Variability in the accumulation of hepatitis B virus S antigen in storage roots and leaves of individual transgenic carrot plants
We transferred Gene env, encoding one of the proteins of the viral envelope, such as short S-antigen under control of the 35S promoter of cauliflower mosaic virus (CaMV), into the genome of carrot plants. The level of HBsAg in leaves and roots was examined as the ratio of HBsAg in the leaves and roots of thirteen carrot plants was determined by ELISA accumulation individually, and especially HBsAg accumulation in various plant organs. As a binary vector for plant transformation we used pBINPLUS/ARS plasmid (Fig. 1а), kindly provided by Dr. RW Hammond (USDA, USA). At sites of restriction endonucleases Hi«dIII-Acc65I we inserted HBsAg (GenBank V00867.1) coding sequence, under control of CaMV35S (435 bp) promoter and restricted at the 3'-end by the polyadenilation signal sequence мРНК CaMV (polyA, 202 bp), in pBINPLUS/ARS (Fig. 1b). Carrot cells (Daucus carota L.) were transformed by means of agrobacterium mediation. Genomic DNA of plants was isolated by the method of Murray MG and Thompson WF. The presence of the transformed gene in genomic DNA of carrot plants was confirmed by PCR using primer pairs (Fig. 1b) We observed variability both in leaves and roots of the transgenic carrot in HBsAg accumulation. The HBsAg content in the leaves of the plants chosen for this comparative analysis varied from 0.1 to 20.69 ng/g of fresh weight, amounting on the average to 9.43 ng/g, and in the storage roots, from 0.34 to 12.07 ng/g, with an average of 4.57 ng/g. HBsAg content in the plant leaves selected for this comparative analysis varied from 0.1 to 20.69 ng/g fresh weight, amounting, on average, to 9.43 ng/g, and in the storage roots, from 0.34 to 12.07 ng/g, with an average of 4.57 ng/g. Among analyzed plant carrots there are only two (№ 3 and № 25) where the relationship between HBsAg accumulation in leaves and roots was close to equal. Other plants were divided into two groups, the first group is where the ratio of HBsAg leaves prevailed over the number in the roots (eight plants) and the second group where the roots of HBsAg dominated the quantity in the leaves (three plants). We discussed possible reasons for the lack of a direct correlation between the level of HBsAg accumulating in the tissues of roots and leaves of the transgenic carrot. We found no direct correlation between the level of the target protein accumulation in leaves and storage roots of individual plants.