WO1997020058A2 - PLANT GENE EXPRESSION VECTOR FAMILY BASED ON THE REGULATORY DNA SEQUENCES OF AN ALFALFA H3 HISTONE GENE VARIANT (MsH3g1) - Google Patents

Abstract

Disclosed herein are nucleic acid molecules comprising sequence regions participating in the regulation of the expression of the H3g1 gene of alfalfa (Medicago sativa). A plant gene expression vector family based on the regulatory DNA sequences of said histone gene variant (MsH3g1) is also disclosed. The invention also provides transformed cells, transgenic plants and parts thereof comprising the nucleic acid sequences of the invention.

Description

Plant gene expression vector family based on the regulatory DNA sequences of an alfalfa H3 histone gene variant (MsH3gl)

The present invention relates to nucleic acid molecules comprising sequence regions participating in the regulation of the expression of the H3gl gene of alfalfa ( Medicago sa ¬ ti va ) and a gene expression vector family based on the nu¬ cleic acid molecules of the invention. More specifically, the invention relates to nucleic acid molecules comprising sequences presented on Fig. 1. The invention also provides transformed cells, transgenic plants and parts thereof com¬ prising the nucleic ac d sequences of the invention.

The present invention s useful in expressing foreign genes in different plants and in altering the measure or spatial and temporal pattern of the expression of different endogenous plant genes .

With respect to the present specification and claims, the foregoing technical terms will be used n accordance with the below given definitions. With regard to the inter¬ pretation of the present invention, it shall be understood that the below defined terms are used in accordance with the given definitions even if said definitions might not be in perfect harmony with the usual interpretation of said technical term.

A "homologue"' or a "variant" of a nucleic acid sequence is defined as a sequence that is at least 50 % identical to the sequence m question. A "functi onal varian t '' of a sequence is every sequence having the same type of biological activity even f the measure of the biological activity of tne functional vari¬ ant is significantly different from that of the original sequence (e.g., the transcriptional activity of tne func¬ tional variant of a promoter can either be smaller or larger than that of the original promoter) .

A nucleic acid molecule is regarded "hybridizable" with another nucleic acid molecule if it can specifically be bound to the other molecule (i.e., the binding can give rise to a signal that is distinguishable from the oac - ground noise and from the signal caused by the aspecific binding of any random sequenced nucleic acid molecule) .

A regulatory sequence is " opera ti vely linked" to struc- tural gene withm a DNA construct if the regulatory se¬ quence is able to influence the expression rate or manner of said structural gene under conditions suitable for the expression of said structural gene and for the functioning of said regulatory sequence. The first scientific papers disclosing so called trans¬ genic plants - prepared by transformation and comprising foreign genes - were published m the early 1980' s [ Fraley, R.T. et al., Proc. Natl. Acad. Sci. USA 80: 4803 (1983)] . Since that time transformation methods has been elaborated for almost all economically important industrial crops [ for review see Hinchee et al . , Plant Cell and Tissue Culture, pages 231-270, ed. Vasil, J.K. and Thorpe, T.A., Kluwer Academic (1994)] . Transgenic techniques on one hand became generally applied research tools m the field of experimen¬ tal plant biology and, on the other hand, transformed plants are now getting used worldwide in plant breeding systems and in the seed-corn industry. In 1993, statistics account for 320 announceα field experiments establishing the widespread application of transgenic plants. There is a commercially available transgenic tomato line in the USA, that is advantageously marketable because of its better taste and applicability for long term storage. There are a lot of factors influencing the advantageous applications of transgenic plants, one of the most impor¬ tant of which - with respect to the establishment of new technologies - is the appropriate choice of the so called "agronomic" genes (genes causing positive effects) and the regulatory elements directing their expression (e.g., pro¬ moters, introns and 3' regulatory regions) . A very narrow range of promoters has been used so far for the expression of foreign genes n plants . The cauliflower mosaic virus αeπved CaMv35S promoter is most often used for the con- struction of plant expression vectors. With regard to the limitations of such expression systems there is a rapidly growing need for the preparation of further vector systems based on different promoters. In a wide range of possible applications it would be advantageous to use so called con- stitutive vector constructions ensuring high expression rate in every cells and tissue types of the transformed plant. It would also be preferred to use plant promoters instead of virus derived promoters to avoid possiole bio- hazard.

