Background Art

Barley (Hordeum vulgare) is an economically important plant that is used mainly for the production of malt, and it is furthermore a crop used in food production, feeding, and pharmaceutical industry. Modification of its genetic properties may lead to a significant improvement of agricultural parameters of this cereal.

The architecture of the plant may be specifically modified by changing the levels of cytokinins, plant hormones (Mok DWS and Mok MC. (2001), Ann Rev Plant Physiol Plant Mol Biol. 52:89-118) that participate in the regulation of a number of key processes occuring in plants, such as for example division and differentiation of cells (Bianco MD, Giustini L, Sabatini S. (2013), New Phytol. 199:324-38), resistance to stress (Dobrev PI, Vankova R. (2012), Methods Mol Biol. 913:251-61; Kuppu S, Mishra N, Hu R, Sun L, Zhu X, Shen G, Blumwald E, Payton P, Zhang H. (2013), PLoS One. 8:e64190), and delayed senescence (Khan M, Rozhon W, Poppenberger B. (2013), Gerontology, (in print)). The individual forms of cytokinins and their derivatives differ in respect of their biological activity. According to the cited publications, it is apparent that these hormones are continuously intensively studied for their key influence on the growth and development of the plants, which can be used in growing agriculturally significant crops for example to increase the yield (Powell AF, Paleczny AR, Olechowski H, Emery RJ. (2013), Plant Physiol Biochem. 64:33-40) or to improve the resistance to biotic stress (Reusche M, laskova J, Thole K, Truskina J, Novak O, Janz D, Strnad M, Spichal L, Lipka V, Teichrnann T. (2013), Mol Plant Microbe Interact. 26:850-60) and abiotic stress (Dobrev PI, Vankova R. (2012), Methods Mol Biol. 913:251-61). With respect to the increase in world population, there are ongoing efforts to utilize land for agricultural purposes also in the areas that are currently not suitable for growing crops. It is also believed that the negative changes of climate, including those caused by human activity, will result in longer dry periods and temperature rise. Efforts to increase the resistance of important plants to drought stress while using different approaches are therefore considerable in recent years and they correspond to the seriousness of the situation (Grayson M. (2013), Agriculture and drought. Nature Outlook 01:S1).

Degradation of cytokinins is ensured in each plant by enzyme activity of cytokinin dehydrogenase (EC 1.5.99.12; CKX) (Galuszka P, Frebort I, Sebela M, Sauer P, Jacobsen S, Pec P. (2001), Eur J Biochem.268:450-61), which occurs in the plants in various forms acting in various cell compartments (Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmulling T. (2003), Plant Cell. 15:2532-50; Schmulling, T. _s Werner, T., Riefler, M, Krupkova, E. and Bartrina y Manns, I. (2003), J Plant Res. 116:241-52; Liu Z, Lv Y, Zhang M, Liu Y, Kong L, Zou M, Lu G, Cao J, Yu X. (2013), BMC Genomics. 14:594 (in print); Tsai YC, Weir NR, Hill K, Zhang W, Kim HJ, Shiu SH, Schaller GE, Kieber JJ. (2012), Plant Physiol. 158:1666-84) in various stages of development of the plants (Powell AF, Paleczny AR _S Olechowski H, Emery RJ. (2013), Plant Physiol Biochem. 64:33-40). The individual forms of CKX have also different substrate specificity, which is determined by a different localization of cytokinins in the plant tissue. In the plant A. thaliana, the substrate specificity of CKX (Galuszka P, Popelkova H, Werner T, Frebortova J, Pospisilova H, Mik V et al. (2007), J Plant Growth Regul. 26:255-67) and the localization of the individual CKX is described most thoroughly. A. thaliana contains two vacuolar CKX (AtCKXl and AtCKX3), one cytosolic CKX (AtCKX7), and the remaining four CKX are probably apoplastic (Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmulling T. (2003), Plant Cell. 15:2532-50). It has been found out that the secreted (apoplastic) forms of CKX prefer free cytokinin bases as substrates, vacuolar forms prefer N-glycosyl cytokinins (Galuszka et al. (2007), J Plant Growth Regul. 26:255-67). It is interesting that vacuolar CKX prefer di- and triphosphates of cytokinins that are primary products of the de novo cytokinin biosynthesis in the plants (Kowalska M, Galuszka P, Frebortova J, Sebela M, Beres T, Hluska T, Smehilova M, Bilyeu KD, Frebort I. (2010), Phytochemistry. 71:1970-8). Based on this, we can deduce different biological functions of the individual forms of CKX. It follows that by the combination of the desired form of the enzyme, signal sequence for protein targeting to the individual compartments, and suitable tissue-specific promoter, we can specifically influence the resulting phenotype of the plant.

The study of the genetically modified dicotyledonous plants with overexpressed CKX was started in 2001 (Werner T, Motyka V, Strnad M, Schmiilling T (2001), Proc Natl Acad Sci U S A. 98:10487-92). The phenotype of such plants is called cytokinin- deficient, and it is characterized mainly by reduced aerial part and productivity and further by the increase of the root system (Werner T, Motyka V, Strnad M, Schmulling T (2001), Proc Natl Acad Sci U S A. 98:10487-92; Galuszka P, Frebortova J, Werner T, Yamada M, Strnad M, Schmiilling T, Frebort I. (2004), Eur J Biochem. 271 :3990-4002). In particular, prepared genetically modified plants A. thaliana with overexpression of AtC Xl, 2, 3, 4, 5 or 6 have similar cytokinin-deficient phenotype which was the most significant in the plants with overexpression of AtCKXl, 3 and 5 (Werner et al. (2003), Plant Cell. 15:2532-50). Later, Arabidopsis plants with root-specific overexpression of AtCKX3 were prepared, in which the mass of roots was increased by as much as 40% without any negative influence to the fertility of the plants, and the modified plants were showing a higher resistance to drought stress (Werner T, Nehnevajova E, Kollmer I, Novak O, Strnad M, Kramer U, Schmulling T. (2010), Plant Cell. 22:3905-20). A similar phenotype was observed also in tobacco plants with AtCKXl overexpression, where the resistance of these plants to drought was also confirmed (Mackova H, Hronkova M, Dobra J, Tureckova V, Novak O, Lubovska Z, Motyka V, Haisel D, Hajek T, PraSil IT, Gaudinova A, Storchova H, Ge E, Werner T, Schmulling T, Vankova R. (2013), J Exp Bot. 64:2805-15).

Unlike dicotyledonous plants, monocotyledonous plants are more difficult to transform, mainly as a result of insufficient regeneration of the plants from non-differentiated tissue. To obtain stable lines, the same transformation as in the dicotyledonous plants is used - A. tumefaciens mediated transformation (Harwood WA _} Bartlett JG, Alves SC, Perry M, Smedley MA, Leyland N, Snape JW. (2009), In Methods in Molecular Biology, Vol. 478 (Jones H. D, Shewry P. R. ed.), pp. 137-147, Humana Press, New York, USA). Based on published data, the manipulation with CKX genes in a monocotyledonous plant was performed only once, namely for barley, in our laboratory. The gene was a corn gene ZmCKXl (Mrizova , Jiskrova E, Vyroubalova &, Novak O, Ohnoutkova L, Pospisllova H, Frebort I, Harwood WA, Galuszka P. (2013), Accepted for publication to PLOS ONE). Unlike the overexpression of the CKX protein in dicotyledonous plants, the cytokinin- deficient phenotype in the plants of barley with CKX under the constitutive promoter was too strong, which was manifested by a reduced capability of regeneration of transgenic plants, their sterility, and premature senescence. For this reason, β-glucosidase promoter was used, which has the strongest activity in the roots (Gu R, Zhao L, Zhang Y, Chen X, Bao J, Zhao J, Wang Z, Fu J, Liu T, Wang J, Wang G. (2006), Plant Cell Rep. 25:1157- 65), for the regulation of expression of ZmCKXl (unpublished data). These plants also had a reduced capability of regeneration and strong cytokinin-deficient phenotype (Fig. 1). 5 independent transgenic lines of barley were obtained, in which as much as 10-times increased activity of CKX was detected. These plants were sterile due to the introduction of ZmCK l and did not provide any progeny. The conclusion from these data is that the results obtained for CKX overexpression in dicotyledonous plants cannot be applied to monocotyledonous cereals, where the regulation of metabolism of cytokinins may occur in a different way (Hirose N, Makita N, Kojima M, Kamada-Nobusada T, Sakakibara H. (2007) Plant Cell Physiol. 48-.523-39).

Further available data about monocotyledonous plants with overproduced CKX are presented in the document US 2009/0165174. They relate to overexpression of corn CKX in corn. According to our data shown above, the use of the corn ZmCKXl in barley is unsuitable and it is therefore impossible to apply the results obtained by the study of genetically modified corn to barley.

