Extract from vegetable somatic embryos, method for preparing and applying extract, diagnostic scheme based on extract and its applying

FIELD: biotechnology, biochemistry.

SUBSTANCE: invention relates to extracts prepared from vegetable somatic embryos for the cell-free translation system and/or the coupled transcription-translation system. Method involves preparing embryonic callus from the primary material and the embryonic suspension culture. After induction of the secondary somatic embryogenesis extract is prepared from somatic embryos. Based on the extract the diagnostic system is developed for detection of biologically active compounds. Invention provides overcoming the species limitations and strain specificity and to attain the high effectiveness of the cell-free translation system and the coupled transcription-translation system also.

EFFECT: improved preparing method, valuable biological and biochemical properties of system.

49 cl, 5 dwg, 2 tbl, 9 ex

 

The technical field to which the invention relates.

The invention relates to molecular biology, genetic engineering and biotechnology. In particular, in the invention disclosed is a method of obtaining an extract from plant somatic embryos, which can be used for protein synthesis, including paired with the process of transcription in a cell-free system that provides different ways posttranslational modification of the protein product, characteristic of eukaryotic cells. Obtained using the invention of the protein products can be used in medicine, veterinary, agriculture, food industry, such as therapeutic or prophylactic uses, for immunization, for creating diagnostic products (based on antibodies, synthetic antigens, allergens), developments of biologically active compounds of protein nature, obtaining enzymes used in food production, as well as for scientific research.

The level of technology

In recent years there has been a growing interest in biologically active polypeptides in connection with the opening of a brand new their biological functions. For example, such an extremely interesting and important objects, as the peptide activators of the immune system, peptide neurotransmitters and tra is smithery, peptides regulators salt metabolism, peptides humoral immune response with anti-bacterial properties and many others. The availability of these peptides from natural sources is practically zero. The price of the tremendous efforts of many researchers, generally using genetic methods, in the best case, you are able to establish their structure and to trace their Genesis. In most cases, these peptides are synthesized in the body in the form of a relatively large protein precursor, resulting in a complex multi-stage processing into the final biologically active regulators of those or other intracellular processes. As a consequence, dramatically increased the interest in the creation of new and improvement of existing methods of producing polypeptides. Despite progress in recent years, progress in the use of chemical synthesis for the preparation of polypeptides, and the use of recombinant techniques, a number of important and interesting biologically active polypeptides, first of all peptides with a length of 30 to 100 amino acid residues, remains hitherto unavailable or unaffordable for wide use. Disadvantages of chemical synthesis is due, mainly, a number of technological and economic limitations. To technological limitations which include racemization, incomplete elongation of polypeptide chain at each stage, difficulties with the choice of protective groups and their disposal and, most importantly, difficulties with the implementation of functionally active conformation released in quite a long polypeptide. Economic constraints related to the fact that the cost of the final product of the synthesis grows exponentially with increasing length of polypeptide chain.

Methods the expression of foreign genetic material in living cells also have some limitations, especially in relation to the polypeptides of relatively short length (up to 50-60 amino acid residues). Restrictions are reduced, mainly to the difficulty of obtaining the genetic material, because many of these objects unknown biogenesis, and the final active peptides are, in most cases, products of complex processing predecessors. As a result, there are formidable difficulties in the creation of genetic constructs, the expression of which in prokaryotic or eukaryotic cells would lead to active products. It should be noted that even with the successful creation of expressing the system encounters a number of difficulties. Foreign genes can be unstable or poorly expressed in the actions of regulatory mechanisms of the host cell. In many the cases the products of foreign genes insoluble and form aggregates in the form of Taurus include, what causes additional difficulties in obtaining biologically active molecules. Other polypeptides are unstable and rapidly degraded under the action of intracellular proteases. Finally, the products of expression in some cases can have a dampening effect on the cell or to be toxic for her and therefore cannot be expressed.

However, the successful development of molecular biology and biochemistry opens up new opportunities and approaches to solving the problem of synthesis of peptides, namely: (i) using a cell-free system broadcast for ribosomal synthesis of biologically active peptides on artificially synthesized or natural matrices and (ii) the use of neriosang multienzyme systems synthesis of peptides.

The possibility of using cell-free systems broadcast for micropreparative synthesis of polypeptides due to the following circumstances:

i) developed efficient methods for the synthesis of defined deoxynucleotide sequences sufficiently large length (Engels J., Uhlmann E., (1988) in Advances in Biochemical Engineering/Biotechnology, v.37 (A.Fichter eds.) pp.75-127; Erlich H.A. (1989) PCR Technology: Principles and Applications for DNA Amplification, Stockholm, New York). (Hereinafter, all references included in the description by reference);

ii) the developed system propagation matrix RNA vplo the ü to preparative quantities using phage polymerases (Campbell J.L., Richardson C.C., Studier F.W. (1978) Proc.Natl.Acad.Sci. USA 75, 2276-2280; Butler E.T., M.J. Chamberlin (1982). J.BioI.Chem. 257, 5772-5778; Melton D.A., Krieg. P.A., Rebagliati M.R., et al. (1984) Nucleic Acids Research 12, 7035-7056);

iii) adjusted prokaryotic and eukaryotic cell-free system broadcast of natural and artificial matrices (Pratt J.M. (1984) in reduced and Translation (Hames B.D., S.J. Higgins, eds) pp. 179-210; Clemons M. (1984) in reduced and Translation (Hames B.D., Higgins S.J. eds) pp.231-270);

iiii) developed an effective system for the allocation of peptides from complex mixtures.

The first systems for cell-free protein synthesis were developed in the laboratory Zamechnic (Hoagland M.B., M.L. Stephenson, Scott J.F., et al. (1958) J.BioI.Chem, 231, 241-257), where it was shown that peptide synthesis goes to the ribosome and requires ATP, GTP and tRNA. The first protein with the completed polypeptide chain is synthesized in the cell-free translation system is based on an extract from E. coli, was protein shell coliphage f2 (Nathans D., Notani, G., Schwartz J.H., N.D. Zinder (1962) Proc.Natl. Acad.Sci.USA, 48, 1424-1431).

Later DeVries and Zubay ((1967) Proc.Natl.Acad.Sci. USA, 57, N 4, 1010-1012) demonstrated the synthesis part of the polypeptide chain of b-galactosidase in the dual system of transcription-translation. Since the first publications (Zubay, G. (1973) Ann.Rev.Genet 7, 267-287; J. DeVries, Zubay, G. (1969) J.Bacterial 97,No. 3, 1419-1425; Gold L.M., Schweiger, M. (1971) In: Methods in Enzymology, v.20, (PTC), (Moldave K., Grossman eds.) Acad. Press.Inc., New York, 537-542) cell free translation system on the basis of the crude cell extracts (S-30) from E. coli or reconstructed the second system based on the extract of the S-100 has undergone small changes (Collins J. (1979) Gene 6,N 1, 29-42; J.M. Pratt, Boulnois G.J., Darby V., et al. (1981) Nucleic Acid.Res. 9, 4459-4474) and is currently widely used for investigation of the mechanisms of translation and its regulation, translocation across or integration into the membrane processes co - and posttranslational protein folding, modification and Assembly of oligomeric proteins and much more.

