KR100271097B1 - Production of male sterile plants using RIP gene of dianthus sinensis L. - Google Patents

Abstract

PURPOSE: Provided is a method for producing male sterile plants by using a RIP gene isolated form Dianthus Sinensis L., thereby mass producing male sterility plants. CONSTITUTION: A method for producing male sterile plants by using a RIP gene isolated form Dianthus Sinensis L. is composed of following steps: (a) identification of a plant producing ribosome-inactivation proteins; (b) cloning cDNA of a RIP gene from Dianthus Sinensis L.; (c) analysis of the RIP gene from Dianthus Sinensis L.; (d) estimating organic system of the RIP gene from Dianthus Sinensis L. from genome, to observe expression of the RIP gene from Dianthus Sinensis L. in E. coli; and (e) inducing male sterility using fertilized RIP gene from Dianthus Sinensis L.

Description

Production of male sterile plants using RIP gene of dianthus sinensis L.}

The present invention relates to a method for separating and identifying ribosomal-inactive protein (RIP) genes from Dianthus sinensis L. and producing male infertile plants using the same.

Ribosome-inactivated proteins were originally known as toxins, ricin and arbin isolated from the seeds of Ricinus communis and Abrusprecatorius. In 1887 dixon first doubted that the main toxicity of castor beans was protein. Stillmark then refined a protein called ricin and attributed its toxicity to the properties of agglutinating erythrocytes (Barbieri et al., 1993). In the late 1960s, Lin et al. (1970) reported that these toxins are more toxic to cancer cells than normal. After this report, much work has been done to define the double chain structure of RIP, and the inhibitory activity on protein synthesis has been clarified. The first single chain of type I RIP, defined, was the constellation antiviral protein (PAP). It was isolated as a major activator of PHYTOLACCA americana leaf extract that prevented transmission of infection of TMV (Wyatt et al., 1969; Obrig et al., 1973) and was purified from spring leaves that inhibited eukaryotic protein synthesis (Irvin, 1975). Endo and Tsurugi (1987) clarified the activity mechanism of RIP by discovering the RNA N-glycosidase activity of these proteins.

Ribosome-inactive proteins are widely distributed and appear to be ubiquitous in the plant kingdom. Various RIPs have been described from about 50 plants covering the 17 groups. (Caryophyllaceae, Cucurbitaceae, Euphorbiaceae, Liliaceae, Phytolaccaceae, Gramineae, Amaranthaceae, Chenopodiaceae, Portulacaceae, Asparagaceae, Nyctaginaceae, Fabaceae, Viscaceae, Passifloraceae, Caprifoliaceae, Leguminoseae, Loranthaceae). Some families include Carophyllales, which has a higher priority than Caryophyllales, and many RIP-producing species, such as the Cucurbitaceae, Euphorbiaceae, and Poaceae. Ecologically, RIP-producing plants are of different types and are found in weeds, tropical trees, annual plants in spring, desert cacti and certain parasites. RIP was detected in most plant cells and tissues, including embryos, fresh fruit, roots, leaves, bark, and latex. RIP was identified and purified from monocotyledonae and dicotyledonae. Translation protein inhibitory activity was detected from 52 tested species belonging to monocotyledonae. And 122 species were detected from 292 dicotyledonae tested (Grasso and Shepherd, 1978; Gasperi-Campani et al., 1985; Stripe and Barbieri, 1986; Barbieri et al., 1993;).

Most of the purified RIPs so far are similar to one chain type I RIP, but not identical to each other and found in different parts of the flora and fauna. Thus, more than one type I RIP has been demonstrated, Phytolacca americana (Irvin, 1975; Irvin et al., 1980; Barbieri et al., 1982; Houston et al., 1983) and Saponaria officinalis (Stripe et al., 1981; Fabbrini et al., 1997) from leaves, roots and seeds. Most species containing RIP have a type RIP of type l or type II except for Sambucus ebulus (dwarf elder). Interestingly, the leaves of dwarf elder produce both type I and type II RIPs (Benito et al., 1995).

RIP is found in all plant tissues, including leaves, roots, fruits, and seeds. And other organisms produce RIP in the same plant (Gasperi-Campani et al., 1985; Barbieri et al., 1993). Dianthin30, isolated from Dianthus caryophyllus, was found in plants containing seeds. But dianthin 32 was found only on leaves and growing young branches. Dianthins are much higher in older leaves, which represent proteins that can extract 1-3% than young leaves (Reisbig and Bruland, 1983). RIP can be divided into two classes, B-chain with lectin activity and dependent on the presence or absence of polypeptide chains. Type l, which consists of single-chain polypeptides, is more diverse than type II RIP, most of which are glycoproteins. Double-chain type II RIPs consist of two or four polypeptide chains having a molecular mass of approximately 60,000 or 120,000. One of its chains is the A chain (region of the catalyst) with RNA N-glycosidase activity and the one is the B chain (cell binding region) with galactose specific binding activity. Type II RIP binds to cells by the interaction between cells and surface galactosides. Subsequent internalization and delivery of the A chain to the cytosol cause cell death. Because the cells are bound and allow the activity of galactose-specific lectin regions, and type II RIP is much more toxic than intact cells against intact cells.

