REGULATORY NUCLEIC ACID MOLECULES FOR ENHANCING GENE EXPRESSION IN PLANTS

Abstract

The present invention is in the field of plant molecular biology and provides methods for production of high expressing promoters and the production of plants with enhanced expression of nucleic acids wherein nucleic acid expression enhancing nucleic acids (NEENAs) are functionally linked to said promoters and/or introduced into plants.

Claims

1. A method for enhancing expression derived from a plant promoter comprising functionally linking to a promoter one or more nucleic acid expression enhancing nucleic acid (NEENA) molecule heterologous to said promoter comprising i) a nucleic acid molecule having the sequence of SEQ ID NO: 1 or 2, or ii) a nucleic acid molecule having a sequence with an identity of at least 90% to SEQ ID NO: 1 or 2, which has expression enhancing activity as the corresponding nucleic acid molecule having the sequence of SEQ ID NO: 1 or 2 or iii) a nucleic acid molecule hybridizing under stringent conditions to a nucleic acid molecule having a sequence of SEQ ID NO: 1 or 2, or iv) a fragment of 30 or more consecutive bases of a nucleic acid molecule of i) to iii) which has expression enhancing activity as the corresponding nucleic acid molecule having the sequence of SEQ ID NO: 1 or 2, or v) a nucleic acid molecule which is the complement or reverse complement of any of the previously mentioned nucleic acid molecules under i) to iv).

2. A method for producing a plant or part thereof with, compared to a respective control plant or part thereof, enhanced expression of one or more nucleic acid molecule comprising the steps of a) introducing into the plant or part thereof one or more NEENA molecule comprising a nucleic acid molecule as defined in claim 1 i) to v) and b) functionally linking said one or more NEENA molecule to a promoter and to a nucleic acid molecule being under the control of said promoter, wherein the NEENA molecule is heterologous to said promoter.

3. The method of claim 1 comprising the steps of a) introducing the one or more NEENA molecule into a plant or part thereof and b) integrating said one or more NEENA molecule into the genome of said plant or part thereof whereby said one or more NEENA molecule is functionally linked to an endogenous promoter heterologous to said one or more NEENA molecule and optionally c) regenerating a plant or part thereof comprising said one or more NEENA molecule from said transformed cell.

4. The method of claim 3 wherein the one or more NEENA molecule is integrated into the genome of a plant or part thereof by applying genome editing technologies.

5. The method of claim 4 wherein the genome editing technology comprises the introduction of single or double strand breaks at the position the one or more NEENA molecule is to be integrated into the genome using nucleic acid guided nucleases, TALEN, homing endonucleases or Zink finger proteins and the introduction of a DNA repair template comprising the NEENA molecule and at its 3′and 5′ end sequences essentially identical or complementary to the sequences upstream and downstream of the single or double strand break facilitating recombination at the position of the single or double strand break.

6. The method of claim 4 wherein the genome editing technology comprises introduction of point mutations in the genome of the plant or part thereof thereby introducing the sequence of the NEENA in the plant genome.

7. The method of claim 1 comprising the steps of a) providing an expression construct comprising the one or more NEENA molecule functionally linked to a promoter heterologous to said one or more NEENA molecule and b) integrating said expression construct comprising said one or more NEENA molecule into the genome of said plant or part thereof and optionally c) regenerating a plant or part thereof comprising said one or more expression construct from said transformed plant or part thereof.

8. The method of claim 1 wherein said one or more NEENA molecule is functionally linked to a promoter upstream or downstream of the translational start site of the nucleic acid molecule the expression of which is under the control of said promoter.

9. The method of claim 1 wherein said one or more NEENA molecule is functionally linked to a constitutive promoter within the 5′UTR of the nucleic acid molecule the expression of which is under the control of said promoter.

10. The method of claim 1 wherein said one or more NEENA molecule is functionally linked to a tissue specific, developmental specific or inducible promoter within the 5′UTR of the nucleic acid molecule the expression of which is under the control of said promoter.

11. A recombinant expression construct comprising a NEENA molecule selected from the group of i) the nucleic acid molecule having a sequence of SEQ ID NO: 1 or 2, and ii) a nucleic acid molecule having a sequence with an identity of at least 90% to SEQ ID NO: 1 or 2, which has expression enhancing activity as the corresponding nucleic acid molecule having the sequence of SEQ ID NO: 1 or 2 and iii) a nucleic acid molecule hybridizing under stringent conditions to a nucleic acid molecule having a sequence of SEQ ID NO: 1 or 2, and iv) a fragment of 30 or more consecutive bases of a nucleic acid molecule of i) to iii) which has expression enhancing activity as the corresponding nucleic acid molecule having the sequence of SEQ ID NO: 1 or 2, and v) a nucleic acid molecule which is the complement or reverse complement of any of the previously mentioned nucleic acid molecules under i) to iv), functionally linked to one or more promoter and one or more expressed nucleic acid molecule wherein the promoter is heterologous to said one or more NEENA molecule.

