The Study Of The Enhanced Salt Tolerance In Tobacco By Transgene Pyramiding

Salt stress is one of the major abiotic stresses in nature,and can severely affect the productivity of crops all over the world.Improving the salt tolerance of plants through genetic engineering is one of the most effective strategies to solve this problem.Until now,improved salt tolerance of plants has been achieved by genetic transformation of salt tolerance-related genes.The tolerance to salt stress of plants is a complex trait that is modulated by multiple genes and requires the coordination of many genes.Therefore,the transformation of single gene into a plant can only enhance salt tolerance partially.In general introducing genes from different salt responses into a single plant by gene pyramiding is considered as an efficient approach for engineering extreme salt tolerance,which could be achieved either by cross-breeding plants containing different salt-tolerant genes or by transformation with multiple genes.The betA gene from Escherichia coli,which encodes chroline dehydrogenase (CDH),could increase the betaine content in transgenic plants;the TsVP gene from salt cress(Thellungiella halophila),which encodes vacuolar H~+-translocating inorganic pyrophosphatase,would enhance the transport across the vacuolar membrane;the AtNHX1 gene from Arabidopsis thaliana,which encodes vacuolar Na~+/H~+ antiporter,could sequestrate excessive Na~+ into vacuoles.It has been reported that overexpressing each gene alone can improve the salt tolerance of plants.In this study,the beta gene,TsVP gene and AtNHX1 gene transformed tobaccos were named BL,TL and AL,respectively.The transgene pyramiding tobacco plants were obtained by sexual crossing,including two genes that undergo different mechanisms of salt tolerance(betA×TsVP(named BT),betA×AtNHX1(named BA)). PCR,Southern blot,RT-PCR,and the determination of betaine content,V-H~+-PPase hydrolytic activity and Na~+ concentration indicated that the two genes were integrated into the genome and expressed functionally in transgene pyramiding tobacco plants. The analysis of salt tolerance at the whole plant level and cellular level were performed,which confirmed the consistency between the cellular level and the whole plant level.The transgene pyramiding plants showed higher salt tolerance than single gene transgenics,though the extent was not great.The main results of this study were summarized as follows:Studies on salt tolerance of betA×TsVP(BT)tobaccoOn the medium containing the same concentration of NaCl,the transgene pyramiding tobacco line(BT)showed the highest percentage of seed germination,the best growth and the highest seedling fresh weight.In contrast those of the single gene transgenic lines(BL and TL)were moderate and those of the non-transgenic control line were the lowest.These results indicated that transgene pyramiding could further enhance tobacco salt tolerance than the transformation of single gene at the seed germination and seedlings growth stage.Under normal conditions,the biomass of betA-transgenics(BL)and the wild-type(WT)plants were similar.The fresh weight and dry weight of BL were both higher than those of WT after the treatment of 250 mM NaCl for 14 days.The TsVP-transgenics and the transgene pyramiding plants showed more developed shoot and root systems compared with WT under both normal and salt stress conditions,and the biomass of betA×TsVP plants was the highest.The TsVP-transgenic tobacco plants (TL and BT)showed significantly higher net photosynthetic rate than wild-type and betA-transformed plants under normal conditions.There was no significant difference on the photosynthesis between TL and BT,and the photosynthesis of WT and BL plants were also comparable in the absence of NaCl.Salt stress decreased the photosynthetic activities of both transgenic and wild-type plants,while the transgenic plants exhibited higher photosynthetic rate and Fv/Fm ratio than wild-type,implying the higher photosynthetic efficiency in transgenies.The results of the measurement on other photosynthesis features also indicated the transgenic plants had higher photosynthetic capacity than the wild-type ones under salt stress conditions,but the difference in photosynthesis between transgene pyramiding plants and single gene transgenics was not significant.Salinity stress increased the Na~+ and Cl~- concentrations in all plants,but the TsVP-transgenics(TL)and the transgene pyramiding plants(BT)accumulated significantly more Na~+ and Cl~- than wild-type and betA-transgenic plants.There was no significant difference on Na~+ and Cl~- contents between BT and TL plants.The accumulation of Na~+ in vacuoles was measured using Sodium Green fluorescent indicator and laser scanning confocal microscopy.Higher fluorescent signals(i.e. higher Na~+ concentration)were observed in both transgenic and wild-type cells under salt stress than in the absence of NaCl.It was found that the Ts VP-transgenic and the transgene pyramiding cells accumulated significantly higher Na~+ than wild-type and betA-transgenic cells under salinity stress,but there was no obvious difference on Na~+ accumulation between the transgene pyramiding cells and TsVP-transgenic