The dna fragment, determining sterility oqura and reporting zitoplazmaticescuu male sterility if present in the mitochondrial genome of plants and the sample dna for hybridization with the given fragment

 

(57) Abstract:

The DNA fragment is intended to give the cytoplasmic male sterility in CMS at its introduction into the genome of plants and can be used to create transgenic plants with CMS. To highlight or identify this fragment uses a sample of DNA that has at least 10 consecutive nucleotides labeled with radioactive or non-radioactive means. Given the nucleotide sequence of the DNA fragment and DNA samples. 2 S. p. f-crystals, 14 tab., table 2.

The present invention relates to a biological material possessing male sterility, which is used to develop hybrid varieties of crops of agricultural importance.

It concerns, in particular, plants belonging to the crucifer family, in which the cytoplasm contains organelles, such nucleotide sequence that tells male sterility and high agronomic characteristics.

The development of hybrid varieties can be accelerated or may result from the use of cytoplasmatic system of male sterility. Hybrids obtained by lane is such. One of the obstacles that occur when it is desirable to obtain hybrid varieties of the same quality at a sexual crossing sampledatabase species is the ability of plants to self-pollination. System of male sterility ensure reception of the female plants, which are not capable of samoobsledovaniu and after pollination can be directly taken seeds that are all hydrides, without using time-consuming techniques, such as castration colors.

Genetic determinants of male sterility include that carries the cytoplasm. Each produced sexually generation they were carried solely by the mother. Thus, each gene generation is obtained 100% of the offspring of male sterility and the system cytoplasmatic male sterility (CMS). These genetic determinants are carried by the genome of the mitochondria.

Suitable cytoplasma system of male sterility in cruciferous is defined by the following characteristics:

1. Male sterility must be complete, i.e. there should not be opisannogo product regardless of the conditions of cultivation, regardless of the line, which is desirable DSI, will occur partly from self-fertilization and, therefore, they will not hybrid seeds F1.

2) Getting these seeds should be implemented, taking advantage of natural pollinated vectors, that is, in the case of these species pollinated with the help of Hymenoptera, Diptera and fertilized by pollen transported by wind. Pollen must be transferred from pollinating plants to male sterile (female) plants. Almost in the mustard family, such transfer may be made only insects.

Female plants, thus, should be sufficiently attractive to insects, which fly to them and collect nectar. Morphology of flowers should be such that insects accomplished this task through the top of the flower so that their breast was in contact with the stigma of the pistil. In practice, this leads to a situation in which the base of the petals should form a certain type of straw around the base of the pistil.

3) the Morphology of the female organs (pestle) must be identical to the morphology of the fertile plants, especially plants with one petal in the flower, and to be rectilinear in shape. Male sterility is often cited olnie flowers. Sometimes formed in this way, the pistil or pistils also deformed. All these deviations from the norm does not allow to obtain good seeds and it can be considered as kb somewhat female sterility.

4) For hybrid varieties F1 in those species, which are collected seeds, such as rapeseed or mustard, it is important that the male parent of the hybrid completely excluded the effect of male sterile cytoplasm, so that the hybrid plants were easily pilalis.

The first case of male sterile cytoplasm or CMS in the mustard family, was described by Ogura (1968 ) in radish Raphanus sativus. Bannerot (1974, 1977) moved the Ogura cytoplasm in Brassica, thus generating plants, with cytoplasmatic male sterility. These same plants do not possess the required agronomic characteristics (chlorosis at lower temperatures, weak female fertility), which results in low yields, therefore they are unsuitable for commercial use.

To correct chlorosis in plants cruciferous nuclear and chloroplasts genomes of the same kind should be combined in the same cell. Thus, plants of the species Brassica with one of chlorine show full cytoplasma male sterility, but, however, the flowers will have abnormal morphology, which makes it impossible for them pollination natural vectors.

In addition, for those species, the seeds are interested, it is very important to restore male fertility in hybrid species that marketroid by nuclear genes, known as genes reductants.

It is difficult to restore the male fertility of the plant with all the fullness of the mitochondrial genome Ogura, since it is necessary the simultaneous participation of several genes reductants.

The authors conducted with the aim of obtaining a suitable system of male sterility by deleting the genes responsible for desirable characteristics of the Ogura cytoplasm with preservation of male sterility, which is effective and easily restored.