Disclosure of the Invention

Subject of the present invention is a method of preparation of barley plants resistant to drought, wherein a nucleotide sequence is inserted into the barley genome, said nucleotide sequence containing a sequence with at least 80% identity, preferably with at least 90% identity, more preferably with at least 95% identity, to the sequence of the gene AtCKXl (cytokinin dehydrogenase originated from the plant Arabidopsis thaliana) selected from the group comprising the sequences SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, or said nucleotide sequence containing a sequence with at least 90% identity, preferably with at least 95% identity, to the nucleotide sequence encoding a polypeptide selected from the group comprising the sequences SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9. The inserted nucleotide sequence encodes an enzyme with cytokinin dehydrogenase activity. In a preferred embodiment, the inserted sequence is controlled by β-glukosidase promoter. In a preferred embodiment, the sequence is inserted using the Agrobacterium tumefaciens.

The 80% identity, the preferred 90% identity, the more preferred 95% identity with the above-listed sequences is achieved for example by extension or shortening of the sequence or by point mutations in the sequence, especially such mutations which do not change the encoded amino acid due to degeneration of the genetic code.

Within the framework of the present invention, it has been found out that for the preparation of the genetically modified plants resistant to drought, it is suitable to use the vacuolar AtCKXl (nucleotide sequence SEQ ID NO. 1; natural form; Werner et al. (2001), Proc Natl Acad Sci U S A. 98:10487-92, corresponding amino-acid sequence SEQ ID NO. 7), apoplastic AtCKXl (nucleotide sequence SEQ ID NO. 2, signal peptide from AtCKX2, corresponding amino acid sequence SEQ ID NO. 8) or the cytosolic form AtCKXl (nucleotide sequence SEQ ID NO. 3, without signal peptide, corresponding amino acid sequence SEQ ID NO. 9), in a preferred embodiment under the β-glukosidase promoter with the highest rate of expression in the root (Gu et al, (2006), Plant Cell Rep. 25:1157-65). Using A. tumefaciens, these sequences can be integrated to the genome of barley in order to obtain transgenic plants in which a higher resistance to drought was observed. The increased resistance of the plants to drought increases the chances of survival of the barley plants growing in adverse climatic conditions.

Subject of the present invention is further an expression cassette which contains promoter-^iCJCX -terminator. AtCKXl is a sequence with at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, to a sequence selected from the group containing the sequences SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, or AtCKXl is a sequence with at least 90% identity, preferably at least 95% identity, to a nucleotide sequence encoding the polypeptide selected from the group containing the sequences SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9. The promoter is preferably β-glucosidase promoter, the terminator is preferably NOS terminator. In one preferred embodiment, the expression cassette contains: β-glucosidase vacuolar::NOS terminator, and it has a sequence with at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, to the sequence SEQ ID NO.4.

SEQ ID NO.4:

Positions of the individual functional sections:

β-glucosidase promoter: 50 bp - 950 bp

Vacuolar signal sequence: 966 bp - 1076 bp

AtCKXl (excluding the signal sequence): 1077 bp - 2693 bp

NOS terminator: 2721 bp - 2974 bp

I I I 1 I I I I I I I I I.... I I I

1 I I I I I I I \ I I I ....I I I I ...I I I I I I I I I ] ] t I I I I I

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I .... I 1 I I I I .... I I I I ! I I I I ...,|....| I ! I I I I [ I I I I I I

960 970 9SO ] I I I I I I I I— I — I— I — I— I — I— I — I— I — I— I

1110 1120 1130 1140 1150 1160 1170 1180 1190 1200

GAATTACCTT CTTCAAAT-CC TTCAGATATT CGTTCCTCAT TA3TTTCACT AGATTTGGAG GGTTATATAA GCTTCGACGA TGTCCRCAAT GTGGCCAAGG

1210 1220 1230 1240 1250 1260 1270 1280 1290 1300

ACTTTGGCAA CAGATACCAG TTACCACCTT TGGCAATTCT ACATCCAAGG TCAGTTTTTG ATATTTCATC GATGATGAAG CATATAG AC ATCTGGGCTC

....I.... I ....I.... I ....I.... I ....I....I ....I.... I .... ί .... J ....]....! ....]..,.[ ....I,... I ....I.... I

1310 1320 1330 1340 1350 1360 1370 1380 1390 1400

CACCTCAAAT CTTACAGTAG CAGCTAGAGG CCAIGGTCAC TCGCTTCAAG GACAAGCTCT AGCTCATCAA GGTGTTGTCA TCAAAAIGGA GTCACTTCGA

I i t I I I I ! 1 I I I I I I I i t i I

1410 1420 1430 1440 1450 1460 1470 1480 1490 1500

AGTCCTGATA TCAGGATTTA TAAGGGGAAG CAACCATATG TTGATGTCTC AGGTGGTGAA ATATGGATAA ACATTCIACG CGAGACTCTA AAATACGGTC

.... I I ! .... I I I I I I I I 1 I I I I I I I I

1510 1520 1530 1540 1550 1560 1570 1580 1590 1600

TTTCACCAAA GTCCTGGACA GACTACCTTC ATTTGACCGT TGGAGGTACA CTATCTAATG CTGGAATCAG CGGTCAAGCA TTCAAGCATG GACCCCAAAT I I I 1 I I I I I I I I I I I I I I I I

1610 1620 1630 1640 1650 1660 1670 1680 1690 1700

CAACAACGTC TACCAGCTAG AGATTGTTAC AGGGAAAGGA GAAGTCGTAA CCTGTTCTGA GAAGCGGAAT TCTGAACTTT TCTTCAGTGT TCTTGGCGGG

1710 1720 1730 1740 1750 1760 1770 1780 1790 1800

CTTGGACAGT TTGGCATAAT CACCCGGGCA CGGATCTCTC TTGAACCAGC ACCGCAIATG GTTAAATGGA TCAGGGTACT CTACTCTGAC TTTTCTGCAT

— I I — I— I — I— I — I— i ]— i — I— I — I— I — I— I — i— ι — i— i

1810 1820 1830 1840 1850 I860 1870 1880 1890 1900

TTTCAAGGGA CCAAGAATAT CTGATTTCGA AGGAGAAAAC TTTTGATTAC GTTGAAGGAT TTGTGATAAT CAATAGAACA GACCriCTCA ATAATTGGCG

....(.... I ....|.... I ....I.... I . I .... I ....I.... I ....I....I ....I.... I ....I.... I ....I.... I

1910 1920 1930 1940 1950 1960 1970 I960 1990 2000

ATCGTCATIC AGTCCCAACG ATTCCACACA GGCAAGCAGA IICAAGTCAG ATGGGAAAAC TCTTTATTGC CTAGAAGTGG TCAAATATTT CAACCCAGAA

— t I — i— J — I— I ....I— I — I— I ....I.... I ....I— I ....I— I — I— ι — ι— ι

2010 2020 2030 2040 2050 2060 2070 2030 2090 2100

GAAGCTAGCT CTATGGATCA GGAAACTGGC AAGTTACTTT CAGAGTTAAA TTATATTCCA TCCACTTTGT TTTCATCTGA AGTGCCATAT ATCGAGTITC

2110 2120 2130 2140 2150 2160 2170 2180 2190 2200

TGGATCGCGT GCATATCGCA GAGAGAAAAC TAAGAGCAAA GGGTTTATGG GAGGTTCCAC ATCCCTGGCT GAATCTCCTG ATTCCTAAGA GCAGCATATA

I.... I ....I I I I I I ....I I I I I I I....I I I

2210 2220 2230 2240 2250 2260 2270 2230 2290 2300

CCAATTTGCT ACAGAAGTII TCAACAACAT TCTCACAAGC AACAACAACG GTCCTATCCT TATTTATCCA GTCAATCAAT CCAAGTGGAA GAAACATACA

2310 2320 2330 2340 2350 2360 2370 2380 2390 2400

TCTTTGATAA CTCCAAATGA AGATATATTC TATCTCGTAG CCTTTCTCCC CTCTGCAGTG CCAAATTCCT CAGGGAAAAA CGATCTAGAG TACCTTTTGA

2410 2420 2430 2440 2450 2460 2470 2480 2490 2500

AACAAAACCA AAGAGTTATG AACTTCTGCG CAGCAGCAAA CCTCAACGTG AAGCAGTATT TGCCCCATTA TGAAACTCAA AAAGAGTGGA AATCACACTT

I t ! I I....I I 1 I i I I I I I i ( ) ! I

2510 2520 2530 2540 2550 2560 2570 2580 2590 2600

TGGCAAAAGA TGGGAAACAT TTGCACAGAG GAAACAAGCC TACGACCCTC TAGCGATTCT AGCACCTGGC CAAAGAATAT TCCAAAAGAC AACAGGAAAA

....I I I I I I t 1 I t I 1 I I 1 I I I I I

2610 2620 2630 2640 2650 2660 2670 2660 2690 2700

TTATCTCCCA TCCAACTCGC AAAGTCAAAG GCAACAGGAA GTCCTCAAAG GTACCATTAC GCATCAATAC TGCCGARACC TAGAACIGTA TAAGICGACT 1 I I I I I I.... I ....I I I I I I I I I.... I

2710 2720 2730 2740 27S0 2760 2770 2790 2790 2800

CGAGTCTAGA AGTCGAAGCA GATCGTTCAA ACATTTGGCA ATAAAGTTTC TTAAGATTGA ATCCTGTTGC CGGTCTTGCG ATGATTATCA TATAATTTCT

....I.... I .... I .... I I ....I....! ....I.... I ....I.... I .... I .... I

2810 2820 2830 2840 2B50 2860 2870 2880 2890 2900

GTTGAATTAC GTTAAGCATG TAATAATTAA CATGTAATGC ATGACGTTAT TTATGAGATG GGTTTTTATG ATTAGAGTCC CGCAATTATA CATTTAATAC

1.... I I I I I I I I I [ ! I I I ] ! I I t

2910 2920 2930 2940 2950 2960 2970 2980 2990 3000

GCGATAGAAA ACAAAATATA GCGCGCAAAC TAGGATAAAT TATCGCGCGC GGTGTCATCT ATGTTACTAG ATCGACCGGC AAGCAAGCTG ATATGCGGCC

3010 3020 3030 3040 30S0 3060 3070 3030 3090 3100

GCACTCGAGA TAICTAGACC CAGCTTTCTT GTACAAAGTG GTTGATGGGC TGCAGGAATT CGATATCAAG CTTATCGATA CCGTCGACCT CGAGGGGGAT

...,| ,...| ....I ....I ...

