Cloning And Function Analysis Of Seed-specific Promoter SACPD-Cp From Soybean

Soybean is an important grain and economic crop, its grains are rich inunsaturated fatty acid, vitamin, trace elements and protein. In the plant geneengineering seed-specific transgene expression in soybean for the production of novelcompounds of industrial or pharmaceutical has became the important target. How toexpress effectively exogenous genes in plants? The first consideration is to choose theright promoter. Seed-specific promoter can regulate the exogenous genes to expressexclusively in the seed, and expression product concentrate in the seeds. Soseed-special promoter is a very important tool in the soybean quality improvementproject.In this research, the flanking sequences of soybean seed specific gene SACPD-Cwas cloned by Tail-PCR. Then the studies focused on the characterization of tissueexpression and expression intensity of the promoter sequences through transientexpression in the soybean roots, stems, leaves and seeds for instantaneous expressionand in arabidopsis transgenic plants in stable expression by Agrobacteriumtumefaciens mediated method. The core regulatory regions were analysed, whichwould be useful for the soybean transgenic engineering. The main results as follow:1. The SACPD-C gene previously has been shown to be expressed specifically in thesoybean seeds. A fragment was cloned from genomic DNA by Tail-PCR, which wasthe partial upstream sequence of the SACPD-C gene, the length was2081bp,namedSAC-Cp. Bioinformatics analysis showed that SAC-Cp sequence possesses typicalfeatures of the seed-specific promoter and includes kinds of typical seed-specificelements such as E-box, AACA, ACGT, RY-repeat, SEF1-motif and so on.2. SAC-Cp deletion analysis experiment was carried out. Replacing CaMV35Spromoter of pCAMBIA1301with these deletion fragments, binary expression vectorsnamed pCAM-SAC-Cp, pCAM-SAC-Cp1, pCAM-SAC-Cp2, pCAM-SAC-Cp3, pCAM-SAC-Cp4, pCAM-SAC-Cp5were constructed.3. These binary expression vectors were introduced into soybean and Arabidopsis byAgrobacterium transformation. The transient expression and stable integrationexpression of GUS gene were detected by histochemical GUS staining and fluorescentquantitative assay and their results were almost same.1) The results show thatpCAM-SAC-Cp, pCAM-SAC-Cp1and pCAM-SAC-Cp2could promote theexogenous genes to express specially in the seed. GUS activity was detected in root,stem and leaf from pCAM-SAC-Cp3transgenic, the region between SAC-Cp2andSAC-Cp3(-1526bp~-1060bp) was supposed to contain positive regulatory elementsrelated to seed-specific expression.2) Compared with pCAM-SAC-Cp4, the GUSactivity of pCAM-SAC-Cp5could not be detected in every organizations, the resultsprove that the region between SAC-Cp4and SAC-Cp5(-433bp~-61bp) was essentialto this promoter.3) Compared with pCAM-SAC-Cp3, the GUS activity ofpCAM-SAC-Cp4was very low, so suggested that the region between SAC-Cp3andSAC-Cp4(-1059bp~-434bp) contains cis-elements related to the promoter.