WO1991008292A1

WO1991008292A1 - Dna molecules improving cold-resistance

Info

Publication number: WO1991008292A1
Application number: PCT/FI1990/000284
Authority: WO
Inventors: Marianne Franck; Sirpa Kurkela
Original assignee: Kemira Oy
Priority date: 1989-11-23
Filing date: 1990-11-23
Publication date: 1991-06-13
Also published as: JPH04503013A; FI895614L; FI85286C; EP0497892A1; FI85286B; CA2044606A1; FI895614A0

Abstract

The present invention relates to a novel gene which was isolated from the plant Arabidopsis thaliana L. More particularly, the present invention relates to a DNA molecule which comprises a structural gene which encodes a cold hardening protein or a protein which has similar biological properties and is substantially homologous with the said protein, and the regulating region of the structural gene.

Description

DNA--molecules improving cold-resistance

The present invention relates to a. novel cryoprotective gene which has been isolated from the plant Arabidopsis thaliana L.

In spite of the large amount of biochemical and physiological data available on the cold resistance of plants, what really happens to a plant in cold is not yet fully understood. For example, it is not known why certain plants tolerate freezing and others do not, or what the primary reason for cold damage is. An ability to improve the cold resistance of plants would provide considerable advantages for agriculture in cold regions of the globe.

The cold resistance of a plant is not a constant state but develops when the plant is exposed to low non-freezing tempera¬ tures (= acclimation). Although a number of cold acclimation -specific changes have been observed in mRNA and in polypeptide profiles, very little is known about the action of these cold inducible proteins or about the mechanisms regulating these changes. So far, acclimation specific genes have not been iso¬ lated.

The object of the present invention is thus to isolate a cold hardening gene which can be transferred by genetic engineering methods into some crop plant and be induced to produce a pro¬ tein which will enhance the resistance of the plant to cold.

In order to obtain additional information regarding the genetic and molecular bases of the cold hardening process of plants, cold acclimation specific genes from Arabidopsis thaliana were investigated in connection with the present invention. It was observed that Arabidopsis is cold resistant, and that this cold resistance is associated with a number of changes at the level of gene expression. The present invention relates to a novel gene which was iso¬ lated from the plant Arabidopsis thaliana L. More particularly, the present invention relates to a DNA molecule comprising a structural gene which codes for a cold hardening protein or for a protein which has similar biological properties and is sub¬ stantially homologous to the said protein, and its regulating region which responds to a drop in temperature.

It was observed that the gene kinl was induced in six hours at +4 °C and acted as long as the plant was kept in cold and be¬ came inactive in 12 h when the plant was transferred back to the control temperature. The gene is also inducible by water stress and salt stress and by abscisic acid (ABA) . ABA is a plant hormone which functions as a mediator in various plant stress situations, and it has been shown also to contribute to the cold acclimation of plants. ABA (50 μM) has been observed to raise the induction level to as high a level as does cold.

A genomic library was constructed in EMBL3, and cold inducible genes were identified by differential hybridization. One gene, the one which was induced most clearly, was selected for further characterization. A genomic clone was used as a probe for finding the corresponding cDNA clone in the enriched libra¬ ry which had been constructed in the plasmid pUEXl. Both clones were sequenced, the transcription initiation site was deter¬ mined 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 assumed regulating region of the gene is underlined, starting from the base pair 720 and ending with the base pair 2132. After this base pair there begins the actual structural gene of the DNA molecule. The cDNA sequence and the corresponding amino acid sequence are shown underlined in Figure 2.

The longest open reading frame (ORF) of the cDNA sequence, starting from the first ATG downstream from the transcription initiation site, encodes a protein of 65 amino acids having 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 the kinl clone hybridizes to mRNA(s) which encode a polypeptide of similar size. The protein is rather hydrophilic, and it has a high amount of α-helical structure. Its amino acid composition is rather unusual: 22.4 % Ala, 13.4 % Gly and 13.4 % Ser. The gene contains the 5' and 3' untranslatable regions of the 62 and 117 nucleotides, respectively, and two introns.

The invention is described below in greater detail.

1) Isolation of a cold-inducible gene from the genomic library of the plant Arabidopsis thaliana

For the genomic library, the total DNΛ of the plant Arabidopsis thaliana was fragmented partially by means of SAU3a restriction enzyme, and fragments of 15-20 kb were isolated from agarose gel. The fragments were ligated to BamHl enzyme restricted arms of the vector ΛEMBL3, and the ligation mixture was packed in vitro inside the λ particles by a method known per se (Maniatis et al., Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982). The E. coli host was infected with λ particles, and the recombinant phases which contained the cold-inducible gene were identified by so-called differential hybridization (on the dish, the phages formed plaques from which the DNA was transferred, by the method of Maniatis et al. , to the filters used in the hy¬ bridization) . The probes used in the differential hybridization consisted of cDNA made from RNA isolated from the control pi ts. For further experiments, a recombinant was selected which yielded in the hybridization a signal with cold cDNA but not with control cDNA. The fragment containing the gene was transferred from the λ vector to the pUC18 vector, and the more precise location of the gene in the fragment was determined by differential hybridization. The 4369 kb fragment was sequenced.