3110 3120

CACCACTTTG TACAAGAAAG CTG

Protein AtCKXl vacuolar (575 amino acids, SEQ ID NO. 7)

AUG GGA UUG ACC UCA UCC UUA CGG UUC CAU AGA CAA AAC AAC AAG

Met Gly Leu Thr Ser Ser Leu Arg Phe His Arg Gin Asn Asn Lys

ACU UDC CDC GGA AUC UUC AUG AUC UUA GUU CUA AGC UGU AUA CCA

Thr Phe Leu Gly lie Phe Met lie Leu Val Leu Ser Cys lie Pro

GGU AGA ACC AAU CUU UGU UCC AAU CAU UCU GUU AGU ACC CCA AAA

Gly Arg Thr Asn Leu Cys Ser Asn His Ser Val Ser Thr Pro Lys

GAA UUA CCU UCU UCA AAU CCU UCA GAU AUU CGU UCC UCA UUA GUU

Glu Leu Pro Ser Ser Asn pro Ser Asp lie Arg Ser Ser Leu Val

UCA CUA GAU UUG GAG GGU UAU AUA AGC UUC GAC GAU GUC CAC AAU

Ser Leu Asp Leu Glu Gly Tyr lie Ser Phe Asp Asp Val His Asn

GUG GCC AAG GAC UUU GGC AAC AGA UAC CAG UUA CCA CCU UUG GCA

Val Ala Lys Asp Phe Gly Asn Arg Tyr Gin Leu Pro Pro Leu Ala

ADU CUA CAU CCA AGG UCA GUU DUU GAU AUU UCA UCG AUG AUG AAG

lie Leu His Pro Arg Ser Val Phe Asp He Ser Ser Met Met Lys

CAU AUA GUA CAU CUG GGC UCC ACC UCA AAU CUU ACA GUA GCA GCU

His He Val His Leu Gly Ser Thr Ser Asn Leu Thr Val Ala Ala

AGA GGC CAU GGU CAC UCG CUU CAA GGA CAA GCU CUA GCU CAU CAA

Arg Gly His Gly His Ser Leu Gin Gly Gin Ala Leu Ala His Gin

GGU GUU GUC AUC AAA AUG GAG UCA CUU CGA AGU CCU GAU AUC AGG

Gly Val Val He Lys Met Glu Ser Leu Arg Ser Pro Asp He Arg AUU UAU AAG GGG AAG CAA CCA UAO GUU GAU GUC UCA GGU GGU GAA He Tyr Lys Gly Lys Gin Pro Tyr Val Asp Val Ser Gly Gly Glu AUA UGG AUA AAC AUU CUA CGC GAG ACU CUA AAA UAC GGU CUU UCA

He Trp He Asn He Leu Arg Glu Thr Leu Lys Tyr Gly Leu Ser

CCA AAG UCC UGG ACA GAC UAC CUU CAU UUG ACC GUU GGA GGU ACA

Pro Lys Ser Trp Thr Asp Tyr Leu His Leu Thr Val Gly Gly Thr

CUA UCU AAU GCU GGA AUC AGC GGU CAA GCA UUC AAG CAU GGA CCC

Leu Ser Asn Ala Gly He Ser Gly Gin Ala Phe Lys His Gly Pro

CAA AUC AAC AAC GUC UAC CAG CUA GAG AUU GUU ACA GGG AAA GGA

Gin He Asn Asn Val Tyr Gin Leu Glu He Val Thr Gly Lys Gly

GAA GUC GUA ACC UGU UCU GAG AAG CGG AAU UCU GAA CUU UUC UUC

Glu Val Val Thr Cys Ser Glu Lys Arg Asn Ser Glu Leu Phe Phe AGU GUU CUU GGC GGG CUU GGA CAG UUU GGC AUA AUC ACC CGG GCA

Ser Val Leu Gly Gly Leu Gly Gin Phe Gly He He Thr Arg Ala

CGG AUC UCU CUU GAA CCA GCA CCG CAU AUG GUU AAA UGG AUC AGG

Arg He Ser Leu Glu Pro Ala Pro His Met Val Lys Trp He Arg

GUA CUC UAC UCU GAC UUU UCU GCA UUU UCA AGG GAC CAA GAA UAU

Val Leu Tyr Ser Asp Phe Ser Ala Phe Ser Arg Asp Gin Glu Tyr

CUG AUU UCG AAG GAG AAA ACU UUU GAU UAC GUU GAA GGA UUU GUG

Leu He Ser Lys Glu Lys Thr Phe Asp Tyr Val Glu Gly Phe Val

AUA AUC AAU AGA ACA GAC CUU CUC AAU AAU UGG CGA UCG UCA UUC

He He Asn Arg Thr Asp Leu Leu Asn Asn Trp Arg Ser Ser Phe AGU CCC AAC GAU UCC ACA CAG GCA AGC AGA UUC AAG UCA GAU GGG

Ser Pro Asn Asp Ser Thr Gin Ala Ser Arg Phe Lys Ser Asp Gly

AAA ACU CUU UAU UGC CUA GAA GUG GUC AAA UAU UUC AAC CCA GAA

Lys Thr Leu Tyr Cys Leu Glu Val Val Lys Tyr Phe Asn Pro Glu

GAA GCU AGC UCU AUG GAU CAG GAA ACU GGC AAG UUA CUU UCA GAG

Glu Ala ser Ser Met Asp Gin Glu Thr Gly Lys Leu Leu Ser Glu

UUA AAU UAU AUU CCA UCC ACU UUG UUU UCA UCU GAA GUG CCA UAU

Leu Asn Tyr He Pro Ser Thr Leu Phe Ser Ser Glu Val Pro Tyr ADC GAG UUU CUG GAU CGC GUG CAU AUC GCA GAG AGA AAA CUA AGA lie Glu Phe Leu Asp Arg Val His He Ala Glu Arg Lys Leu Arg

GCA AAG GGU UUA UGG GAG GUU CCA CAU CCC UGG CUG AAU CUC CUG

Ala Lys Gly Leu Trp Glu Val Pro His Pro Trp Leu Asn Leu Leu

AUU CCU AAG AGC AGC AUA UAC CAA UUU GCU ACA GAA GUU UUC AAC

He Pro Lys Ser Ser He Tyr Gin Phe Ala Thr Glu Val Phe Asn

AAC AUU CUC ACA AGC AAC AAC AAC GGU CCU ADC CUU AUU UAU CCA

Asn He Leu Thr Ser Asn Asn Asn Gly Pro He Leu He Tyr Pro

GDC AAU CAA UCC AAG UGG AAG AAA CAD ACA DCO UUG AUA ACD CCA

Val Asn Gin Ser Lys Trp Lys Lys His Thr Ser Leu He Thr Pro

AAU GAA GAU AUA UUC UAU CUC GUA GCC UDU CDC CCC UCU GCA GUG

Asn Glu Asp He Phe Tyr Leu val Ala Phe Leu Pro Ser Ala Val

CCA AAU UCC UCA GGG AAA AAC GAU CUA GAG UAC CUU UUG AAA CAA

Pro Asn Ser Ser Gly Lys Asn Asp Leu Glu Tyr Leu Leu Lys Gin.