Thus, the present invention relates to a DNA sequence, which hereinafter will be treated as a DNA sequence with Ogura sterility, which differs in that:

a) it carries the DNA sequence, linked nucleotides 928 and 2273, as shown in Fig. 1-9, or

b) it is at least 50% homologous to the sequence shown, mentioned www male sterility specified plant.

In particular, an object of this invention is the DNA sequence with Ogura sterility, characterized in that:

(c) represents the sequence between nucleotides 928 and 1569, as shown in Fig. 1-9, or

d) at least 50% homologous to the sequence specified in (c),

moreover transcribed into RNA in mitochondria of plants with male sterility.

Next, specify the accompanying description of the figures:

Fig. 1-9: the Nucleotide sequence of the mitochondrial DNA fragment of Ogura radish, bearing the sign of the CMS.

Fig. 10: Restriction map of the mitochondrial DNA fragment described in Fig. 1-9.

Fig. 11: Electrophoresis of mitochondrial DNA after cleavage BglI (3a) and NruI (3b). The detected bands correspond hybridization with a sample of CoxI (Hiesel and others, 1987).

Fig. 12: Electrophoresis of mitochondrial DNA after cleavage SalI.

The detected bands correspond hybridization with a sample containing the sequence between nucleotides NN 389 and 1199 of the sequence described in Fig. 1-9.

Fig. 13: Fruit cabbage plants carrying different cytoplasma genomes.

Fig. 14: Electrophoresis of mitochondrial RNA. Found the, related to ORFB.

The DNA sequence with Ogura sterility is defined in terms of consistency between rooms 1 and 2428, as shown in Fig. 1-9. She tolerated transcribed sequence in which the 3' and 5' ends are connected broken line in Fig. 10 and which is observed only in plants with male sterility. ORFB corresponds to an open reading frame; this designation is given on the basis of the observed homology with the sequence described Brennicke. In Fig. 10, the sequence corresponding to one of the transfer RNA genes, darkened.

The DNA sequence between nucleotide number 928 and 2273, as shown in Fig. 1-9 corresponds to the transcript, which can be visualized by molecular hybridization (1, 4), as shown in Fig. 14. In Fig. 14, each cell corresponds to a fertile plant (F) or plants with male sterility (S). Only plants with male sterility synthesize transcript of approximately 1400 bases. This transcript begins with the provisions 928 (10 bases) the sequence shown in Fig. 1-9 and ends at position 2273 (5) (start and end of transcription can occur in different positions in the plant alnost DNA with not less than 50% homology with the sequence between nucleotides 928 and 2273, Fig. 1-9, indicating signs of a CMS, or concerns of cytoplasm containing a DNA sequence having at least 50% homology with the sequence between nucleotides 928 and 1569, Fig. 1-9, and transcribed into RNA, indicating the sign of the CMS and characterized in that:

- contains the chloroplasts of the same species as the nuclear genome or other species, but which is compatible with the nuclear genome, is not contains does monohedral genome Ogura, as defined below:

* carrying one of the two formylmethionine gene transfer RNA that is used to initiate translation.

* carrying CoxI gene encoding subunit N 1 cytochromoxidase.

The absence of these fragments, referred to as "junk sequence", is necessary in order to obtain mitochondrial genomes, the corresponding male sterility high quality, corresponding to the four defined above characteristics.

According to a further aspect, the present invention relates to a recombinant plant or a kernel mitochondrial genome, characterized in that it contains posledovatelnosti, it is shown in Fig. 1-9, or

b) which is at least 50% homologous to the sequence shown, referred to in (a),

and reports in the case of the presence in the cytoplasm of plants cytoplasma male sterility specified plant.

In particular, one of the objects of this invention is the recombinant plant core or mitochondrial genome, characterized in that it contains a DNA sequence with Ogura sterility,

c) which represents the sequence between nucleotides numbered 928 and 1569, Fig. 1-9, or

d) which is at least 50% homologous to the sequence shown, referred to in c),

and reports in the case of the presence in the cytoplasm of plants and transcription into RNA cytoplasma male sterility specified plant.

Nuclear or mitochondrial genome, which meets the present invention can vary the fact that this recombinant mitochondrial genome is devoid of all or part of the genomic fragments Ogura:

- carrying one of the two genes formylmethionine transfer RNA used for initiating broadcast,

- carrying CoxI gene encoding subunit N 1 of cytochrome C oxidase, or in which the specified freeney, may be different:

1) the fact that he is deprived of all or part of approximately 10,7 kb after evaporation with or BglI fragment of approximately 11 kb after digestion with NruI, which (fragments) are CoxI gene.