The concentration of a given gene product in a cell is influenced by a multilevel system of molecular mechanisms. Highly important factors of such mechanisms are the speed of transcription, the term of half life of the produced mRNA molecules and the stability of said molecules. These parameters are mainly dependent on the characteristics of the 5' promoter region and can also be influenced by the introns present in the coding region and the 3' non trans¬ lating regions. Accordingly, the above regulatory elements of a plant gene of interest can be used for constructing transformation vectors ensuring high expression levels of agronomic genes. For the isolation of constitutive promoters, it is ad¬ visable to consider genes participating in basic functions of the cells . One such type of genes can be the histone gene family. The present inventors has conducted intensive research in the field plant histone genes since the mid 1980' s, primarily using experimental systems based on al¬ falfa. Our publication, in which we have demonstrated the presence of H3 histone gene variants in -alfalfa, was pub¬ lished in 1988 (Wu, S.C, Bogre, L., Vincze, E-, Kiss, G.B. and Dudits, D., Plant Mol. Biol. 11: 641) . In this paper - by applying cDNA clones - we have demonstrated the presence of a cell cycle dependent (H3.1) and a constitutive gene variant (H3.2) . We have isolated the genomic clone of the H3.1 variant from a gene bank and characterized tne cell cycle dependent functioning of its promoter [ Kapros, T. et al., Plant Physiol. 98: 621 (1992)] . We have also isolated the genomic counterpart of the constitutive cDNA variant and characterized the regulatory elements of this gene that make us possible to use said regulatory elements for the development of new transformation vector molecules.

It is, thus, an object of the present invention to con¬ struct a wide host range constitutive plant gene expression vector family based on the regulatory regions of the ISO- lated MsH3gl gene variant.

The object of the invention was achieved by first de¬ termining the whole nucleotide sequence of the MsH3gl gene. Based on the gained sequence information it became possible to determine the location of the 5' promoter region (from -482 to -1 bp with respect to the transcription initiation site; nucleotides 1-482 of the sequence shown on Fig. IA) , of three introns (nucleotides 555-668, 746-962, 1053-1174; Fig. IB) and of the 3' non translating region (nucleotides 1346-1676; Fig. IC) . The above regulatory regions are suitable - alone or in combination - for constructing novel vector molecules mak¬ ing possible the functioning of foreign genes in plant cells or the alteration of the spatial and/or temporal ex¬ pression pattern of endogenous plant genes . The present invention provides single or double stranded DNA molecules comprising - wholly or in part - the sequence regions of the H3gl gene of alfalfa (Medicago sa ¬ ti va ) shown on Fig. IA (nucleotides 1-482), Fig. IB (nucleotides 555-668, 746-962 and 1053-1174) and/or Fig. IC (nucleotides 1346-1676), or their homologues or functional variants .

The nucleic acid molecules of the invention are advan- tageously at least 80 % homologue to the sequences shown on Fig. 1 or parts thereof.

The invention also concerns nucleic acid molecules hy- bridizable to the nucleic acid molecules of the invention. The invention further provides transformation vectors comprising the nucleic acid molecules according to the in¬ vention, cells and transgenic plants transformed with sucn vectors, and reproducible parts of said transgenic plants.

The invention is further illustrated by the attached figures the short description of which is as follows: Fig. 1 shows the regulatory regions of the MsH3gl gene (Fig. IA depicts the sequence of the promoter region; Fig. IB shows the sequences of introns I, II and III; and Fig IC shows the 3' non translating region) .

Fig. 2 shows the schemes of vector constructions pre- pared using the regulatory regions according to the inven¬ tion (see also Ex. 2) .

Fig. 3 shows the schemes of further vector construc¬ tions according to the invention (see also Ex. 2) .

