Function Studies On PeTIP1;2 Gene And Its Promoter Effects Of Tonoplast Aquaporin In Populus Euphratica

Aquaporin(AQP)is a highly conserved protein with a certain selectivity and high water permeability.Its relative molecular mass is generally between23-31 Ku.It belongs to membrane instrinsic protein(major instrinsic protein,MIPs)superfamily and widely exists in various organisms,such as plants,animals,and microorganisms.More than 150 MIPs have been found in these organisms.It plays an important role in the regulation of various physiological processes such as water transport,small molecule solute,gas and heavy metal transport,root water absorption,cell elongation and differentiation,and plant reproductive growth.Under abiotic stress conditions,plant aquaporin proteins can also respond to abiotic stress,including abiotic stresses such as salt stress,drought stress,cold stress and heavy metal stress.In this experiment,Populus euphratica,Populus tomentosa,and tobacco were used as materials to clone the AQP gene Pe TIP1;2 from Populus euphratica;the transgenic plant of Pe TIP1;2gene was studied under heavy metal stress,salt stress and drought stress conditions.Then the 35Spro::Pe TIP1;2-RNAi vector construction and knockout to explore.And then subcellular localization of Pe TIP1;2 proteins by Arabidopsis thaliana protoplasts..Finally,the Pe TIP1;2 promoter was cloned and the plant expression vector Pe TIP1;2pro::GUS was constructed to transform poplars.Tissue expression of the promoter and tissue expression differences induced by salt and mannitol.From the aspects of functional analysis and expression regulation,the role of this gene in the stress-resistance mechanism of Populus euphratica was studied.The research results obtained are as follows:1.Pe TIP1;2 gene cloning and identification of transgenic plantsIn this study,we cloned the Pe TIP1;2 gene of Populus euphratica and the Populus tomentosa and tobacco were transformed by leaf disc transformation.we obtain 18 tissue trees.The positive trees have 16,and the rate of positive was88.88%.The expression level was analyzed by Real-time quantitative PCR.Real-time quantitative PCR analysis showed that the highest expression was 59.7times than wild type Populus tomentosa and the lowest was 0.20 times;and we obtain 24 tissue tobacco,have 23 positive tobacco,the positive rate was 95.8%.In this study,transgenic tobacco plants with heterologous expression of Populus euphratica Pe TIP1;2 genes were treated with different abiotic stress treatments.The root length of transgenic tobacco on 1/2MS medium containing200 mmol/L Na Cl was significantly higher than that of wild type tobacco.In the soil culture experiment of tobacco,We found that both transgenic tobacco and wild-type tobacco can grow normally under normal watering.When 200 mmol/L Na Cl solution is poured,the wild type and transgenic tobacco leaves begin to yellow,but the wild type phenotype is more obvious and the leaves become wilted.which indicated that heterologous overexpression of Pe TIP1;2 gene increased the resistance of tobacco to salt;the root length of transgenic tobacco on 1/2 MS medium containing 300 mmol/L Mannitol(mannitol)was significantly longer than that of wild type tobacco,after 7 days of natural drought treatment of tobacco,we found that the wild type tobacco became wilted and the transgenic tobacco grew well,which indicated that the heterologous overexpression of the Pe TIP1;2 gene can increase the resistance of tobacco to drought;then the treatment of transgenic tobacco with 50 mmol/L Cu SO4·5H2O solution found that wild type tobacco wilted,It was found that wild type tobacco was markedly wilted and seriously injured while transgenic tobacco was less harmed and there was no significant wilting.Which indicated that the heterologous overexpression of Pe TIP1;2 gene increased the resistance of tobacco to heavy metal copper sulfate stress.In this study,Arabidopsis protoplasts were used for subcellular localization.The results showed that the localization of Pe TIP1;2 protein mainly located the vacuolar membrane.Based on the genomic data of Populus euphratica,the promoter of Pe TIP1;2 was cloned and the plant expression vector Pe TIP1;2pro::GUS was constructed to transform Populus tomentosa.The tissue expression of the promoter and tissue expression induced by 200 mmol/L Na Cl and 300 mmol/L Mannitol were analyzed.It was initially shown that the Pe TIP1;2 gene is expressed inducibly,ti is mainly expressed in the leaves,a small part is expressed in the roots and almost no expression in the stems3.RNAi interference experimentIn this study,we constructed 35Spro::Pe TIP1;2-RNAi vector.We used leaf disc transformation method to transform Populus tomentosa and obtained 92.2.Populus euphratica Pe TIP1;2 gene functionRNAi-positive Populus tomentosa trees.