In addition to cell-free systems based on bacterial cells for the translation of eukaryotic messenger RNA extracts used a many cell cultures and transplantable cells of ascitic tumors. The most widely lysates of rabbit reticulocytes and extracts of wheat germ (Clemons M. (1984) in reduced and Translation (Hames B.D., Higgins S.J. eds) 231-270; B.E. Roberts, B.M. Paterson (1973) Proc.Natl.Acad.Sci. USA 70, No. 4, 2330-2334).

However, different ways acellular broadcast system described so far have the common disadvantage is the extremely low efficiency of synthesis of the polypeptide. Usually with one molecule of mRNA is synthesized not more than 2-3 molecules of the polypeptide. This fact greatly reduces the potential expressing cell-free system to perform those studies for which the necessary significant amount of study of the polypeptide.

For expanding the capabilities of expressing cell-free system it is possible to implement the VA approach. The first is to develop such methods that would allow to obtain information about, for example, the structure of newly synthesized polypeptide using nanogramme quantity (Fedorov A.N., Dolgikh D.A., Chemeris et al. (1992) J.Mol.Biol.225, 927-931). The second approach is to modernize the cell-free system broadcast order the sharp increase its effectiveness.

After being shown the ability to work a cell-free system broadcast in a continuous mode, it is possible to speak about its biotechnological application and primarily for toxic to cells, but important from the point of view of practical use, biologically active peptides. Recently it has become possible using a cell-free system broadcast for preparative developments proteins for their structural studies (NMR, x-ray analysis, and so on).

Among the currently used systems cell-free system from wheat germ is more affordable and cheaper compared to the system from rabbit reticulocytes and more preferable compared to the S30 system E. coli, as it allows in many cases to successfully solve the problem of the correct folding of eukaryotic proteins. Using this system, mainly solved scientific problems that tie is installed with the study of protein biosynthesis.

One of the main reasons behind the limited use of cell-free systems based on plant zygotic embryos for protein synthesis in a long time, is the presence of impurity nucleonic and protease activities. In native seeds these activities are usually confined to storing tissues or specialized bodies zygotic sardesai, such as cotyledons. Recent advances in the field of culture of plant cells open new possibilities for obtaining somatic embryos without storing tissues of seeds and induction of germination of the resting state. In this regard, to obtain extracts for long-term cell-free protein synthesis dehydrated somatic embryos seem to be the most attractive and promising source material. An additional benefit of this approach is the possibility of stable, does not depend on the time of year and weather conditions receive a fresh homogeneous material for preparation of extracts.

The success of the use of somatic embryos to obtain extracts for cell-free protein synthesis depends on two circumstances. First, it is necessary that obtained somatic embryos were morphologically high quality. Secondly, it should be solved the problem of induction with the situation of peace, inextricably associated with the synchronization of the development of the embryo.

To obtain in vitro a large number of somatic embryos the most promising approach is based on the use of callus and suspension cultures. This approach is actively developed recently in the Annex to this dicotyledonous plants, carrots, alfalfa, tobacco, potatoes (Gray DJ., Purohit A. 1991 Critical Reviews in Plant Sci 10 (1): 33-61; Mc Kersie B.D., Senarata So, Bowley S.R et al. (1989) In vitro Cell. Dev. Biol. 25: 1183-1188; Janic J., S.L. Kitto, Yong-Hwan. (1989) In vitro Cell. Dev. Biol. 25: 1167-1172; Carman JG. (1990) In vitro Cell. Dev. Biol. 26: 746-753).

In the case of monocotyledonous plants such as wheat, receiving the suspension cultures, capable of continuous reproduction of somatic embryos is extremely laborious and time consuming. Only in a limited number of laboratories around the world have developed such protocols for wheat (Redway F.A., V. Vasil, D. Lu & I.K. Vasil, 1990 Plant Cell Rep. 8:714-717; W.C. Wang and H.T. Nguyen 1990 Plant Cell Rep. 8: 639-642; Yang Y.M., He DG, Scott KJJ. 1994 Plant Cell Reports 13: 176-179), but in many cases they turned out to be irreproducible and provided extremely low output separate well-formed bipolar somatic embryos. In all these protocols, the source material to obtain a suspension culture served as a long-term cultivated highly embryogenic callus, which itself can serve as the source of the om for somatic embryos. This callus can be obtained from various tissues, such as immature zygotic embryos, inflorescence primordia and Mature seeds. Maintenance of embryogenic ability in callus culture of wheat for a long time depends on many factors, including genotype, type nutrient, hormonal composition, conditions and duration of cultivation.

Immature zygotic embryos are a favorite source material for preparation of somatic embryos in vitro, and to obtain long-term supported embryogenic callus (Maddock, S.E., VA Lancaster, R. Risiott & J. Franklin, 1983. J Exp Bot 34: 915-926; Borelli, G.M., Lupotto, E., Locatelli, F. and Wittmer, G. (1991) Plant Cell Rep. 10: 296-299; Vasit V, Redway FA & Vasil IK (1990) Bio/TechnoIogy 8: 429-434; Mohamand A.S., M.W. Nabors 1991. Plant Cell Tissue Organ Cult 26: 185-187; Felfoldi K., and Purnhauser 1. 1992. Cereal Res Comm 20: 273-277). However, the explants such available only for a limited period during the year, or donor plant should be grown in greenhouse conditions, which increases the complexity. Usually this cultivation takes 3-4 months. In that case, when using the rudiments of inflorescences, duration greenhouse cultivation is reduced to only 2.5-3 weeks. In Mature seeds, which are very promising source material in connection with their unlimited availability regardless of time of year, use the Finance limited is still very low embryogenic capacity (Fennell, S., N. Bohorova, M. van Ginkel, J. et al. 1996. Theor Appl Genet 92: 163-169; Bartok T., and F. Sagi 1990. Plant Cell Tissue Organ Cult 22:37-41;Sancak, C. (1998) Plant Cell Reports 18:331-335).

The invention

The present invention provides extracts from somatic plant embryos obtained under conditions of artificial cultivation in vitro, cell-free systems broadcast and/or coupled transcription-translation. Obtaining somatic embryos in vitro based on the ability of plant tissue to regenerate organs and whole plants from somatic cells. This principle is universal for a large number of plant species, regardless of their belonging to dicots or monocots. In particular, the invention provides extracts from somatic embryos of wheat, barley and maize.

In accordance with another aspect of the present invention provides a method of producing extracts from somatic plant embryos for cell-free systems broadcast and/or coupled transcription-translation, including:

a). the primary material from somatic tissues;

b). the induction of somatic embryogenesis;

c). preparation of extract from somatic embryos.

Derived from somatic tissues of plants primary material (Explant) placed on a nutrient medium for the Indus the functions of somatic embryogenesis in vitro. Under cultivation of explants formed embryogenic callus, from which, as cultivation is formed somatic embryos. During the development of embryos to the stage of forming the first sheet to separate from the callus and used for preparation of extracts.