AAC CAA AGA GUU AUG AAC UUC UGC GCA GCA GCA AAC CUC AAC GUG

Asn Gin Arg Val Met Asn Phe Cys Ala Ala Ala Asn Leu Asn Val

AAG CAG UAU UUG CCC CAU UAU GAA ACU CAA AAA GAG UGG AAA UCA

Lys Gin Tyr Leu Pro His Tyr Glu Thr Gin Lys Glu Trp Lys Ser CAC UUU GGC AAA AGA UGG GAA ACA UUU GCA CAG AGG AAA CAA GCC

His Phe Gly Lys Arg Trp Glu Thr Phe Ala Gin Arg Lys Gin Ala

UAC GAC CCU CUA GCG AUD CUA GCA CCU GGC CAA AGA AUA DUC CAA

Tyr Asp Pro Leu Ala He Leu Ala Pro Gly Gin Arg He Phe Gin

AAG ACA ACA GGA AAA UUA UCU CCC AUC CAA CUC GCA AAG UCA AAG

Lys Thr Thr Gly Lys Leu Ser Pro He Gin Leu Ala Lys Ser Lys

GCA ACA GGA AGU CCU CAA AGG UAC CAU UAC GCA UCA ADA CDG CCG

Ala Thr Gly Ser Pro Gin Arg Tyr His Tyr Ala Ser He Leu Pro

AAA CCU AGA ACU GUA UAA

Lys Pro Arg Thr Val End In another preferred embodiment, the expression cassette contains: β-glucosidase apoplastic::NOS terminator, and it has a sequence with at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity to the sequence SEQ ID NO.5.

SEQ ID NO.5:

Positions of the individual functional sections:

β-glucosidase promoter : 68 bp - 968 bp

Apoplastic signal sequence: 999 bp - 1067 bp

AtCKXl (excluding the signal sequence): 1068 bp - 2684 bp

NOS terminator: 2712 bp -2965 bp

....I....I ....|.... I ,...!....( ....|.... I .... [....! ....(.... ί ....I.... I ....I.... I

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1 I I I I I [ I I ( I 1 I I I i I I I )

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I.... I I I i ! I I I I I ! 1 I i ! I I I I

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....I I I I 1 I I I I I I I I I I I I I I 1110 1120 1130 1140 11S0 1160 1170 ¹¹⁸ ° 1190 1200

TCTTCAAATC CTTCAGATAT TCGTTCCTCA TTAGTTTCAC TAGATTTGGA GGGTTATATA AGCTTCGACG ATGTCCACAA TGIGGCCAAG GACTTTGGCA

I....I I I I I I I I I I I

1210 1220 1230 1240 1250 1260 1270 1260 1290 1300

ACAGATACCA GTTACCACCT IIGGCAATTC TACAICCAAG GTCAGTTTTI GATATTTCAT CGATGATGAA GCATATAGTA CATCTGGGCT CCACCTCAAA I I I I I I I

1310 1320 1330 1340 1350 1360 1310 1330 1390 1400

TCTTACAGTA GCAGCTAGAG GCCATGGTCA CICGCTTCAA GGACAAGCIC TAGCTCATCA AGGTGTTGTC ATCAAAATGG AGTCACTACG AAGTCCTGAT I I I I I I I I I I I

1410 1420 1430 1440 1450 1460 1 70 1480 1450 1500

ATTAGGATTT AIAAGGGGAA GCAACCATAT GTTGATGTCT CAGGTGGTGA AATATGGATA AACATTCTAC GCGAGACTCT AAAATACGGT CTTTCACCAA I I— I — I— I — I I I — I I— I

1510 1520 1S30 1540 1550 1560 1570 1580 1590 1600

AGTCCTGGAC AGACTACCTT CATTTGACCG TTGGAGGTAC ACTATCTAAT GCTGGAATCA GCGGTCAAGC ATICAAGCAT GGACCCCAA& TCAACAACGT I ....I.,.. ....|....I ....|.... j ....!.... I ....I

1610 1620 1630 1640 1650 1660 1670 1680 1690 1700

CTACCAGCTA GAGATTGTTA CAGGGAAAGG AGAAGTCGIA ACCTGITCTG AGAAGCGGAA CTCTGAACTT TTCTTCAGTG TTCTTGGCGG GCTTGGACAG

1710 1720 1730 1740 1750 1760 1770 1780 1790 1800

TTTGGCATAA TCACCCGGGC ACGGAICTCT CTTGAACCAG CACCGCATAT GGTTAARTGG ATCAGGGTAC TCTACTCTGA CTITTCTGCA TTTTCAAGGG

.... I I [ I I I I I I I I I .

1B10 1820 1330 1840 1850 1860 1870 1880 1890 1900

ACCAAGAATA TCTGATTTCA AAGGAGAAAA CTTTTGATTA CGTTGAAGGA TTTGTGATAA TCAATAGAAC AGACCTTCTC AATAATTGGC GATCGTCATT I I I ! I [ I I I I

1910 1920 1930 1940 1950 196D 1970 1980 1990 2000

CAGTCCCAAC GATTCCACAC AGGCAAGCAG ATTCAAGTCA GATGGGARAA CTCTTTATTG CCTAGAAGTG GTCAAATATT TCAACCCAGA AGAAGCTAGC

2010 2020 2030 2040 2050 2060 2070 2080 2090 2100

TCTATGGATC AGGAAACTGG CAAGTTACIT TCAGAGTTAA ATTATATTCC ATCCACTTTG TTTTCATCTG AAGTGCCATA TATCGAGTII CTGGATCGCG I I I I I I I I I I

2110 2120 2130 2140 2150 2160 2170 2180 2190 2200

TGCATATCGC AGAGAGAAAA CTAAGAGCAA AGGGTTTATG GGAGGTTCCA CATCCCTGGC TGAATCTCCT GATTCCTAAG AGCAGCATAT ACCAATTTGC

....! I I I ....I I I I I I I

2210 2220 2230 2240 2250 2260 2270 22Θ0 2290 2300

TACAGAAGTT TTCAACAACA TTCTCACAAG CAACAACAAC GGTCCTATCC TTATTTATCC AGTCAATCAA TCCAAGTGGA AGAAACATAC ATCTTTGATA

I ! ! I I I I I I. ...I I I I

2310 2320 2330 2340 2350 2360 2370 2380 2390 2400

ACTCCAAAIG AAGATATAII CTATCTCGTA GCCTTTCTCC CCTCTGCAGT GCCAAAITCC ICAGGGMAA ACGATCTTGA GTACCTTTTG AAACAAftftCC

2410 2420 2430 2440 2450 2460 2470 2430 2490 250O

AAAGAGTTAI GAACTTCTGC GCAGCAGCAA ACCTCAACGT GAAGCAGTAT TTGCCCCATT ATGSAACTCA AAAAGAGTGG AAATCACACT TTGGCAAAAG I I I I I I I I I I I I

2510 2520 2530 2540 2550 2560 2570 2580 2590 2600

ATGGGAAACA TTTGCACAGA GGAAACAAGC CTACGACCCT CTAGCGATTC TAGCACCTGG CCAAAGAATA TTCCAAAAGA CAACAGGAAA ATTATCTCCC

2610 2620 2630 2640 2650 2660 2670 2680 2690 270O

AICCAACTCG CAAAGTCAAA GGCAACAGGA AGTCCTCAAA GGIAICAITA CGCATCAATA CTGCCGAAAC CTACAACTGT ATAAGTCGAC TCGAGICIAG

2710 2720 2730 2740 2750 27S0 2770 2780 2790 2800

AAGTCGAAGC AGATCGTTCA AACATTTGGC AATAAAGTTT CTTAAGATTG AATCCTGTTG CCGGTCTTGC GATGATTATC ATATAATTTC TGTTGAATTA i

2810 2S20 2830 2840 28S0 2860 2B70 2380 2890 2900

CGTTAAGCAT GTAMAATTA ACATGTAATG CATGACGTTA TTTATGAGAT GGGTITTTAT GATTAGAGTC CCGCAATTAT ACATTTAATA CGCGAIAGAA

2910 2920 2930 2940 2950 2960 2970 2980 2990 3000

A&CAAAATAT AGCGCGCAAA CTAGGATAAA TTATCGCGCG CGGTGTCATC TATGTTACIA GAICGACCCG CAAGCAAGCT GATATGCGGC CGCACTCGAG

....I.. ... I

3010 3020 3030 3040 3050 3060 307O 3080 3090 3100

ATATCTAGAC CCAGCTTTCT TGTACAAAGT GGTTGATGGG CTGCAGGAAT TCGATATCAA GCTTATCGAT ACCGTCGACC TCGAGGGGGA TCACCACTTT

3110

GTACAAGAAA GCTG

Protein AtC Xl apoplastic (557 amino-acids, SEQ ID NO. 8)