This is illustrated, in particular, as shown in Fig. 11, molecular hybridization with sample carrier CoxI sequence,

2) the fact that he is deprived of the whole or part of a 5.1 kb after digestion with SalI, or a fragment of approximately 15 kb after digestion with NruI, or fragment about 18,5 kb after digestion with BglI, which (fragments) are one of the two genes formylmethionine transfer RNA.

This is demonstrated, in particular, as shown in Fig. 12, molecular hybridization with sample related nucleotides NN 389 and 1199 of the sequence described in Fig. 1-9.

In Fig. 11 and 12 genotypes, designated by the numerals correspond to plants with a suitable system of male sterility (see tab. A).

In addition, the presence of characteristic CMS high quality necessitates the presence of DNA sequences that can be identified by hybridization of DNA/DNA handling restrictase. Thus, the present invention relates to posledovatelnostyu fragment of 2.5 kb after splitting McoI, gives a fragment of 6.8 kb after splitting NruI and gives a fragment of 4.4 kb after SalI cleavage.

This sequence can also be identified by hybridisable on total RNA of plants with male sterility. Illustrated transcript of approximately 1400 bases. It is absent in plants, returning to fertility.

The definition of "unwanted" nucleotide sequences and nucleotide sequences "essential" for sterility according to this invention allows to choose plant material carrying the chloroplasts, which are compatible with the nuclear genome and mitochondria are of high quality, by DNA hybridization, a method well known to specialists in this field, not expecting this adult plant species and the appearance of flowers and fruits.

Thus, the consumer has a highly effective tool for selection of plants having the cytoplasm male sterility with high agronomic characteristics.

According to a further aspect, the present invention relates to mitochondrion, characterized in that it comprises the nucleotide sequence corresponding to the DNA, imagazine in Fig. 1-9, and encoding cytoplasma male sterility Ogura; or for mitochondrion, including the DNA sequence between nucleotides 928 and 1569, Fig. 1-9, or has 50% homology with this sequence, which is transcribed into RNA in mitochondria of plants with male sterility. This DNA may have, in addition, properties that are defined above, in particular, it might not undesirable sequence.

The present invention also concerns the cytoplasm cruciferous, characterized in that it comprises the DNA sequence with the "Ogura sterility"; the cytoplasm includes, in addition, the chloroplasts of the same or other species, but which are compatible with the nuclear genome.

The sequence with Ogura sterility is different because:

a) is contained in the DNA sequence at 2428 base pairs, as shown in Fig. 1-9.

b) is located between nucleotides 928 and 2273, shown in Fig. 1-9, and corresponds to the transcript, which in Fig. 10 shows a broken line, and visualized by molecular hybridization (1, 4), as shown in Fig. 14,

c) is not less than 50% homology with the specified sequence in (b) and if it prnu, or

(d) represents the sequence between nucleotides 928 and 1569, and it is transcribed into RNA in mitochondria sterile plants, or

e) is not less than 50% homology with the sequence described in (d), and is transcribed into RNA in mitochondria sterile plants.

The present invention also concerns plants of the family Brassicaceae, characterized in that it contains chloroplasts and nuclei of the same species or compatible, and mitochondria carrying the gene, indicating the sign of a CMS, as defined above.

In particular, the present invention also concerns plants belonging to the Brassica family (cabbage), characterized in that it contains chloroplasts and nucleus Brassica and mitochondria carrying the gene, giving a sign of CMS, as defined above.

These mitochondrial genomes should also be a certain number of genes in plants of the Brassica family. This is achieved by recombination between the genome Ogura and Brassica genome.

The present invention relates, in particular, plants belonging to the species Brassica napus, characterized in that it contains in itself the core of Brassica, and the fact that the cytoplasm contains chloroplasts and sterile Brassica male m the and also the largest part of the mitochondrial gene of Brassica napus (18S, Atp9, Atp6, CoxII, ndh1, cob). Brassica napus corresponds to rape or canola and kb.

The present invention relates to plants of the species Brassica oleracea, characterized in that it comprises the core of Brassica and the fact that the cytoplasm contains chloroplasts Brassica and mitochondria that encloses the DNA sequence encoding the sign of the CMS, which is defined above.