2) Preparation of cDNA — cDNA corresponding to the genomic clone was captured from a cDNA library prepared from the cold RNA. The synthesis of cDNA from messenger RNA and its cloning into the vector pUEXl were carried out using Amersham kits (RPN 1256 and RPN 1282). The probe used in the hybridization was a genomic clone.- The cDNA was sequenced (Figure 2).

3) Southern blot analysis of DNA

Southern blotting was carried out by the method of Maniatis et al. (1982). The results of the genomic Southern blot test and the sequence analysis of cross-hybridizing cDNA clones shows that the gene is present in two copies in the genome of Arabi¬ dopsis.

4) Northern blot analysis of RNA

-The cold-inducibility of the gene kinl was demonstrated by Northern blotting. The northern blot analysis was carried out by the method of Maniatis et al. (1982). Northern blotting was used for analyzing the steady state levels of kinl mRNA during acclimation. Total RNA was isolated from the control plants and from the plants kept at a low temperature, by using the method described in Jones et al., High level expression of introduced chimeric genes in regenerated transformed plants, EMBO J. 4, pp. 2411-2418. The total RNA was analyzed carefully by the method of Maniatis et al., 1982. The results are shown in Fig¬ ure 4. The figure shows that at low temperatures the amount of RNA corresponding to the kinl gene is approx. 15-20 times the amount that is present at the control temperature, +22 °C, which means that the gene is cold inducible.

kinl mRNA was detectable 6 hours after the transfer to a low temperature, and the level remained high throughout the 7-day acclimation phase. It was observed that the induction was cb^~ld specific, i.e. when the plants were transferred back to the control temperature, the amount of kinl mRNA dropped in 12 hours.

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

Since the adding of exogenous abscisic acid (ABA) induces cold resistance in a number of plant species which are cold resis¬ tant, and an increase of endogenous ABA levels has been ob¬ served during this time, an investigation was made as to whether the kinl gene would respond to ABA. The experiment was carried out as in point 4 above, by Northern blot analysis. The probe used was kinl cDNA. Figure 5 shows the results of the effect of ABA (10 mM and 100 mM) on the expression of the kinl gene. It was observed that both the E >raying of Arabidopsis with 100 μM ABA and watering it with 10 μM ABA caused kinl induction. This was the first time that direct molecular evi¬ dence of the role of ABA in cold acclimation was observed.

b) Water stress

An investigation was made as to whether the gene kinl is in¬ ducible by water stress. The lowering of the cell water poten¬ tial is common to this stress. It has been demonstrated that the damage caused to spinach leaves by wilting is similar to the dehydration caused by freezing. It has also been thought that tolerance of freezing is due to avoidance or tolerance of the dehydration caused by freezing. The dehydration is caused by ice formed in the extracellular space "sucking" the water out from inside the cell. It has also been noted that cold resistance can be induced in plants by mere desiccation stress, without exposure to low temperatures. When plants were exposed to desiccation of different degrees, it was observed that wilt¬ ing really did induce the kinl gene.

c) Salt stress

A high intercellular salt concentration results in water leav- ing the cell, with dehydration resulting from it. An investiga¬ tion was made whether the kinl gene was inducible by salt stress (300 mM NaCl). The result of a Northern blot analysis shows that the kinl gene is inducible also by salt stress (Fig¬ ure 6) .

6) Making of DNA constructs which are capable of action in plants, and their transfer into plants

The activity of the kinl gene was investigated in cold-sensi¬ tive, non-acclimating tobacco plants (Nicotiana tabacum SRI) and Arabidopsis, by using two different DNA constructs. The first construct (p35S-sense-kinl) contains the kinl gene with the cDNA in the correct orientation (so-called sense), reg¬ ulated by a strong, continuously acting cauliflower mosaic virus 35 S transcript regulating region (p35S; Fromm M. et al., 1985. Proc. Natl. Acad. Sci. USA, 82:5824-5828). In this manner the activity of the kinl gene is in all^"parts of the plant independent of the temperature and the gene is also active in other plant species. In the other construct (p35S-antisense- kinl) , the cDNA of the kinl gene is in the wrong orientation (so-called antisense) , regulated by p35S. The activity of the kinl gene can be eliminated by transferring this construct into Arabidopsis, from which kinl has been isolated. Thus it can be determined whether^'or not the kinl gene is indispensable in the acclimation and cold resistance of Arabidopsis.