AUG AUC ACU UUA AUC ACG GUU UUA AUG AUC ACC AAA UCA UCA AAC

Met lie Thr Leu lie Thr Val Leu Met lie Thr Lys Ser Ser Asn

GGC AUG CUU UCG AAU CAU UCU GUU AGU ACC CCA AAA GAA UUA ecu

Gly Met Leu Ser Asn His Ser Val Ser Thr Pro Lys Glu Leu Pro

UCU UCA AAU ecu UCA GAU AUU CGU UCC UCA UUA GUU UCA CUA GAU

Ser Ser Asn Pro Ser Asp He Arg Ser Ser Leu Val Ser Leu Asp

UUG GAG GGU UAU AUA AGC UDC GAC GAU GUC CAC AAU GUG GCC AAG

Leu Glu Gly Tyr He Ser Phe Asp Asp Val His Asn Val Ala Lys

GAC UUU GGC AAC AGA UAC CAG UUA CCA ecu UUG GCA AUU CUA CAU

Asp Phe Gly Asn Arg Tyr Gin Leu Pro Pro Leu Ala He Leu His

CCA AGG OCA GUU UUU GAU AUO UCA UCG AUG AUG AAG CAU AUA GUA

Pro Arg Ser Val Phe Asp He Ser Ser Met Met Lys His He Val

CAU CUG GGC UCC ACC UCA AAU CUU ACA GUA GCA GCU AGA GGC CAU

His Leu Gly Ser Thr ser Asn Leu Thr Val Ala Ala Arg Gly His

GGU CAC UCG CUU CAA GGA CAA GCU CUA GCU CAU CAA GGU GUU GUC

Gly His Ser Leu Gin Gly Gin Ala Leu Ala His Gin Gly Val Val

AUC AAA AUG GAG UCA CUA CGA AGU CCU GAU AUU AGG AUU UAU AAG

He Lys Met Glu Ser Leu Arg Ser Pro Asp He Arg He Tyr Lys GGG AAG CAA CCA UAU GUU GAU GUC UCA GGU GGU GAA AUA UGG AUA

Gly Lys Gin Pro Tyr Val Asp Val Ser Gly Gly Glu He Trp He

AAC AUU CUA CGC GAG ACU CUA AAA UAC GGU CUU UCA CCA AAG UCC

Asn He Leu Arg Glu Thr Leu Lys Tyr Gly Leu Ser Pro Lys Ser UGG ACA GAC UAC CUU CAU DUG ACC GUU GGA GGU ACA CUA UCU AAU Trp Thr Asp Tyr Leu His Leu Thr Val Gly Gly Thr Leu Ser Asn

GCU GGA ADC AGC GGD CAA GCA UUC AAG CAU GGA CCC CAA AUC AAC

Ala Gly lie Ser Gly Gin Ala Phe Lys His Gly Pro Gin He Asn

AAC GUC UAC CAG CUA GAG AUU GUU ACA GGG AAA GGA GAA GUC GUA

Asn Val Tyr Gin Leu Glu He Val Thr Gly Lys Gly Glu Val Val

ACC UGU UCU GAG AAG CGG AAC UCU GAA CUU UUC UUC AGU GUU CUU

Thr Cys Ser Glu Lys Arg Asn Ser Glu Leu Phe Phe Ser Val Leu

GGC GGG CUU GGA CAG UUU GGC AUA AUC ACC CGG GCA CGG AUC UCU

Gly Gly Leu Gly Gin Phe Gly He He Thr Arg Ala Arg He Ser

CUU GAA CCA GCA CCG CAU AUG GUU AAA UGG AUC AGG GUA CUC UAC

Leu Glu Pro Ala Pro His Met Val Lys Trp He Arg Val Leu Tyr

UCU GAC UUU UCU GCA UUU UCA AGG GAC CAA GAA UAU CUG AUU UCA

Ser Asp Phe Ser Ala Phe Ser Arg Asp Gin Glu Tyr Leu He Ser

AAG GAG AAA ACU UUU GAU UAC GUU GAA GGA UUU GUG AUA AUC AAU

Lys Glu Lys Thr Phe Asp Tyr Val Glu Gly Phe Val He He Asn

AGA ACA GAC CUU CUC AAU AAU DGG CGA UCG UCA UUC AGU CCC AAC

Arg Thr Asp Leu Leu Asn Asn Trp Arg Ser Ser Phe Ser Pro Asn

GAU UCC ACA CAG GCA AGC AGA UUC AAG UCA GAU GGG AAA ACU CUU

Asp Ser Thr Gin Ala Ser Arg Phe Lys Ser Asp Gly Lys Thr Leu

UAU UGC CUA GAA GUG GUC AAA UAU UUC AAC CCA GAA GAA GCU AGC

Tyr Cys Leu Glu Val Val Lys Tyr Phe Asn Pro Glu Glu Ala Ser

DCU AUG GAU CAG GAA ACU GGC AAG UUA CUU UCA GAG UUA AAU UAU

Ser Met Asp Gin Glu Thr Gly Lys Leu Leu Ser Glu Leu Asn Tyr

AUU CCA UCC ACU UUG UUU UCA UCU GAA GUG CCA UAU AUC GAG UUU

He Pro Ser Thr Leu Phe Ser Ser Glu Val Pro Tyr He Glu Phe

CUG GAU CGC GUG CAU AUC GCA GAG AGA AAA CUA AGA GCA AAG GGU

Leu Asp Arg Val His He Ala Glu Arg Lys Leu Arg Ala Lys Gly

UUA UGG GAG GUU CCA CAU CCC UGG CUG AAU CUC CUG AUU CCU AAG

Leu Trp Glu Val Pro His Pro Trp Leu Asn Leu Leu He Pro Lys AGC AGC AUA DAC CAA UUU GCU A A GAA GUU UOC AAC AAC AUU CUC

Ser Ser lie Tyr Gin Phe Ala Thr Glu Val Phe Asn Asn lie Leu

ACA AGC AAC AAC AAC GGU CCU AOC COD AUU UAU CCA GUC AAU CAA

Thr Ser Asn Asn Asn Gly Pro lie Leu lie Tyr Pro Val Asn Gin

UCC AAG UGG AAG AAA CAU ACA UCU UUG AUA ACU CCA AAU GAA GAU

Ser Lys Trp Lys Lys His Thr Ser Leu lie Thr Pro Asn Glu Asp

AUA UUC UAU CUC GUA GCC UUU CUC CCC UCU GCA GUG CCA AAU UCC

lie Phe Tyr Leu Val Ala Phe Leu Pro Ser Ala Val Pro Asn Ser

UCA GGG AAA AAC GAU CUU GAG UAC CUU UUG AAA CAA AAC CAA AGA

Ser Gly Lys Asn Asp Leu Glu Tyr Leu Leu Lys Gin Asn Gin Arg

GUU AUG AAC UUC UGC GCA GCA GCA AAC CUC AAC GUG AAG CAG UAU

Val Met Asn Phe Cys Ala Ala Ala Asn Leu Asn Val Lys Gin Tyr

UUG CCC CAU UAU GAA ACU CAA AAA GAG UGG AAA UCA CAC UUU GGC

Leu Pro His Tyr Glu Thr Gin Lys Glu Trp Lys Ser His Phe Gly

AAA AGA UGG GAA ACA UUU GCA CAG AGG AAA CAA GCC UAC GAC CCU

Lys Arg Trp Glu Thr Phe Ala Gin Arg Lys Gin Ala Tyr Asp Pro

CUA GCG AUU CUA GCA CCU GGC CAA AGA AUA UUC CAA AAG ACA ACA

Leu Ala lie Leu Ala Pro Gly Gin Arg He Phe Gin Lys Thr Thr

GGA AAA UUA UCU CCC AUC CAA CUC GCA AAG UCA AAG GCA ACA GGA

Gly Lys Leu Ser Pro He Gin Leu Ala Lys Ser Lys Ala Thr Gly

AGU CCU CAA AGG UAU CAU UAC GCA UCA AUA CUG CCG AAA CCU AGA

Ser Pro Gin Arg Tyr His Tyr Ala Ser He Leu Pro Lys Pro Arg

ACU GUA UAA

Thr Val End

In yet another preferred embodiment, the expression cassette contains: β-glucosidase promoter: :AtCKXl cytosolic::NOS terminator, and it has the sequence with at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, to the sequence SEQ ID NO. 6. SEQ ID NO.6:

Positions of the individual functional sections:

β-glucosidase promoter : 68 bp - 968 bp

AtCKXl (excluding the signal sequence): 999 bp - 2615 bp

NOS terminator: 2643 bp - 2896 bp I ! f ! I I I I I I I I I I I I I I I I

....I I I I .... I I f I i I I I I I I I I I ! I

.... I ] I I I I I I I I I .... I .... I .... I I I I I I I

.... [....I ....]....( ....I.... I .... |....| ....I.... I .... |...,| ..,.|....| .... I .... I .... I.... I ....I....I

....I....I .... J .... I ....I..., I ,...|.... I ....I,... I

I 1 ] 1 I I I I ...,|....| .,..|....| ....I I I I I I I I I I f I I I I I I I ! ! I I I I I I

....[.... I ...,|....| .... ! .... I .... I .... I ..-. I .... I ....I.... I .... I .... I ...,Ι.,,.Ι ....I, ...I

I I I I I I ! I I 1 1 I I I I I i f j I

....I I ....I I I 1 I I I I [ ( I I ! f I I I ! 1410 1420 1430 1440 1450 1460 1470 1430 1490 1500

CGCGAGACTC TAAAATACGG TCTTTCACCA AAGTCCTGGA CAGACTACCI TCATTTGACC GTTGGAGGTA CACTATCTAA TGCTGGAAIC AGCGGTCAAG

— I— [ — I— I — I— I — I— I — I— I — I— I .... I— [ — I— I — I— I — I— I

1510 1520 1530 1540 1550 1560 1570 1580 1590 1600

CATTCAAGCA TGGACCCCAA ATCAACAACG ICTACCAGCT AGAGATTGTT ACAGGGAAAG GAGAAGTCGT AACCTGTTCT GAGAAGCGGA ACTCTGAACT

....I.... I ....|.... I .... ! .... I ....I....I ....I.... I ....I.... I ....I.... I ....|....I ...,|....I

1610 1620 1630 1640 1650 1660 1670 1680 1690 1700

TTTCTTCAGT GTTCTTGGCG GGCTTGGACA GTTTGGCATA ATCACCCGGG CACGGATCTC TCTTGAACCA GCACCGCATA IGGTTAAATG GATCAGGGIA

I.... I ....I I I I I I ....I....I I I I I I I I .... I I

1710 1720 1730 1740 1750 1760 1770 1780 1790 1800

CTCTACTCTG ACTTTTCTGC ATTTTCAAGG GACCAAGAAT ATCTGATTTC AAAGGAGAAA ACTTTTGATT ACGTTGAAGG ATTTGTGATA ATCAATAGAA I I I I I I ....I I I I I I I I .... I .... I