cells, which was consistent with the measurement result of Na~+ in the leaves.These results suggested that the functional expression of TsVP enhanced the sequestration of Na~+ in vacuoles.Salt stress resulted in membrane injury and caused MDA content and electrolyte leakage rising in both transgenic and wild-type plants.The wild-type plants exhibited higher MDA content and relative conductance than the transgenics under saline conditions,suggesting the higher salt tolerance in transgenic tobacco plants.The transgene pyramiding plants showed the highest salt tolerance for the lowest MDA content and ion leakage of leaves under salinity stress.Such results were in line with the observations of cell viability and the integrity of mitoehondrial membrane at the cellular level.The transgenic tobacco cells showed higher cell viability and better integrity of mitochondrial membrane than wild-type cells under the same NaCl treatment,which also suggested the improved salt tolerance in transgenic cells.The cytoplasmic and vacuolar pH values were recorded by means of the pH-sensitive,cell-permeant fluorescent indicator BCECF-AM.The transgenic cells, especially TsVP transgenic cells,showed the less change of pH,which suggested that they could maintain the intracellular environment homeostasis better than wild-type under salt stress.The vacuolar pH values in TL and BT cells were lower than those in wild-type ones.The membrane potential produced by pH difference across the vacuolar membrane(△Ψ(H~+))was calculated using Nernst equation.The△Ψ(H~+) values of TsVP-transgenic and transgene pyramiding cells were significantly higher than those of wild-type and betA-transgenic cells,both before and after salt stress. These results suggested that the V-H~+-PPase could acidify vacuoles and maintain higher proton electrochemical gradient across the vacuolar membrane in TsVP transgenic cells.The higher△Ψ(H~+)would provide more motive force for the V-Na~+/H~+ antiporter to sequestrate Na~+ within vacuoles,which was proved by the determination of sodium ion in vacuoles.Studies on salt tolerance of BA(betA×AtNHX1)tobaccoThe transgenic tobacco lines exhibited higher percentage of seed germination.In addition,they grew better than the wild-type when placed on medium supplemented with the same concentration of NaCl.The seed germination percentage of the transgene pyramiding line(BA)was the highest,and the BA fresh weight per seedling was slightly higher than single gene transgenics(BL and AL),suggesting improved salt tolerance in the transgene pyramiding line.The AtNHX1-transgenic plants(AL)and the betA×AtNHX1 transgene pyramiding plants(BA)accumulated more Na~+ content than wild-type and betA-transgenic plants under salinity stress.Salt stress resulted in increasing Na~+ accumulation in the AtNHX1 transgenie cells(AL and BA)compared with that in WT or BL cells measured with Sodium Green,and the difference was significant. Alternatively,there was no distinct difference between AL and BA cells in the Na~+ content.The results were in agreement with the findings of Na~+ in leaves,indicating that the heterologous expression of AtNHX1 accelerated the sequestration of Na~+ within vacuoles.The calculation of cytoplasmic pH,vacuolar pH and the△Ψ(H~+) across the vacuolar membrane also indicated that the transgenic cells could maintain the homeostasis of intracellular environment better than WT ones.The transgenic cells showed higher protoplast viability and better mitochondrial integrity than wild-type cells under saline conditions.But no significant difference was found between the transgene pyramiding and single gene transgenic cells.Salt stress increased MDA content and relative conductance in all plants.However,the values of transgenic plants measured were much lower than those of wild-type.These results indicated that the transgenic cells showed enhanced salt tolerance compared with wild-type under salt stress conditions,but the improved salt tolerance of betA×AtNHX1 transgene pyramiding plants was not greater than that of single gene transgenics(BL and AL).In transgene pyramiding plants,the transgenes increased the salt tolerance of tobacco by different tolerance mechanisms,respectively.But the improved salt tolerance of transgene pyramiding plants compared with single gene transformed plants was much less than the additive effects of the two genes alone.It is presumably because plant salt tolerance is a polygenie trait which requires the cooperation of many genes,and some elements restricted the sensitivity of plants to salinity stress. Although the pyramiding of genes involved in different salt tolerance mechanisms is an important approach for enhanced plant salinity tolerance,it is necessary to deeply understand the metabolic and regulatory mechanisms for plant tolerance to salt stress. As a consequence,strategies for plant stress-tolerance genetic engineering could be made with better pertinency and maneuverability.Furthermore,our work implied that the roles of genes might be enlarged due to the damage of regulatory networks when their biological functions were studied by mutants.Therefore,functional differences of target genes should be considered under different genetic backgrounds.