Expression Analysis Of PtLFY Promoter And Its Application In"Gene-Deletor" System

In order to study the mechanisms of flowering in Populus tomentosa Carr., and regulate the flowering of P. tomentosa, a5’flanking sequence of PtLEAFY (PtLFY) gene was cloned by PCR from P. tomentosa genomic DNA. The expression characteristics of the promoter was analysed by both transient and stable expression methods in tobacco and poplar. Contraposing the security problems of genetically modified crop, professor Li Yi developed "Gene-deletor" technology, which fused two recombination systems, obtaining the breakthrough in this field. We use PtLFY promoter to construct the "Gene-deletor" system, and investigate the excision efficiency of "Gene-deletor" system by transforming tobacco and poplar, which lay a foundation for studying the mechanisms of flowering in P. tomentosa and the application of " Gene-deletor" system in tobacco and P. tomentosa. Main results are as follow.1. A5’ flanking sequence of PtLFY gene, with a length of about1.6kb was amplified from genomic DNA of P. tomentosa by PCR. The results analyzed by PLACE and PlantCARE showed that the sequence contains TATA-BOX,CAAT-BOX and other regulatory elements, such as MYB binding site involved in drought-inducibility, cis-acting regulatory element involved in the ABA responsiveness, light-responsive elements and other transcription factor-binding sites. It was supposed that the expression of PtLFY was regulated by drought, ABA and light et al. Compared the PtLFY promoter with that of other5species by FootPrinter, both conservation and diversity were found in these promoter sequences. It indicated LEAFY homologues genes perform a similar function in Plants. Furthermore, a plant expression vector carrying PtLFYp::GUS, called PtLFYp1304.2. Transient expression was performed by Agrobacterium-mediated transformation using PtLFYpl304constructs. Immunohistochemical staining indicated that PtLFY can drive GUS gene expression in root, stem, leaf and floral organs of tobacco and P. tomentosa. However, compared with CaMV35S promoter, only slight expression of GUS gene was detected in roots, stems and leaves, whereas strong GUS expression driven by PtLFY promoter in the floral organs.3. The expression vector PtLFY1304was introduced into the P. tomentosa clone TC1521and tobacco by Agrobacterium-mediated transformation. Immunohistochemical staining indicated that results of stable expression were consistent with that of transient expression. GUS gene was detected expressing in roots, stems, leaves and floral organs. But only slight expression were detected in roots, stems and leaves, strong GUS expression were detected in floral organs.4."Gene-deletor" system containing PtLFY promoter, fusing two recombination systems were constructed, which can greatly improve the excision efficiency. PtLFY promoter drives recombinase gene FLP, which can excise the foreign genes by site-specific recombination.5. The "Gene-deletor" system was introduced into the P. tomentosa clone TC1521and tobacco by Agrobacterium-mediated transformation. Immunohistochemical staining indicated that the "Gene-deletor" system can excise foreign gene in root, stem, leaf and floral organs.The results mentioned above do not only contribute to elucidating the molecular mechanism of poplar flowering, but also lay the foundation for the application of "Gene-deletor" technology in forestry breeding..