Removal of genes using tissue-specific promoters to control the expression of genes of cell destruction is an effective technique for killing specific cells. Removal techniques help to examine cells for cellular interactions as well as for more applied investigations (Goldman et al., 1994; Strittmatter et al., 1995), such as regulation of fertility and disease resistance (Day et al., 1995). Cell-breaking genes encode proteins that, when expressed intracellularly, can cause cell death. Many different proteins can cause cell death, and some are used for the removal of genes in plants and animals, including diphtheria toxin A (DTA), exotoxin A, RNase T1 and barnase. Both DTA and exotoxin A cause NAD ⁺ -dependent ADP-ribosylation of elongation factor-2 and inhibit translation. Ricin A toxin also inhibits protein synthesis by catalyzing the depurination of 28S ribosomal RNA. Barnase and RNase T1 have RNase activity and interfere with protein synthesis by RNA degradation in cells (Day and Irish, 1997). In animals, ricin A is a potent toxin used to remove genes (Moffat et al., 1992) but has not been demonstrated to have toxic effects in plant cells (Day and Irish, 1997). Ricin A chains are mutated in yeast to produce temperature-sensitive and cold-sensitive mutations. One cold-sensitive mutation produces temperature-dependent clearance of eye cells in Drosophila when expressed under the control of an eye-specific sev enhancer (Moffat et al., 1992). For efficient gene removal, it is important to have suitable promoters to regulate the spatial and transient expression of cytotoxic genes. One problem with the use of tissue-specific promoters for gene removal is that the basal level of expression is still sufficient to kill all cells (Stritmatter et al., 1996). Nevertheless, many plant promoters have rigorous spatial and temporal properties and have been successfully used to regulate cytotoxic gene expression specifically in anther, seed, ovule and floral meristematic tissues (Mariani et al., 1990; Czako et al. , 1992; Goldman et al., 1994; Day et al., 1995).

Improvement of crop plants through the production of hybridized varieties is an important objective of plant improvement. Hybrids between natural lines often become descendants with high yields, increased resistance to disease, and increased performance in various environments compared to their parental lines. This is called hybridization growth and plays an important role in agriculture. The production of a large range of hybridized seeds is stimulating. Because many crops have the same vegetative organs, stamens and pistils in the same flower or in separate flowers for the same plant. This arrangement produces a large range of designated crosses between natural lanes that are high levels of magnetization and difficult to achieve. To ensure that crossbreeding takes place to produce hybridized seeds, the breeders were removed manually or mechanically from a single parent line, using natural incompatibility to interfere with the pollination, or male infertility to disrupt pollen development. Mutations were developed. In high vegetation, male propagation requires some coordinated evolutionary events; Iii) assimilation of structural male organs from afferent meristems, ii) evolution of half of the gametes in anther, iii) release of the male gametes to the female regenerated organs, and finally iii) male and female gametes for acquired fertilization. Mutual awareness. Mutations that disrupt some male production cause male infertility. There are a large number of spontaneous male infertility mutants that fail the normal production of microsporogenesis. In tomato mutant msll, tapetal decay was too early, so both tapetal cells and spontaneous tissues were affected. Male-infertility variants can be found in mutations that pertubate structural male fertility. In Arabidopsis, the homeotic mutant pistillata reduces two and three rotifers, and the resulting plant is functionally male sterile and fertile (Bowman et al., 1989). The genetic basis of male infertility also varies. Male infertility, controlled by gene, nuclear, or genetic male infertility in the nucleus, first occurs in many crops (GMS). Similarly, male infertility is regulated by cytoplasmic genes, mainly mitochondria, and cytoplasmic male infertility (CMS) is known in many species (Frankel and Galan, 1977; Kaul, 1988).

The tapetum consists of a single layer of cells containing the deepest layer of anther wall developed in tetrad microsporogenesis. tapetum Provides a source of nutrients to the developing pollen blasts surrounding the sporogenous tissue. It has also been reported that tapetum produces a callase that hydrolyzes Carlos, which affects the dissolution of tetrad walls. During the post-meiotic period, sporopollenin, pollenkit substances and tryphine were released from the collapsed tapetum and the outer wall of the released vesicles (Mascarenhas, 1990). None of the alterations in this tissue, tapetum, functioned as a male regenerative agent, causing infertility.

Although the most widely known male infertility lines are naturally occurring, male infertility can also be induced by radiation, chemical treatment, protoplast lysis and genetic manipulation. Several approaches have been initiated using recombinant DNA technology to achieve male sterility. The first study to make successful male infertility plants was to induce male infertility in oilseed rape by the chimaeric ribonuclease gene under the control of the tapetum specific promoter by Tobacco and Mariani et al. (1990). They fused the 1.5-kb TA29 gene regulatory fragment with two different ribonucleases to selectively destroy the tapetal cell line. 10% of the TA29-RNase T1 transformant (2/20) and 92% of the TA29-barnase transformant (106/115) failed to shed pollen. This male infertility plant can be restored to male infertility. This was obtained by crossover with chimeric tapetal-cell-specific ribonuclease-inhibitors, basters and male fertility plants that could be modified (Mariani et al., 1992). Worrall et al. (1992) produced tobacco plants by manipulating the release of modified β-1,3-glucanase from tapetum before callase activity appeared in the locule. In tapetum cells of transgenic plants, the microsporocyte callase wall was prematurely degraded by transformants showing reduced male fertility. Pseudomonas aeruginosa exotoxin A has also been used to induce male infertility (Koning et al., 1992). In addition to the aforementioned experiments, several attempts have been made to induce male infertility by genetic regulation (Denis et al., 1993; Tsuchiya et al., 1995; Zhan et al., 1996). Recently, a system has been developed for inducible male infertility based on the non-toxic compound N-acetyl- _L- phosphinothricin (N-ac-Pt). The argE genetic product of E. coli, -acetyl- _L -ornithinedeacetylase, was converted to -ac-Pt for the cytotoxic compound L-phosphinothricin (Pt). In transgenic plants that owns the TA29 promoter -argE construct, N application of -ac- Pt induces empty anther which the male sterile plants. However, N-ac-Pt unless the treated plants are completely fertility (Kriete et al., 1996)

An object of the present invention is to conceive and identify the RIP gene from dianthus in consideration of the prior art and to mass produce male infertile plants using the same.

