SU1546022A1 - Method of identification of genotype of perenneal grasses of the wheat - Google Patents

Abstract

The invention relates to biotechnology, in particular, the problem of protein labeling of plant genetic systems and the use of protein markers in addressing topical issues of applied botany, genetics, and selection. The aim of the invention is to increase the accuracy of analysis of small seeds of perennial forage crops. The method consists in that the prolamins are removed from the seeds by soaking the grains in distilled water, separating the germ with the dissecting needles, squeezing the semi-liquid endosperm, followed by extraction with 5-10 µl of extraction solution, and the electrophoresis is carried out in vertical plates of 1 mm polyacrylamide gel and the extract is applied to the start pocket with a layer of 0.5-0.8 mm. 1 tab.

Description

(21) 16 / 31-13

(22) 11/02/87

) 02.28.90. Bul If 8

(71) Central Siberian Botanical Garden, Siberian Branch of the Academy of Sciences of the USSR

(72) A.V. Agafonov and O.V. Agafonov (53) 575.633.112.1 (088.8)

(56) Sozinov A.A. Protein polymorphism and its significance in genetics and selection. P., Science, 1985.

(METHOD OF IDENTIFICATION OF GENOTYPES OF MULTI-YEAR CEREALS WHEAT TRIBES (TRITICEAE)

(57) The invention relates to biotechnology, in particular to the problem of protein labeling of genetic systems.

plants and the use of protein markers in addressing topical issues of applied botany, genetics, selection. The aim of the invention is to increase the accuracy of analysis of small seeds of perennial forage crops. The method consists in the fact that prolamins are extracted from the seeds by soaking the kernels in distilled water, separating the germ with the dissecting needles, squeezing the semi-liquid endosperm, followed by extraction with 5-10 µl of extracting solution, electrophoresis is carried out in vertical plates of full acrylamide gel 1 mm, and the extract is applied to the start pocket with a layer of 0.5-0.8 mm. 1 tab.

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The invention relates to biotechnology, in particular, the problem of protein labeling of plant genetic systems and the use of protein markers in addressing topical issues of applied botany, genetics, and selection.

The purpose of the invention is to increase the accuracy of the analysis of small seeds.

The method is carried out as follows.

Peeled from floral scales grains soaked in distilled water during the day. The embryo is removed with a dissection needle and the semi-liquid mass of the endosperm is squeezed out using a thin rod, rolling it from the top of the weevil to its base. The obtained isolated endosperm with

weighing 0.2-1 mg, separated from the shell and the embryo, contains all the spare proteins without impurities, which are formed during grinding of the weevil and constituting 20-60% of its mass. The absence of impurities makes it possible to carry out the extraction of proteins under more severe conditions for 12–18 h at 40 ° C in 5–10 µl of 70% ethane or 50% isopropanol in sealed microtubes. This sequence of operations allows for the most complete extraction of the prolamin fraction of storage proteins. After centrifugation, microliter of concentrated extract is obtained, which is mixed in a 2: 1 ratio with an electrode buffer of an electrophoretic system containing 60% sucrose, and for the second time the centrifuge.

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rigged. The prepared extract is subjected to electrophoretic separation in a vertical plate of a 1-millimeter full-acrylamide gel, while the high concentration of prolamine proteins in the extract makes it possible to put a thin layer of extract of 0, 8 mm into the starting pocket. This achieves a significant increase in the resolution of the electrophoretic method. The width of the starting pocket does not play a significant role and can vary within mm. Thermostating a gel plate during electrophoresis at 8–12 ° C makes it possible to increase the strength of the electric field to V / cm and reduce the separation time for proteins in a plate 12 cm long to 2.5 hours. The method allows for individual analysis of small perennial kernels forage cereals with a mass of less than 5 mg to identify varieties and species, ecotypes and forms. In addition, it is possible to analyze the electrophoretic spectra of prolamins in herbarium specimens collected tens of years ago, which becomes necessary when establishing phylogenetic relationships. Soaking the weevil in distilled water, removing the embryo with a dissecting needle and squeezing out the semi-liquid mass of the endosperm with a thin rod provides an analysis of individual kernels of small dimensions. An isolated endosperm obtained in this way, containing all spare proteins without impurities, is suitable for extraction and further procedures associated with the separation of proteins in a modified electrophoretic system.

The method is illustrated by the following examples.

Example 1. There are seeds of fodder slag from the grass of the New England (wheat rootless) variety Pervomaysky and seed samples from natural populations of two taxonomic species of the grass tree (rough rough and French and British), differing by an insignificant morphological sign (degree of pubescence spike joint). A preliminary morphological analysis of varietal seeds shows that among any part should be attributed to one

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a part, and a part to another, and also to allocate a number of intermediate forms with a distinct species.