a) p35S-sense-kinl

The BamHI-BclI fragment of the pUEXl-cDNA clone was ligated to BamHI-digested plasmid pHTT203. The result was the plasmid pSKHlOO (Figure 7), in which the kinl cDNA was in the correct orientation under p35S regulation and which had the known neo- mycin phosphotransferase 2 nopaline synt ase gene (pnos-npt2), used as the selectable marker, for fusion to the regulating region, and the known marginal areas of T-DNA which are needed for the transfer of DNA to a plant. b) p35S-antisense-kinl

The Ncol fragment of t -.EXl-cDNA clone was ligated to BamHI digested plasmid pHTT2υ.,. The result was the plasmid pSKH107 (Figure 8), in which kinl cDNA was in the wrong orientation under p35S regulation and which had the pnos-npt2 gene used as the selectable marker and the known marginal regions of T-DNA.

The p35S-sense-kinl construct was transferred into tobacco - plants and the p35S-antisense-kinl construct was transferred to Arabidopsis plants by using a known Agrobacterium-mediated transfer method (Hernalsteens J.P. et al., 1980, Nature 287: 654-656; Valvetens D. et al. , 1988, Proc. Natl. Acad. Sci. USA, 85:5536-5540).

7) Investigation of the action of the cold gene a) In tobacco

Tobacco does not have the kir'l gene nati"-ully. In transgenic plants into which the construct p35S-s^r --kinl has been trans¬ ferred it was observed that the transfe..--:ed recombinant gene is present and is detectable by the Northern blot analysis (Figure 9). In cold resistance tests, ion bleedir of tobacco leaf fragments is measured as a function of tht- temperature, by a known method (Sukumaran & Weiser, 1972, Hortscience 7:467-468). The plants are regarded as dead when a 50 % ion bleeding oc¬ curs. It was observed that the transgenic tobacco had a cold resistance approximately 1.2 degrees greater than had the con¬ trol plant (Figure 10). This indicates that, when transferred into a cold-sensitive plant, the kinl gene improves the cold resistance of the plant concerned.

b) In Arabia >psis p35S-antiseι..*5e-kinl constructs were investigated in Arabidop¬ sis. The cold resistance test on the transgenic plants (Figure 11) shows that the acclimation ability of p35S-antisense-kinl plants is significantly lowered. These observations demonstrate that the kinl gene affects the cold resistance of Arabidopsis and is indispensable for it.

8) Investigation of the regulating region

A genomic clone fragment Hind3-Bsml which contained regulation region was ligated to the glucuronidase gene (gusA; Jefferson R.A. et.al., 1987, J. EMBO 6:3901-3907), and this fusion was transferred into a known vector which contained the selectable marker puos-npt2 and the marginal regions of T-DNA. The thus obtained construct pkinl-gusA in the plasmid pMEG3 (Figure 12) was transferred into tobacco plants by Agrobacter mediation.

The presence of the construct in a tobacco plant, and its ac¬ tivity, were investigated by determining the gus activity by a known method. Table 1 shows that pkinl acts as the regulating region in tobacco, and its activity is higher at a low tempera¬ ture.

Table 1

Claims

1. A DNA molecule, characterized in that it comprises a structural gene which encodes a cold hardening protein, and the regulating region of the gene.

2. A DNA molecule according to Claim 1, characterized in that the structural gene encodes a cold hardening protein iso¬ lated from the plant Arabidopsis thaliana L. , or a protein which has similar properties and is substantially homologous with the said protein.

3. A DNA molecule according to Claim 1 or 2, characterized in that it is derived from Arabidopsis thaliana L.

4. A DNA molecule according to any of the above claims, characterized in that it has the base sequence shown in Figure 1, or part thereof.

5. A DNA molecule according to any of the above claims, characterized in that its structural gene has a base sequence which corresponds to the bases 2136-3100, or a part thereof, in Figure 1.

6. A DNA molecule according to Claim 1, 3 or 4, charac¬ terized in that its regulating region has a base sequence which corresponds to the bases 720-2132, or a part thereof, in Figure 1.

7. A DNA molecule according to any of the above claims, characterized in that its structural gene encodes a protein the amino acid sequence of which is depicted in Figure 3, or a protein of equivalent biological activity, having substantially the amino acid sequence depicted in Figure 3.

8. A DNA molecule according to Claim 5 or 6, characterized in that additional bases are ligated to the 5' and 3 ' ends of ^{' ' ■"} . ^' 10

the said base sequence.

9. A cDNA molecule, characterized in that it has the base sequence depicted underlined in Figure 2.

10. A crop plant or decorative plant, characterized in that a DNA molecule according to any of Claims 1-8 has been incorpo¬ rated into it by genetic engineering methods.

11. A crop plant or decorative plant, characterized in that a cDNA molecule according to Claim 9 has been incorporated into it by genetic engineering methods.