In order to achieve the above object, the present invention identifies plants that produce ribosomal inactive protein, clones cDNA of RIP gene from dianthus, analyzes expression of dianthus RIP gene and estimates organic tissue of dianthus RIP gene in RH genome. After the expression of the dianthus RIP gene in E. coli was observed, male fertility was induced using the modified dianthus RIP gene.

Hereinafter will be described in detail the specific configuration and operation of the present invention.

Figure 1 shows the CMC-Sepharose chromatography of the crude extract from dianthus leaves, Figure 1a shows the absorbance, NaCl concentration, inhibition rate of TMV infection at 260nm and Figure 1b shows the SDS-PAGE pattern of fractions from 34 to 49 1C shows local legion for virus infection.

Figure 2 shows the electrophoresis and Southern blot analysis of the dianthus RIP by PCR, Lane 1, 2, 3 are PCR products amplified using primers 1 and 2, Lane 4, 5, 6, 7, 8 is PCR products amplified using Primer 1 and the Xba1-primer adapter are shown.

FIG. 3 shows Southern blot analysis of the dianthus RIP gene, where 20 μg of leaf genomic DNA was digested with various restriction enzymes, isolated on a 0.8% agarose gel, transferred to a nylon membrane, and the membrane was labeled [α- ³² P]. RIP clone hybridized to DsPCRIP5.

4 shows Northern blot analysis of total RNA extracted from several tissues of dianthus. Membrane hybridized with dianthus RIP clone DsPCRIP5 labeled [α- ³² P].

FIG. 5 shows the size of positive cDNA clones isolated from the dianthus leaf cDNA library, FIG. 5A shows electrophoresis of positive cDNA clones amplified by PCR using universal primers, and FIG. 5B shows hybridization of RIP clone DsPCRIP5. Electrophoresis.

FIG. 6 shows cross-hybridization of cDNA clones, FIG. 6A is a small fragment of cDS302 clone and FIG. 6B is a large fragment of cDS302 clone, and the 5′-site comprising the 5′-UTR of cDS203 clone is in the same membrane. It is used as a probe.

Figure 7 shows the multiple alignments of the complete sequences of cDS203, cDS1202 and cDS2401. The start codon ATG and the stop codon TGA are shown as bold faces, the shaded bars represent other residues in these clones and the putative active site of the RIP is the box. Treated with. The number at the 3 'end is the number of A residues present in the cloned poly (A) tail.

FIG. 8 compares the derived amino acid sequences of cDS203, cDS1202 and cDS2401 with other RIP genes, the shaded bar is the base residue for N-glucosidase activity of RIP, and the box is the site for the putatively active site of this enzyme. And Asterisk represent different residues in RIP cDNA clones isolated from dianthus, the putative N- and C-terminal signal peptides are shown in bold and the N-glucosylation signal is indicated by arrows.

Figure 9a shows the construction of the dianthus RIP gene clone with the 5 'and / or 3' end removed by PCR, Figure 9b is the electrophoresis of the PCR product on the agarose gel, Lane 1 is P ₁ and P ₄ ( DsRIP), Lane 2 is P ₁ and P ₃ (3 ' Δ DsRIP), Lane 3 is P ₂ and P ₅ (5' Δ DsRIP), Lane 4 is P ₂ and P ₃ (5 ', 3' Δ DsRIP) It is about the product.

FIG. 10 shows vector construction for expression of modified cDS1202 clones DsRIP, 5 ′ Δ DsRIP, 3 ′ Δ DsRIP, 5 ′, 3 ′ Δ DsRIP in E. coli PQE vectors.

Figure 11 shows the growth analysis of E. coli cells containing a modified cDS1202 clone inserted into the pQE expression vector, OD ₆₀₀ of the culture was measured at 0, 60, 120, 180, 240 minutes after IPTG induction.

Figure 12a is a PCR amplification result of the pTA29 promoter portion from the tobacco leaf, Figure 12b shows the nucleotide sequence of the cloned promoter, the base sequence in the box shows the primer sequence used to amplify the promoter site.

Figure 13 shows the construction of a vector for male sterile plant production using the dianthus RIP gene.

FIG. 14 shows regeneration of tobacco plants transformed with pMTA2914 / LBA4404, pMTA2913 / LBA4404, pMTA2924 / LBA4404, pMTA2923 / LBA4404 and pMTA29gus / LBA4404, shoots (a) obtained from inoculated tobacco leaf discs, and into soil in a greenhouse Migration (b), normal growth of the entire plant (c) is shown.

FIG. 15 shows the flowers of male sterile tobacco plants, FIG. 15A shows the transformed and control plants, FIG. 15B shows the removal of half of the corollas from this flower to expose the internal male and female rotifers, FIG. 15c magnifies this.

Figure 16 shows a dissecting micrograph of anther obtained from male sterile flowers (b) of transformed tobacco plants expressing normal flowers (a) and Δ 3'DsRIP.

Fig. 17 compares the fruit capsules obtained from untransformed tobacco plants and the seed and transformed pMTA2913.

FIG. 18 shows Southern blot analysis of transformed male infertility pMTA2913, where Lane 1 is an untransformed tobacco plant and Lane 2-10 is a transformed plant comprising pTA29-3 ′ Δ DsRIP (pMTA2913). Indicates.

