The Study Of The Gene Pyramid BetA And TsVP Improves The Stress Tolerance Of Maize

In the face of a global increase of population and scarcity of water resources and the decreasing area under cultivation,it is more and more important to exploit and utilize arid and saline-alkali soils.Lots of the genic engineering of improving the stress tolerance of plants are the introduction of single gene into plants to gain engineering plants,and the enhanced level of the stress tolerance is still awaiting to incresae.The plant resitance to abiotic stress is controlled by polygene,so multi-gene assembling and transformation technology emerged as a new strategy.One of the most effective pathway to express multi-transgene in plants appears to be gene stacking.First,individual gene is transformed into parent plants,respectively,and then,the plants with different transgene from one genetype are hybridized each other to generate multi-transgene plants when they are homozygote transgenes.This approach is relatively taking time compered with the introduction of several genes into a plants at one time,but the gene expression is comparatively stabilization and it is more easy to obtain the transgenic plants with high-expression multi-trangene.In our experiment,the betA(coding glycine betaine of E.coil)and TsVP(coding ppase of Thellungiella salsugniea)genes were pyramidsed into maize using cross-pollinated method.The transgene pyramiding plants of the F1 generation were identified by PCR and Southern blotting analysis.And the stable expression of two target transgenes in the pyramiding plants were confirmed using real-time RT-PCR analysis.We chosed the transgene pyramiding lines,and their perent lines that each had a target transgene,and non-transformed DH4866 as wild type control to analysis the tolerance to drought and salt in experimental conditions.Analysis of drought tolerance of the transgene pyramiding plantsThe seeds were planted in unified flowerpots,the drought stress was carried out at the ten leaves stage.Soil water content was maintained at 15-16%.The drought stress was lasted for 16 days before restoring watering the plants.In the period of drought stress,the transcriptional quantity of the two targart genes in the transgene pyramiding plants were distinctive higher than the single transgene.After the drought stress,the transgene pyramiding lines appeared difference with their parent lines and wild type control line in the traits of plant height,branches of tassels,anthesis, anthesis-silking interval,pollen viability,ear length,100 grain weight and so on.The wild type plants had less branches of tassels,abnormal stamen,the sterility of pollen, long anthesis-silking interval,and less grain weight per plant,while the transgene pyramiding plants showed obviously improved drought tolerance,the normal pollen development on the whole,the grain weight per plant was substantially higher than that of their parents,and much higher than that of wild type plants.So the transgene pyramiding plants exhibited significantly improved drought tolerance and grew better under drought sress.The examination of physiology displayed that the decrease rate of relative water content in the leaves of transgene pyramiding plants were lower than that of their parents in the same environment during drought stress,so they had better water capacity.And the soluble carbohydrate and proline content were accumulated more in transgene pyramiding maize than that in their parents and wild type control,and this indicated the transgene pyramiding plants had lower solute potential during drought stress,and had lower ion leakage and MDA content,indicating less damage of cell membrane under drought stress.These results suggested the transgene pyramiding plants could maintain normal metabolism by accumulated osmotic substance under drought stress condition,and undergo moderate damage,so the enhanced drought tolerance is displayed.Analysis of salt tolerance of the transgene pyramiding maizeThe seeds from different lines were planted in unified sand plots and then watered with different concentration NaCl solution.After 30 days,we counted the ratio of seeds germinated and the livability of seedling.The transgene pyramiding seeds had germinated in a relatively higher frequencies under high NaCl condition, and the livabilities and growth of transgene pyramiding seedlings were better than that of their parents,and the biomass of the transgene pyramiding ones were higher too.In the transgene pyramiding seedlings Na⁺ and Cl^- contents had increased more comparing with their parents,but the K⁺ and Ca²⁺contents had little change.These results suggested that the transgene pyramiding plants have obviously enhanced the accumulation of Na⁺ in vacuoles to reduce the toxicity of excessive Na⁺ in cytoplasm to the cell.After that,the maize seedlings growing in Hoagland solution were examined for their tolerance to salt stress.The transgene pyramiding plants grew better than their parents and wild type control,and had lower cell membrane ion leakage and MDA contents.The decrease rate of chlorophyll contents were less than that of their parents and wild type control.The levels of transgene expression were higher than that of their parents under salt stress.We inferred that the two transgenes in the transgene pyramiding plants could co-expressed to produce a relatively better conditions in cells that was beneficial to the genes expression stably,accordingly the plant stress tolerance was improved.Above all,the betA and TsVP co-expression in plants could effectively improve the drought and salt tolerance in transgenic maize.Under drought stress,the transgene pyramiding plants could accumulate more solutes including organic and inorganic ions solute in cell,which would benefit the capability of water uptake at low soil solution water potential to maintain cell turgor.Under salt stress,the TsVP gene in transgene maize could enhance the accumulation of Na⁺ in vacuoles to reduce the toxicity of excessive Na⁺ to the cell,the betA gene could increase ability of stress tolerance,the cooperatioin of the two genes boosted up the plant salt resitence. Besides,compared with the wild type plants,the single gene transgenics had a higher extent stress tolerance.The transgene pyramiding plants showed a moderate improvement in drought and salt stress tolerance compared with the single gene transgenic plants,namely their parents.This results indicated that the improving of maize plant stress tolerance were also restricted by other metabolic pathways.This study has created some valuable maize materials for maize drought and salt tolerance breeding,that would make contribution to maize production and utilization of large areas of arid and saline-alkali soils in China.On the other hand,this study produces some important informations for better understanding the molecular mechanisms in maize drought and salt tolerance.