The experiment aims to compare the prolamin spectra of two taxonomic species, each of the material from the May Day variety and from two distant wild growth sites in order to assess the legitimacy of the separation of these species based on minor morphological differences.

Samples of grains from each sample indicated in the table, after separation of the flower scales, were placed in water chambers for 24 hours.

In addition, for the objective comparison of the electrophoretic spectra obtained in a series of experiments, 2 grains of Siberian grassland with a known component composition of the spectrum are analyzed as a standard marker.

Excess water is removed from the soaked grains with filter paper, the embryo is cut off with a dissecting needle, and a semi-liquid endosperm is squeezed out through a hole in the casing, rolling it from the opposite end of the grains with a thin rod of stainless steel. With the same rod, an isolated endosperm, constituting in a rootless 0, 6 mg, is placed in a polyethylene microtube with 8 µl of 50% isopropanol, homogenized, sealed with a tight stopper and placed in a thermostat with a temperature of AOC C for 12-15 hours. the microtubes are cooled to room temperature, the sediment is stirred up with a blunt needle and centrifuged for 5 minutes at 12,000 and, the Supernatant is as completely as possible sucked off with a micropipe and mixed in a 2: 1 ratio with an aluminum-lactate buffer pH 3.1 containing 60% sucrose and red Itel pyronin, after which the samples are centrifuged again for 15 min at 12000g.

For electrophoretic separation of the prolamins, a homogeneous gel-buffer system with vertical plates of 1 mm 9% polyacrylamide gel on aluminum-lactate buffer pH 3.1 is used. At the same time, 2k samples are analyzed, which are deposited under a layer of electrode buffer in an amount of 2 µl. Electrophoresis is carried out under voltage

 Yo in for 2.5 h, cooling the gel plate by overlaying plexiglass coolers with circulating water at 10-12 ° С. Gels are fixed in 12% trichloroacetic acid (TCA) and stained with 0.1Ј Kumasi dye - 250 ml. in 12% TCA for 18-20 hours, not removed from the second glass. Colored gels are differentiated with 7% acetic acid and photographed in transmitted light.

In the spectra of the studied samples of grassland there are Fri 13 to 19 components of different intensity. Moreover, about half of the components with a certain frequency occur in all studied natural and varietal samples.

A number of components are randomly distributed, regardless of the morphology of the spike joint, some components of the spectrum are characteristic only of a certain natural ecotype.

Based on the previously established patterns of distribution of the prolamin components in the wild cereals of the wheat tribe, it can be considered that representatives of different biological species cannot have identical sets of components in the composition of the prolamin spectra. Thus, all the studied specimens of the couch grass should be attributed to one species - the pinned bush.

Example 2. There is a lot of seeds of a long-term wheat cereal forage with a lost variety certificate. The wheat tribe includes 9 genera of perennial cereals, including the most important in terms of fodder, such as wheatgrass, prairie grass, cornwood, corn stalk, shredder, etc. The similar structure of the kernel allows the use of the described method for identifying the components of prolamin (the predominant storage protein of the kernel), based on the proposed method, as one for all

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representatives of the tribe. To accurately determine the variety, it is necessary to carry out an electrophoretic separation of the endosperm protein extract from a series of seeds of an unknown sample and compare the obtained spectra with similar spectra of known varieties.

Thus, the proposed method makes it possible to carry out certification of existing varieties of forage crops of the wheat tribe. It also controls all stages of the selection process, testing and zoning when creating new valuable forms, significantly reducing the economic costs of many procedures, reliably analyze natural ecotypes of wild plants. wheat cereals that are of great interest for introduction, as well as to identify herbarium samples containing seeds (regardless of SHELF LIFE) for the purpose of refinement intra- and interspecific phylogenetic bonds in the tribe.

Claims (1)

Invention Formula A method for identifying the genotypes of perennial cereals of the wheat tribe (Triticeae), including preparing an extract of prolamins, electrophoretic separation of prolamins in the vertical plates of a full-acrylate-amide gel, characterized in that, in order to increase the accuracy of analysis of small seeds, the extract of prolamins is prepared from an isolated endosperm obtained by soaking the seeds in distilled water, followed by removing the germ, squeezing out the semi-liquid mass of the endosperm and extracting µl of extraction solution, Electrophoresis was carried out on prolamine gel plates, thickness 1 mm with the application of the starting extract pocket layer 0 and 8 mm.