Cloning E8 Promoter From Tomato And Analysis Expression Of E8 Gene In Transgenic Arabidopsis Thaliana

The availability of fruit specific promoters makes great benefits to modify the fruit quality by genetic transformation. As a kind of fruit specific promoters,E8 promoter have been use into fruit of transgene tomato. But it can't be used in any other plants in which the pro-source-relationship are farther with tomato. To make it clear that if the fruit-specfic promoter E8 can be used to regulate the gene specifically in Arabidopsis , the fruit-specific promoter E8 was cloned successfully and its regulation function in Arabidopsis siliqus was identified by transient expression and genetic transformation system. The main results were summarized as following:(1) A polymerase chain reaction(PCR) amplification was used to isolation a fruit-specific expression promoter E8 from tomato genomic DNA. Sequence analysis revealed that E8 was 1088bp and shared significant nucleotides identity 99.8% with GenBank database, containing E8 binging protein region and TATA box. Intermediate vector pCAMBIA-E81.1 was constructed by inserting the promoter in pCAMBIA2300,where the original CaMV35S promoter was replaced by the E8 promoter.(2) The research chooses tomato genomic DNA as material,we obtained E8 gene fragment by PCR technology ,which is 517bp in length and highly homologous 100% with reported in NCBI. The result of amino acid analysis indicated that it include an integrated ORF and have identical with sequence of amino acid reported in NCBI. Plant expression vector were construct through replaced the CAMV35S promotor by E8 promoter and E8 gene fragment. The PCR and restriction enzymes confirmed E8 promoter and E8 gene were fused into pCAMBIA2300.(3) The construct was transformed to Arabidopsis via Agrobacterium tumefaciens using floral dipping method. Transgenetic plants were obtained by screening with ampicillin and kanamycin resistance in the 1/2 murashige and skoog (MS) medium. The result of PCR and dotting blotting detection suggested our aim DNA had been conformed into Arabidopsis genomic. The expression pattern of the E8 gene in Arabidopsis was analyzed by RT-PCR, which showed the presence of E8 gene transcripts only in siliques. The RT-PCR analysis revealed the specific-expression of E8 gene in Arabidopsis siliques. The achievement of transgenetic plant would provide a scienfic basis for the transformation of promoter E8 into other fruit plant.