1810 1820 1830 1B40 1B50 1860 1870 1880 1890 1900

CAGACCTTCT CAATAATTGG CGATCGICAT TCAGTCCCAA CGATTCCACA CAGGAAGCA GATTCAAGTC AGATGGGAAA ACTCTTTATT GCCTAGAAGT

....I.... I .... I .... I ....I.... I ,...|....| ...,|....I ....|....I

1910 1920 1930 1940 1950 1960 1970 1380 1990 2000

GGTCAAAIAT TTCAACCCAG AAGAAGCTAG CTCTATGGAT CAGGAAACTG GCAAGTTAC1 TICAGAGITA AATTATATTC CATCCACTTT GTTTTCATCT I I I I I I ....I.... I I I I I

2010 2020 2030 2040 2050 2060 2070 2080 2090 2100

GAAG1GCCAI ATATCGAGIT TCTCGATCGC GTGCATATCG CAGAGAGAAA ACTAAEAGCA AAGGGTTTAT GGGAGGTTCC ACATCCCTGG CTGAATCTCC

....I.... I ....I.... I ....|.... I ....).... I .. . . I . . . . I ....!....! I I . . . . I ....I ....!....(

2110 2120 2130 2140 2150 2160 2170 2180 2190 2200

TGATTCCTAA GAGCAGCATA TACCAATTTG CTACAGAAGT TTTCAACAAC ATTCTCACAA GCAACAACAA CGGTCCTATC CTTATTTATC CAGTCAATCA

.... I .... I ....|....I .... I .... j ....I.... I . I .... I ....(....! ....I....I

2210 2220 2230 2240 2250 2260 2270 2280 2290 2300

ATCCAAGTGG AAGAAACATA CATCTTTGAI AACTCCARAT GAAGATATAT TCTATCTCGT AGCCTTTCTC CCCTCIGCAG TGCCAAATTC CTCAGGGAAA

! ! I I I I f t ! I I I I I I I I I I

2310 2320 2330 2340 2350 2360 2370 2380 2390 2400

AACGATCTTG AGTACCTTTT GAAACAAAAC CAAAGAGTTA TGAACTTCTG CGCAGCAGCA AACCTCAACG TGAAGCAGIA TTTGCCCCAT TATGAAACTC I I I I I I I ! I I I ....I I I I 1 I I I

2410 2420 2430 2440 2450 2460 2470 2480 2490 2500

AAAAAGAGTG GAAATCACAC TTTGGCAAAA GATGGGAAAC ATTTGCACAG AGGAAACAAG CCTACGACCC TCTAGCGATI CTAGCACCTG GCCAAAGAAT I I t I I .... I I I ! ] I I I.... I ....I I ! [ ] I

2510 2520 2530 2540 2550 2560 2570 2580 2590 2600

ATTCCAAAAG ACAACAGGAA AATTATCTCC CAICCAACTC GCAAAGTCAA AGGCAACAGG AAGTCCTCAA AGGTATCATT ACGCATCAAT ACTGCCGAAA

2610 2620 2630 2640 2650 2660 2670 2680 2690 2700

CCTAGAACTG TATAAGTCOA CTCGAGTCTA GAAGTCGAAG CAGATCGTTC AAACATTTGG CAATAAAGTT TCTTAAGATT GAATCCTGTT GCCGGTCTTG

....I....I ....I,,.. I .... I .... I ....I.... I ....I,... I , . . . | . . . . t . . . . J . . . . I .... .... ^! ....I.... I .... I .... I

2710 2720 2730 2740 2750 2760 2770 2780 2790 2800

CGATGATTAT CATATAATTT CTGTIGAATT ACGTTAAGCA TGTAATAATT AACATGTAAT GCATGACGTT ATTTATGAGA TGGGTTTTTA TGATTAGAGT

.... I .... I ..-.I I I I I I I I I 1 i I I t I I I I

2810 2B20 2830 2840 2850 2860 2870 2880 2890 2900

CCCGCAATTA TACATTTAAT ACGCGATAGA AAACAAAATA TAGCGCGCAA ACTAGGATAA ATTATCGCGC GCGSTGTCAT CTATGTTACT AGATCGACCG

.... I .... I I I I I I I I ! I I I I I I t I I I

2910 2920 2930 2940 2950 2960 2970 2980 2990 3000

GCAAGCAAGC TGATATGCGG CCGCACTCGA GATATCTAGA CCCAGCTTTC TTGTACAAAG TGGTTGATGG GCTGCAGGAA TTCGATATCA AGCTTATCGA 3010 3020 3030 3040

TACCGTCGAC CTCGAGGGGG ATCACCACTT TGTACAAGAA AGCTG

Protein AtCKXl cytosolic (538 amino-acids, SEQ ID NO. 9)

AAU CAU UCU GUU AGU ACC CCA AAA GAA UUA CCU UCU UCA AAU CCU

Asn His Ser Val Ser Thr Pro Lys Glu Leu Pro Ser Ser Asn Pro

UCA GAU AUU CGU UCC UCA UUA GUU UCA CUA GAU UUG GAG GGU UAU

Ser Asp lie Arg Ser Ser Leu Val Ser Leu Asp Leu Glu Gly Tyr

AUA AGC UUC GAC GAU GUC CAC AAU GUG GCC AAG GAC UUU GGC AAC

lie Ser Phe Asp Asp Val His Asn Val Ala Lys Asp Phe Gly Asn

AGA UAC CAG UUA CCA CCU UUG GCA AUU CUA CAU CCA AGG UCA GUU

Arg Tyr Gin Leu Pro Pro Leu Ala He Leu His Pro Arg Ser Val

UUU GAU AUU UCA UCG AUG AUG AAG CAU AUA GUA CAU CUG GGC UCC

Phe Asp He Ser Ser Met Met Lys His He Val His Leu Gly Ser

ACC UCA AAU CUU ACA GUA GCA GCU AGA GGC CAU GGU CAC UCG CUU

Thr Ser Asn Leu Thr Val Ala Ala Arg Gly His Gly His Ser Leu

CAA GGA CAA GCU CUA GCU CAU CAA GGU GUU GUC AUC AAA AUG GAG

Gin Gly Gin Ala Leu Ala His Gin Gly Val Val He Lys Met Glu

UCA CUA CGA AGU CCU GAU AUU AGG AUU UAU AAG GGG AAG CAA CCA

Ser Leu Arg Ser Pro Asp He Arg He Tyr Lys Gly Lys Gin Pro

UAU GUU GAU GUC UCA GGU GGU GAA AUA UGG AUA AAC AUU CUA CGC

Tyr Val Asp Val Ser Gly Gly Glu He Trp He Asn He Leu Arg

GAG ACU CUA AAA UAC GGU CUU UCA CCA AAG UCC UGG ACA GAC UAC

Glu Thr Leu Lys Tyr Gly Leu Ser Pro Lys Ser Trp Thr Asp Tyr

CUU CAU UUG ACC GUU GGA GGU ACA CUA UCU AAU GCU GGA AUC AGC

Leu His Leu Thr Val Gly Gly Thr Leu Ser Asn Ala Gly He Ser

GGU CAA GCA UUC AAG CAU GGA CCC CAA AUC AAC AAC GUC UAC CAG

Gly Gin Ala Phe Lys His Gly Pro Gin He Asn Asn Val Tyr Gin

CUA GAG AUU GUU ACA GGG AAA GGA GAA GUC GUA ACC UGO UCU GAG

Leu Glu He Val Thr Gly Lys Gly Glu Val Val Thr Cys Ser Glu AAG CGG AAC UCU GAA CUU UUC UUC AGU GUU CUU GGC GGG CUU GGA Lys Arg Asn Ser Glu Leu Phe Phe Ser Val Leu Gly Gly Leu Gly CAG UUU GGC ADA AUC ACC CGG GCA CGG AUC UCU CUD GAA CCA GCA