Brassica oleracea covers various types of cabbage plants: cabbage with round shape of the head of cabbage, Brussels sprouts, kohl-rabi, Broccoli, fodder cabbage and cauliflowers.

The present invention also concerns plants varieties of cabbage (Brassica campestris), characterized in that it comprises the core of Brassica and the fact that the cytoplasm contains chloroplasts Brassica, which is compatible with the nuclear genome and mitochondria, including the DNA sequence encoding the sign of the CMS, which is defined above.

Brassica campestris corresponds rapeseed, turnip and Chinese, Chinese and Japanese cabbage.

The present invention also concerns plants, selected from the group including varieties of B. juncea, B. nigra, B, hirta and B. carinata, characterized in that contain the core of Brassica, and the fact that the cytoplasm contains chloroplasts Brassica, which somestyle above.

According to the following aspects of the object of the present invention is a plant belonging to the cabbage family (Brassica), whose nuclear genome is comprised of a sequence with Ogura sterility, which is defined above as well as the active elements on the expression and transport of product broadcast in mitochondrion. This plant, in particular, belong to one of the following species: B. napus, B. oleracea, B. campestrus, B. nigra, B. juncea, B. hirta, B. carinata.

The presence of sequences with Ogura sterility" is necessary and sufficient to induce the complete absence of pollen in the absence of genes reductants. The pollination of these plants is usually the result of good production of nectar.

The morphology of the female organs normal and fruits (pods), which are formed, contain the normal number of seeds. In Fig. 13 shows the morphology observed in normal control plants (z), plants with abnormal morphology, with the complete genome Ogura (z(6)) and chloroplasts Brassica oleracea cabbage plants carrying the Brassica napus chloroplasts and mitochondria from male sterility, with the genes of Brassica napus (z(A)), and in plants carrying the Brassica oleracea chloroplasts and recombinant mitochondria characteristics (see table. B).

Genotypes z(A) and z(6) no longer have the need cytoplasmatic system with male sterility.

Such plants can be obtained, for example, by fusion of protoplasts or in other ways, which is good recombination between mitochondrial genome data of the species and the mitochondrial genome Ogura. These plants fertility is restored by a single gene reductant, which is referred to as Rf1, the source of which is a radish that does not happen in plants, which are completely undesirable mitochondrial genome. Such plants can be obtained by natural or artificial reproduce sexually.

Plants that have mitochondrial genome, corresponding to this invention, can be obtained by gene transfer in mitochondrion.

In all cases, these plants have desirable CMS, namely:

- complete male sterility,

- morphology, allowing for good pollination and good production of seeds, as illustrated in the table. 1 and 2.

Thus, the present invention relates to a method of producing hybrid plants, clickindia.com or nuclear genome, able to interbreed with normal plant in the case of food or feed crops, or plant, giving a restoring fertility gene Rf1, when should be collected seeds. The present invention relates to hybrid plants obtained by the given path.

Generally speaking, better agronomic quality is achieved in the case of plants with male sterility, with chloroplasts of the same species as the nucleus or mitochondria with an appropriate system of male infertility.

In table. 1 shows the productivity of cabbage lines with different cytoplasm (suitable are the genotypes z9 and z17). In table. 2 shows the productivity of rapeseed lines with different cytoplasm (suitable are the genotypes Fu27, Fu58 and Fu85).

Factory default setting: the Number of pods on m2- WISq: Weight per pod (mg) - NSdPSq: Number of seeds per pod

NSd: Number of seeds per m2- WISd: Weight per seed (mg) - TPM: Total dry weight (g/m2)

HI - harvest Index (%) - YLD: Yield (qx/ha); HT: Height (cm)

These plants often have deformed fruits (pods).

The subject of this invention is also a sample comprising a sequence of at least 10 frames, predpochtite machina, for example, radioactive or other means, for example by means of fluorescence. This sample can be used to demonstrate male sterility and can be used, in particular, for the selection of clones.

