Physiological Mechanisms Of Salt Tolerance And Cloning And Sequence Analysis Of Na~+/H~+Antiporter Gene On Vacuole Membrane Of Glehnia Littoralis

Soil salinization is an important environmental issues facing humanity, and it constitute aserious threat to the sustainable development of agriculture and animal husbandry （Ma Qing,2011）. Understand the mechanism of salt tolerance in plant and cultivate salt tolerant cropswith a certain economic value of resources have great significance to curb land salinizationand salt wasteland.In this study, the salt-tolerance evaluation system in seed germination and seedlinggrowth period has been established based on the seed dormancy breaking of Glehnia littoralis,mean while, the salt-tolerance ability was identified and the type of salt-tolerance and saltstress physiological response mechanisms were cleared.We obtained tonoplast Na⁺/H⁺antiporter geneand its expression characteristics and theeffect in resistance to salt stress wereanalyzed. This study applies the basis of preliminary work to further improve plantsalt-tolerance mechanisms and the development and utilization of salt-tolerant plantresources. The main findings are as follows:1. The seed of Glehnialittoralis has strong dormancy characteristics. Under the conditionof temperature-accelerated stratification, solution of100mg·L^-16-BA may significantlypromote the seed germination. The germination rate was up to63.3%in the first90days.Germination in vitro experiments showed that the factor of endosperm inhibited seedgermination of Glehnialittoralis, and the germination rate of the seed with one thirdendosperm was the highest, could reach31%. Treated by TDZ、6-BA and GA3had no effecton seed dormant breaking, but easily lead to deformity seeding.2. It possesses better salt resistence during the stage of Glehnia littoralis seedgermination, and the median lethal concentration（LC50） and the lethal concentration is0.68%and1.52%respectively treated by NaCl. The germination of seed is significantly restrainedwhen the concentration of NaCl is higher than1.5%. NaCltreatmen can extend the initial timeof the seed germination and reduce the whole germination percent of seed. Rehydrationexperiment showed that Glehnialittoralis seed keep higher germination potential which wassignificantly positive correlation with treatment concentration.3. Glehnia littoralis seedling possess strong salt resistence in the growth period. Underthe solution culture condition, the growth status and increment of seedling was normal whentheNaClconcentration was100mmol·L^-1, the growth had been restrained when theconcentration was higher than200mmol·L, the plants began to die when the concentrationwas300mmol L^-1, and the survival rate was ablut50%.4. The related physiological signs of Glehnia littoralis seedling showed that, solublesugar played a major regulatory role firstly as penetration regulating substances under lowNaClconcentration,and antioxidant enzyme systerm gradually started with the increasing ofNaCl concentration and treated time, however, When the NaCl concentration exceeded theregulating ability of the plant, the unit quantity of reaction center was dropped significantly,thus the growth of the plant was affected. Multivariate statistical analysis showed that theeffective evaluation system of salt resistance indexes was consisted by Malondialdehydecontent、Fv/Fm、ETo/ABS、Chlorophyll content DIo/RC and RC/CSo together.5. Under the soil potted condition, Glehnialittoralis growth situation changedsignificantly with the increase of NaCl concentration. Glehnia littoralis slightly affected when treated by0.5%and1.0%NaCl, and when soil NaCl concentration was up to1.5%, the ratedry matter accumulation dropped evidently, individual plants appeared death.6. The determination result of Na⁺、K⁺、Ca²⁺and Mg²⁺in different organs of Glehnialittoralis. showed that petiole was the main organ for Na⁺storage in Glehnia littoralis. Inordinary conditions, Na⁺content in petiole is2.7times and79times of leaves and rootsrespectively. Be different from Na⁺, NaCl treatment had smaller effect to K⁺、Ca²⁺and Mg²⁺contents in root, which were more stable and not exist significant differences with contrast. K⁺in petioles and leaves was decreased with NaCl concentration increasing, on the contrary,Ca²⁺content showed an increasing trend. Mg²⁺content in petiole was reduced with theincrease of NaCl concentration and Mg²⁺contents in leaves changed little. The rate of Na⁺/K⁺increased obviously under1.5%NaCl treatment, exiting a significant difference comparedwith other treatments.7. The results of selective absorption and transport of K⁺、Na⁺、Ca²⁺and Mg²⁺whenGlehnia littoralis. treated by NaCl showed that the ability of soil selective absorption of K⁺、Ca²⁺and Mg²⁺increased at first and then decreased during NaCl treatment, however, thesethree ions were always increasing during the selective transport from roots to the leaf and K⁺and Mg²⁺ obvious decrease, while the Ca²⁺content not much changed as the selectivetransport from petiole to leaf.8. NaCl had great impact on Glehnia littoralis. Stomatal limitation resulted inphotosynthetic efficiency reduction under0.5-1.0%NaCl treatment whereas thephotosynthetic efficiency was mainly affected by the reduction of mesophyll cellphotosynthetic activity under1.5%NaCl treatment. The pathway of both substrate oxidationelectron transport had shown the characteristics to adapt to the saline environment.9. The determination results of respiratory rate and respiratory pathway showed that, therespiration rate of Glehnia littoralis. was more stable under0-1.0%NaCl treatment whenNaCl concentration increased to1.5%, the respiration rate decreased significantly.EMP+TCA are the main substrate oxidation pathway. With the concentration increasing byNaCl treatment, both of the proportion increased significantly while the PPP, by contrast,decreased significantly. CP is the main way of electron transport, and NaCl treatment hadlittle effect on CP and AP pathway.10. A full length cDNA of NHX1gene in Glehnia littoralis．was cloned using RT-PCRand RACE，and this fragment was subsequently cloned，sequenced and analyzed．The datashows that and the full-length of GiNHX cDNA was2553bp,5’ non-translation District for531bp in,3’non-translation District for361bp, and an open reading frame was1662bp,encoding554amino acids and its predicted molecular weight is60.059kD.