Bacterial Strains and Culture Media

Escherichia coli strain JM109 was used for general transformation and amplified the plasmid as host cells. XL1-BlueMRF 'and SOLR strains were used for infection and reproduction of bacteriophage and cleavage of recombinant phagemid in vivo, respectively. coli M15 strain was used for the expression of the pQE expression vector. Agrobacterium tumefaciens strain LBA4404 was used for plant transformation. E. coli strains were cultured in LB medium supplemented with 37 ° C., 12.5 μg / ml tetracycline, 50 μg / ml kanamycin or 50 μg / ml ampicillin according to the experimental purpose. M15 cells were cultured in LB medium supplemented with 100 μg / ml ampicillin and 25 μg / ml kanamycincells at 37 ° C. JM109 host strains used for transformation were obtained in M9 minimal medium. Agrobacterium strains were incubated in 28 ° C. YEP medium.

Hereinafter, the specific configuration and operation of the present invention will be described in detail with reference to the embodiments so that those skilled in the art can easily perform the scope of the present invention is not limited only to these embodiments.

Example 1 CM-Sepharose Column Chromatography of Raw Extracts Obtained from Leaves of Dianthus

50 g of Dianthus sinensis leaves are ground to a fine powder with LN ₂ , 300 ml of 1 × extraction buffer (0.14 MNaCl, 5 mM sodium phosphate pH 7.2, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 14 mM β-mercaptoethanol Homogenized). The extract was filtered through six layers of cheese colth. And centrifuged for 15 minutes at 10,000 rpm. Supernatant was filtered through 6 layers of Miracloth, and natural extracts were dialyzed against 5 L of 5 mM sodium phosphate buffer (pH 6.6). And centrifuged to remove the precipitate formed during dialysis. Supernatants were dialyzed again against 5 Lof 10 mM sodium phosphate buffer (pH 6.6) with three changes in buffer. The dialysis extract was centrifuged as described above to remove the precipitate. Supernatants were applied to columns of (20-cm × 1.6-cm, BIO-RAD) of CM-sepharose fast flow column chromatography. The column was washed with> 200 ml of 1 × column buffer (5 mM sodium phosphate pH 6.6) to adjust the base line for 12 hours. Absorbed materials were 1 × column buffer. Fractions (1 mL) were eluted with 100 mM lineargradient of 0-0.5 M NaCl and collected. 1 ml Fractions were measured by the Bradford method at absorbance A ₂₈₀ .

As a result, CM-Sepharose ion exchange chromatography was used to purify antiviral protein from dianthus. Leaf extracts were dialyzed and applied directly to the column. The column was washed with column washing buffer and the protein eluted with lineargradient NaCl in the column buffer (FIG. 1A). Fractions were collected and analyzed on SDS-PAGE (FIG. 1B) and analyzed for antiviral activity against TMV infection in N. glutinosa. Three series of fragments were bound to 11 tubes and each tube was analyzed for inhibitory activity. Fractions bound from # 5 to # 11 showed at least 90% inhibitory activity (FIG. 1C). There is no major peak for inhibitory activity, but instead possible antiviral proteins were obtained from a wide range of salt gradients. The experiment clearly shows dianthus containing possible inhibitors of viral infection.

Experimental Example 1. Identification of RIP gene from dianthus by PCR

To confirm that the dianthus contains ribosomal inactive protein (RIP) in its genome, primers were designed from the amino acid sequences of nucleotides, dianthin30 and saporin. PCR reactions were carried out using genomic DNA, the first strand cDNA synthesized by annealing Xba1-primeradaptor on total RNA extracted from dianthus leaves, and cDNA of dianthus leaves. D. caryophyllus was used as a control. PCR conditions were as follows; Denaturation of 35 cycles (94 ℃, 1 minute), annealing (55 ℃, 1 minute) and polymerization (72 ℃, 2 minutes). It can be expected that nearly 870 bp DNA fragments can be amplified using dianthus cDNA in PCR. The expected size of the amplified DNA fragments was observed after agarose gel electrophoresis even in the presence of weak 'background' products (FIG. 2A). The same gel hybridized with 870bp DNA fragment blotted and reamplified (FIG. 2B). To confirm the identity of the 870 bp DNA fragment, it was cloned and sequenced by the dideoxy-chain termination method. Results from sequence homology analysis showed that 870bp DNA fragments were 80% homologous to diathin 30 and saporin genes and 50-60% homologous to PAP gene. This fragment was named DsPCRIP5.

Experimental Example 2 Southern Blot Analysis of Dianthus RIP Gene

Total genomic DNA was extracted from dianthus leaves and digested with restriction enzymes, Bam HI, BglII, Eco Ri, HindIII, ScaI and DraI. The cleavage sites of Bam HI and BglII are present in cloned RIP cDNAs and the other four enzymes digest the 5 'and 3' flanks of the dianthus RIP gene. Digested genomic DNA was concentrated in the optimal volume loaded in each well and separated by electrophoresis for more than 48 hours at 30V on 0.8% agarose gel. After electrophoresis, the gel was rinsed with distilled water and depurinated for 15 min at 0.25 N HCl. The gel was then immersed by shaking for 90 minutes at 0.4 N NaOH at room temperature. Denatured DNAs were transferred to Hybond N ⁺ nylon membrane (Amersham) using 0.4 N NaOHtransfer buffer. At the end of the transfer, the membrane was rinsed with membrane washing buffer (0.2 M Tris-HCl, pH 7.5, 2X SSC) and high salt hybridizationbuffer (5XSSC, 70 mM Na ₂ HPO ₄ , 30 mM Na ₂ HPO ₄ for 4 hours at 58 ° C). , 2.5 mM EDTA, 0.6% SDS adjusted to pH 7.2). The membranes were hybridized with [α- ³² P] dCTP labeled DsRIP for 24 hours at 58 ° C in the same buffer. After hybridization, the membrane was subjected to 2X SSC / 0.1% SDS for 30 minutes at 40 ° C, 2X SSC, 0.1% SDS for 60 minutes at 63 ° C, 0.5X SSC, 0.1% SDS for 60 minutes at 50 ° C, and finally at 63 ° C. Washed continuously with 0.5X SSC, 0.1% SDS for 30 minutes. At each washing step, the membrane was exposed to X-rayfilm.