Some distinctive features and advantages of this invention will become more clear from the description of the following examples,

EXAMPLE 1: ILLUSTRATION OF A DNA SEQUENCE RESPONSIBLE FOR CYTOPLASMA MALE STERILITY OGURA

1. Plant. (referring to the hybrid in the future. mean shape obtained by fusion of isolated protoplasts and subsequent regeneration of whole plants. This method of production allows to obtain information about the cytoplasm from both parents who are simultaneously in the cell. Cybrid N 13 obtained from plants up to 820, regenerated mergers protoplasts Ogura-cms, resistant to triazine of cybrid B. napus (offspring from Cybrid 77, described in the publication Pelletier and others, 1983 and Chetrit and others , 1985 ) and are sensitive to triazine and fertile varieties Brutor. The test for resistance to triazine (Ducruet and Gasquez, 1978) was carried out on samples of leaves each regenerant giving the type of chloroplast (resistant to triazine holdem is to be determined. Carried out the cultivation of these plants, and observed the following stages of flowering. Plants exhibiting a non-parent combinations (as sensitive male sterility, and persistent / male fertility) were selected as cibreo (Cybrids). Cybrid N 13 was sensitive male sterility zimrida. Cybrid 1 was resistant / male fertility zimrida.

2. Isolation of nucleic acids.

Full DNA was isolated from leaves from four-week plants according to the method described by Dellaporta (1983), Mitochondrial DNA was extracted from leaves of 8-week-old plants as described Vedel and Mathieu, 1982, with the following options:

The mitochondria were not purified on sucrose gradient before lysis and lysis was performed in 4% sarkosyl containing 0.5 mg/ml proteinase (Bochringer Mannheim GmbH) in 50 mmol. Tris, pH 8, 20 mmol. ethylenediaminetetraacetic acid (EDTA). After deposition of mitochondrial DNA was purified by centrifugation on a gradient italianbased/caesium chloride (Method 1 - Vedel and Mathieu 1082 g) in test tubes from polyallomer.

Total RNA was isolated from leaves or flower buds according to the method of Logemann and others, 1987

Methandrostenlone analyses of mitochondrial DNA and electrophoresis on agarose gel.

These procedures were carried out as described by Pelletier and others, 1983 Full or mitochondrial RNA was introduced into gels for electrophoresis containing formaldehyde as described Sambrook and others, 1989

4. Hybridization

Carried out the transfer of DNA or RNA to nylon filters (Hybond-N, Amersham) by capillary absorption with 6 x SSC or 10 x SSPE, respectively, according to the manufacturer's instructions. Pre-hybridization and hybridization were carried out according to Amersham using samples labeled by a labeling system multiprimer DNA (Amersham) after purification in a column with Sephadex G50 (Sambrook and others, 1989).

5. Cloning of mitochondrial DNA

There were built two genomic libraries cebrenia lines with male sterility (13 - 7) and revertant (13 - 6) in the phage vector EMBL3, kultivirovanii restriction in the strain E. coli Nm539 (Frischauf and others, 1983). Was approximately 2,5104clones g mitochondrial DNA.

Mitochondrial library DNA were analyzed and were inoculated to isolate colonies, which were transferred to nylon filters, as described in Sambrook and others , 1989 Hybridization probe used for screening two libraries mitochondrial Politowaniem procedures gene cleanupTM(BIO 101 INC.) from the product of cleavage of mitochondrial DNA by preparative agarose gel. Allerona DNA was then marked, as has already been described.

Extraction of lambda DNA, subclavian fragment of 2.5 NcoI in the NcoI site of pTrc99A (Amann and others, 1988) and extraction of DNA plasmids was carried out according to the procedure of Sambrook and others, 1989 Recombinant plasmids were introduced into strain E. coli NM522 (Gough and Murray, 1983).

6. Genetic study of cybrid 13 and its progeny.

In the first generation progeny obtained by pollination of cybrid 13 through Brutor, which included 13 plants, five plants were completely male sterile (including plants 13-2 and 13-7), one was male fertility (N 13-6), 7 plants were completely sterile with multiple flowers with male fertility.

Fertile plant 13-6 were self-pollinated and was crossed with Brutor. In both cases it was only fertile plants (respectively 42 and 43).

In crosses of plants with male sterility N 13-7 and Brutor, 24 offspring were completely sterile and 6 plants had several of fertile florets, a result similar to that observed in cybrid. Plants 13-2 crossed with the line is realnosti (Chetrit and others, 1985). Offspring from this mating consisted of 53 plants with male sterility, 37 plants with male fertility and 9 plants, which were almost completely sterile, although they had several of fertile florets. These results confirm that plants with male sterility of the family of cybrid 13 incorporate the determinants Ogura, as well as other Tubridy studied previously with easier recovery profile (Chetrit and others, 1985).

