FI85286C - Cold resistance enhancing recombinant DNA molecules - Google Patents

Description

1 85286

Cold resistance is enhanced by the recombinant DNA molecule

The present invention relates to a novel cold-protective gee-5 isolated from the plant Arabidopsis thallana L.

There is a lot of biochemical and physiological information on the cold resistance of plants, but nevertheless it is not yet fully understood what actually happens to plants in cold weather. For example, what makes some plants resistant to freezing and others do not is unknown or what is the main cause of cold damage. If the cold resistance of plants could be improved, considerable advantages in agriculture would be achieved in the cold regions of the world.

15

The cold resistance of a plant is not a permanent state, but develops when the plant is exposed to low temperatures above the freezing point (= acclimatization). Although several changes specific to cold acclimatization have been observed in mRNA and polypeptide profiles, very little is known about the function of these cold-inducible proteins or the mechanisms that regulate these changes. To date, no acclimatization-specific genes have been isolated.

It is therefore an object of the present invention to isolate a gene that promotes cold resistance, which can be transferred to a useful plant by genetic engineering methods, and to induce the gene to produce a protein that enhances the plant's resistance to cold.

30

In order to obtain further information on the genetic and molecular basis of the cold resistance process in plants, cold acclimatization-specific genes from Arabidopsis thaliana were studied in the context of the present invention. It was found that Arabi-35 dopsis is cold-resistant and that this cold-resistance is associated with several changes at the gene expression level.

2 85286

The present invention relates to a novel gene isolated from the plant Arabidopsls thaliana L. More particularly, the present invention relates to a DNA molecule comprising a structural gene encoding a cold tolerance promoting protein or a protein having similar biological properties and substantially homologous to said plant. protein, as well as its regulatory region, which responds to a decrease in temperature. The essential features of the invention are set out in the appended claims.

10

The gene, kinl, was found to be induced at + 4 ° C for six hours and to function as long as the plant is kept cold and turned off at 12 h when the plant is brought back to the control temperature. The gene is also induced by water-15 and salt stress as well as abscisic acid (ΑΒΑ). ABA is a plant hormone that acts as a mediator in various plant stress situations and has also been shown to play a role in cold acclimatization of plants. ABA (50 μΜ) has been found to raise the induction level as high as cold.

20

A gene library was constructed in λ EMBL3 and cold-inducible genes were identified by differential hybridization. One gene, the one most clearly induced, was selected for further characterization. The genomic clone was used as a probe-25 to find the corresponding cDNA clone from the enriched library constructed on plasmid pUEX1. Both clones were sequenced, the transcription start site was determined by the "primer extension" method, and the polyadenylation site from the cDNA sequences. The nucleotide sequence of the genomic clone is shown in Figure 1. The putative regulatory region of the gene is underlined starting at base pair 720 and ending at base pair 2132. After this base pair, the actual structural gene of the DNA molecule begins. The cDNA sequence and the corresponding amino acid sequence are shown in Figure 2 as underlined.

The longest open reading frame (ORF = * open reading frame) of the cDNA sequence, from the first ATG downstream of the transcript 3 85286, encodes a 65 amino acid protein with a predicted molecular mass of 6478 and an isoelectric point of 7.2. The amino acid sequence of the protein is shown in Figure 3. Hybrid selection experiments showed that cin1 clone 5 hybridizes to mRNAs encoding a polypeptide of the same size. The protein is quite hydrophilic and has a lot of α-helical structure. Its amino acid composition is quite unusual: 22.4% Ala, 13.4% Gly and 13.4%

Ser. The gene contains the 5 'and 3' untranslated regions of 62 and 117 nucleotides, respectively, and two introns.

The invention is described in more detail below.

1) Isolation of a cold-inducible gene from the genid library of Arabidopsis thaliana 15

For the genomic library, total DNA of Arabidopsis thaliana was partially digested with Sau3a restriction enzyme and fragments of 15-20 kb were isolated from an agarose gel. The fragments were ligated into the BamHI-digested arms of the EMBL3 vector, and the ligation mixture was packaged inside the in vitro particles using a method known per se (Maniatis et al., Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982). particles infected E. coli host, and recombinant phages containing the cold-inducible gene were searched for so-called by differential hybridization (phages formed plaques on a plate from which DNA was transferred according to Maniatis et al. to filters used in hybridization). CDNA made from RNA isolated from control plants was used as probes in the differential hybrid-30 dissection. Further, a recombinant was selected that gave a signal for cold cDNA in hybridization but not for control cDNA. The fragment contained in the gene was transferred from the vector to the pUC18 vector, and the exact location of the gene in the fragment was determined by differential hybridization. The 4369 kb fragment was sequenced.