As a result of Southern blot analysis of divalent genomic DNA using ³² P-labelled DsPCRIP5 as a probe, hybrid bands were digested with Eco RI, Hind III, Sca I, Dra I, BglII and BamH I and detected in each lane as shown in FIG. 3. Labeled DsPCRIP5 probes hybridized with 1-4 fragments according to restriction enzymes. Four fragments hybridized in DraI and only one fragment hybridized in Eco RI digestion.

Experimental Example 3. Northern blot analysis of dianthus from other tissues

RNA samples were treated with formaldehyde and formamide according to Sambrook et al. Total RNA was electrophoresed on 1.0% agarose gel containing 0.67 M formaldehyde and delivered to Hybond N ⁺ nylon membrane. The membranes hybridized at 42 ° C. for 18 hours in 0.1% SDS, 5X Denhardt's solution, 6X SSC and 100 μg / ml sheared and denatured herring spermDNA containing 50% formamide. DNA probes were labeled with DsRIP with [α- ³² P] dCTP using a random primer labeling method. Membranes were washed with 2x SSC for 15 minutes at 42 ° C. in 0.1% SDS and once at 0.5 × SSC for 10 minutes at 52 ° C. in 0.1% SDS. The membrane was exposed to X-ray film at -70 ° C.

As a result, RIP mRNA was detected only from dianthus leaf tissue when hybridized with DsPCRIP5 as a probe (FIG. 4). Low background signals at the same leaf tissue were also detected in the roots and stems. It is clear from this experiment that the expression of the dianthus RIP gene as another RIP is tissue-specific and predominant in leaf tissue.

Example 2. cDNA Cloning of Dianthus RIP Gene

Experimental Example 1. Analysis of positive clones isolated from dianthus cDNA library

To build the dianthus cDNA library, 2.65 mg of total RNA was prepared from 10 g dianthus leaves. 10 μg of mRNA was purified from 2.65 mg of total RNA and 62.5 ng of double-stranded cDNA purified from 5 μg of mRNA was synthesized using a λ-ZAPII cDNA synthesis kit. 62.5 ng of cDNA was bound to 1 μg of Uni-ZAP XR vector and packaged with Gigapack III Gold packaging extract. From 62.5 ng of cDNA, 5.67 × 10 ⁵ recombinations were obtained. After amplification, 9.0 × 10 ⁵ plaques were screened with DsPCRIP5 fragments labeled [α- ³² P] (approximately 3.5 × 10 ⁴ plaques / plate). 44 positive plaques were obtained at the first screening, and finally 38 actual clones were cloned from pBluescriptII SK phagemide through the second screening.

PCR was applied to determine the insertion size of 38 positive clones. The size ranged from 250 bp to 1750 bp for akarose gel electrophoresis (FIG. 5A). Gels were blotted and hybridized with DsPCRIP5. Thirty clones out of 38 show strong hybridization bands and eight clones show low background signal (FIG. 5B).

Results obtained from cross-hybridization showed that the seven clones were the full length cDNA of the dianthus RIP (FIG. 6C). The cDS302 clone contained the longest insert. The small fragment hybridized to all clones, while the large fragment hybridized to four clones similar to cDS302 in its insertion (Figure 6a, b). Through subcloning and the arrangement of small and large fragments, cDS302 becomes a rare recombinant clone. It is composed of CP12 (large fragment) and partial RIP gene (small fragment). Large fragments of the cDS302 clone (1,150 bp) have an initiation codon, polyA tail in it, and 85% homology with other CP12 genes. The characteristics of the cDNA clones are summarized in Table 1. The description of the cDNA clones is based on the results from the array data of each cDNA clone and analysis by the NCBIBLAST survey program.

Characteristics of cDNA clones longer than 1 kb in size Serial clonenumber clone size (bp) Characteristics of partial sequenced cDNAs One cDS201 930 Partial cDNA clone shows ～ 80% homologyto Dianthin 30 and saporin genes 2 cDS203 1260 Full length cDNA clone shows ～ 80% homologyto Dianthin 30 and saporin genes 3 cDS302 1750 Hybrid cDNA clone of which 5'-region presents 67% homology to P. sativum mRNA for CP12 and 3'-region shows ～ 80% homology to Dianthin 30 and saporin genes 4 cDS901 1180 Full length cDNA clone shows ～ 80% homologyto Dianthin 30 and saporin genes 5 cDS1102 1150 Full length cDNA clone shows ～ 80% homologyto Dianthin 30 and saporin genes 6 cDS1202 1190 Full length cDNA clone shows ～ 80% homologyto Dianthin 30 and saporin genes 7 cDS1401 1750 Hybrid cDNA cloneof which 5'-region presents 67% homology to P. sativum mRNA for CP12 and 3'-region shows ～ 80% homology to Dianthin 30 and saporin genes 8 cDS1201 960 Partial cDNA clone shows ～ 80% homologyto Dianthin 30 and saporin genes 9 cDS2302 1180 Full length cDNA clone shows ～ 80% homologyto Dianthin 30 and saporin genes 10 cDS2303 1750 Hybrid cDNA clone of which 5'-region presents 67% homology to P. sativum mRNA for CP12 and 3'-region shows ～ 80% homology to Dianthin 30 and saporin genes 11 cDS2401 1200 Full length cDNA clone shows ～ 80% homologyto Dianthin 30 and saporin genes 12 cDS2402 1170 Partial cDNA cloneof which 5'-region contains ∼ 200 nt of TC repeats and 3 'shows -80% homology to Dianthin 30 and saporin genes 13 cDS2403 1200 Full length cDNA clone shows ～ 80% homologyto Dianthin 30 and saporin genes 14 cDS2601 1040 Partial cDNA clone shows ～ 80% homologyto Dianthin 30 and saporin genes 15 cDS2701 1750 Hybrid cDNA clone of which 5'-region presents 67% homology to P. sativum mRNA for CP12 and 3'-region shows ～ 80% homology to Dianthin 30 and saporin genes 16 cDS2702 1160 Partial cDNA clone of which 5'-region contains ∼ 200 nt of TC repeats and 3 'shows -80% homology to Dianthin 30 and saporin genes

