INHIBITION OF BOLTING AND FLOWERING OF A BETA VULGARIS PLANT

Abstract

The present invention provides means for inhibiting the bolting and flowering of a Beta vulgaris plant, including an isolated nucleic acid, which can be used to produce a transgenic Beta vulgaris plant, where bolting and flowering is inhibited after vernalization. Furthermore, the invention discloses vectors, transgenic and non-transgenic, non-bolting plants and parts thereof, and methods for producing such plants.

Claims

1-15. (canceled)

16. A method for producing a Beta vulgaris plant, where the bolting and flowering is inhibited after vernalization, comprising the following steps: (I) mutagenizing one or more parts of a Beta vulgaris plant and subsequently regenerating one or more Beta vulgaris plants from one or more parts to yield a regenerated plant, or mutagenizing on or more Beta vulgaris plants to yield a mutagenized plant; (II) identifying a regenerated or mutagenized plant of (I) that exhibits one or more mutations in an endogenous DNA sequence; (III) generating a Beta vulgaris plant in which the one or more mutations in the endogenous DNA sequence is homozygous; wherein the endogenous DNA sequence has a nucleic acid sequence identical to a sequence that A) exhibits a sequence comprising the coding sequence of SEQ ID NO: 4, B) comprises a nucleotide sequence exhibiting at least 98% sequence identity to the coding sequence of SEQ ID NO: 4; C) is complementary to SEQ ID NO: 4; or D) encodes a protein comprising an amino acid sequence exhibiting at least 98% sequence identity to SEQ ID NO: 6.

17. A Beta vulgaris plant, produced by the method of claim 16.

18. A Beta vulgaris plant, wherein bolting and flowering is inhibited after vernalization, wherein the plant comprises one or more mutations in an endogenous DNA sequence comprising a nucleic acid sequence identical to a sequence that a) comprises a sequence comprising the coding sequence of SEQ ID NO: 4, b) comprises a nucleotide sequence exhibiting at least 98% sequence identity to the coding sequence of SEQ ID NO: 4, c) is complementary to SEQ ID NO: 4, or d) encodes a protein comprising the amino acid sequence of SED ID NO: 6 or encodes a protein comprising an amino acid sequence exhibiting at least 98% sequence identity to SEQ ID NO: 6, wherein the one or more mutations cause a reduction of the activity or stability of the protein or polypeptide encoded by the endogenous DNA sequence compared to a non-mutagenized wild type plant.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0061] FIG. 1: Schematic structure of cloning vector pAB70S-1 35S Ataap6 RNAi

[0062] FIG. 2: Schematic structure of cloning vector pAB70S-1 35S Ataap6 FUL AP1 including partial sequences of AP1 and FUL

[0063] FIG. 3: Schematic structure of binary Ti-plasmid pZFN d35S RNAi FUL-AP1 including partial sequences of AP1 (212 bp) and FUL (184 bp) used in Agrobacterium-mediated transformation

[0064] FIG. 4: Schematic representation of cloning vector pAB70s-1 d35S Ataap6 RNAi viI1-AP1-FUL LF including partial sequences of AP1, FUL and VIL1

[0065] FIG. 5: Schematic representation of the binary Ti-plasmid pZFN d35S RNAi vil1-FUL-AP1 including partial sequences of AP1, FUL and VIL1 used in Agrobacterium-mediated transformation

[0066] FIG. 6: Alignment of the amino acid sequences of the protein AP1 from Beta vulgaris (SEQ ID NO: 5) and from Arabidopsis thaliana (SEQ ID NO: 41) based on the EMBOSS Needle algorithm; BvAP1 =Beta vulgaris AP1, AtAP1=Arabidopsis thaliana AP1

[0067] FIG. 7: Alignment of the amino acid sequence of the protein FUL from Beta vulgaris (SEQ ID NO: 6) and from Arabidopsis thaliana (SEQ ID NO: 42) based on the EMBOSS Needle algorithm; BvFUL=Beta vulgaris FUL, AtFUL=Arabidopsis thaliana FUL

EXAMPLES

[0068] 1. Inhibition of bolting and flowering by RNAi constructs targeted to AP1 and FUL

[0069] Identification/isolation and characterization/annotation of complete cDNAs of sugar beet for inhibition of bolting and flowering:

[0070] By analysis within a specially created proprietary sugar beet EST database, the 780 base pairs (bp) long cDNA (SEQ ID NO: 2) of BvAP1 as well as the 735 bp long cDNA (SEQ ID NO: 4) of BvFUL have been identified. In addition, corresponding genomic DNA sequences could be identified. An alignment of genomic DNA with cDNA shows the structures of the entire DNAs. AP1 consists of 8 exons and 7 introns, FUL of 8 exons and 7 introns.

[0071] A comparison of the resulting full-length DNA and the translated protein sequence shows only low sequence similarity with Arabidopsis homologs AtAP1 and AtFUL. At protein level the identity over the entire sequence length to AtAP1 is at 65.6% (see also FIG. 6) and to AtFUL is at 57.3% (see also FIG. 7), at cDNA level the identity AtAP1 is 71% and to AtFUL is 72% (see Table 2).

