Optimization Of Tobacco Genetic Transformation System And Functional Verification Of Dehydrin Gene(SsDHN) Promoter

Genes that control all physiological and biochemical phenomena in plants are regulated by related promoters.Promoters play important roles in regulating gene expression,even play decisive roles.When plants face stress environments,in order to improve their own resistance,they will improve the expression of genes relating to environmental facts.The expression of these genes is controlled by the promoter at the spatial and temporal level.So the study of promoter's function can make us better understand the regulatory mechanism of plants in response to stress.In this study,wild type tobacco NC89 was used as experimental material.Basing on the establishment of NC89 tissue culture system and Agrobacterium-mediated tobacco genetic transformation system,we gained some transgenic callus and plantt of the deletion fragment of Suaeda salsa dehydrin gene?Ss DHN?promoter and using the PCR and GUS??-glucuronidase gene?staining to verify materimals.Furthermore,these transgenic materials was treated with methyl jasmonate,salicylic acid and abscisic acid to initially verified the function of the fragment of Ss DHN promoter,The research results are as follows:1.Establishment of tobacco tissue culture systemHormone ratio for callus induction process:IBA 0.1 mg/L+6-BA 0.5 mg/L+6-KT 0.3mg/L.Hormone ratio for callus proliferation process:NAA 0.15 mg/L+6-BA 0.6 mg/L+6-KT0.3 mg/L.Hormone ratio for bud induction process:IBA 0.6 mg/L+6-BA 0.6 mg/L+6-KT 0.5 mg/L.Hormone ratio for bud proliferation:IBA 0.6 mg/L+6-BA 1.0 mg/L+6-KT 0.6 mg/L.Hormone ratio required for rooting:IBA 0.3 mg/L+NAA 1.2 mg/L.2.Optimization conditions of Agrobacterium-mediated Transformation System of TobaccoDuring the screening process of transgenic tobacco,the concentration of Hygromycin was30 mg/L,when the bacterial solution was OD₆₀₀=0.6.It was diluted 5 times for incubation.The time of infiltration was 5 min.3.Identification of transgenic tobaccoCallus of DM 1,DM 2,DM 3,DM 4 containing truncated fragments of Suaeda salsa dehydrin promoter was successfully identified by GUS staining and PCR;DM 2,DM 3transgenic tobacco was screened,T1 plants of transgenic tobacco Successfully identified.4.Ss DHN promoter function verificationSA can significantly induce the expression of GUS gene.The-851?-404 bp of the DM 1promoter contains an unknown SA response element that can positively regulate the expression of GUS gene;this indicates that the Ss DHN promoter-289?-1 bp has some unknown element that responds to the SA signal or the presence of SA,interacts with other hormones to indirectly affect the amount of GUS expression.Me JA is positively regulating the expression of GUS,especially in the presence of the DM 1 promoter of the Me JA response element;there may be some elements located on the region of-404?-1 bp of the Ss DHN promoter directly or indirectly responded to the Me JA signa.ABA can positively regulate the expression of GUS,ABA plays an important role in the regulation of GUS expression.The ABA response element is likely to work with other elements to regulate the expression of GUS.