Experimental Example 2 Base and Amino Acid Sequences of cDS203, cDS1202, and cDS2401

From the 5 'and 3'-alignment results, the seven full-length cDNAs of the dianthus are divided into three groups according to their base sequence. Three of the seven clones, cDS203, cDS1202 and cDS2401, were selected to determine their complete base sequence. The cDS203, cDS1202 and cDS2401 were 1,167 bp, 1,138 bp and 1,170 bp in length and had an open reading frame of 882 bp. These clones do not have consensus polyadenylation signals in their 3′-untranslated portion (FIG. 7). Their theoretical pl and Mw are 9.71 and 33 KDa, respectively. The deduced amino acid sequence has a putative signal peptide of 23 amino acid residues at the N-terminus and a putative signal peptide of 18 amino acid residues at the C-terminal and a coding sequence of 291 amino acid residues (FIG. 8). Computer analysis showed that these cDNA clones were 79% identical to dianthin 30 and saporin, while 59% identical to PAP and MAP. There is some heterogeneity in the sequencing of DsRIPs'. 4, 8 and 13 nucleotide exchanges were found in the 5'-untranslated region, coding region and 3'-untranslated region, respectively.

Nucleotide mismatch among the three DsRIP clones causes a change in protein sequence. The 10 amino acid alterations are due to 18 nucleotide changes in the coding region (FIG. 8). Changes in the amino acid sequence are preferential in adjacent regions of the putative N-terminal signal peptide and the enzyme active moiety. Despite the microheterosity in the amino acid sequence, the sequence of 'IQMVSEAARFKYI', the enzymatic active part of the RIPs, was conserved (FIG. 8).

Example 3. Expression of E. coli of Modified Dianthus RIP Gene

Experimental Example 1. Construction of Dianthus RIP gene with 5 'and / or 3' ends removed

In order to limit the transcription of RIP in the cytosol, the CDS1202 clone was modified by removing the 5'- and / or 3'-signal peptides of cDS1202. For PCR amplification, two upstream primers were designed to anneal in the arrangement of amino acid residues from 1 to 7 (primer1, p1) and from 24 to 31 (primer2, p2). Two downstream primers hybridized to cDS1202 amino acid residues from 269 to 276 (primer3, p3) and 287 to 294 (primer4, p4). At the 5 'end introducing the' ATG 'initiation codon of this sequence, the NcoI or RcaI portion was integrated and at the 3' end of this sequence introducing the 'TGA' stop codon. This sequence was introduced to incorporate a convenient restriction portion, Kpn I, Sac I and Hind III. RcaI produces a compatible end of Nco1 so that the RcaI fragment can be ligated to the end of the NcoI digested fragment. By using these four primers, four different structures were generated by PCR. PCR conditions were predenaturated at 94 ° C for 3 minutes, denaturated at 94 ° C for 1 minute, polymerized at 72 ° C for 2 minutes, and annealed at 55 ° C for 1 minute. The total number of cycles was 36 and PCR products were isolated on 1.2% agarose gel.

As a result, the cDS1202 clone was modified by removal of the 5 'and / or 3' signal peptide to limit the modified product in the cytosol when the modified structure was introduced and expressed in fortified plants. Computer analysis of the amino acids of cDS203, cDS1202 and cDS2401 found that cDS1202 contains an estimated 23 N-terminal amino acid residues (N-sp) and 18 C-terminal amino acid residues (C-sp, Figure 8). The former also assumes that the protein is responsible for delivering it to the Golgi apparatus, endoplasmic reticulum, and extracellular space, and the rest is related to delivery to the vacuoles. For PCR amplification, sequences of two unstream primers are produced. And from 1 to 7 (p1) and from 24 to 31 (p2). The two downstream primers hybridized with cDS1202 amino acid residues from 269 to 276 (P3) and from 287 to 294 (p4). At the 5 'end of this sequence, the NcoI or RcaI portion representing the' ATG 'initiation codon was integrated and a' TGA 'stop codon was seen at the 3' end of the sequence. The sequence was shown to incorporate convenient inhibitory moieties, Kpn l, Sac l and HindIII (FIG. 9A). PCR using P1 and P2 can produce complete sequences containing both N-terminal and C-terminal signal peptides. This structure is expected to limit intracellular space, vacuoles, and what is termed DsRIP. The definition of each structure expressed in the transformed plant is described below the diagram. PCR was performed according to the previous example. This PCR amplification gives a specific pattern of one major band corresponding to the measured size. The expected size of each PCR product is 882 bp, 828 bp, 813 bp and 759 bp for DsRIP, 3 ' Δ DsRIP, 5' Δ DsRIP and 5'3 ' Δ DsRIP, respectively (FIG. 9B). Four different amplified structures of the PCR product of cDS1202 were purified and digested with Kpn I and Hind III. These fragments were conjugated to pQE30 or pQE32 as expression vectors of E. coli. Recombinant plasmids were named pQE3214 (DsRIP), pQE3213 (3 ' Δ DsRIP), pQE3024 (5' Δ DsRIP) and pQE3023 (5'3 ' Δ DsRIP). E. coli strain M15 was transformed with these plasmids pQE30 and pQE32. Each transformant was named pQE3214 / M15, pQE3213 / M15, pQE3024 / M15, pQE3023 / M15, pQE30 / M15 and pQE32 / M15.

