Gene Cloning,Expression And Physiological Function Of PagPIP1;2 From Populus Abla × P.glandulosa

Drought is the leading factor restricting forest productivity in northern of China,and the degree of drought may intensify under global climate change in the future.The water use efficiency of tree leaves is an important feature that characterizes the relationship between transpiration water consumption and photosynthetic carbon sequestration.Improving leaf water use efficiency is of great significance to the adaptation and survival of trees in arid environments.Aquaporins(AQPs)belong to the major intrinsic proteins(MIPs),which can specifically permeate water molecules and CO2 and other small molecules in both directions physiological process,and participate in water absorption and transportation.Among them,the aquaporin that can permeate CO2 may increase the CO2 conductivity of mesophyll,thereby achieving the purpose of improving leaf water use efficiency.Based on the known gene sequence of Populus trichocarpa PIP1;2,the c DNA sequence of its PagPIP1;2 gene was cloned from P.abla × P.glandulosa,and 35S:: PIP1;2 and 35S::PIP1;2-e GFP overexpression vector;the amino acid sequence,three-dimensional structure and cell location of PagPIP1;2protein were analyzed by software;the gene was obtained in the model plant Arabidopsis thaliana and N.tabacum overexpression lines have verified their subcellular localization and studied the effect of this gene on the drought resistance of A.thaliana and N.tabacum.The main results are as follows:1.PagPIP1;2 aquaporin is composed of 288 amino acids,the theoretical isoelectric point(p I)is 8.83,the instability coefficient is 24.52,the average hydrophilicity coefficient is 0.377,and the lipid coefficient is 96.25.It contains a typical pfam00230 conserved domain and belongs to the MIP family member.Mainly exists in the cell plasma membrane and endoplasmic reticulum,is a typical PIP aquaporin gene,mainly composed of α-helix,extended chain,β-helix and random coils,in a tetrameric structure.PagPIP1;2 water channel amino acid sequence is highly conserved and protein differentiation is low.Overexpression of PagPIP1;2 water channel may increase the mesophyll conductance and hydraulic conductance of plants,and enhance the stress resistance of the whole plant.2.Laser confocal microscope observation of N.benthamiana leaf cell membrane and chloroplast membrane that transiently express p CM1300-PIP1;2-GFP Agrobacterium and overexpression of PagPIP1;2-GFP N.tabacum on the root hair,epidermis,and cortical cell membranes obviously green fluorescence.3.Under full light conditions(24 hours light),overexpression of PagPIP1;2 tobacco lines are no different from wild type in normal water supply.The stomata of transgenic N.tabacum are closed,stomatal conductance(Gs)and intercellular CO2 concentration(Ci)are significantly reduced,stomatal restriction(Ls)is enhanced,photosynthetic rate per unit area(Pn)is reduced,and there is basically no significant difference in chlorophyll fluorescence.High CO2 restriction may be a key factor affecting the photosynthesis of functional leaves of tobacco lines overexpressing PagPIP1;2.Height and stem thickness of transgenic N.tabacum plants increased significantly under drought stress,leaf number increased,leaf area increased(LA),and investment in rhizome organs increased.Overexpression of PagPIP1;2 gene is beneficial to regulating root and crown tissue at the entire plant level.Under light / dark conditions(16h light / 8 h dark),the leaf soluble sugar content,electron transfer rate(ETR)of the OE lines under drought increased,and the leaf area,organ dry weight,and root-to-shoot ratio increased,which is conducive to improving water transport capacity and stabilizing photosynthesis.4.A.thaliana lines overexpressing PagPIP1;2 increased the single leaf area and rosette diameter,the number of leaves increased,and the dry weight of the shoot,the total dry weight is significantly increased.Drought treatment of transgenic plants increases the root to shoot ratio,adjusts plant morphology,and enhances soil water absorption and drought tolerance.The PagPIP1;2 gene is a typical PIP1 aquaporin.Overexpression of PagPIP1;2 gene is conducive to leaf Non-structural sugar(NSC)accumulation.By regulating the root and crown tissue morphology and hydraulic conductivity,it directly affects leaf transpiration,promotes plant growth,and significantly improves drought resistance of plant.