2) preparation of cDNA

The cDNA corresponding to the genomic clone was captured from 40 cDNA libraries of cold RNA. Synthesis of cDNA from messenger RNA 4 85286 and cloning into the pUEX1 vector was performed using Amersham kits (RPN 1256 and RPN 1282). A genomic clone was used as a probe for hybridization. The cDNA was sequenced (Figure 2).

3) DNA "Southern blotting" analysis "Southern blotting" analysis was performed according to Maniatis et al.

(1982) method. The results of the genomic "Southern blotting" experiment and the sequence analysis of the cross-hybridizing cDNA clones show that the gene is present in two copies in the Arabidopsis genome.

4) "Northern blotting" analysis of RNA The cold inducibility of the kin1 gene was demonstrated using "Northern blotting" analysis. Northern blotting analysis was performed according to Maniatis et al. (1982) method.

The "Northern blotting" method was used to analyze the "steady-state" levels of kinl mRNA during acclimatization. Total RNA was isolated from control and refrigerated plants by the method described in Jones 20 et al., High level expression of introduced chimeric genes in Regenerated transformed plants, EMBO J. 4, pp. 2411-2418. Total RNA was carefully analyzed according to the method of Maniatis et al., 1982. The results are shown in Figure 4. The figure shows that in the cold there is about 15-20 times more RNA corresponding to the kin1 gene in the cells than at the control temperature of + 22 ° C, which means that the gene is induced in the cold.

kinl mRNA was detectable 6 hours after cold transfer and the level remained high throughout the acclimatization phase at 30 days. The induction was found to be cold-specific, i.e. when the plants were returned to the control temperature, the amount of kinl mRNA dropped in 12 hours.

5) Investigation of the induction properties of the cold gene 35 a) Abscisic acid (ABA)

Because external addition of abscisic acid (ABA) induced cold resistance in several plant species that are cold-resistant, and an increase in endogenous ABA-858528 levels has been observed during this time, the response of the kin1 gene to ABA was investigated.

The experiment was performed as in step 4 using "Northern 0 blotting" analysis. Kin1 cDNA was used as a probe. Figure 5 shows the results of the effect of ABA (10 mM and 100 mM) on kin1 gene expression. It was found that both spraying Arabidopsis with 100 μΜ ΑΒΑ and irrigating it with 10 μΜ ΑΒΑ caused induction of the kinl gene. For the first time, direct molecular evidence for the role of ABA in cold acclimatization was observed.

10 b) Water stress

It was investigated whether the kin1 gene is induced by water stress. Common to this stress is a decrease in the water potential of the cell. It has been shown that withering causes damage to the tin-15 tin leaves similar to dehydration due to freezing. It has also been thought that resistance to freezing is due to the avoidance or persistence of dehydration caused by freezing. Dehydration is due to the fact that the ice formed in the intercellular space "absorbs" water from inside the cell 20. It has further been found that cold resistance can be induced in plants by dry stress alone without exposure to low temperatures. When plants were exposed to varying degrees of drought, it was found that withering indeed induced the kin1 gene.

25 c) Salt stress

High external salinity results in the removal of water from the cell as a result of dehydration. It was examined whether the kin1 gene is induced by salt stress (300 mM NaCl). The result of the "Northern blot-30 Ting" analysis shows that the kin1 gene is also induced by salt stress (Figure 6).

6) Construction of plant DNA constructs and transfer to plants The function of 35 kin1 genes was studied in cold-sensitive non-acclimatizing tobacco (Nicotiana tabacum SRI) and Arabldopsis using two different DNA constructs. The first construct (p35S-sense-kin1) contains the cDNA of the kin1 gene upstream of the 85 S transcript regulatory region of the potent ever-acting cauliflower mosaic virus 35 p transcript (p35S; Fromm M. et al., 1985. Proc. Natl. Acad. Sci. USA, 82: 5824-5828). In this way, the effect of the kin1 gene is functional in all parts of the temperature-5 ton plant as well as in other plant species. In another construction (p35S-antisense-kin1), the cDNA of the kin1 gene is upside down (so-called antisense) regulated by p35S. By transferring this construct to an Arabidopsis from which kin1 has been isolated, the effect of the kin1 gene can be eliminated. 10 Thus, it becomes clear whether the kinl gene is essential for Arabldopsis acclimatization and cold tolerance.