Experimental Example 2 Construction of Expression Vectors for Modified Dianthus RIP Genes

The pQE vector for the expression of the dianthus RIP gene is composed of a tac5 promoter, a riboson binding moiety, an initiation codon (ATG), a 6 × His-tag, a multicloning site, and a termination codon (FIG. 10) as an expression vector of E. coli. Depending on their encryption capabilities, there are three kinds of vectors: pQE30, pQE31and pQE32. The modified cDS1202 clone, 5 ' Δ DsRIP and 5'3' Δ DsRIP were bound to pQE30 and produced pQE3024 and pQE3023, respectively (FIG. 10A). DsRIP and 3 ′ Δ DsRIP are associated with pQE32 and produce pQE3214 and pQE3213, respectively (FIG. 10B). This recombinant plasmid was transformed with the E. coli host, M15 and was specifically designed to express virulence genes under strong inhibitory regulators, a safety device that protects E. coli from virulence genes.

Experimental Example 3 Growth of E. coli Cells Containing the Modified Dianthus RIP Gene

To investigate the cytotoxicity of the cloned dianthus RIP gene against E. coli, E. coli strain M15 containing DsRIP, 5 ' Δ DsRIP, 3'ΔDsRIP or 5'3' Δ DsRIP, was treated with 100 μg / ml of ampicillin. 25 μg / ml of kanamycin was incubated for 12 hours in 3 ml LB medium. 50 μl of primary culture was transferred to 20 mL of medium and the OD ₆₀₀ was measured immediately. At the time of OD ₆₀₀ = 0.2, 1 mM of IPTG was added and the growth rate of each transformant was observed by absorbance at 600 nm.

pQE32 vector alone grew well for IPTG induction, whereas pQE32, including DsRIP (pQE3214) and 3 ′ Δ DsRIP (pQE3213), did not grow in IPTG induction (FIG. 11). Like pQE32, pQE30 alone showed normal growth rates and pQE30, including 3 ' Δ DsRIP (pQE3024) and 5'3' Δ DsRIP (pQE3023), inhibited their growth by induction of IPTG. The transcriptional product of the gene has a toxic effect on E. coli ribosomes and it indicates that it produces at least functional proteins in E. coli despite removal of N- and / or C-terminal signal peptides.

Example 4 Induction of Male Infertility Using Modified Dianthus RIP Gene

Experimental Example 1. Nicotiana tabaccum cv. Amplification of the pTA29 Promoter Part from samsun

N. tabaccum cv. Two primers were designed to obtain anther specific promoter portions from Samsun's genomic DNA. The sequence of the 5 'terminal primer is 5'-dCGGGGTACCAAGC TTATCATCGATTATATTAGGG-3' (34-mers) and the 3 'end is 5'-CTACCATGGCTAATTTCTT TAAG-3' (26-mers). The 5'-terminal primer has restriction enzyme sites of kpnI and HindIII and the 3'-terminal primer has a Ncol site. The NcoI site is produced by substituting A ₁₅₂₅ and A ₁₅₂₆ for G ₁₅₂₅ and G ₁₂₂₆ , respectively. The underlined portion shows the beginning of the pTA-29 promoter (EMBL accession no. X52283). The PCR reaction mixture contains a master mix (2.5 mM MgCl ₂ ) and Taqpolymerase 1 unit. The template concentrations are 2.5 ng, 50 ng and 200 ng, with each template binding to primers with varying concentrations of 50 pmoles, 25 pmoles, 10 pmoles and 2.5 pmoles per primer. PCR reaction is carried out according to the following conditions. That is, predenaturation was performed at 95 ° C. for 3 minutes, denaturation at 94 ° C. for 1 minute, annealing at 50 ° C. for 2 minutes, and polymerization at 72 ° C. for 2 minutes 30 seconds. The total number of cycles was 36. PCR products were electrophoresed on 0.8% agarose gel. The nucleotide sequence of the promoter separated by the electrophoresis is shown in Figure 12a. The base sequence of the promoter region is shown in the box as shown in Figure 12b. In FIG. 12B the base sequence in the box shows the primer used to amplify the promoter and the parenthesis shows the sequence missing from the amplified pTA29 promoter.

Experimental Example 2. Construction of a vector for the production of male sterile plants using dianthus RIP gene

The pTA29 promoter and pLAT52-7 obtained by PCR in Experimental Example 1 were digested with restriction enzymes, Kpn I and Nco I and then recombined. This recombinant plasmid was named pLTA29-gus with pTA29 protomer, GUS site and NOS-terminator. pLTA29-gus was digested with NcoI and SacI to remove the GUS site and bound to fragments of pQE3214, pQE3213, pQE3024and pQE3023 digested with NcoI (or RcaI) and Sac l. These plasmids were named pLTA2914, pLTA2913, pLTA2924 and pLTA2923. These structures were digested with restriction enzymes, HindIII and Sac I and bound to pMBP1, a plant expression vector. The final structure was named pMTA2914, pMTA2913, pMTA2924 and pMTA2923 (Figure 13).