Gin Phe Gly He He Thr Arg Ala Arg He Ser Leu Glu Pro Ala

CCG CAO AUG GUU AAA UGG AUC AGG GUA CUC UAC UCU GAC UUU UCU

Pro His Met Val Lys Trp He Arg Val Leu Tyr Ser Asp Phe Ser

GCA UUU UCA AGG GAC CAA GAA UAU CUG AUU UCA AAG GAG AAA ACU

Ala Phe Ser Arg Asp Gin Glu Tyr Leu He Ser Lys Glu Lys Thr

UUU GAU UAC GUU GAA GGA UUU GUG AUA AUC AAU AGA ACA GAC CUU

Phe Asp Tyr Val Glu Gly Phe Val He He Asn Arg Thr Asp Leu

CUC AAU AAU UGG CGA UCG UCA UUC AGU CCC AAC GAU UCC ACA CAG

Leu Asn Asn Trp Arg Ser Ser Phe Ser Pro Asn Asp Ser Thr Gin

GCA AGC AGA UUC AAG UCA GAU GGG AAA ACU CUU UAU UGC CUA GAA

Ala Ser Arg Phe Lys Ser Asp Gly Lys Thr Leu Tyr Cys Leu Glu

GUG GUC AAA UAU UUC AAC CCA GAA GAA GCU AGC UCU AUG GAU CAG

Val Val Lys Tyr Phe Asn Pro Glu Glu Ala Ser Ser Met Asp Gin

GAA ACU GGC AAG UUA CUU UCA GAG UUA AAU UAU AUU CCA UCC ACU

Glu Thr Gly Lys Leu Leu Ser Glu Leu Asn Tyr He Pro Ser Thr

DUG UUU UCA UCU GAA GUG CCA UAU AUC GAG UUU CUG GAU CGC GUG

Leu Phe Ser Ser Glu Val Pro Tyr He Glu Phe Leu Asp Arg Val

CAU AUC GCA GAG AGA AAA CUA AGA GCA AAG GGU UUA UGG GAG GUU

His He Ala Glu Arg Lys Leu Arg Ala Lys Gly Leu Trp Glu Val

CCA CAU CCC UGG CUG AAU CUC CUG AUU CCU AAG AGC AGC AUA UAC

Pro His Pro Trp Leu Asn Leu Leu He Pro Lys Ser Ser He Tyr

CAA UUU GCU ACA GAA GUU UUC AAC AAC AUU CUC ACA AGC AAC AAC

Gin Phe Ala Thr Glu Val Phe Asn Asn He Leu Thr Ser Asn Asn

AAC GGU CCU AUC CUU AUU UAU CCA GUC AAU CAA UCC AAG UGG AAG

Asn Gly Pro He Leu He Tyr Pro Val Asn Gin Ser Lys Trp Lys

AAA CAU ACA UCU UUG AUA ACU CCA AAU GAA GAU AUA UUC DAU CUC

Lys His Thr Ser Leu He Thr Pro Asn Glu Asp He Phe Tyr Leu

GUA GCC UUU CUC CCC UCU GCA GUG CCA AAU UCC UCA GGG AAA AAC

Val Ala Phe Leu Pro Ser Ala Val Pro Asn Ser Ser Gly Lys Asn GAU CDU GAG UAC CUU OUG AAA CAA AAC CAA AGA GUD AUG AAC UUC

Asp Leu Glu Tyr Leu Leu Lys Gin Asn Gin Arg Val Met Asn Phe

OGC GCA GCA GCA AAC CUC AAC GUG AAG CAG UAU UUG CCC CAU UAU

Cys Ala Ala Ala Asn Leu Asn Val Lys Gin Tyr Leu Pro His Tyr

GAA ACU CAA AAA GAG UGG AAA OCA CAC UUU GGC AAA AGA UGG GAA

Glu Thr Gin Lys Glu Trp Lys Ser His Phe Gly Lys Arg Trp Glu

ACA UUU GCA CAG AGG AAA CAA GCC UAC GAC CCU CUA GCG AUU CUA

Thr Phe Ala Gin Arg Lys Gin Ala Tyr Asp Pro Leu Ala lie Leu

GCA CCU GGC CAA AGA AUA UUC CAA AAG ACA ACA GGA AAA UUA UCU

Ala Pro Gly Gin Arg He Phe Gin Lys Thr Thr Gly Lys Leu Ser

CCC AUC CAA CUC GCA AAG UCA AAG GCA ACA GGA AGU CCU CAA AGG

Pro He Gin Leu Ala Lys Ser Lys Ala Thr Gly Ser Pro Gin Arg UAU CAU UAC GCA UCA AUA CUG CCG AAA CCU AGA ACU GUA UAA

Tyr His Tyr Ala Ser He Leu Pro Lys Pro Arg Thr Val End

Subject of the present invention is further the use of the above-mentioned expression cassettes for the preparation of barley plants showing the resistance to drought.

Brief description of Drawings

Fig. 1: Phenotype of the plants with overexpression of ZmCKXl under the β-glukosidase promoter. 1. Control WT barley, 2., 3. and 4. genetically modified lines of barley.

Fig. 2: Number of AtCKXl transcripts in 1 ng RNA at 1, 2, 5, 8 and 14 weeks old transgenic plants.

Fig. 3: Determining the relative contents of water in leaves in 3-months old plants of barley, of which half of the plants were stressed for 2 months by drought. Transgenic plants contain vacuolar form of AtCKXl . Flag leaves were taken from five plants from the oldest stem after being exposed to drought for the miiiimum of 24 hours.

Fig. 4: Photo documentation of the effect of drought stress to transgenic and control WT barley plants. A) WT barley, B) transgenic barley with vacuolar AtCKXl.

Fig. 5: Photo documentation of the effect of drought stress to transgenic and control WT plants of barley. A) WT barley, B) transgenic barley with apoplastic AtCKXl . Fig. 6: Photo documentation of the effect of drought stress to transgenic and control WT of the plants of barley. A) WT barley, B) transgenic barley with cytosolic AtCKXl.

Examples of carrying out the Invention

Example 1: Preparation of genetically modified barley

1. Preparation of binary construct for introduction of foreign DNA to barley

3 constructs with vacuolar cytosolic or apoplastic AtCKXl form were prepared (originally from the plant Arabidopsis thali na). Each construct contains regulation sequences - β-glukosidase gene promoter and NOS terminator. The majority of functional segments was synthesized by a commercial company Mr. Gene GmbH (Regensburg, Germany): the sequence of β-glukosidase gene promoter (Gu et al. (2006), Plant Cell Rep. 25:1157-65), the AtCKXl gene (Kowalska et al. (2010), Phytochemistry. 71:1970-8) with artificially created restriction site for cleavage of the signal sequence, the signal secretion sequence of the AtCKX2 gene (Werner et al. (2003), Plant Cell. 15:2532-50) and the NOS terminator (Bevan M, Barnes WM, Chilton MD. (1983), Nucleic Acids Res. 11 :369-85). Further DNA fragments introduced to barley were acquired by purchasing binary T-DNA plasmid pBRACT209 (John Innes Centre, Norwich, Great Britain) and a plasmid construct with natural form of AtCKXl was obtained from Dr. T. Werner (Freie Universitat Berlin, Germany), the preparation of which is shown in Werner et al. (2001), Proc Natl Acad Sci USA. 98:10487-92.

The functional segments were - using the restriction endonucleases and T4-DNA ligase (Sarabrook J, Russell DW Molecular cloning: A Laboratory Manual (third ed.), 3:A2.2, Cold Spring Harbor, New York 2001) - introduced into the vector pENTRl A (purchased from the company Invitrogen, Life Technologies Czech Republic s.r.o., Prague), which serves as a donor vector for introduction of the required DNA fragment to the target vector pBRACT209 using the so-called Gateway cloning (performed according to the instructions of the company Invitrogen). The resulting constructs were verified by commercial sequencing and used for the transformation of the bacteria Agrobacterium tumefaciens (strain AGL1), which was obtained from Plant Breeding and Acclimatization Institute (Blonie, Poland). Transformation of A. tumefaciens was carried out according to the protocol shown in the Sambrook J, Russell DW Molecular cloning: A Laboratory Manual (third ed.), 3:A2.2, Cold Spring Harbor, New York 2001.

2. Transformation of barley using Agrobacterium tumefaciens

Transformation of barley using Agrobacterium tumefaciens was performed according to published procedure shown in Harwood et al. (2009), In Methods in Molecular Biology, Vol. 478 Transgenic Wheat, Barley and Oats (Jones H. D, Shewry P. R. ed.), pp. 137-147, Humana Press, New York, USA. The only modifications consisted in the use of 0.2 mM acetosyringone in agrobacterial suspension before the actual infection of the donor material, which were barley scutella. Further modification was the addition of 6- benzylaminopurine into the regeneration medium (0.4 mg/1 medium) to ensure a sufficient level of cytoki ins in the regenerating plants.

