Fractional composition of proteins from seeds of transformed (Trn) Gossypium hirsutum

Abstract

We studied seeds of Gossypium hirsutum L. (Namangan-77 variety) and G. arboreum L. (D-1331 variety) and seeds of cotton transformed forms (Trn 1 and Trn 2 ) that were produced by micro-injection of sperm cells isolated from sprouted G. arboreum L. pollen tubes into ovaries of G. hirsutumm flowers. Pollen tubes were obtained by sprouting fresh pollen in liquid medium. Sprouted pollen tubes were disintegrated in buffer (pH 7.3) containing saccharose (20%), EDTA (5 mM), and Triton X-100 (0.05%). The whole mixture was filtered through a nylon filter and layered into Percoll [3] or saccharose [4–6] gradients. The layer in which sperm cells collected was determined using a microscope. Changes were made to the literature method [3] for the samples that we used. Thus, nutrient medium was supplemented with Triton X-100 (0.05%), EDTA (5 mM, pH 5.3), and CaCl 2 (0.1 M). This mixture was mixed gently for 30 min. Then, the suspension was filtered through a 20–50 μm filter and rinsed with saccharose solution (20%). Each portion of filtrate (5 mL) was treated with Percoll (500 μL). The whole mixture was layered into a gradient of 15% and 40% Percoll in 20% saccharose. The gradient was centrifuged at 9,000 g and 4°C for 60 min. Starch grains, remnants of pollen grain coatings, cell organelles, and other contaminants were precipitated. Sperm cells that accumulated in the 15% Percoll layer were collected, washed, and concentrated by centrifugation at 3,000 g (4°C, 15 min). The viability of the isolated sperm cells was determined. The resulting material was introduced by microinjection into the ovaries of preliminarily castrated flowers. Seeds from ripe pods were used for electrophoretic analysis of proteins. Seeds were ground to a powder. The powder was defatted with acetone. Proteins were dissolved in PS-buffer and heated for 3 min on a boiling water bath. Bromphenol blue (0.01%) was added. Samples were placed on a gel in a minimum volume (30 μL). Electrophoresis of proteins was carried out at room temperature calculated at 5 mA per tube in a PAAG linear concentration gradient from 10 to 18% [7]. The gel was fixed after the electrophoresis was finished in coumassie P-250 solution (0.1%) in EtOH:AcOH:H 2 O (25:5:70). The excess of dye was removed with acetic acid (7%). Bovine albumin (67,000 Da), ovalbumin (45,000 Da), and cytochrome C (11,700 Da) were used as marker proteins. Molecular weights of subunits were calculated from the relative electrophoretic mobility of standard and studied proteins. Our goal was to compare electrophoresis of total proteins from seeds of parent forms and first and second generation transformed plants (Trn 1 and Trn 2 ). Fertile plants grown from seeds that ripened after microinjection of G. arboretum sperm cells into G. hirsutum flower ovaries had a monotypic morphology for the vegetative and generative organs that was intermediate between the initial parent forms [8]. Electrophoretic analysis showed significant differences among fractions of parent specimens (G. hirsutum and G. arboreum) with respect to the distribution of major fractions (Table 1).