Another aspect refers to the extract from somatic plant embryos, obtained by the method according to the invention in accordance with the previous aspect.

Another aspect of the invention provides a method of producing extracts from somatic plant embryos for cell-free systems broadcast and/or coupled transcription-translation, including:

a). the primary material from somatic tissues;

b). getting vysokoobrazovannogo callus;

c). obtaining embryogenic suspension culture;

d). the induction of secondary somatic embryogenesis;

e). preparation of extract from somatic embryos.

Accordingly, another aspect provides an extract from somatic plant embryos, obtained by the method of the present invention in accordance with the previous aspect.

The following aspect of the invention provides for the use of extract from plant somatic embryos obtained under conditions of artificial cultivation in vitro, cell-free system Tran the ablation and/or coupled transcription-translation. In particular, provision for the use of the extract according to the invention for cell-free synthesis of oligopeptides, polypeptides, proteins.

Another aspect of the invention provides for the use of extract from plant somatic embryos obtained in an artificial in vitro cultivation for the production of diagnostic systems for the detection of biologically active compounds with modulating activity on the processes of transcription and/or translation, and/or for screening for such compounds.

Another aspect of the invention relates to a diagnostic system based on an extract from plant somatic embryos for the detection of biologically active compounds with modulating activity on the processes of transcription and/or translation, and/or for screening for such compounds.

The following aspect of the invention provides the use of such a diagnostic system based on an extract from plant somatic embryos for the detection of biologically active compounds with modulating activity on the processes of transcription and/or translation, and/or for screening for such compounds.

Compared to the standard extract, which is defined as an extract prepared from Mature zygotic embryos of wheat seeds, which this is currently the most widely used eukaryotic system broadcast of plant origin, the present invention provides the following advantages:

1. Obtaining extracts from plant somatic embryos grown in vitro, allows to overcome specific limitations upon receipt of such extracts from zygotic embryos of different cultures.

2. Obtaining somatic embryos may be year-round, which allows to overcome the pronounced seasonality in obtaining extracts from zygotic embryos.

3. A method of obtaining a plant somatic embryos present invention provides a receiving embryos morphologically high quality. The present invention solves the problem of obtaining an extract of guaranteed high quality by inducing quiescence formed embryos, providing synchronization of the development of the embryo.

4. Using embryogenic suspension cultures can overcome the large material and labor costs for cultivation and preparation of large quantities of high quality seed material. Embryogenic suspension culture of plants allows to obtain a large number of somatic embryos.

5. The extract of the invention provides a more efficient broadcast and coupled transcription-translation in a cell-free system. Extract from somaticheskih embryos allows to increase the output synthesized in a cell-free system product broadcast at least 30%.

6. The extract of the invention changes the kinetics of cell-free protein synthesis, making it especially effective in the early stages of biosynthesis, thereby reducing the response time.

7. The activity of the extract obtained from somatic embryos, equally high regardless of the sort, that allows to overcome the pronounced varietal specificity of the extract prepared from zygotic embryos.

8. Derived from somatic embryos extract successfully works with a variety of matrices; synthesized proteins are full size and have a functional biological activity.

List of figures

Figure 1. Translation of mRNA obelin the luminescent protein (80 pmol/ml) in different extracts.

Figure 2. The kinetics of the mRNA obelin the luminescent protein (80 pmol/ml).

Figure 3. Synthesis of obelin the luminescent protein in extracts prepared from different wheat varieties (duration broadcast 60 minutes).

Figure 4. Kinetics of synthesis of green fluorescent protein GFP.

Figure 5. Kinetics of GFP fusion protein in the dual system of transcription-translation.

Information confirming the possibility of carrying out the invention

In the present description are common in this area terms and concepts. However, in order to ensure their interpretation in the sense and to the extent as it is in the context of the present description, the following are the ODA is the division of the terms used.

Under "somatic tissues" in the present description refers to tissue of vegetative plant organs (leaves, roots, stems, and other).

"Zygotic embryos (embryos)is the germ, resulting fusion of gametes during fertilization.

"Somatic embryos (embryos)" - germs of plants, shaped by simple cell division (mitosis) somatic tissues without fusion of gametes.

Mature embryos (embryos)" - this term means a fully formed embryos, are capable of further self germination under natural conditions without creating additional conditions.

Immature embryos (embryos)" - this term refers to the immature embryos are not capable of germination without creating additional conditions.

Under "callous" in the present description refers to undifferentiated plant tissue, formed as a result of the simple cell division.

Under "primary somatic embryogenesis" is meant the formation of somatic embryos (embryos) from primary tissue explants in a relatively short period (up to 2.5 months.)

Under "secondary somatic embryogenesis" is meant the formation of somatic embryos (embryos) from long supported the on callus (3-4 months or more), originally formed from the tissues of primary explants.

The term "induction" is understood in the sense that it is changing the terms of the cultivation of plant tissues with the purpose of artificial stimulation of the processes of somatic embryogenesis.

Methods extraction of plant embryos of the present invention provide for obtaining primary material (explants) from somatic plant tissues. Pre-plant tissue surface treated with sterilizing agents, known in the field. Examples of sterilizing agents, but not limited to, can serve 70% ethanol and/or 0.5% sodium hypochlorite. The selection of primary material from plant tissue is performed under a binocular microscope.

As the primary material (explants) to obtain callus in vitro can serve a variety of somatic tissue is immature zygotic embryos, the embryos of Mature seeds, the beginnings of the inflorescence, the base of young leaves, anthers, and other tissues, depending on the plant species. In the preferred embodiment of the present invention as explants used immature zygotic embryos derived from immature seeds.

Primary material can be obtained from a variety of dicotyledonous and monocotyledonous plants In the preferred embodiment of the invention such plants are wheat, barley and corn.

Explant is placed on a nutrient medium for the induction of somatic embryogenesis in vitro. Under cultivation of explants produced primary embryogenic callus, from which, as cultivation is formed somatic embryos. During the development of embryos to the stage of forming the first sheet they are separated from the callus and are used to prepare extracts. This process can be carried out year-round, which allows to overcome the pronounced seasonality in obtaining extracts from zygotic embryos.

Induction of somatic embryogenesis (both primary and secondary) occurs under controlled conditions on an artificial nutrient medium, regardless of external conditions. This allows to overcome climatic, geographical and seasonal factors, leading to unpredictable variability in the quality of the extract obtained from zygotic embryos.

Artificial nutrient media used at different stages of obtaining somatic embryos can be different or the same, depending on the applied method of producing extracts from plant somatic embryos, plant species and other factors. The nutrient medium may be liquid, semi-liquid or solid and includes several components: mine is real salt including trace elements, organic additives of different composition and concentration (sugar, amino acids, vitamins, protein hydrolysates, osmoregulatory, etc), phytohormones, and if necessary, agar and its natural or synthetic analogs.