The subject of the invention will be further lllus- trated by the experimental examples described below, how¬ ever, the scope of the invention will by no means be lim¬ ited to the specific embodiments described in the examples. Example 1

Determination of the nucleic acid sequence cf the MsH3gl gene isolated from alfalfa (Medicago sa tiva ) , iden¬ tification of functional elements of the gene We have isolated a clone comprising the MsH3gl gene from phage λ clones (Alfalfa Genomic Library, supplied by Clontech Laboratories, Inc.) carrying alfalfa genomic DNA applying a colony hybridization method known m the art [ Sambrook et al . , Molecular Cloning, A Laboratory Manual, 2. ed., Cold Spring Harbor, N.Y. (1989)] . A PvuII/Hmdlll fragment of the previously isolated pH3cll cDNA clone [ Wu, S.C. et al., Nucleic Acids. Res. 17: 3057 (1989)] was em¬ ployed as the hybridization probe. Sequencing of the iso¬ lated H3 histone gene was also done using techniques known in the art [ Schuwmann, R., et al., Biotechmques 10: 185 (1991) ; Seto, D., Nucleic Acids . Res. 18: 5905 (1990); Man- fioletti, G. et al . , Nucleic Acids. Res. 16. 2873 (1988)] . The nucleotide sequence of the 5' promoter region of the isolated alfalfa H3 histone gene - that is located between nucleotides -482 and -1 - is shown in Fig. 1 (the numbering of this sequence on the figure is 1-482) . This DNA segment comprises all the functional elements needed for the ex¬ pression of a gene of interest. The isolated alfalfa his¬ tone gene comprises three non coding intron regions shown on Fig. IB. The 3' non coding region is comprised of 230 nucleotides (Fig. IC) . Examp l e 2

Preparation of a transformation vector family based on the functional regulatory elements of the MsH3gl gene

The vector molecules snown on F g. 2 and Fig. 3 were prepared using DNA manipulation methods generally known in the art [ Sambrook et al . : Molecular Cloning, A Laboratory Manual, 2. ed., Cold Spring Harbor, N.Y. (1989)] . The fol¬ lowing fragments of the MsH3gl genomic clone were used to construct said vector molecules: the promoter region flanked by 5' AccI and 3' Ncol restriction sites, the in¬ tron I region flanked by Nsil/Ncol sites and the transcrip¬ tion termination region flanked by SacI/NotI restriction cleavage sites. The applied multiple cloning sites (MCS) region was derived from pBluescript II SK+ (Stratagene; GenBank #X52328[ SK(+)] , designated as plasmid pBSK on the figures) . For the construction of vectors pHEX-N and pHEXHO, we have also used the termination signal of the bacterial nopaline synthase gene [ Depicker et al . , J. Mol. Appl. Genet. 1: 561 (1982)] . We summarize the construction of the above vector mole¬ cules n the following steps .

1. The promoter region was cut from plasmid construc¬ tion pHEX-N (Fig. 2) by AccI and Ncol restriction cleavage, overhangs were filled in with Klenow enzyme (E . col i DNA polymerase I, Large Fragment, Biolab' s) , then the isolated promoter fragment was ligated into plasmid pBluescript II SK+ previously cut with enzymes Xbal and BamHl and simi- larly filled in with Klenow enzyme, resulting in plasmid pBH.

2. The NOS terminator was isolated on a KpnI/SacI frag¬ ment and inserted into the KpnI/SacI sites of plasmid pBH yielding plasmid pBH . Plasmid pBHN comprises the following construction: - NotI - Xbal - H3.2 promoter - Ncol pBluescript II MCS (from BamHl to Kpnl) - Sacl - NOS termi¬ nator - NotI -.

3. Plasmid pBluescript II SK+ was cleaved with enzymes Kpnl and Sacl and a synthetic NotI linker was inserted into the cleaved plasmid, resulting in plasmid pBNot. The se¬ quence of the inserted NotI linker was the following: upper strand: 5' CCCGCGGCCGCCCCAGCT 3' lower strand: 3' CTAGGGGCGCCGGCGGGG 5' . 4. Plasmid pBHN was cut with NotI and the isolated fragment comprising the - H3.2 - promoter - MCS - NOS ter¬ minator - construction was ligated into the NotI site of plasmid pBNot, resulting in plasmid pHEX-N2.