a) The BamHI-BclI fragment of the p35S-sense-kin1 pUEX1 cDNA clone was ligated into the BamHI-15 digested plasmid pHTT203. The result is plasmid pSKHIOO (Figure 7), in which the kin1 cDNA is upstream under p35S regulation and in which the known neomycin phosphotransferase 2 to the regulatory region of the nopaline synthetase gene for fusion (pnos-npt2) and 20 known T-DNAs are used as selectable markers. areas required for DNA transfer to a plant.

b) The NcoI fragment of the p35S antisense kin1 pUEX1 cDNA clone was ligated into the BamHI-Diges-25 fed plasmid pHTT203. The result is plasmid pSKH107 (Figure 8), in which the kin1 cDNA is upside down under p35S regulation and has the pnos-npt2 gene to be used as a selectable marker and known T-DNA flanking regions.

The p35S-sense-kin1 construct was transferred to tobacco and the p35S-antisense-kin1 construct to Arabidopsis plants using a known Agrobacterium-mediated transfer method (Hernalsteens JP et al., 1980, Nature 287: 654-656; Valvetens D. et. et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 5536-5540). 35 7 85286 7) Investigation of cold gene function a) In tobacco

Tobacco does not have the kinl gene naturally. In transgenic plants grafted with the construct p35S-sense-kin1, it was found that the transferred recombinant gene was present and expressed by Northern blot analysis (Figure 9). In the cold endurance test, the ion leakage of tobacco leaf pieces is measured as a function of temperature by a known method (Sukumaran & Weiser, 1972, Hortscience 7: 467-468). Plants are considered dead when 50% ion leakage occurs. It was found that transgenic tobacco has about 1.2 degrees greater cold resistance than control plants (Figure 10). This indicates that the kin1 gene, when transferred to a cold-sensitive plant, improves the cold tolerance of that plant.

B) In Arabidopsis p35S antlsense-kinl constructs were examined in Arabidopsis. The cold resistance test of transgenic plants (Figure 11) shows that the acclimatization capacity of p35S antisense kinl plants is significantly reduced. These findings indicate that the kin1 gene affects and is essential for the cold hardiness of Arabidopsis.

8) Investigation of the regulatory region A Hind3-BsmI fragment containing the regulatory region of a genomic clone was inserted into the glucuronidase gene (gusA; Jefferson RA et al., 1987, J. EMBO 6: 3901-3907) and this fusion was transferred to a known vector containing a selectable gene. the edge regions of the marker Puos-npt2 and T-DNA. The construct 30 thus obtained in plasmid pkinEG-gusA pMEG3 (Figure 12) was transferred to tobacco via Agrobacterium.

The presence of the construct in tobacco and its functionality were examined by determining gus activity by a known method.

- 35 Table 1 shows that pkin1 acts as a regulatory region in tobacco and has a higher activity at low temperatures.

β 85286

Table 1 A415nm

+ 22 ° C + 4 ° C

control 0.01 0.01 5 pkinl-gusA 0.281 0.830

Claims (5)

9 85286 A recombinant DNA molecule comprising the structure of a protein comprising a protein of 30 and a base sequence of base 2136-3100 in Figure 1, further comprising a base sequence of base sequence 720-2132 in Figure 1. A recombinant DNA molecule, characterized in that it comprises a structural gene encoding a cold-tolerance-promoting protein having a base sequence corresponding to 5 bases 2136-3100 in Figure 1 and a regulatory region having a base sequence corresponding to bases 720-2132 in Figure 1. . 2. A recombinant DNA molecule according to claim 1, wherein the recombinant DNA molecule comprises a structural coding and a protein for isolating the protein from Arabldopsis thaliana L. 10 85286 Recombinant DNA molecule according to Claim 1, characterized in that the structural gene encodes 10 Cold tolerance protein isolated from Arabldopsis thaliana L. 3. A recombinant DNA molecule according to any one of claims 1 or 2, which comprises the basal sequence of Figure 1. Recombinant DNA molecule according to Claim 1 or 2, characterized in that it has the base sequence shown in Figure 15. Recombinant DNA molecule according to any one of the preceding claims, characterized in that its structural gene encodes a protein having the amino acid sequence 20 shown in Figure 3, or a protein having an equivalent biological activity, having the amino acid sequence shown in Figure 3. 5. A cDNA molecule, characterized in that it has the 25 base sequence shown underlined in Figure 2. 4. A recombinant DNA molecule having the appearance of a structurally encoded protein, the amino acid sequence of which is shown in Figure 3, or having a protein having equivalent activity to that of the biological activity, and having the same amino acid in Figure 3. -10 syrasequence. 5. A cDNA molecule that has been shown in Figure 2 as a subcutaneous bass sequence.