As the mineral component of the nutrient medium to obtain somatic embryos can be used in different compositions of salts used for the culture of cells and tissues of plants (Chu C.C., Wang, C.C., Sun, C.S., et al. 1975. Sci. Sin. 18:659-668; Gamborg O. L., Miller R. A., Ojima K. 1968. Expt. Cell. Res. 50: 151-158). In the preferred embodiment of the present invention uses a composition of salts on Murashige and Scoog (Murashige T., Skoog F. 1962 Physiol. Plantarum. 15: 473-497), however, other known from the prior art compositions can be equally well used in the present invention.

In addition to the standard compositions of salts in an artificial environment, depending on the plant species may be added various trace elements. For example, induction of primary callus in the case of barley in a nutrient medium preferably add CuSO4.

As vitamins you can use various compositions. The preferred composition of vitamins in the present invention is the prescription proposed in (Murashige T., Skoog F. 1962 Physiol. Plantarum. 15: 473-497), however, other known from the prior art recipe can be equal to us who ehom used in the present invention.

As Sakharov, one of the main components of the nutrient medium can be used various types of mono - and disaccharides, and combinations thereof. In the preferred embodiment of the present invention are glucose at a concentration of from about 1 to about 3%, maltose at a concentration of from about 2 to about 5%, or sucrose at a concentration of from about 2 to about 4%. The most preferred of the present invention is the use of glucose or sucrose at a concentration of from about 2 to about 3%, or maltose at a concentration of from about 2 to about 5%.

As amino acids are used in a variety of natural L-amino acids, such as, but not limited to, Proline, asparagine.

As protein hydrolysates can be used in a variety of enzymatic or acid hydrolysates of proteins, for example, known under the trade names Tripton, Peptone, Kazarinova acid. Preferred according to the present invention is the use of hydrolyzed casein.

The hormones necessary for the directed induction of somatic embryogenesis. As major inducers of embryogenesis in vitro use auxins, for example: 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 3,6-dichloro-O-anisic acid (Dicamba) and p-chloroperoxidase acid (CPA), as well as the cytokinins kinetin and 6-benzylaminopurine. In a preferred embodiment of the present invention using 2,4-D at a concentration of about 2 mg/l nutrient medium.

To increase the efficiency of somatic embryogenesis in the environment also add other hormones, such as cytokinins and gibberellins, and others In the preferred embodiment of the present invention use Daminozide at a concentration of about 25 mg/L.

The efficiency of embryogenesis may also depend on osmotic shock, which can be created by introducing into the composition of the culture medium components such as polyethylene glycol, or an increase in the concentration of sugars. In the preferred embodiment of the present invention is polyethylene glycol 6000 (PEG 6000) at a concentration of from about 0.5 to about 1 g/L.

To impart lubricating properties of the medium can be used bacteriological agar, agarose and artificial substitutes agar (gelrite, plant agar).

Tissue culture in vitro can be directed to control the quality of the obtained extract by artificial induction of quiescence in somatic embryos. Translation of actively growing somatic embryos in a state of rest can be done by modifying the culture conditions to simulate the conditions associated with the formation of seeds in nature, in particular, by increasing the temperature cultiv the simulation, changes the osmotic balance of the medium, forced drying of somatic embryos and other Alternative way is the use of phytohormones. In the preferred embodiment of the present invention is applied abscisic acid (ABA) and its analogues. Abscisic acid and its analogues injected into the culture medium after a certain period of time, in particular after already occurred tab somatic embryos in embryogenic callus. In the preferred embodiment of the present invention abscisic acid (ABA) at a concentration of about 1 mg/l is introduced into the nutrient artificial environment for approximately 15 days before the separation of somatic embryos from callus.

Provided by one of the methods of the present invention using high-tech methods cell culture and plant tissue in vitro allows to overcome the large material and labor costs for cultivation and preparation of large quantities of high quality seed material. This preferred method of the present invention is an embryogenic suspension culture of plants, allowing a large number of somatic embryos, which can be formed either during the cultivation of cells in a liquid nutrient medium, and through the stage PE the Enos callous, formed in suspension culture on solid medium and induction of secondary embryogenesis.

The procedure for preparation of the extract of somatic embryos in their primary stages repeats classic (B.E. Roberts, Paterson V.M. (1973) Proc. Natl. Acad. Sci. USA 70,No. 4, 2330-2334; Erickson, A.H., BIobel, G. (1983) Meth. EnzymoL 96, 38-50) using Mature zygotic embryos of wheat and includes:

stage 1 - homogenization of embryos frozen in liquid nitrogen

stage 2 extraction buffer solution,

stage 3 - centrifugation,

stage 4 - chromatography.

Activity derived from somatic embryos extracts checks in the standard cell-free system of the mRNA. For comparison, use the extract from Mature zygotic embryos of wheat. The optical density of the extracts was approximately the same (A260=100-150 TH). As test matrices monitoring broadcast may be used any suitable artificial RNA or natural mRNA. To facilitate the monitoring broadcast can be used, for example, mRNA CA2+- activated fotobanka obelin the luminescent protein or green fluorescent protein (GFP). The synthesis of these proteins in the cell-free translation system allows you to monitor the progress of broadcast not only on the inclusion of a radioactive label, but luminescent reaction (in the case of obelin the luminescent protein) is whether the fluorescence of GFP. All comparisons extracts is carried out at equal concentrations of mRNA in the broadcast system.

Extract from somatic embryos in accordance with the present invention allows to increase the output synthesized in a cell-free system product broadcast at least 30%. The resulting extract was equally successful in the synthesis of various proteins (GFP, obelin), all products broadcast panoreserve and possess the biological activity. The presence of functional biological activity in the cell-free product broadcast confirms the correct spatial laying polypeptide chain folding and Assembly of the enzyme in an active conformation. Thus, the conformation of the product broadcast and/or coupled transcription-translation obtained in a cell-free system of the present invention, is identical or substantially identical to its native conformation.

The extract of the present invention, obtained from plant somatic embryos obtained in culture conditions in vitro, changes the kinetics of synthesis of these proteins, making it more efficient compared to the standard extract. The most noticeable difference in the efficiency of the mRNA in the initial stages of biosynthesis (up to 20 minutes).

The activity of the extract of the present invention, obtained from the soma is practical germ, equally high regardless of the sort, that allows to overcome the pronounced varietal specificity of the extract prepared from zygotic embryos.

Obtained in accordance with the present invention extracts from plant somatic embryos can be, in particular, applied to preparative developments proteins, polypeptides, oligopeptides. This system allows to overcome the technological and economic limitations of chemical synthesis and recombinant techniques used currently. In particular it becomes possible to obtain for a wide use of peptides in length 30-100 amino acid residues. Thus it becomes possible to overcome the incompleteness of elongation of polypeptide chain, to avoid difficulties with the choice of protective groups and their disposal and, most importantly, difficulties with the implementation of functionally active conformation in a long polypeptide. This in turn will allow you to get relatively inexpensive end product. The system also allows us to overcome a number of difficulties arising from the creation of genetic constructs for expression in prokaryotic or eukaryotic cells for the production of biologically active molecules associated with instability or poor expression as a result of regulatory, milling the Dom of the host cell, instability and degradation of the formed aggregates or their toxicity to cells.

