grape growth and downy mildew resistance,this experiment optimized the prokaryotic expression conditions of the core peptide of harpins protein,sprayed the crude harpins protein on the leaf surface,and verified its effect on grape growth and downy mildew resistance.The main research findings are as follows:1.Four harpins proteins were used,among which the prokaryotic expression conditions of the harpins protein Hrp Z＿K＿2 × 3 were optimized.The optimal prokaryotic induction conditions of harpins protein Hrp E1 are: 28℃,6 h,1 mmol/L.The harpins protein Hrp E2 has not screened out the best solubility conditions.The optimal prokaryotic induction conditions of harpins protein Hrp Z＿K＿2＿2×3 are: 28℃,6 h,0.5 mmol/L.2.Injecting harpins protein into tobacco leaves and vacuumizing grape leaves induced allergic reactions.After injection from the back of tobacco leaves,when the harpins proteins Hrp Z＿K＿2×3 and Hrp Z＿K＿2＿2×3 were diluted 100 times,significant allergic necrosis appeared.When diluted to 500 folds,harpins proteins with different treatments induced allergic reactions in tobacco leaves,resulting in a large area of allergic necrosis and withered spots.Collectively,the optimal dilution concentration of harpins protein is 500 times,and the allergic necrosis reaction of harpins protein Hrp Z＿K＿2＿2×3 is the most.3.Harpins protein promoted the growth of grapes.The variety of potted grapevines is‘Midknight Beauty’,The variety of field grapevines ‘Flame seedle’.By measuring the growth index of potted seedlings,the results show that harpins protein can promote the thickening growth of potted seedlings stems and promote plant height.Among them,the stem diameter effect of harpins protein Hrp E1 is the most significant;The effect of harpins protein Hrp Z＿K＿2×3 on plant height is the most significant.By measuring the photosynthetic index and chlorophyll fluorescence index of potted and field seedlings,the results show that harpins protein can promote the photosynthesis of grape leaves.The Fv/Fm andΦPSⅡof leaves treated with harpins protein Hrp Z＿K＿2×3 and Hrp Z＿K＿2＿2×3 were the most effective.By measuring the chlorophyll content of grape leaves in pot seedlings and field seedlings,the results show that harpins protein can increase the chlorophyll content of grape leaves.Among them,the chlorophyll content of potted seedlings and field seedlings treated with harpins protein Hrp Z＿K＿2×3 was the highest,which increased by 85.21% and 23.41%respectively compared with the control.4.By counting the natural diseased leaves of potted grape downy mildew and the in vitro inoculation test of potted grape seedlings,it can be known that the incidence index of downy mildew of grape decreased significantly after spraying harpins protein Hrp Z＿K＿2×3.The peroxidase,superoxide dismutase and catalase in the leaves of potted and field seedlings were determined.The results showed that the activities of POD,SOD and CAT in the leaves of potted and field seedlings were more significant after the treatment of harpins protein Hrp Z＿K＿2×3and Hrp Z＿K＿2＿2×3.The MDA content of grape leaves was measured after inoculation in vitro,and the MDA content of harpins protein Hrp Z＿K＿2＿2×3 was low,which indicated that harpins protein could alleviate the degree of membrane lipid peroxidation in grape leaves and improve the resistance of grape to downy mildew.