12. A recombinant expression vector comprising one or more recombinant expression construct of claim 11.

13. A cell or plant or part thereof comprising a recombinant expression vector as claimed in claim 12.

14. The cell, plant or part thereof of claim 13, selected or derived from the group consisting of bacteria, fungi, yeasts or plants.

15. A cell culture, seed, parts or propagation material, comprising a recombinant expression construct of claim 11.

16. (canceled)

17. (canceled)

Description

FIGURES

[0151] FIG. 1: Impact of various parts of the CaMV 35S promoter on promoter activity in transiently transformed wheat protoplasts. Horizontal axis legend: 1st row: promoter (35S2: 528-nt long CaMV 35S promoter; -46: minimal 35S promoter); 2nd row: enhancer (35S enhancer coordinates or lambda phage sequence). GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pKA63 plasmid.

[0152] FIG. 2: Impact of candidate wheat enhancers on promoter activity in transiently transformed wheat protoplasts. Horizontal axis legend: 1st row: promoter (35S2: 528-nt long CaMV 35S promoter; min35S: minimal 35S promoter); 2nd row: absence or presence of the −208 to −65 35S enhancer; 3rd row: identity of the candidate wheat enhancer. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pKA63 plasmid.

[0153] FIG. 3: The ALMT1B fragment is sufficient for full enhancer activity in transiently transformed wheat protoplasts. Horizontal axis legend: 1st row: promoter (35S2: 528-nt long CaMV 35S promoter; min35S: minimal 35S promoter); 2nd row: enhancer sequences. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pKA63 plasmid.

[0154] FIG. 4: The ALMT1B fragment is needed for full enhancer activity in transiently transformed wheat protoplasts. Horizontal axis legend: 1st row: promoter (35S2: 528-nt long CaMV 35S promoter; min35S: minimal 35S promoter); 2nd row: enhancer sequences. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pKA63 plasmid.

[0155] FIG. 5: The ALMT1B and HMW-GS-43 fragments increase activity of a 35S promoter in transiently transformed wheat protoplasts. Horizontal axis legend: 1st row: minimal 35S promoter; 2nd row: −208 to −65 35S enhancer; 3rd row: enhancer sequences. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pKA63 plasmid.

[0156] FIG. 6: The ALMT1B and HMW-GS-43 enhancers increase activity of the wheat T6PP promoter in transiently transformed wheat protoplasts. The horizontal axis legend indicates the location of the enhancer insertion sites relative to the translation start site. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pKA63 plasmid.

[0157] FIG. 7: The ALMT1B and HMW-GS-43 enhancers increase activity of the wheat ACCase promoter in transiently transformed wheat protoplasts. The horizontal axis legend indicates the location of the enhancer insertion sites relative to the translation start site; (i) indicates that the insertion site is located within the first intron. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pKA63 plasmid.

[0158] FIG. 8: The reverse complement of the ALMT1B enhancer increases activity of the wheat T6PP promoter in transiently transformed wheat protoplasts. The enhancer was inserted 200 nt upstream of the translation start site. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pKA63 plasmid.

[0159] FIG. 9: The HMW-GS-43 enhancer increases promoter activity in infiltrated Nicotiana benthamiana leaves.

[0160] FIG. 10: Impact of the ALMT1B enhancer on activity of the wheat T6PP promoter in transiently transformed wheat protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis legend shows which enhancer fragment was used: the reverse complement (ALMT1B RC), 2 copies (2xALMT1B) or 1 copy (ALMT1B) of SEQ ID 2. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pKA63 plasmid. Activity of the promoter without enhancer (none) was set at 1.

[0161] FIG. 11: Impact of the ALMT1B enhancer on activity of the wheat ACCase promoter in transiently transformed wheat protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis legend shows the location of the enhancer insert within the promoter, relative to the translation start site. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pKA63 plasmid. Activity of the promoter without enhancer (none) was set at 1.

EXAMPLES

Chemicals and Common Methods

[0162] Unless indicated otherwise, cloning procedures carried out for the purposes of the present invention including restriction digest, agarose gel electrophoresis, purification of nucleic acids, Ligation of nucleic acids, transformation, selection and cultivation of bacterial cells were performed as described (Sambrook et al., 1989). Sequence analyses of recombinant DNA were performed with a laser fluorescence DNA sequencer (Applied Biosystems, Foster City, Calif., USA) using the Sanger technology (Sanger et al., 1977). Unless described otherwise, chemicals and reagents were obtained from Sigma Aldrich (Sigma Aldrich, St. Louis, USA), from Promega (Madison, Wis., USA), Duchefa (Haarlem, The Netherlands) or Invitrogen (Carlsbad, Calif., USA). Restriction endonucleases were from New England Biolabs (Ipswich, Mass., USA) or Roche Diagnostics GmbH (Penzberg, Germany). Oligonucleotides were synthesized by Eurofins MWG Operon (Ebersberg, Germany).