As the messenger RNA for cell-free protein synthesis product can be used natural mRNA, synthetic RNA, total RNA, and RNA transcripts produced in vitro. To obtain RNA transcripts in vitro can be used well-known approaches based on the use of high-activity and high-affinity RNA-polymerases of phages T3, T7 or SP6. To achieve this, the DNA sequence encoding a protein product in the upstream position (against the direction of transcription) enter a sequence of appropriate promoters, recognizable fagbemi RNA polymerase.

Cell-free extract of the present invention may be equally well used for the implementation of the coupled transcription-translation. In this case, the system as a matrix introducing exogenous DNA encoding a protein product, which may be represented, for example, a DNA fragment, the product of a polymerase chain reaction (PCR), legirovannye mutually overlapping oligonucleotides. Such encoding a protein product of the DNA sequence must contain signals of transcription and translation. A prerequisite is the presence of effective transcriptional promoter. For example, DNA can in order to keep the sequence of the 35S-promoter RNA of cauliflower mosaic virus, cloned in upstream position relative to the coding sequence. Alternatively, the promoter comprising DNA-matrix can be represented by a promoter of RNA-polymerases of phages T3, T7 or SP6, and encoding a protein product of the DNA sequence is located downstream of transcription. In the latter case, to ensure efficient transcription DNA-matrix system coupled transcription-translation on the basis of cell-free extract of the present invention add an exogenous enzyme - RNA polymerase of phage T3, T7 or SP6, which is available from commercial sources or can be easily obtained in laboratory conditions. If necessary, DNA-matrix can also contain a polyadenylation signal.

Very promising seems to be another area of practical use systems cell-free protein synthesis - development and production-based cell-free system broadcast and/or coupled transcription-translation of new diagnostic test-systems for detection of various biologically active substances capable of modulating the processes of transcription and/or translation, and/or for screening for such compounds. The possibility of the production of such test systems is based on the fact that different factors of biological and chemical nature, modulating, i.e. Inga is yousie or activating, the process of protein biosynthesis at various stages (transcription, translation), can be identified using a cell-free system of biosynthesis at the level of the output broadcast or coupled transcription/translation. These compounds include, for example, various poisons, toxins, antibiotics, heavy metals, etc. for Example, the toxic effect of a whole range of fungal toxins (the most prominent representative of which is α-amanitin) due to the fact that they completely block transcription under the action of eukaryotic RNA polymerases II and III. When screening aimed at compounds capable of modulating the processes of transcription and/or translation, can be used, for example, but not limited to, natural mixtures, extracts, extracts, medium after culturing various microorganisms artificially created libraries of individual compounds, including combinatorial libraries. It is obvious that this kind of test systems based on the extract of the present invention can find wide application in biology, medicine, pharmacology, toxicology, ecology, etc.

As an example of such a diagnostic system based on cell-free translation, can be described Gorokhovatsky and co-authors (Biotechnol. Appl. Biochem. (1998) 27, 259-263) is iofilizirovanny system, derived from wheat germ, which was successfully used for analysis of various compounds, inhibiting the process of protein biosynthesis at various stages of diphtheria toxin and antibiotics - tetracycline, kanamycin and puromycin. Specialist it is obvious that the same image can be analyzed and substances which activate the process of protein biosynthesis at various stages. Among the advantages of such diagnostic systems based on cell-free systems broadcast the authors note that when using bioluminescent method of detection of the resulting product broadcast, i.e. when the reaction product is a natural fluorescent protein, such as activated CA2+photoblog obelin, the system provides extremely high sensitivity that exceeds three orders of magnitude the sensitivity of radioisotope method detection product. Additionally it can be noted that the use of fluorescent proteins (such as, for example, obelin the luminescent protein, green fluorescent protein GFP and its variants, and the like) as detected products broadcast also provides another advantage, namely the ecological safety of the diagnostic system.

Examples.

The following examples are given to demonstrate the ability to implement the ia of the invention, however, any person skilled in the art it is clear that they are given as an illustration and may not be used to limit the scope of the claims of the applicant.

Example 1. The primary material from immature zygotic embryos.

Example 1.1. The primary material from immature zygotic embryos of wheat.

Zygotic embryos isolated from immature seeds of wheat (stage translucency). To do this, collect seeds with emerging young ears in 12-15 days after release of the anthers. Immature seeds are surface treated with sterilizing agents (ethanol 70%, sodium hypochlorite 0.5%), and then immature germ separates from the seed under a binocular microscope and placed on an artificial nutrient medium for the induction of embryogenic callus.

Example 1.3. The primary material from immature seeds of corn.

Immature zygotic embryos of maize inbred line # 91 1.5-2.5 mm extracted from immature seeds collected from young corn cobs in 9-12 days after release of the anthers. Immature seeds are surface treated with sterilizing agents (ethanol 70%, sodium hypochlorite 0.5%), and then immature germ separates from the seed under a binocular microscope and placed on an artificial nutrient medium for the induction of callus.

Primer. Induction of somatic embryogenesis.

Example 2.1. Induction of somatic embryogenesis in wheat

Example 2.1.1. Artificial nutrient medium to obtain somatic embryos of wheat includes the following components: mineral salts on Murashige and Scoog (Table 1), 3% sucrose, 150 mg/l asparagine, vitamins (Table 2), phytohormones: auxin 2,4-D at a concentration of 2 mg/l environment, abscisic acid (ABA) concentration of 1 mg/l environment (add to encourage dormancy in Mature somatic embryos 15 days after induction of embryogenesis), 0.7% agar bacteriological. The cultivation of material in vitro is carried out in a period of 25-30 days at a temperature of 26°in the dark. When the embryos beginning stage of forming the first sheet of their separated under a binocular microscope from embryogenic callous and stored frozen at a temperature of 70°C.

Table sostav mineral salts on Murashige and Scoog (1962)
ComponentConcentration
Potassium nitrate1900 mg/l
Ammonium nitrate1650 mg/l
Potassium phosphate one-deputizing170 mg/l
Calcium chloride anhydrous332,2 mg/l
2Aboutof 16.9 mg/l
Boric acid6,2 mg/l
Zinc sulphate · 7H2About8.6 mg/l
Potassium iodide0,83 mg/l
Copper sulfate · 5H2About0.025 mg/l
Sodium molybdenate · 2H2About0.25 mg/l
Cobalt chloride · 6N2O0.025 mg/l
Iron sulfate · 7H2Aboutof 27.8 mg/l
Na2EDTA·2H2O37,26 mg/l
Table sostav and the ratio of vitamins on Murashige and Scoog (1962)
ComponentConcentration
Nicotinic acid0.5 mg/l
Pyridoxine0.5 mg/l
Thiamine0.5 mg/l
Glycine2 mg/l

Example 2.1.2. Induction of somatic embryogenesis occurs under the same conditions, with one exception: in the composition of the nutrient medium include glucose instead of sucrose.

Example 2.1.3. Induction of somatic embryogenesis occurs under the same conditions, with one exception: in the nutrient medium composition includes maltose instead of sucrose.