3. Growing transgenic barley

After approximately thirty days of growing in the basic medium (Harwood et al. (2009), In Methods in Molecular Biology, Vol. 478 Transgenic Wheat, Barley and Oats (Jones H. D, Shewry P. R. ed.), pp. 137-147, Humana Press, New York, USA), the plants were moved to peat discs and were grown for one week in phytotron (16-hour-day - 15 °C / 8- hour-night - 12 °C; illumination 300 μΜ/ιη ² /3 on the level of the growing vessel). In the end, the plants were replanted to a mixture of soil : commercial substrate : perlite (ratio 1:1:1) and grown in the phyto chamber under the same conditions until the stage of maturity.

In the Tl generation of transgenic barley, the lines with one integration of T-DNA were selected, in which the presence of functional transgene was confirmed and verified on the level of gDNA, RNA and protein level. In the T2 generation, homo2ygotic lines of transgenic barley were selected and then further characterized and studied.

4. Study of expression of AtCKXl driven by β-ghikosidase promoter and study of the expression of 35S::hpt

The number of transcripts of AtCKXl was detected in 1 ng of RNA for 1, 2, 5, 8 and 14- week old transgenic barley plants with AtCKXl vacuolar. The number of transcripts of hpt was detected in 1 ng of RNA for 1 , 2, 5 and 8-week old transgenic barley plants with AtCKXl vacuolar. Quantification was performed using calibration curves, for the preparation of which clean plasmids with the corresponding genes in a known concentration were used, by means of Sybr green qPCR (StepOnePlus™ Real-Time PCR System, Life Technologies Czech Republic s.r.o., Prague). Primers were designed using the program Primer Express 3.0. As endogenic control, the genes for β-actin, barley cyclophilin and elongation factor 1 were used.

High-quality RNA was isolated using the RNAqueous® kit (Ambion, Life Technologies Czech Republic s.r.o., Prague), treated by Turbo DNAse (Life Technologies Czech Republic s.r.o., Prague), and purified using magnetic balls (Agencourt RNA-CLEAN XP, Beckman Coulter, Prague). Transcription to cDNA followed, using the reversion transcriptase RevertAid H Minus M-MULV (ThermoFischer SCIENTIFIC, Pardubice).

To confirm the integration of the required functional cassette to the barley genome and thus also to confirm the genetic modification of the plants, the selection hpt gene was monitored on the genome level as well as on the RNA level. In all transgenic plants, the presence and the transcription of the monitored hpt gene was confirmed (Tab. 1), which is under the 35S promoter (part of the binary vector pBRACT209).

Table 1 : Number of hpt transcripts in 1 ng of RNA in 1, 2, 5 and 8-week old transgenic plants.

For the quantification of expression of AtCKXl controlled by β-glukosidase promoter, the number of AtCKXl transcripts in 1 ng of RNA was determined in the aerial part and in the roots in 1, 2, 5, 8, and 14/16-week old transgenic barley plants (Fig. 2). The strongest expression was observed in 4-week old plants, in the roots of barley. In comparison with the expression driven by 35S promoter (Tab. 1), which is considered as moderate promoter for use in the cereals, we can consider the β-glucosidase promoter as a weak promoter for the expression of the target gene in monocotyledonous plants. Example 2: Application of drought stress

A. Drought stress was applied on 3 -month old plants that were grown in a greenhouse in 1 kg of homogenous mixture of soil and perlite in the ratio 2:1. The soil was allowed to dry out in regular intervals, which corresponded to 1 to 3 waterings of 200 ml per 1 kg of soil per week, depending on the age of the plant and temperature conditions. The unstressed plants were watered every day. 30 transgenic barley plants with vacuolar form of AtCKXl and 30 control WT were stressed. In the 3-month old plants the relative water content ( WC) was determined in the six youngest, however fully developed leaves for the tested line (Turner NC, Abbo S, Berger JD, Chaturvedi SK, French RJ, Ludwig C, Mannur DM, Singh SJ, Yadava HS. (2007) J Exp Bot. 58:187-94).

B. Study of effects of the stress to 4-week old, hydroponically grown barley plants growing in the phytotron (16 hours day - 23 °C / 8 hours night - 20 °C; illumination 100 μΜ/ιη ² /8 on the level of the growing vessel, 300 μΜ/ιη ² /3 on the level of the growth apex of the 6-week old plants). The stress was applied by pouring the nutrient medium off the growing vessel (Hoagland DR and Arnon DI. (1950), California Agricultural Experiment Station Circular 347:1-32), slight dripping off and returning the plants to the vessel, where they were further grown for 24 hours. During this time, the leaves were regularly collected (always 5 per one line of barley) to determine the relative water contents, and photo documentation of the stressed plants was further performed.

C. Study of effects of the stress to 4-week old barley plants that were grown in a minimum quantity of soil in phytotron (16 hours day - 22 °C / 8 hours night - 20 °C; illumination 150 μΜ/ηι ² /8) to evaluate the capability of revitalization of the genetically modified plants. The plants were left for 2 days without watering. The measurement of relative water content in the leaves confirmed a strong stress by drought. The control of revitalization took place 24 hours from the renewal of watering by leaf collection (always 5 leaves per one line of barley) to determine the relative water content.

A. Increased resistance of transgenic plants of barley grown in 1 kg of growing substrate, long-term exposed to drought stress.

Transgenic plants with vacuolar form of AtCKXl were grown in 1 kg of growing substrate in a greenhouse. After two months of drought, flag leaves of the grown plants were taken to determine the relative water contents. It was determined that compared to the control WT plants, the transgenic plants have by more than 10% higher water contents during the application of long-term stress (Fig. 3). Compared to the control conditions (i.e. without application of stress), it is apparent that no significant reduction of water in the leaves (Fig. 3) occurred during the stress in the genetically modified plants, in contrast to WT barley plants.

B. Increased resistance to drought stress in hydroponically grown plants

The plants of barley (transgenic lines and WT barley) were hydroponically grown in Hoagland nutrient solution to ensure optimum uptake of nutrients and water through the roots, which is ideal for determining the studied parameters (RWC, total dry mass of the leaves and roots after the two- week long regeneration of plants) during the drought stress and revitalization. During the experiment, the phenotype of the plants was monitored. Due to a different place of action of ectopically produced C X, different results were obtained for transgenic plants with vacuolar, cytosolic, or apoplastic CKX.

Barley with AtCKXl vacuolar

During the stress and revitalization of the hydroponically grown plants, increase of RWC was not observed, compared to WT barley. After the two-week regeneration, the transgenic plants had the weight of the aerial part increased by 32% compared to WT, which was given by a higher number of tillers. From the phenotypic point of view, faster regeneration is apparent in the transgenic plants already after 4 hours of revitalization (Fig.4).

Barley with AtCKXl apoplastic

During the stress of the hydroponically grown plants, an increase of RWC by up to 19% was detected after 20 hours without growth medium compared to the control WT plants; after 24 hours of stress by up to 12% higher RWC compared to WT barley. The weight of the aerial part or roots was not higher after the application of stress than in the stressed WT plants. From the point of phenotype, there is an apparent resistance of the transgenic plants during the stress as well as faster regeneration of these plants compared to the WT barley (Fig. 5).

Barley with AtCKXl cytosolic

During the two-hour revitalization of the hydroponically grown plants the relative water content detected in leaves was higher by 18% than in the control WT plants. After the two-week regeneration of the plants, the transgenic plants had by 17 % higher mass of the aerial part and by 17% higher mass of the roots than WT. The number of tillers was comparable to the WT barley, the plants were higher. From the point of phenotype, a higher resistance of transgenic plants to the stress is apparent during the entire 24 hours, and fast revitalization already after 2 hours after addition of nutrient medium to the plants (Fig. 6).

C. Increased revitalization capability of transgenic plants of barley grown in a minimum quantity of the growing substrate

Transgenic plants with vacuolar, cytosolic, or apoplastic form of AtCKXl were grown in a minimum quantity of the growing substrate in phytotron. After the application of drought (termination of watering) and during revitalization (restoration of watering), the youngest, however fully developed leaves of the plants were taken to determine the relative water contents. With respect to the fact that the plants were growing only in a limited quantity of soil thus being considerably spatially and nutritionally limited, they could not fully utilize their potential during the stress. This experiment was thus focused on the capability of revitalization of the genetically modified plants after previous application and confirmation of drought stress. After the watering was restored, the transgenic plants showed, compared to WT barley, a significantly faster revitalization of the aerial part of the plants (Tab. 3). The line with cytosolic AtCKXl was confirmed to have by 25% higher water contents in the leaves than the WT barley. In the plants with apoplastic AtCKXl, the RWC was by 17% higher; in the line with vacuolar AtCKXl, the value of RWC was by 15% higher than in the WT barley. Transgenic plants with ectopically overexpressed AtCKXl thus had a higher capability to quickly deliver the necessary amount of water to the aerial parts of the plant in the environment with sufficient quantity of watering.

Table 3: Determining Relative Water Contents (RWC) in the leaves during revitalization of transgenic and WT barley plants after the application of drought stress.

Barley line RWC in % during 24-hour revitaiization

WT 78

AtCKXl Cytosolic 98 Barley line RWC in % during 24-hour revitalization

AtCKXl Apoplastic 92

AtCKXl Vacuolar 90