Example 1: Characterization of Wheat Enhancer Sequences

[0163] The impact of three wheat promoter elements on promoter activity was tested by transient expression in wheat mesophyll protoplasts. To identify expression vectors that are suitable for testing enhancer activity, various derivatives of plasmid pBay01160 (SEQ ID NO 6), containing the GUS coding sequence under control of the constitutive CaMV 35S2 promoter (Odell et al (1985). Nature 313(6005), 810-2) and the rice actin-1 intron (McElroy et al (1991). Mol Gen Genet. 231(1), 150-60), were tested in wheat protoplasts. To correct for differences in introduction efficiency, GUS activities of wheat transfected protoplasts were divided by the luciferase activities from a cointroduced control vector (pKA63, SEQ ID NO 9) having the firefly luciferase gene under control of the constitutive maize ubiquitin promoter (Christensen et al (1992). Plant Mol Biol. 18(4), 675-89). Wheat protoplast preparation and PEG transfection of wheat protoplasts was performed according to Shang et al. ((2014), Nature protocols 9(10), 2395-2410).

[0164] The vector pBay01697 (SEQ ID NO 7), containing only the minimal 35S promoter (nt −46 to −1) as well as its derivative pBay01704 (SEQ ID NO 10), having a 144-nt lambda phage sequence upstream of the minimal 35S promoter, showed strongly reduced promoter activity compared to the fully active 35S2 promoter of pBay01160 (FIG. 1). Plasmid pBay01701 (SEQ ID NO 8) that has −208 to −65 enhancer sequence of the 35S promoter upstream of the minimal 35S promoter showed promoter activity that is close to the 35S2 promoter of pBay01160 (FIG. 1). This shows that these vectors are suitable for testing enhancer activity in wheat protoplasts. Vector pBay01697 was further used to test the impact of the putative enhancers of SEQ ID NO: 1, 2 and 3 on a promoter that has minimal activity, whereas vector pBay01701 was used to assess the impact on a promoter that has already good activity.

[0165] To test the enhancer activity of the wheat promoter elements, the Vrn-D1 175-nt insertion (SEQ ID NO 5) (Zhang et al (2015). Front Plant Sci 6, 470), a 99-nt sequence (SEQ ID NO 11) containing the 43-nt HMW-GS 1Bx7OE promoter insertion (SEQ ID NO 1) (Geng et al (2014). PLoS ONE 9(8), e105363), and sequences corresponding to the ALMT1 AB and BC blocks (SEQ ID NO 2 to 4) (Ryan et al (2010). The Plant Journal 64, 446-455) were inserted upstream of the 35S minimal promoter in pBay01697 as well as upstream of the −208 to −65 35S enhancer in pBay01701. When introduced in wheat protoplasts, the ALMT1 AB enhancer showed the strongest expression increase (FIG. 2). Expression of the minimal 35S promoter was increased up to 60% of the fully active 35S2 promoter whereas in the presence of the 35S enhancer, promoter activity was increased 3.3-fold above that of the 35S2 promoter. The HMW-GS sequence increased expression of the 35S enhancer-containing promoter to a level that was 2-fold above that of the 35S2 promoter whereas no positive effect of this enhancer was observed on the minimal 35S promoter. In contrast, the Vrn-D1 sequence did not show a clear expression increase for both the minimal and the fully active 35S promoter.

Example 2: Deletion Analysis of ALMT1AB Enhancer

[0166] To determine the active fragment of the ALMT1AB enhancer, various deletion mutants were tested in combination with the minimal 35S promoter. The B fragment alone showed the same enhancer activity as the complete AB fragment (FIG. 3). In contrast, any 35-bp deletion within the B fragment destroyed enhancer activity whereas the A fragment did not show any enhancer activity (FIG. 4). This maps this enhancer activity to the 107-nt long B fragment.

Example 3: Validation of Active Enhancer Fragments with a Fully Active 35S Promoter

[0167] Next, the ALMT1B enhancer fragment and various variants of the 1Bx7OEHMW-GS fragment were tested with a fully active 35S promoter (FIG. 5). In this experiment, the ALMT1 B fragment showed a 2.9-fold increase of expression compared to the control plasmid (35S enhancer only), which was clearly higher than that of the AB fragment. From the HMW-GS fragments that were tested, the 43-nt fragment (SEQ ID NO 1) gave the best expression enhancement (2.5-fold). Two copies of the insert did not result in an enhanced activity. Combination of the 1Bx7OEHMW-GS 43-nt fragment with the ALMT1 B fragment did not result in a further expression enhancement.

