Study On Regulatory Element Of Xylem Specific Promoter From Poplar

On this study,we would be based on the early screening of xylem specific promoterJCesAP,which is drived from Populus canadensis-“1”,using5’ end series deletionanalysis method, model plant (tobacco) as the transgenic system, analysis levels intransgenic plants of this promoter in different sections driven GUS gene and GFP geneexpression. In order to clarify the relationship regulatory between the element results andfunction, determine the regulatory elements of control xylem tissue-specific and providenew information for the regulation of plant gene expression. If the regulatory element isuse to regulate the expression of insect resistance gene in the xylem specific, they canimprove the level of gene expression, while reduce the development of resistance to insects.The main results were as follws:(1) Construction of xylem specific strong promoter5’ deletion fragments ZU2, ZU3,ZU4For the convenience of insert pBI121and Pcambia1302expression vector, we needto design the different primers based on the selective strong promoter and add therestriction sites before each primer for PCR amplification. The PCR amplification productpurified was used the gel extraction kit methods. We were obtained different size of5’deleted promoter fragments (named ZU2, ZU3, ZU4).The above fragments were clonedinto the vector and transformed into E. coli DH5α, obtained positive clones by PCRamplification and enzyme digestion analysis.(2) Construction of plant expression vectors harboring5’ deletion fragments andtransformation to Agrobacterium tumefaciensThe fragment ZU2, ZU3, ZU4were respectively replaced the CaMV35S promoter ofplant expression vector pBI121and Pcambia1302,so that each fragment couldrecombinant GUS reporter gene and GFP reporter gene. The plant expression vector wastransferred into Agrobacterium tumefaciens Agl L by electroporation.(3) Genetic transformation of Agrobacterium mediated plant modelThe construction of the plant expression vector p2CesAP-GUS、p3CesAP-GUS、p4CesAP-GUS were transformed into tobacco by leaf disc method. The cefotaxime (CTX)critical concentration was400mg/L for the tobacco transformation. Co-culture medium: MS+0.2mg/L6-BA+0.1mg/L NAA,differentiation medium: MS+0.5mg/L6-BA+0.05mg/L NAA；selective medium: MS+0.5mg/L6-BA+0.05mg/L NAA+400mg/LCTX+50mg/L Kan;The rooting medium: MS medium supplemented with antibiotics.Preliminary PCR testing, gene was integrated into the tobacco genome,excluded negativeplants based on PCR results,the complete regeneration resistant tobacco of plants wereobtained.(4) Detection the function of fusion GUS reporter gene and GFP reporter geneTransgenic plants were tested by GUS protein staining、enzyme activity of GUSquantitative analysis and GFP report gene detection,showed that ZU2promoter had veryweak or no expression function, both ZU3and ZU4promoter could regulate report geneshowed strong tissue specificity.ZU4promoter was the strongest function, had strongxylem tissue specificity.