Example 2.1.4. Induction of somatic embryogenesis occurs when those who e conditions, with one exception: in the nutrient medium composition include auxin instead of Dicamba 2,4-D.

Example 2.1.5. Induction of somatic embryogenesis occurs under the same conditions, with one exception: in the nutrient medium composition optionally containing 1% PEG 6000.

Example 2.1.6. Induction of somatic embryogenesis occurs under the same conditions, with one exception: in the nutrient medium composition optionally containing Daminozide (succinic acid mono(2,2-dimethylhydrazide)) at a concentration of 25 mg/L.

Example 2.1.7. Induction of somatic embryogenesis occurs under the same conditions, with one exception: in the nutrient medium composition include Daminozide at a concentration of 200 mg/l instead of AVA.

Example 2.2. Induction of somatic embryogenesis in barley

Example 2.2.1 Induction of somatic embryogenesis in barley consists of two consecutive kultivirovanii:

A. Induction of primary callus

B. Induction of embryogenesis from primary callus.

For the induction of primary callus of immature zygotic embryos of barley placed on a nutrient medium of the following composition: mineral salts on Murashige and Scoog and (PL. 1), CuSO4at a concentration of 0.5 mm, vitamins (Table 2), 3% maltose, the auxin 2,4-D at a concentration of 2 mg/l medium, 0.7% agar bacteriological. Immature zygotic embryos were cultured for 3 weeks at a temperature of 26°in the darkness.

For the induction of somatic is about embryogenesis dense pockets of callus, formed as a result of the cultivation of immature zygotic embryos, move on nutrient medium of the following composition: mineral salts on Murashige and Scoog (Table 1), 3% sucrose, vitamins (Table 2), the cytokinin kinetin at a concentration of 1 mg/l environment, 0.7% agar bacteriological. The cultivation of material in vitro is carried out in a period of 25-30 days at a temperature of 26°in the dark. When the embryos beginning stage of forming the first sheet of their separated under a binocular microscope from embryogenic callous and stored frozen at a temperature of 70°C.

Example 2.2.2 Induction of somatic embryogenesis occurs under the same conditions, with one exception: in the nutrient medium composition for embryogenesis include auxin NAA.

Example 2.2.3 Induction of somatic embryogenesis occurs under the same conditions, with one exception: in the nutrient medium composition for embryogenesis include cytokinin BA instead of kinetin.

Example 2.3. Induction of somatic embryogenesis in maize.

For the induction of embryogenic callus of immature zygotic embryos of corn is placed on a nutrient medium of the following composition: mineral salts on Murashige and Scoog (Table 1), vitamins (Table 2), 2% sucrose, casein hydrolysate at a concentration of 100 mg/l environment L-Proline at a concentration of 3 g/l environment, the auxin 2,4-D at a concentration of 2 m is/l environment, 0.7% bacteriological agar. Immature zygotic embryos were cultured for 5 weeks at a temperature of 26°in the dark. The resulting callus cultured every 4 weeks on fresh medium of the same composition. When the embryos beginning stage of forming the first sheet of their separated under a binocular microscope from callus and stored frozen at a temperature of 70°C.

All shown in example 2 variants of embodiment of the invention indicate that the invention is equally well feasible in the whole volume of claims, as it is set forth in the claims.

Example 3. Obtaining embryogenic suspension culture

Example 3.1 Obtaining embryogenic suspension cultures of wheat

Example 3.1.1. To obtain embryogenic suspension use vysokoobrazovannyj callus derived from immature zygotic embryos. Callus cultured on medium induction of embryogenesis (see Example 2) at least 1.5-2 months. Fresh embryogenic callus, crushed into small pieces with a size of 0.2-0.5 cm, transferred into a flask of Erlenmeyer 250 ml In each flask is filled with 50 ml of liquid nutrient medium which comprises mineral salts on Murashige and Scoog (Table 1), 3% sucrose, vitamins (Table 2) and the auxin 2,4-D at a concentration of 2 mg/l medium and add about 1 g of callus. The primary suspension is cultivated is within 4 weeks. After that, the upper phase medium (25 ml) with young in dividing cells is transferred into a new flask containing 25 ml of fresh nutrient medium. Every 10-14 days of conducting the update suspension culture by dilution with fresh medium at a ratio of 1:1. Flasks were cultured in the dark at 26°C on a rotating shaker (120 rpm). After the establishment of suspension culture (2,5-3 months.) formed in the culture medium of cell aggregates larger than 1 mm is transferred to Petri dishes for the induction of secondary somatic embryogenesis. To do this, use a nutrient medium of the following composition: mineral salts on Murashige and Scoog and (Table 1), 3% sucrose, vitamins (Table 2), the auxin 2,4-D at a concentration of 1 mg/l environment), 0.7% agar bacteriological. Aggregates cultured for 30-45 days at a temperature of 26°in the dark. As the formation of somatic embryos on the calli formed from the suspension units, separated under a binocular microscope from embryogenic callous and stored frozen at a temperature of 80°C.

Example 3.1.2. Obtaining embryogenic culture occurs under the same conditions, with one exception: in the composition of the liquid nutrient medium include maltose instead of sucrose.

Example 3.1.3. Obtaining embryogenic culture occurs under the same conditions, with one exception: W is dcoi nutrient medium include auxin 2,4,5-T instead of the auxin 2,4-D.

Example 3.1.4. Obtaining secondary somatic embryos from callus formed from the suspension units, occurs under the same conditions, with one exception: for the induction of secondary embryogenesis in the nutrient medium composition include auxin Dicamba instead of the auxin 2,4-D.

Example 3.1.5. Obtaining secondary somatic embryos from callus formed from the suspension units, occurs under the same conditions, with one exception: for the induction of secondary embryogenesis in the nutrient medium composition include auxin 2,4,5-T instead of the auxin 2,4-D.

Example 3.2. Obtaining embryogenic suspension culture of maize

To obtain embryogenic suspension use vysokoobrazovannyj callus corn, coltiviruses on the environment induction of embryogenesis (see Example 2.3.) not less than 3 months. Fresh embryogenic callus, crushed into small pieces with a size of 0.2-0.5 cm, transferred into a flask of Erlenmeyer 250 ml In each flask is filled with 50 ml of liquid nutrient medium which comprises mineral salts on Murashige and Scoog (Table 1), 3% sucrose, vitamins (Table 2), casein hydrolysate at a concentration of 200 mg/l Inositol concentration of 100 mg/l L-Proline at a concentration of 3 g/l and the auxin 2,4-D at a concentration of 2 mg/l medium, and add about 1 g of callus. Primary suspension cultured for 7 weeks, carrying out regeneration of the culture throughevery 7 days. After that, the upper phase medium (25 ml) with young in dividing cells is transferred into a new flask containing 25 ml of fresh nutrient medium. Every 7-10 days spend upgrading suspension culture by dilution with fresh medium at a ratio of 1:1. Flasks were cultured in the dark at 26°C on a rotating shaker (120 rpm). After the establishment of suspension culture (2,5-3 months.) formed in the culture medium of cell aggregates larger than 1 mm is transferred to Petri dishes for the induction of secondary somatic embryogenesis. To do this, use a nutrient medium described in example 2.3.1. Aggregates cultured for 30-45 days at a temperature of 26°in the dark. As the formation of somatic embryos on the calli formed from the suspension units, separated under a binocular microscope from embryogenic callous and stored frozen at a temperature of 70°C.