At this stage can be considered two possibilities: Either cybrid 13 contains a mixture monohedral genomes "male fertility and female sterility, and it is possible to select two additional phenotype, or cybrid 13 contains recombinant mitochondrial genome with unstable structure, which becomes more persistent "fertile" configuration, and already it is impossible to save phenotype with homogeneous male fertility in subsequent generations.

Plants with male sterility obtained from progeny plants with male sterility N 13-7, cultivated by cutting cuttings and breeding sexually with Brutor. After different numbers of generations (1 - 5), carried out in both ways, all families gave the sterile plants.

In light of these results we can conclude that it is the second proposed above explanation, i.e. that cybrid 13 carries unstable mitochondrial genome, which loses the determinants Ogura-cms during this process, leading to "fertile" configuration without the possibility of returning to the sterile phenotype.

7. Comparison of mitochondrial DNA revertants offspring with male sterility and fertility. Isolation of fragments, specific for plants with male sterility.

Mitochondrial DNA was extracted from leaves of offspring with male sterility 13-7 and fertile revertants (13-6 or 13-7 offspring) and was boiled with some restriction enzymes to compare their restriction profiles. Mitochondrial genomes of these two types are very similar, because no differences were observed between the restriction profile of the mitochondria with the male sterile and fertile revertants using different enzymes. However, it was discovered restriction enzyme to 6.8 kb in the restriction profile of the mitochondrial DNA of plants with male sterility treated with NruI, and he was never observed in the respective profiles fertiltiy and was used as a test for the restriction profiles of mitochondrial DNA NruI: broad signal, corresponding to 6.8 kb was observed in the offspring from the all-male sterility of cybrid 13, while no fragment of this size was not hybridisable with this breakdown in the genomes of mitochondria fertile revertants. In addition, the sample N 6,8's hybrid with a fragment of 6.8 kb in mitochondrial DNA Ogura, boiled with NruI, but not in B. napus cv Brutor that shows the origin of this fragment from Ogura.

The lambda library, containing extracts from mitochondrial DNA originating from plants with male sterility (13-7), was tested with elyuirovaniya labeled fragment, and from 8 hybridizers clones were isolated 2 recombinantly phage containing the full fragment N 6.8 and neighboring sequences. Was obtained detailed restriction map of this zone. Hybridization of restriction profiles of mitochondrial DNA, derived from fertile and sterile offspring of cybrid 13 N 6,8 as samples, suitable for zone-specific genotype of male infertility, should be limited to a fragment of 2.5 kb.

Fragment of 2.5 kb NcoI were marked and used as the sample in relation to mitochondrial DNA originating from the seed of 13-7 and 13-6, boiled with NcoI. Not counting signal corresponding to 2.5 is expressed as in fertile revertant, and in the profiles of male infertility; these fragments are fragments of 2.2, 10 and 14 kb. Fragments of 2.7 kb NcoI strongly hybridize in the mitochondrial genome of fertile offspring, but not in the genome sterile offspring. The analysis of this profile hybridization allowed us to conclude that the fragment of 2.5 kb NcoI, although specific for mitochondrial DNA from male sterility, but encompasses sequences that are repeated elsewhere in the mitochondrial genome (2.2 - 10 - and 14-kb fragments after NcoI), and these repeated sequences are also present in the mitochondrial DNA of fertile revertants, not counting the specific fragment of 2.7 kb.

Total RNA is extracted from the leaves or buds of the seed of zibido 13, or zibido male sterility or fertility (obtained in other experiments merger), and line Brutor. Was carried out by Northern-blotting and replicas were hybridisable with the sample corresponding to the insertion of the lambda clone containing N 6,8 described in example 3. The primary transcript of 1.4 kb was detected in all Zebidah male infertility, including cybrid 13-7, while no transcript of this size were not found in the line Brutor or two verse with this breakdown, which is absent or present in very low concentrations in all tested hybrids with male sterility.

Some transcripts that are common to all samples hybridize weakly with this breakdown due to the large size of the labeled insert. This confirms that mitochondrial transcripts can be detected in samples of total RNA by Northern hybridization of the same replica DNA fragment comprising a gene sequence atpa.

