Expression Analysis Of PtSEP2, PtSEP3-1, PtAP1-2 And PtMCP Promoters In Nicotiana Tabacum And Populus Tomentosa

Considering problems of flying catkin and the shedding pollen with Populus tomentosa Carr., a 5' sequence fragment of PtSEP2 was cloned from its genomic DNA in this study, based on retrieval of promoters of PtSEP3-1, PtAP1-2 and PtMCP. Tissue expression character of this four promoters was researched by transforming tobacco and Populus tomentosa, laying a foundation for genetically modifying traits of flying catkin and shedding pollen. The main results are as follow.1. A 5'flanking sequence of SEPALLATA like gene PtSEP2, with a length of 2.3 kb was amplified from genomic DNA of Populus tomentosa. Sequence analysis on PlantCARE showed many conserved fragment and light responded motif existing in this sequence, which was therefore predicted as PtSEP2 promoter. An expression vector was constructed using GUS as reporter gene, named PtSEP2::GUS.2. Transient expression research was performed by Agrobacterium-mediated transformation using four constructs which containing promoter::GUS, taking root, stem, leaf, flower as the recipient. Immunohistochemical staining indicated that GUS activity only existed in anther, PtAPl-2 weakly expressed at sepal and petal, GUS activity driven by PtSEP3-1 was detected in no tissues, GUS activity driven by PtMCP was detected in every tissue, but with an obvious weaker expressed strength than that of 35S promoter, Thus inferring PtMCP as a mild and constitutive promoter.3. The Populus tomentosa clone TC1521 and tobacco were used for genetic transformation.The positive results verified by PCR include respectively 37and 88.GUS Staining of root, stem and leaf from genetically modified tobacco showed a consistent result with that of transient expression. Staining of the whole plantlet of genetically modified Populus tomentosa suggested that PtSEP, PtSEP3-1 and PtAP1-2 all expressed in no tissues.4. GUS activity assay on root, stem, leaf and flower of transgene tobacco indicated a weaker activity of PtMCP compared with pBI121, its activity are 21.9%,5.3%,5.3%,17.6% of GUS activity with pBI 121 respectively. Whereas, GUS activity was not detected in any tissues of wild type tobacco.Through the above analysis, conclusions can be drawn that, PtSEP2 promoter can drive expression of GUS gene in anther only, and initially speculated that it belongs to anther-specific promoter; weak expression of GUS driven by PtAPl-2 was only found in sepals and petals only, flower tissue-specific promoter may be inferred; PtMCP promoter can drive GUS gene express in tobacco roots, stems, leaves and flower tissues, with an expression strength significantly weaker than that of 35S promoter, therefore it is a mild constitutive promoter type.The above-mentioned results do not only make a profound theoretical sense for illuminating molecular mechanism of poplar flowering, but also provide potential practical value for gene engineering addressing the flying catkin-caused problem.