Cloning And Regulation Analysis Of DR8 Gene Promoter In Tomato

DR8(Development Regulation 8)-a new gene about fruit ripening,has higher homology with Aux/IAA in the Arabidopsis. DR8 gene as an auxin-induce gene,is also negatively regulated by ethylene and positively regulated by auxin,it suggests DR8 gene has important regulation effect on fruit ripening and the relation between auxin and ethylene probably. At present,DR8 gene's full long sequence was inserted into vector-pGA643,and was transformed into tomato genome by Agrobactera transform technology,DR8 over-expression and under-expression transgenic plants were obtained. In order to clarify the expression and regulation mechanism about DR8 gene and the response of ethylene and auxin,in the research intending to amplify DR8 gene's promoter,according to analysis the function of DR8 gene promoter,confirming the regulated region and exact regulated site,establishing the base for researching DR8 gene's function and promoter's application. Using genome walking PCR technology amplified promoter,designed and constructed three 5'delete pGreen-GFP fluorescent expression vectors,and transformed into tobacco protoplast,after treated with 2,4-D and fluorescent detecting,the results showed that there was one auxin positively regulated site in 250 bp of 3'end . Meanwhile,the seven pBI121 plant expression vectors were construced,preparing for the transformation work of DR8 promoter in tomato.The main results of research in this thesis are followed:①The 1031 bp promoter region of DR8 gene from tomato was amplified by genome walking PCR technology.②Promoter sequence analysis showed that this cloned fragment has lots of AT rich-regions,TATA-box,CAAT-box,G-box,they are the base function region in all promoters. Meanwhile,there are some other regulation regions in this promoter sequence,such as ERE is ethylene response element,HSE is hot response element,3-AF1 binding site is light response element, TCA-element is salicylic response element.③Constructing three fluorescence vectors,the constructed fragment were 250 bp,500 bp,1031 bp. ④The expression vectors were transformed into tobacco protoplast by PEG technology and were treated with 2,4-D,fluorescence analysis showed that the auxin positive regulation region likely in the 250 bp of 3'end.⑤Constructing seven pBI121 plant expression vectors.