Electrophoretic analysis of proteins from seeds of transformed cotton hybrids

Abstract

Herein we report the electrophoretic analysis of the action of xenotypic DNA ( H. syriacus) on cotton (G. hirsutum L.) and the nature of its succession in hybrid generations. The goal of the analysis was to reveal changes in protein synthesis in seeds of hybrids that occur after the genetic transformation. Distribution spectra of water-soluble proteins (DSWSP) from seeds of hybrids obtained by hybridization of a transformed cotton form designated PG with variety Yulduz (G. hirsutum L.) in direct and reverse crossing combinations and their F 1 —F 3 generations were investigated. The PG form was obtained by application of a suspension of cotton pollen with DNA isolated from H. syriacus pollen. Ground seeds were defatted with acetone. The water-soluble protein fraction was extracted with distilled water at a 1:10 powder:water ratio over 30 min. The extract was centrifuged at 8000 rpm [3]. Before electrophoresis, the protein extract s were denatured with heat for 3 min and centrifuged at 8000 rpm. Wells were charged with 10 µL each. Electrophoresis was carried out in 12% PAAG using the standard Laemmli and Favre method [4]. Eight bands of varying color intensity were seen visually in the DSWSP from seeds of the parental form PG (P 1 ) (Fig. 1a). These included fractions with electrophoretic mobility Rf 0.42, 0.60, and 0.82, which were the most intensely colored and differed significantly in amount of protein compared with the other fractions. The polypeptide fractions with R f 0.145, 0.21, and 0.29 were moderately colored; with Rf 0.88 and 0.90, weakly colored. The fraction with R f 0.90 was characteristic of P 1 and was absent for P 2 . The DSWSP from seeds of the second parental form P2 (Yulduz variety) differed in that it had 16 fractions (Fig. 1b), among which were fractions with Rf 0.04, 0.51, and 0.77 and weakly colored fractions with R f 0.66, 0.82, 0.98, and 0.99 that occurred only for the second parental form and were absent for the first (Table 1). Polypeptide fractions with R f 0.145, 0.21, 0.29, 0.42, 0.60, 0.82, 0.86, and 0.92 that were present in spectra of both parents were classified as the major fractions; fractions with Rf 0.64, 0.82, and 0.92, which were weakly colored, as minor