5. Plasmid pHEX-N2 was digested with enzymes Ncol and Sacl and a NcoI/SacI fragment of plasmid pLP140

[ Szczyglowski, K. et al . , The Plant Cell 6: 317 (1994)] comprising the coding sequence of the GUS gene was inserted into the cleaved plasmid, yielding plasmid pHEX-N: :GUS.

6. Intron I of the H3.2 genomic clone was amplified by PCR and the PCR product was introduced to the Ncol site of plasmid pBHN2, resulting in plasmid pHEXHO. ^~ι . The GUS coding sequence comprised on a NcoI/SacI fragment was then inserted into the NcoI/SacI sites of plasmid pHEXHO, yielding plasmid pHEXHO : :GUS .

8. The NOS terminator in pHEXHO vector was replaced by the H3.2 terminator amplified by PCR using the pH3.2 genomic clone as template and applying techniques well known in the art, resulting in plasmid pHEXlll. The se¬ quences of the primers used in the PCR reaction were as follows : upstream primer:

5' GAG CTC TAG GTA GGT AGC ATT CGC GGT GAA CGT GCT 3' downstream primer:

5' GCG GCC GCT GTC ACC GAT AGA CAA ACT ACC 3' . The PCR product was polished with Klenow fragment of DNA Polymerase I and then it was subcloned into the T4 po- lymerase-blunted KpnI/SacI restriction sites of plasmid pBluescrιptSK+ . The SacI/SacI fragment of the resulting plasmid was isolated and inserted into the SacI/SacI sites of plasmid pHEXHO replacing the NOS terminator by the al- falfa histone terminator. This resulting construction was designated pHEXlll.

9. The GUS coding region was then inserted to the NcoI/SacI sites of pHEXlll, yielding plasmid pHEXlll : :GUS . Example 3

Functioning of the pHEX expression vectors in transient experiments performed after transforming alfalfa and maize protoplasts

With the purpose of checking the proper functioning of the transformation vectors shown on Fig. 2, we have also prepared their variants comprising reporter genes. The scheme of vectors carrying the β-glucuronidase (GUS) re- porter gene is shown on Fig. 3.

Protoplasts isolated from A2 alfalfa cell suspension

[ described in Magyar et al . , The Plant Journal 4: 151

(1993)] and from HE89 maize cell suspension [ Mόrocz et al . ,

Theor. Appl. Genet. 80: 721 (1990)] were used in the ex- periments. To 2x10 protoplasts, 20 μg of plasmid DNA was added and DNA uptake was induced by treatment with 40 % PEG [ Oznirulleh, S. et al . , Gene transfer to plants, ed. Po- trykus and Spangenberg, G., Springer, pages 99-105 (1995)] . Treated protoplasts were cultured in K3-medιum [ Nagy, J.I. et al., 2. Pflanzenphssiol . 78: 453 (1976)] . For measuring the activity of the β-glucuronidase enzyme, 4x10 proto¬ plasts each was used on the 1th, 2nd and 3rd days of culti¬ vation [ Martin, T. et al . , GUS Protocols, Academic Press, pages 23-59 (1992)] . In the case of maize cell suspensions, GUS activity values determined on the first day did not significantly differ from background values. Table 1 summa¬ rizes specific activity values determined in two parallel experiments using alfalfa protoplast derived cell suspen- sions originating from two independent transformations (data given are mean values from three parallel measure¬ ments) . Compared to the activity of the virus derived pro¬ moter (CaMV35S : :GUS) , the functioning and higher promoter activity of the pHEX expression vectors is definitely dem¬ onstrated. Especially the vector pHEX110: :GUS provided ex¬ tremely high expression rates. The viral vector used as reference (pIDS211) is described in Stefanov et al., Acta Biologica Hungarica 42: 323 (1991) . Very similar results were obtained using the maize pro¬ toplast derived cell suspension. Experimental data are sum¬ marized n Table 2 (data given are mean values from three parallel measurements) .

The proper functioning of the above vectors has also been demonstrated in tobacco protoplast derived cell sus¬ pension (data not shown) .