[0168] The 43-nt HMW-GS and 107-nt ALMT1B fragments showed thus the highest enhancer activity and will be tested in combination with wheat promoters.

Example 4: Impact of the HMW-GS-43 and the ALMT1B Enhancers on Wheat Promoter Activity

[0169] To evaluate the impact of the 1Bx7OEHMW-GS-43 and the ALMT1B enhancers on the activity of endogenous wheat promoters, both fragments were inserted at 4 different sites within 2 wheat promoters: [0170] a 1-kb promoter fragment of the 7A trehalose-6-phosphate phosphatase (T6PP) gene causing constitutive expression (promoter activity in wheat protoplasts about 25% of that of p35S2) (WO/2018/113702). [0171] a 2.95-kb promoter fragment of the B genome ACCase gene causing constitutive expression (contains a 1-kb intron; 2 of the insertion sites are within the intron; expression level in wheat protoplasts about 50% of that of p35S2).

[0172] The sites of insertion (numbers are relative to the translation start codon) were chosen to not overlap with transcription factor binding sites predicted by MotifLocator (Claeys et al (2012). Bioinformatics 28(14), 1931-1932).

[0173] The data in FIGS. 6 and 7 show that each of the enhancers increased activity of both promoters. The level of expression increase depends on the location of the enhancer within the promoter and was the highest with the ALMT1 enhancer. For the T6PP promoter, the increase in promoter activity went up to 7-fold due the ALMT1 enhancer and up to 2-fold for the HMW-GS enhancer. The closer the enhancer was located to the transcription start site (which is located at −126 relative to the translation start codon) the bigger the expression increase.

[0174] For the ACCase promoter, only 2 of the insertion sites were located upstream of the transcription initiation site (nt −1240 relative to the translation start codon). From these 2 insertion sites that were located upstream of the transcription start site the expression increase was highest (2.3- to 2.7-fold increase) for the site that was closest to the transcription start site. However, this site is still about 700 nt upstream of the transcription start site. Therefore, 2 additional insertion sites that are upstream of and closer to the TSS of the ACCase promoter were tested. FIG. 11 shows that the biggest expression increase (about 8-fold) happened when the enhancer was inserted only about 70 nt upstream of the TSS. Insertion in the intron gave a lower expression increase compared to insertion upstream of the transcription start site.

[0175] These results showed that both the ALMT1B and the HMW-GS-43 enhancer can be used to increase expression from wheat promoters by inserting the enhancer at appropriate locations within the promoter or within the first intron.

Example 5: Enhancer Activity is Independent of the Orientation of the Enhancer Fragment

[0176] To test whether the enhancer activity is dependent on the orientation of the enhancer versus the promoter, the impact of the reverse complement of the ALMT1B enhancer on activity of the T6PP promoter was determined. Results showed that the complementary sequence of the ALMT1B enhancer increased expression of the T6PP promoter (FIG. 8) and thus had enhancer activity in both orientations.

Example 6: Impact of the HMW-GS-43 Enhancer on Promoter Activity in Dicotyledonous Plants

[0177] It was evaluated whether this enhancer would work in dicotyledonous plants. For this, the HMW-GS-43 enhancer was inserted immediately upstream of two promoters that are known to have weak constitutive activity in soybean: P-rp113-1.3 (reverse complement of nt 3442-2818 of SEQ ID NO 14) and P-atad1-1.3 (SEQ ID NO 15). The enhancers were inserted into 2 T-DNA vectors that contain a coding sequence for a luciferase-dsRed fusion protein under control of either the P-rp113-1.3 promoter (pBay02771, SEQ ID NO 14) or the P-atad1-1.3 promoter (pBay02773, sequence identical to pBay02771 except for the promoter sequence), resulting in T-DNA vectors pBay02772 and pBay02774, respectively. The 4 T-DNA vectors were transformed into Agrobacterium and the resulting strains were used for infiltration of Nicotiana benthamiana leaves. FIG. 9 shows the levels of luciferase activity that was measured 2 days after infiltration. The vectors containing the wheat HMW-GS-43 enhancer showed 2- to 4-fold increased levels of promoter activity compared to the vectors without enhancer. These results show that the HMW-GS-43 enhancer is also active in dicot plants.

Example 7: Duplication of the ALMT1B Enhancer Results in an Increased Enhancer Activity

[0178] The impact of 2 copies of the ALMT1 enhancer on activity of the wheat T6PP promoter was tested (insertion 200 nt upstream of the translation start site). FIG. 10 shows that two copies of the ALMT1B enhancer (2×ALMT1B) increased activity of the T6PP promoter about 4-fold more than the original enhancer sequence whereas the reverse complement (ALMT1B RC) showed a similar expression increase. This showed that impact of the ALMT1 enhancer is dependent on the copy number but independent of the orientation of the enhancer sequence.