All shown in example 3 variants of embodiment of the invention indicate that the invention is equally well feasible in all of the claims, as they are formulated in the claims.

Example 4. Preparation of extracts from somatic embryos.

5 g of embryos frozen in liquid nitrogen, grind porcelain pestle in a mortar 2 minutes. Put the mortar in the ice, after 10 minutes (poslegarantijnogo thawing) add 1 ml of buffer solution of the following composition: 160 mm HEPES, pH of 7.6, 480 mm potassium acetate, 20 mm magnesium acetate, 24 mm dithiothreitol (DTT). The extraction was carried out for 15 minutes.

After double centrifugation (20 minutes, 30000 g, precipitation drop) supernatant (2.5 ml) is applied onto a column of Sephadex G-25 Medium (NAP 25 Column, equilibrated with buffer containing 40 mm HEPES, pH of 7.6, 120 mm potassium acetate, 5 mm magnesium acetate, 6 mm DTT. The fraction corresponding to the free volume of the column (2.5 ml), collected and centrifuged under the same conditions. The optical density of the extracts is 60-150 TH at 260 nm. Extracts frozen in liquid nitrogen aliquot and store at a temperature of 70°C.

Example 5. Comparison of aktivnosti extracts, obtained from the tissues of wheat.

The activity of extracts obtained from somatic embryos grown in an artificial in vitro cultivation both in the absence and in the presence of abscisic acid test in the standard cell-free system of the mRNA. For comparison, use the extract from Mature zygotic embryos of wheat. The optical density of the extracts approximately equal (A260=100-150 TH). As a test matrix monitoring broadcast choose mRNA CA2+- activated fotobanka obelin the luminescent protein. The synthesis of this protein in the cell-free translation system allows you to monitor the progress of broadcast not only on the inclusion of radioactive m the weave but luminescent reaction. All comparisons extracts is carried out at equal concentrations of mRNA in the broadcast system.

As can be seen in figure 1, the extract of the present invention from somatic embryos obtained in an artificial in vitro culturing in the presence of abscisic acid, has the greatest activity in the cell-free system of the mRNA.

Example 6. The study of the kinetics of protein synthesis using an extract derived from somatic embryos.

Activity derived from somatic embryos extracts checks in the standard cell-free system of the mRNA. For the experiment using somatic embryos obtained in the presence of abscisic acid. For comparison, use the extract from Mature zygotic embryos of wheat. The optical density of the extracts is And260=130 OE for standard extract and a260=152 OE to extract from somatic embryos. As a test matrix monitoring broadcast choose mRNA CA2+- activated fotobanka obelin the luminescent protein. All comparisons extracts is carried out at equal concentrations of mRNA in the broadcast system.

As can be seen from figure 2, the extract from the somatic embryos obtained in accordance with the present invention, provides a higher level of synthesis of the final protein what about the product - obelin the luminescent protein, and the broadcast goes faster that most clearly seen in the initial stages (up to 20-30 minutes).

Example 7. Comparison of extracts obtained from somatic embryos of six varieties of wheat.

Activity derived from somatic embryos extracts checks in the standard cell-free system of the mRNA. For the experiment using somatic embryos of six wheat varieties obtained in the presence of abscisic acid. For comparison, use the extract from Mature zygotic embryos of wheat. The optical density of the extracts approximately equal (A260=90-152 OE). As a test matrix monitoring broadcast choose mRNA CA2+-activated fotobanka obelin the luminescent protein. All comparisons extracts is carried out at equal concentrations of mRNA (80 pmol/ml) in the system broadcast 60 minutes after the beginning of the broadcast.

As can be seen from figure 3, regardless of the sort extracts in accordance with the present invention ensures stable higher efficiency broadcast than the standard extract.

Example 8. The study of the kinetics of the fusion protein GFP using extract. derived from somatic embryos

To confirm the possibility of the synthesis of various proteins using extracts obtained somatic embryos, check the activity in andartes a cell-free system of the mRNA. For the experiment used the somatic embryos obtained in the presence of abscisic acid. For comparison, use the extract from Mature zygotic embryos of wheat. The optical density of the extracts is And260=130 OE for standard extract and a260=152 OE to extract from somatic embryos. As a test matrix monitoring broadcast choose mRNA green fluorescent protein (GFP). All comparisons extracts is carried out at equal concentrations of mRNA (320 pmol/ml) in the broadcast system.

As can be seen from figure 4, the extract of the present invention provides a more efficient translation of mRNA GFP than the standard extract. Together with the results shown in figure 1 and 2, presents the results indicate that the extract of the present invention ensures stable and more efficient translation in a cell-free system compared to a standard extract from Mature zygotic embryos and, thus, confirm that the invention is feasible in all of the claims formulated in the claims.

Example 9. The fusion protein GFP in the dual system of transcription-translation in the CECF mode (continuous-exchange cell-free).

To confirm the protein synthesis in a coupled system of transcription-translation using extracts in the present invention, derived from somatic embryos, the GFP fusion protein in CECF system from somatic embryos of wheat were carried out in a reactor with a fixed reaction volume of 50 µl using a dialysis membrane with a pore size 12000-14000 kDa. The reaction mixture contained an extract from wheat germ (30% weight/volume), 7000 Units/ml T7 RNA polymerase, 100 μg/ml linearized plasmids rtoo-1, 500 Units/ml of RNase inhibitor, 100 μg/ml creatine phosphokinase, 50 μg/ml total yeast tRNA, 0.2 mm of each amino acid, 1 mm ATP, 0.6 mm GTP, 0.4 mm TTF and UTP each, 16 mm creatine phosphate, 40 mm HEPES-KOH pH of 7.6, 4 mm Mg(OAc)2, 100 mm COAs, 3 mm NaN3, 2.5 mm DTT, and 0.25 mm spermidine, 2% glycerol. Nutrient mixture (0.5 ml) contained the same components except for the extract of wheat germ RNA polymerase, plasmid, CPK, RNase inhibitor; the concentration of Mg(OAc)2reduced to 2.5 mm. The reaction was carried out at 25°With stirring. Nutrient mixture was replaced with fresh one every 10 hours. The concentration of GFP protein in the reaction mixture was measured by fluorescence at 510 nm.

As can be seen from Figure 5, the extract of the present invention provides a coupled transcription-translation DNA GFP even with greater efficiency than the standard extract. The obtained results indicate that the extract of the present invention is able to provide Shin is ez different proteins in a cell-free system similar to the standard extract from Mature zygotic embryos and, thus, confirming that the invention is feasible in all of the claims formulated in the claims.

It should be clear that in a similar way, taking into account the disclosure of the invention in the description of the specialist in this area can be obtained from other variants of the invention encompassed by the formula below.

1. Extract from plant somatic embryos obtained in culture conditions in vitro, cell-free systems broadcast and/or coupled transcription-translation.

