Establishment Of An Oil Body-based Expression System And Deletion Analysis Of A Vascular-specific Profilin2 Promoter

The main objectives of this study are (1) to establish an oil body-basedexpression system, by which the isolation and purification process of a target proteincan be simplified; (2) to analyze fragments of a vascular-specific Arabidopsisproflhin2 promoter, aiming at identifying domains and motifs that regulate genes ofinterest specifically expressed in plant vascular bundles. This is particularly importantin the genetic engineering of crops with enhanced resistance to vascular diseases,since the gene product specifically expressed in the vascular system may directlyprovide a defense mechanism to vascular-resided pathogens. Meanwhile, the vascular-specific expression, compared with the constitutive expression, may save energy andreduce risks of transgenic crops.For the establishment of an oil body-based expression system, the followingexperiments were conducted: (1) Cloning of a rape seed (Brassica napus) oleosinpromoter and a sesame structure gene coding for oleosin protein which inserts in thesurface of the oil body. (2) Cloning of a mutated msCT (Ser6, des-leu'9) gene codingfor salmon calcitonin (sCT) protein, an efficient drug used for curing calciumdeficiency, etc. The msCT gene was inserted in the C'-end of the sesame oleosin genedriven by the rape seed oleosin promoter. (3) Construction of a plant expression vectorand transformation of B. napus by Agrobacterium-mediated gene transfer and ofcotton (Gossypium babardense) by pollen-tube pathway. PCR analysis showed thatthe msCT gene was integrated in the rape seed genome. PCR-Southern hybridizationof transgenic cotton was positive and Western analysis of oleosin-calcitonin fusionprotein extracted from cotton-seed oil showed an expected ?9 kD band indicatingthat the msCT gene was specifically expressed in the cotton-seed oil body. At present,44 transgenic cotton lines of 12 generation have been obtained. Since a highconcentration of kanamycin (5,000 ppm) was applied on leaf in the field testing, thetransgenic plant selected should be the one with higher gene expression. As far as weknow, the establishment of an oil body-based system to produce calcitonin protein incotton-seed oil has not been previously reported in the literature.Based on the transient and stable gus gene expression driven by different 5'-deletions of Pfn promoter (Pflil .4, Pfnl .2, Pfhl .0, PfliO.6), the full-length promoter(Pfril.7, -1667 -1 bp) can be divided into 3 parts: (1) Domain A, -1667 ?1380 bp.Deletion of this part, i.e. from Pfnl .7 to Pfnl .4, resulted in the transition of gus gene3expression from vascular-specific to constitutive. It suggested that element(s)responsible for vascular-specific expression might be existed in this region. (2)Domain B, located at -1153 -597 bp, strongly inhibited gus gene expressionindicating there might be negative regulatory element(s) presented. (3) Domain C, -597 -1 bp (PfhO.6) was considered as a basic domain of profilin2, since GUSactivity was not only relatively strong in vascular system but also in parenchyma cells.Detailed sequence analysis indicated that there was a core ACGT motif, the bZIPprotein binding site, both at -1391 ?-1388 bp and -565 ?-562 bp in the profllin2promoter. It is known that bZIP proteins are the most abundant transcription factors inplants. Analysis also shown that there was an AC-I (CCCACCTACC) like sequence at-1647 ?-1640 bp (CCACCTAC) of the profihin2 promoter. The AC-I motif, also as aprotein-binding site, was known to be very important in controlling vascular-specificexpression of the bean phenylalanine ammonia-lyase2 (PAL2) promoter. To clarifywhether the ACGT or AC-I like sequence in profilin2 promoter plays a similarregulatory role in vascular-specific expression, we further constructed following plantexpression vectors contai...