The Research Of Plant Tsransformed By SeNHX1Gene

SeNHX1genes was Na+/H+reverse transport protein gene cloned from Salicornia(Saliconia europaea L.), which played an important role in the transport of Na+in plants andpH regulation and osmotic pressure of cell. Tobacco (Nicotiana tobacum L.) and arabidopsis(Arabidopsis thaliana) as model plants, widely used in the study of gene function analysis,and tobacco, alfalfa (Medicago sativa L.) are important economic crops, having importantsignificance not only for environmental improvement, but for the development of the nationaleconomy. Therefore, this study transformed SeNHX1gene into tobacco, arabidopsis and sixalfalfa cultivars using genetic engineering technology, for tobacco, arabidopsis and alfalfagenetic improvement research of genetic engineering to provide technical support, as well asfor tobacco, arabidopsis and alfalfa breeding of salt tolerance and SeNHX1gene biologyfunction analysis to provide the experimental materials. At the same time, salt toleranceindexes of six alfalfa cultivars in germination stage were analyzed by the use of NaCl stress,and salt resistance of six varieties were evaluated comprehensively, for salt resistancebreeding of alfalfa varieties to provide reference basis.The main results were as follows:1.The research of agrobacterium mediated SeNHX1genetic transformation into tobaccoThe pBin121binary vector carrying reverse transfer protein gene SeNHX1wastransformed into young leaves of tobacco by agrobacterium mediated method, studying somefactors affecting genetic transformation and obtaining regeneration plants. Results showedthat: before the agrobacterium infection young leaves were treated10min ultrasonictreatment, and infection fluid added150μl/L Silwet l-77, bacteria liquid concentration set0.5, the infection time set10min, the resistant callus formation rate is highest; Aftercocultivation, when to take off the bacteria cultivation, using the300mg/L cephalosporinsdrug to inhibit agrobacterium not only can effectively control agrobacterium overgrowth butalso minimize the adverse impacts of bacteriostatic agent on the growth of tobacco explants,and in the process of constantly successive transfer process, cephalosporin concentration downto100mg/L can be meet the inhibition of agrobacterium tumefaciens;When tobacco explantswere selected by kanamycin resistance, kanamycin selection pressure is set to20mg/L, inorder to ensure not seriously affect the normal development and differentiation of explant;After induction, differentiation and regeneration of explants, CTAB method is used to extractgenomic DNA of tobacco leaves, PCR amplification result showed, a positive transformation of tobacco plant that preliminarily judged the exogenous gene SeNHX1had been imported intobacco plants.2. The research of SeNHX1genetic transformation of arabidopsis thaliana by floral dipmethodSeNHX1gene was transformed into arabidopsis living plant by floral dip method, and theinfluence factors of transformation were studied. The results showed that agrobacterium wasrehung in buffer containing sucrose and surfactant silwet L-77, and infection density ataround1.0, infection time in2.5min, resistance germination rate is the highest, and sucroseand surfactants had particularly important impact on conversion rates; Genomic DNA ofresistance plants that were screened30mg/L kanamycin were extracted, and according to theresults of molecular biology identification of emergence plants using PCR technology,exogenous gene SeNHX1had been integrated into the arabidopsis genome.3. The salt resistance comparation of6alfalfa cultivars in seed germination stage underNaCl stressThis test is to Algonquin、Affinity、muge、Sanditi、Victoria、Golden Empress six alfalfavarieties as experimental materials, using the seven concentration gradient of the NaClsolution to deal with seeds, germination potential, germination rate, the radicle length, freshweight, germination index, vigor index and other indicators were statisted and analysed, andthe salt resistance of each varieties seed in germination stage were evaluated through themembership function. Results showed that the low concentration (2‰,4‰)of the NaClsolution had a promoting effect on each type of each indicators generally, as the concentrationof NaCl increased, the test indexes were declined, and in6‰and8‰concentrations theresponsition of different alfalfa varieties to salt concentration were different; The relativegermination rate, relative plant fresh mass, relative length of radicle and seedling of alfalfa inwere declined with the increase of salt concentration, and that was significantly negativelyrelated with the salt concentration, and through the analysis of good value, the critical valueand the ultimate value of the concentration of salt stress of each type, salt stress toleranceability of different varieties were different; Salt resistance comprehensive sorting of sixvarieties were: Affinity "、" Golden Empress "、" muge "、"Sanditi "、"Victoria"、" Algonquin".4. The change of alfalfa transformation induced by hormoneAlfalfa resistant callus of successive transfer culture over a long period of were graduallybrowning and stopping growth, and after that were static cultured in the B5-Ⅳliquid medium90h, a few resume callus restared differentiation, forming embryoid, having similarroot material to form; The result of antagonistic callus by PCR detection was positive,suggesting that foreign genes had been imported into alfalfa callus.