The same specific transcript of 1.4 was found in Ogura mitochondrial RNA extracted from cauliflower, using as a sample fragment of 2.5 NcoI. The extraction limits of this transcript were determined using as a sample fragment subclones of 2.5 NcoI.

8. The study of cybrid 1 and its progeny.

Cybrid 1 was male fertility. In this progeny plant 1.12 was fertile and plant 1.18 was sterile. Plant 1.12 gave in his offspring sterile plants (S3), and fertile plants (RF3). Plant 1.18 gave sterile plants (S2and fertile branch (RF2). Plants S2and S3it was restored by the same nuclear gene that restores do a 2.5 kb NcoI mitochondrial DNA plants S2and S3did not give the signal fragment of 2.5 kb by digestion with NcoI signal or fragment of 6.8 kb by digestion with NruI.

Similarly, hybridization with total RNA (Northern blotting) with the sample, the corresponding sequence ORFB, did not give a signal, corresponding to 1.4 kb, as it happened with sterile zimrida 13. In contrast, the sample corresponding to the sequence between nucleotides 928 and 1569, shown in Fig. 1-9, gave the signal when Northern-blot, corresponding to about 1.3 kb. This signal is absent in RNA plant RF1, RF2, RF3or Brutor. Similarly, you can use this sequence (928 - 1569) as a probe in datamatching total RNA, and in this case will give a signal only plants with male sterility and every one of them.

These results show that plants S2and S3although they and male infertility, but not stored in the nucleotide sequence shown in Fig. 1-9, the original conformation, and they show that in this sequence the plot, linked nucleotides 928 and 1569, is such a plot, which is "specific determinants" Ogura sterility", which gives the plants the male steriles the school homology with the sequence, present in the data Bank.

EXAMPLE 2. ILLUSTRATION OF UNWANTED SOURCES IN THE MITOCHONDRIAL GENOME

Received a collection of hybrid family of B. napus by protoplasts merger between rape, bearing the Ogura cytoplasm, and conventional canola. The first is a plant with a male sterile and lack of chlorophyll at low temperature, while the latter is usually green and fertile. Tubridy were selected from among the regenerated plants and those who had male sterility and were usually green, were adopted.

In the same case, received a collection of cibreo in the family of B. oleracea by protoplasts merger between cabbage, bearing the Ogura cytoplasm and regular cabbage. Tubridy who had male sterility and were usually green, were selected from among the regenerated plants.

These Tubridy crossed with various kinds of rape in the first case and cabbage in the second case. Hybridization was repeated for each generation with the same species, so to get a specific genotype, close to the genotype of a recurring type.

These different types in this way transformed the cytoplasm of various Zubrilov, were subjected to agronomice the interest of producing nectar for the implementation of dusting insects and normal floral morphology with the to this dusting was effective and fruits developed normally.

Collection of Zubrilov, thus, can be divided into two parties:

- the party of zibido with male sterility, suitable for commercial gain seeds.

- the party of Zubrilov, not having all the advantageous characteristics for the proper commercial collection of seeds.

So, for example, rapeseed Tubridy NN 27, 58 and 85 and cabbage Tubridy NN 9, 17, 21, 24 and 27 belong to the first party.

Rape Tubridy NN 23s, 77 and 118 and cabbage Tubridy NN 1, 6 and 14, for example, belong to the second party.

Full DNA of these zibido was processed with SalI, NcoI, NruI, BglI, PST and Cloned. Received replica Southern was hybridizations with different mitochondrial samples Atpa, Cob, Cox1, Atp6, 26s and 18s and two fragments of the genome Ogura 2.5 kb, obtained by splitting NcoI and to 19.4 kb, obtained by splitting NruI.

Both parties of zibido are different because:

a) NN 23s, 55 and 11s in rapso and 1, 16 and 11 in cabbage include the area of the genome Ogura, which surrounds the Cox1 gene, recognized by fragments of 10.7 kb BglI or 11 kb NruI and the area of the genome Ogura, which surrounds one of formylmethionine transport genes RNA recognizable fragments 5e were replaced as a result of recombinations between the genomes of the two parents, which were merged similar areas mitochondrial genome of rapeseed NN 27, 58 and 85 and cabbage in NN 9, 17, 21, 24 and 27c.

From this we can conclude that both this area of the genome Ogura undesirable, if it is desirable to have a system of male sterility for commercial gain seeds.

