Study On The Molecular Mechanism Of POD Overexpression In Tobacco Enhanced Its Al Resistance And Nitrate Absorption

Aluminum toxicity is a crucial factor limiting plants grown and crop production in acidic soils,accounting for 30%of arable land,mostly distributed in tropical and subtropical regions.Acidic soil in China are mainly distributed in Yun Guichuan and tropical,subtropical regions and so on,accounting for nearly 20%arable land of Southern China.In normal soil conditions Al is no harm to plants,but in acidic soil(pH<5)the aluminum released in the form of Al3+,which inducing oxidative damage directly and triggering overproducing of reactive oxygen species(ROS),including H2O2,ect.In order to clear the active oxygen,plants have formed some mechanics,which mainly include non-enzymatic mechanics and enzymatic detoxification mechanics.peroxidases(POD)is one of the enzymatic detoxification mechanics which can clear the H2O2 produced by plants when encounter biotic or abiotic stress and alleviate oxidative damage.What's more,NO3-and NH4+ are the main mineral nutrition of plants,the content of nitrate nitrogen is far more than ammoniuM nitrogen in acidic dry soil,and the absorption and use of nitrate nitrogen plays an important role for the growth of plants.However,aluminum toxicity affects the absorption of nitrate nitrogen in plants,but the mechanism still unknown.In this paper,POD derived from Arabidopsis thaliana over-expression in tobacco,investigated the effects of aluminum on ROS contents and antioxidant defense systems in root tips of transgenic and wild type tobacco under different concentration Al.The aim of the study is to elucidate the role of POD protein in adaptative responses to aluminum toxicity in tobacco plants by analysis the content of H2O2,MDA,soluble protein and H+ pump activity and the uptake of nitrate as well as study the activity of H+-ATPase and its interaction level with 14-3-3 protein.The main results are as follows:1.Got POD transgenic plants.POD gene separated from Arabidopsis thaliana,and got pK2-35S-POD plant expression vectors.Transformed the tobacco using the method of agrobacterium-mediated transformation,and selected the transgenic plants with kanamycin.After the assay of PCR and western,we got the POD transgenic plants.The POD activity in transgenic was 1.2-1.6 folds of WT.2.The POD transgenic plants obtained high Al tolerance compared with WT.The POD transgenic plants and WT were treated with different Al concentrations for 12 h,we found that H2O2 content decreased but the relative root growth(RRG)and the activity of PM H+-ATPase and H+ pump,as well as citrate secretion increased in transgenic plants significantly compared with WT;The Co-immunoprecipitation show that the interaction level of PM H+-ATPase and 14-3-3 protein increased compared with WT.These results indicated that POD transgenic plants increased Al tolerance by clear H2O2 and increased the PM H+-ATPase and H+ pump activity as well as the interaction of protein to promote the citric acid secretion finally endow the transgenic plant higher tolerance to Al in acid soil.Achieve good growth in acid soil further proved POD transgenic has strang Al tolerance.3.The POD transgenic plants promoted the absorption of nitrate nitrogen.The Al-resistance of plants associated with the uptake of NO3-,what's more,absorption of nitrogen nutrition can effect the antioxidant enzyme system of plants.In order to further understand whether over-express POD in tobacco can affect the nutrient absorption of plants,we treated the WT and POD with different Al concentrations for Id and 4d to detect the nitrate uptake of plant.The res?lts suggest that the nitrate uptake decreased remarkably with the increasing Al concentrations.But in same conditions,the nitrate uptake of POD was higher than WT.But compared with treatment 4d with Al,the nitrate uptake of only treated Id was higher.In order to further analysis the molec?lar mechanism of POD promote the nitrate absorption,hydroponic experiments were carried out to study the effect of 1d and 4d exposure to 200 ?mol Al and 15 mmol KNO3 on the activity of H+-ATPase and H+ pump and the interaction between PM H+-ATPase and 14-3-3 protein.The res?lts suggest that Al inhibited the activity of PM H+-ATPase and H+ pump and decreased the levels of the interaction of PM H+-ATPase and 14-3-3 protein,but those in POD were higher than WT under same conditions.Therefore,we conclude that tobacco plants which over-express POD improves plants nitrate uptake by enhancing the interaction levels of 14-3-3 and PM H+-ATPase to increase the activity of PM H+-ATPase and H+pump which play important role in nutrients absorption.4.The promotion of nitrate nitrogen absorption related to 14-3-3 protein and PM H+-ATPase in POD transgenic plants.Experiments were carried out to research whether nutrients absorption promoted by POD transgenic tobacco association with PM H+-TPAase and 14-3-3 protein,the 14-3-3 over-expression plant S23 and inhibite-expression tobacco RP1 as well as PM H+-ATPase over-expression tobacco?GHA2-3 and inhibite-expression RE 10 were treated with different Al concentrations for 1d and 4d.After treatment,we detected the nitrate uptake and PM H+-ATPase activity and H+ pump and the levels of interaction of 14-3-3 and PM H+-ATPase.We found that the nitrate absorption of both PM H+-ATPase overexpression plant ?GHA2-3 and 14-3-3 overexpression plant were higher than WT and inhibited-expression plants when treated 1d and 4d.PM H+-ATPase,H+pump activity and interaction levels between PM H+-ATPase and 14-3-3 in two overexpression transgenic tobacco were higher than WT and inhibit-expression plants when encounter with 200?mol Al and 15mmol KNO3 for 1d,It indicated that PM H+-ATPase and 14-3-3 involve in nitrate absorption mechanism.The results indicated that the POD transgenic plants promoted the secretion of citrate by decreased the contents of H2O2 and increased the activity of H+ pump and PM H+-ATPase and the interaction level with 14-3-3 protein to affect the absorption of nitrate.