Table 1

Transient GUS activity values determined in alfalfa proto- plast derived cell suspension

(pmole MU / mg prot. / mm) Experiment 1 day 1 day 2 day 3 control 0 0 0 pHEX-N: : :GUS 301, .4 2457.8 5270. .0 pHEXHO: : :GUS 4573. .6 18831.7 44434. .0 pHEXlll: : :GUS 434 .5 5384.6 16028 .8 pIDS21 (35S::GUS) 57 [Table continued)

Experiment 2 day 1 day 2 day 3 control 1. .5 0 0 pHEX-N: :GUS 353. .8 6287 .7 15984. .8 pHEXHO: :GUS 1335. .9 23441 .0 42570 .2 pHEXlll : :GUS 208 .8 5268 .4 13891 .4 pIDS21 (35S:: :GUS) 22 .3 665 .2 1044 .0

Table 2

Transient GUS activity values determined in maize proto- plast derived cell suspension

(pmole MU / mg prot. / min)

day 2 day 3 control 0.47 0.49 pHEX-N::GUS 0.46 0.99 pHEX110: :GUS 62.51 50.90 pHEXlll: :GUS 6.64 15.81

Example 4

Stable transformation of pHEX expression vectors into plants

The vectors according to the invention were mcorpo- rated into the widely used Agrobacteri um mediated transfor¬ mation system and stable tobacco transformants were pro¬ duced. The description of the employed Agrobacteri um plas¬ mid designated EHA101 can be found in Hood et al . , J. Bac¬ teriol. 168: 1291 (1986) . Regeneration of the produced transgenic plants carrying the introduced vectors was also according to the above publication. Briefly, the Agrobacte¬ ri um culture co-cultivated with tobacco leaf platelets was further cultured on plant regeneration medium. After elimi¬ nating the bacteria, plants were cultivated in m vi tro culture and in green house and seeds produced were col¬ lected. Proper functioning of the vectors of the invention was established by measuring GUS activity of both primary transformants and progeny seedlings. Production of blue in¬ digo dye was demonstrated in leaf tissues by hystochemical staining according to the method of Jefferson et al . [ EMBO J. 6: 3901 (1987)] . A GUS activity value between 1968 and 3891 pmole MU / mg protein / min was measured in leaves of tobacco plants transformed with vector pHEX-N and this re¬ sult clearly demonstrates the proper functioning of the vectors according to the invention in transgenic plants. In transgenic plants carrying vector construction pHEX110::GUS the measured GUS activity value was between 1598 and 4634 pmole MU / mg protein / mm.

Claims

CLA IMS :

1. Single or double stranded nucleic acid molecule, comprising - wholly or in part - the sequence regions of the alfalfa (Medi cago sa ti va ) H3gl gene presented on Fig. IA, Fig. IB and/or Fig. IC, homologues or functional vari¬ ants thereof.

2. Nucleic acid molecule according to claim 1, compris¬ ing a sequence that is at least 80 % homologous to the se¬ quences or any parts thereof presented in Fig. 1.

3. Nucleic acid molecule accordmg to claim 1, compris¬ ing a sequence - or any part thereof - that is identical with or at least 80 % homologous to the nucleotide sequence presented m Fig. IA (nucleotides 1-482) .

4. Nucleic acid molecule according to claim 1, compris- ing a sequence - or any part thereof - that is identical with or at least 80 % homologous to the nucleotide se¬ quences presented in Fig. IB (nucleotides 555-668, 746-962 and 1053-1174) .

5. Nucleic acid molecule according to claim 1, compris- g a sequence - or any part thereof - that is identical with or at least 80 % homologous to the nucleotide sequence presented in Fig. IC (nucleotides 1346-1676) .

6. Nucleic acid molecule hybπdizable with a nucleic acid molecule according to any one of claims 1-5.

7. Transformation vector according to any one of claims 1-5, comprising sequences presented in Fig. IA, IB and/or IC - parts, homologues or functional variants thereof - op¬ eratively linked to a structural gene.

8. Transformation vector accordmg to claim 7, suitable for transforming plant cells.

9. Transgenic plant or any reproducible part thereof comprising a nucleic acid molecule according to any one of claims 1-7.

10. Plant propagation material prepared from or using a plant according to claim 9.