2. The extract according to claim 1, where the specified somatic embryos are somatic embryos of wheat.

3. The extract according to claim 1, where the specified somatic embryos are somatic embryos of barley.

4. The extract according to claim 1, where the specified somatic embryos are somatic embryos of maize.

5. The method of obtaining extracts from plant somatic embryos according to any one of claims 1 to 4 for cell-free systems broadcast and/or coupled transcription-translation, including:

a) obtaining primary material from somatic tissues;

b) induction of somatic embryogenesis;

C) preparation of extract from somatic embryos.

6. The method according to claim 5, additionally including the induction of quiescence have formed somatics the x embryos.

7. The method according to subparagraph 5 and 6, where for the induction of quiescence use hormones selected from the group comprising abscisic acid (ABA) and its analogs.

8. The method according to pp.5-7, where for the induction of dormancy use ABA at a concentration of about 1 mg/l, which is added to the nutrient medium in approximately 15 days after induction of embryogenesis.

9. The method according to claim 5, where at the stage a) specified primary material derived from somatic tissues of wheat.

10. The method according to claim 5, where at the stage a) specified primary material derived from somatic tissues of barley.

11. The method according to claim 5, where at the stage a) specified primary material derived from somatic tissues of maize.

12. The method according to claim 5, where at the stage a) as the primary material used immature zygotic embryos.

13. The method according to claim 5, optionally, additionally includes the stage of induction of primary callus between the stage of obtaining raw material from somatic tissues (a) and the stage of the induction of somatic embryogenesis (b).

14. The method according to claim 5, where in stage b) use artificial growth medium comprising mineral salts, organic additives of different composition and concentration (sugar, amino acids, protein hydrolysates, vitamins, osmoregulatory and others), phytohormones, as well as agar or artificial or natural counterparts.

Cab on 14 where as sugars use glucose.

16. The method according to 14, where the sugar is used maltose.

17. The method according to 14, where the sugar is used sucrose.

18. The method according to 14, where for the induction of embryogenesis use hormones selected from the group of the auxin, including 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5 trichlorophenoxyacetic acid (2,4,5-T), 3,6-dichloro-O-anisic acid (Dicamba), R-chloroperoxidase acid (CPA), and naphthyl-acetic acid (NAA), and/or groups of cytokines, including kinetin and 6-benzylaminopurine (BA).

19. The method according to 14, where as the main inducer of embryogenesis using 2,4-D.

20. The method according to 14, where for increasing the efficiency of embryogenesis in addition use hormones selected from the group comprising cytokines and gibberellin.

21. The method according to claim 20, where to increase the efficiency of embryogenesis use Daminozide.

22. The method according to 14, where as osmoregulator using polyethylene glycol 6000 (PEG 6000).

23. The method according to claim 5, where somatic embryos under cultivation of cells at the stage b) is formed directly.

24. The method according to claim 5, where somatic embryos under cultivation of cells at the stage b) is formed through the intermediate stage of formation of embryogenic callus.

25. Extract for cell-free systems broadcast and/or conjugate is Oh transcription-translation, obtained by the method according to any of pp.5-24.

26. The method of obtaining extracts from plant somatic embryos according to claims 1 to 4 for the cell-free systems broadcast and/or coupled transcription-translation, including:

a) obtaining primary material from somatic tissues;

b) obtaining vysokoobrazovannogo callus;

C) obtaining embryogenic suspension culture;

d) induction of secondary somatic embryogenesis;

e) preparation of extract from somatic embryos.

27. The method according to p, where at the stage a) specified primary material derived from somatic tissues of wheat.

28. The method according to p, where at the stage a) specified primary material derived from somatic tissues of barley.

29. The method according to p, where at the stage a) specified primary material derived from somatic tissues of maize.

30. The method according to p where to stage a) as the primary material used immature zygotic embryos.

31. The method according to p, where when receiving vysokoobrazovannogo callus stage (b) use artificial growth medium comprising mineral salts, organic additives of different composition and concentration (sugar, amino acids, protein hydrolysates, vitamins and others), phytohormones, as well as agar or artificial or natural counterparts.

32. The way is about p, where as sugars use glucose.

33. The method according to p, where as maltose sugar is used.

34. The method according to p, where the sugar is used sucrose.

35. The method according to p where used phytohormones choose from auxin 2,4-dichlorophenoxyacetic acid (2,4-D), and/or groups of cytokines, including kinetin and 6-benzylaminopurine (BA).

36. The method according to p, where upon receipt of embryogenic suspension culture in stage C) use artificial liquid nutrient medium comprising mineral salts, organic additives of different composition and concentration (sugar, amino acids, protein hydrolysates, vitamins and others), phytohormones.

37. The method according to p, where as maltose sugar is used.

38. The method according to p, where the sugar is used sucrose.

39. The method according to p where used hormones are selected from the group of the auxin, including 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5 trichlorophenoxyacetic acid (2,4,5-T).

40. The method according to p, where induction of secondary embryogenesis in stage d) use artificial growth medium comprising mineral salts, organic additives of different composition and concentration (sugar, amino acids, protein hydrolysates, vitamins and others), phytohormones.

41. The method according to p, where artificial nutrient medium further comprises agar Il is the artificial or natural counterparts.

42. The method according to any of PP-41, where the sugar is used sucrose.

43. The method according to any of PP-41, where used hormones are selected from the group of the auxin, including 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5 trichlorophenoxyacetic acid (2,4,5-T) and 3,6-sodium dichloro-0-anisic acid (Dicamba).

44. Extract for cell-free systems broadcast and/or coupled transcription-translation obtained by the method according to any of PP-43.

45. The use of the extract according to any one of claims 1 to 4, 25, 44 for cell-free system broadcast and/or coupled transcription-translation.

46. The application of § 45 for cell-free synthesis of oligopeptides, polypeptides, proteins.

47. The use of the extract according to any one of claims 1 to 4, 25, 44 for the production of diagnostic systems for the detection of biologically active compounds with modulating activity on the processes of transcription and/or translation, and/or for screening for such compounds.

48. Diagnostic system on the basis of the extract according to any one of claims 1 to 4, 25, 44 for detection of biologically active compounds with modulating activity on the processes of transcription and/or translation, and/or for screening for such compounds.

49. The application diagnostic system p for detection of biologically active compounds with modulating activity processtransaction and/or broadcast, and/or for screening for such compounds.



 

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FIELD: biotechnology, biochemistry.

SUBSTANCE: invention relates to extracts prepared from vegetable somatic embryos for the cell-free translation system and/or the coupled transcription-translation system. Method involves preparing embryonic callus from the primary material and the embryonic suspension culture. After induction of the secondary somatic embryogenesis extract is prepared from somatic embryos. Based on the extract the diagnostic system is developed for detection of biologically active compounds. Invention provides overcoming the species limitations and strain specificity and to attain the high effectiveness of the cell-free translation system and the coupled transcription-translation system also.

EFFECT: improved preparing method, valuable biological and biochemical properties of system.

49 cl, 5 dwg, 2 tbl, 9 ex

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