Cloning Of The Ara H3 Gene Promoter Of Peanut Storage Protein And Localizalation Of ABI3 Homologous Gene That Interact With Ara H3 Promoter

Peanut (Arachis hypogaea L.) belongs to leguminosae which grow widely in China and the production is at a big scale. The content of storage protein in dry peanut seed is about 20%～30%. Multiple kinds of peanut storage protein have been identified as the resource of allergy and can cause serious type I allergic reaction. Storage protein gene of peanut express only in specific developing stage and specific tissue (i.e. the seeds) and this is the result of multiple kinds of cis-acting elements and trans-acting factors act in concert. Promoter is one of the important cis-acting elements and the center of transcriptional regulation. We cloned the promoter of peanut storage protein Ara h3 gene and constructed a plant expression vector GA6 by replacing the CaMV 35S promoter of pBIN-35S mGFP plasmid. GA6 vector was transformed into Arabidopsis by flora dipping with Agrobacteria LBA4404 for testing the Ara h3 promoter's expression intensity and tissue specificity. Analyzing the Ara h3 and Ara h1 promoter sequence, we found that in addition to the basic elements such as TATA-box and CAAT-box they also have specific elements. Both promoters have many DOF(DNA binding with One Finger) binding boxes and Ara h1 have nine DOF binding elements while Ara h3 have five. DOF protein is a kind of single zinc finger transcription factor that is unique in plants. A conserved Dof domain which composed of 52 amino acid residues located in its N-end. Dof protein recognizes the DNA sequence at AAAG (or CTTT) and play important regulating roles in various physiological events of plant. Furthermore, there are two RY (CATGCATG) elements in Ara h1 promoter where located in -235 and -278bp respectively. RY element also exists in Ara h3 promoter. Transcription factors ABI3 (abscisic acid-insensitive 3), LEC2 (leafy cotyledon 2) and FUS3 (fusca3) interact with RY element and controls many events during seed development. The RNA extracted from peanut root, stem, leaf and developing cotyledon were hybridized with probe from the conserved B3 domain of Arabidopsis ABO protein. The micro sections of root, stem, leaf and developing cotyledon (including embryo) of peanut were also hybridized in situ with the same probe. The positive results of hybridization signals only in peanut cotyledon reveal that the homologous gene of ABI3, FUS3 and LEC2 may exist in peanut and they only distributed in the cotyledonand embryo of peanut.