EXAMPLE 3.

This example illustrates the importance of knowledge of the sequence of male sterility Ogura and unwanted sequences for directly carrying out the selection of Zubrilov, without waiting for several years of back-crossing and without agronomic trials.

The protoplasts of Brassica plants carrying the Ogura cytoplasm, were fused with protoplasts of the species Brassica. Colonies from the confluence were cultured in conditions of "in vitro" and were regenerated on medium, which accelerated the formation of pods (see Pelletier and others, 1983).

One gram of fresh material, or callous, or a fragment of regenerated seedlings by the above methods, you can select the entire DNA. After processing SalI hybridization by Southern with a sample representing the sequence between nucleotides NN 389 and 1199 (Fig. 1-9) will give a signal only for a fragment of 4.4 Cox1 gene must be received signal for a fragment other than 11 kb.

These data allow us to predict that a plant that has been received is really sterile and suitable for commercial gain seeds.

EXAMPLE 4.

This example is a variation of example 1, based on the idea of sexual crossing two parents in special circumstances or with certain genotypes instead of implementing protoplastic mergers. The result is that in contrast to known methods of fertilization there is a mixing of protoplasm of ooster and pollen tube or male gametes. If such methods have been described, it could be done earlier screenings already known on young plants, originating from these artificial insemination procedures with the use of these samples and the same criteria as in example 3.

EXAMPLE 5

This example illustrates the importance of knowledge sequence Ogura sterility in the type of genetic manipulation, which has already been described for yeast (Johnston and others, 1988).

Using the usual (normal) plant Brassica, meristem or alternative cells "in vitro" bombarded with particles coated with DNA carrying the sequence Ogura sterility. The plants obtained in the d, if DNA will be able to enter into the mitochondria and become integrated into the genome of these organelles. This procedure eliminates the problems caused by unwanted sequences when there are chloroplasts Ogura radish or defined in this way the sequence of the mitochondrial genome.

EXAMPLE 6.

This example illustrates the importance of knowledge sequence "Ogura sterility" in building a nuclear, not cytoplasmically sterility according to theory of Mendel.

When used as a source of DNA sequence between nucleotides 928 and 1569 can be constructed chimeric gene to be transcribed after genetic transformation of Brassica cells or other gene in the nucleus of the cells get transformed plants. If the chimeric gene includes a preliminary sequence that allows its protein product broadcast be entered in mitochondrion, these transformants will have male sterility and the trait will manifest itself as a dominant Mendelian trait.

References

1. Amann, E., Ochs, B. and Abel K-J. (1988) Gene 69:301-315

2. H. Bannerot, Boulidard L., Y. Cauderon and Tempe, J. (1974) Proc Eucarpia Meeting Cruciferae 25:569:361-366

5. Dellaporta, S. L., J. Wood and J. B. (1983) Plant Mol Biol Rep 1:19-21

6. Ducruet J. M. and J. Gasquez (1978) Chemosphere 8:691-696

7. Frishauf A. M., Lehrach H., Poutska A. and Murray, N. (1983) J Mol Biol 170:827-842

8. Gough J. and Murray, N. (1983) J Mol Biol 166:1-19

9. Hiesel, R., Shobel W., W. Schuster and Brennicke, A. (1987) EMBO J 6: 29-34

10. Johnston S. A., Anziano P. Q., K. Shark, Sanford J. C. and Butow, R. A. (1988) Science 240:1538-1541

11. Logemann, J., Schell, J. and Willmitzer L. (1987) Analytical Biochem 163:16-20

12. Ogura H. (1968) Mem Fac Agric Kagoshima Univ 6:39-78

13. Pelletier G., Primard C., F. Vedel, Chetrit P., Remy R., Rousselle P. and Renard, M. (1983) Mol Gen Genet 191:244-250

14. Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

15. Stern D. B. and Newton, K. J. (1986) Methods Enzymol 118:488-496

16. F. Vedel and Mathieu C. (1982) Anal Biochem 127:1-o

1. The DNA fragment, determinedi Ogura sterility and reporting cytoplasmic male sterility if present in the mitochondrial genome of plants with the following nucleotide sequence 1 shown in C.

2. Sample DNA for hybridization with a DNA fragment under item 1, having at least 10 consecutive nucleotides labeled with radioactive or non-radioactive means, sequence 2, is shown in C.

 

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