Experimental Example 3. Chimeric Gene, pTA29- Δ Transformation of Tobacco Plants with DsRIP

Healthy tobacco leaves (Nicotiana tabacum cv. Xanthi N.N.) from plants grown in a greenhouse about 2 months were carefully rinsed with tap water and soaked for 15 minutes with stirring in 2% sodium hypochlorite. Sterilized tobacco leaves were rinsed four times with 800 ml of distilled water. They were placed on petridish and the bleach-damage site was removed. The leaves were cut into 1 cm 2 using a sterile scalpel blade (# 10) and Agrobacterium was mixed with the dilution. Agrobacterium containing each structure (pMTA2914, pMTA2913, pMTA2923 and pMTA29-gus) was incubated at 28 ° C. for 36 hours and diluted 1:50 in dilution media (MS basal liquid medium). After 2 hours, the leaf disks were transferred to sterile filter paper for drying and darkened for 60 hours without selective antibiotics in the broth medium (MS salts, MS vitamins, 30 g / L of sucrose, 2.0 mg / L of BA and pH 5.8). Placed under conditions. At the end of the incubation period, the inoculated leaf disks were transferred to a sterile plastic tray filled with 50-70 ml of sterile distilled water and rinsed twice. The leaf discs were dried on filter paper and shoots were grown in regeneration medium (MS salts, MS vitamins, 30 g / L of sucrose, 2.0 mg / L of BA, 100 mg / L of kanamycinsulfate, 250 mg / L of carbenicillin, pH 5.8). Incubate until it appears. The shoots from the inoculated leaf discs were cut from callus and trimmed to leave three or five leaves with normal germination and lobe germination. They were inserted into rooting media (MS salts, MS vitamines, 30 g / l of sucrose, 50 mg / l of kanamycin sulfate, 250 mg / l of carbenicillin). After 7 to 10 days, rooted tobacco seedlings were transferred directly to the soil and kept in the shade for 2-3 days.

As a result, shoots were differentiated after 3 weeks from the leaf discs (FIG. 14A) and roots were induced in MS medium containing 50 mg / L kanamycin and 250 mg / L carbenicillin without hormones (FIG. 14B). Seedlings were transferred to soil and grown in greenhouses (FIG. 14C). As a result, 46 pMTA2914, 38 pMTA2913, 58 pMTA2924, 65 pMTA2923 and 20 pMTA29-gus transgenic plants were obtained.

Experimental Example 4. pTA29-3 'Induces Male Infertility in Cigarettes Δ Expression of the DsRIP (pMTA2913) Gene

The 1.5 kb pTA29 gene regulatory fragment was fused to the modified dianthus RIP gene. The ΔDsRIP gene has been modified earlier so that it is not transported into vacuoles or extracellular spaces and that these structures are retained in the cytosol when expressed in tapetum cells (FIG. 9). These structures were introduced into tobacco plants, resulting in 207 pTA29- Δ DsRIP gene transformants. Of the four structures, pTMA2914 and pMTA2913 caused male sterility in tobacco (FIG. 15B). The pMTA2913 transformants were identical in comparison to the control plants that were not transformed in terms of growth rate, height, shape, flower relationships, and flower color patterns. A noticeable decrease in the number of pollen in ten anthers was evident in the pMTA2913 transformant (FIG. 15C). Anther of the pMTA2913 plant was gradual, grayish brown in color, and lacked pollen (FIG. 16B). Pollen-free pMTA2913 plants failed to produce fruit capsules and seeds (FIG. 17). Of the 33 pMTA2913 plants, nine Southern blot analyzes were performed. Three transformants were found to contain only one pTA29-3 ' Δ DsRIP gene and the other had several pTA29-3' Δ DsRIP genes (FIG. 18).

The inventors introduced the genes used in the present invention into bacteria and named them Agrobacterium tumefaciens LBA4404 / pMTA2913 and Escherichiacoli JM109 / pMTA2913. Deposited.

Experimental Example 4. Magnetic Fertility

Cross mating pollination was performed between transformed plants and untransformed control plants to determine whether infertility was limited to others. The magnetic propagation structure of the transgenic plants was not clearly affected by gene substitution. Seed yields were similar to the self-pollinating wild control plants. Table 2 below shows the average weight of pMTA2913 seeds crossed with the control.

Magnetic Fertility of Male Infertile Tobacco Plants (pMTA2913) Number of transgenic plant Average weight of seeds per pod (g) 13M02 0.11 13M03 0.11 13M04 0.08 13M05 0.17 13M07 0.01 13M08 0.07 13M09 0.02 13M10 0.16 Untransformed normal 0.10

As described through the above Examples and Experimental Examples, the present invention has the effect of separating and identifying ribosomal inactive protein genes using leaf extracts to provide a modified gene, and using the same, industrially causing male infertility in tobacco plants. Excellent effect

Claims (5)

The base sequence of the ribosome inactive protein (RIP) coding gene isolated and identified from dianthus. The base sequence of the ribosome inactive protein (RIP) coding gene isolated and identified from dianthus. The base sequence of the ribosome inactive protein (RIP) coding gene isolated and identified from dianthus. Transformed Bacterial Agrobacterium Tumefaciens LBA4404 / pMTA2913 (KCTC 8859P). Transformed Bacteria E. coli JM109 / pMTA2913 (KCTC 8859P).