TABLE-US-00003 TABLE 2 Sequence comparison of BvAP1 and BvFUL with Arabidopsis thaliana (At)-AP1 and -FUL- candidates based on the protein sequence and cDNA. Results given as sequence identity based on the EMBOSS Needle algorithm (www.ebi.ac.uk). AtAP1 protein AtFUL protein AtAP1 AtFUL (SEQ ID NO: 41) (SEQ ID NO: 42) cDNA cDNA BvAP1 protein 65.6% (SEQ ID NO: 5) BvFUL protein 57.3% (SEQ ID NO: 6) BvAP1 cDNA 71% (SEQ ID NO: 2) BvFUL cDNA 72% (SEQ ID NO: 4)

[0072] Production of RNAi constructs targeted to AP1 and FUL and inhibition of bolting and flowering in sugar beet:

[0073] For the production of RNAi constructs the sequences of SEQ ID NO: 19 to 22 were synthesized. Sequence of SEQ ID NO: 19 includes a partial sequence of AP1 according to SEQ ID NO 10 with a length of 184 bp and a partial sequence of FUL according to SEQ ID NO 14 with a length of 212 bp. Sequence of SEQ ID NO: 20 includes a partial sequence of AP1 according to SEQ ID NO 11 with a length of 150 bp and a partial sequence of FUL according to SEQ ID NO 15 with a length of 150 bp. Sequence of SEQ ID NO: 21 includes a partial sequence of AP1 according to SEQ ID NO 12 with a length of 100 bp and a partial sequence of FUL according to SEQ ID NO 16 with a length of 100 bp. Sequence of SEQ ID NO: 22 includes a partial sequence of AP1 according to SEQ ID NO 13 with a length of 50 bp and a partial sequence of FUL according to SEQ ID NO 17 with a length of 50 bp.

[0074] For the further processing the sequences have been amplified by PCR using PCR Primers with SalI/SmaI restriction sites like primers according to SEQ ID NO: 25 (forward) and 26 (reverse) for the amplification of SEQ ID NO 19. The PCR was performed using 10 ng of genomic sugar beet DNA, a primer concentration of 0.2 micron at an “annealing” temperature of 55° C. in a Multicycler PTC-200 (MJ Research, Watertown, USA).

[0075] The PCR products were each integrated into the vector pAB70S-1 35S Ataap6 RNAi (FIG. 1). The vector is designed for the production of “intron-spliced” hairpin structures. The vector contains the d35S promoter for constitutive expression, the ATAAP6 intron from Arabidopsis thaliana and one polyA terminator (nos-T). The ATAAP6 intron is flanked by the interfaces or cleavage sites XhoI/Ecl136II on the 5′ end or by the restriction cleavage sites SmaI/SalI at the 3′-end. This enables the integration of identical fragments in a “sense” and “antisense”, if these fragments have the compatible restriction sites XhoI or SalI, or are stumped on the other end (“blunt end”). For this the original PCR products were reamplified with new PCR primers extended beyond these restriction sites. For further use the PCR fragments were cloned into the TA cloning vector pCR2.1 (TOPO TA Cloning Kit (Invitrogen, Carlsbad, USA)) and transformed in E. coli. A blue-white selection enabled the identification of recombinant plasmids (Sambrook et al. 201, in Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd edition, New York). In the white colonies the expression of ss-galactosidase is suppressed by an insert, which results in white colonies, because the enzyme substrate added to the medium is no longer cleaved. After a subsequent sequencing with M13-fwd/rev-primers, the analysis and the alignment of the sequence data was performed using the program Vector NTI (Invitrogen, Carlsbad, USA).

[0076] The fragments Sal-SmaI and XhoI-SmaI-were each cut from the topovector by SalI/SmaI or XhoI/SmaI and then subsequently first ligated “in sense” with the SalI/SmaI or XhoI/Ecl136II cut pRTRNAi vector. Subsequently, the same fragments were religated for a second time in “antisense” in the compatible XhoI/Ecl136II or SalI/SmaI. The cloning resulted in, for example, pAB70S-1 35S Ataap6 FUL AP1 (FIG. 2).

[0077] Production of transformation constructs and sugar beet transformation:

[0078] For plant transformation the binary vector pZFN was used. The expression cassettes were cut using SfiI to transfer it into the binary vector pZFN to create pZFN d35S RNAi FUL-AP1-212-184 (FIG. 3), pZFN d35S RNAi FUL-AP1-150-150, pZFN d35S RNAi FUL-AP1-100-100, and pZFN d35S RNAi FUL-AP1-50-50. Each of the binary vectors was transformed in Agrobacterium tumefaciens strain GV3101 pMP90 by a direct DNA transformation method (An, G. (1987), Ti binary vectors for plant transformation and promoter analysis, Methods Enzymol. 153, 292-305). The selection of recombinant A. tumefaciens clones was performed using the antibiotic streptomycin (50 mg /1). The transformation of sugar beet and regeneration were carried out according to Lindsey et al. (1990) and Lindsey et al. (1991, “Regeneration and transformation of sugar beet by Agrobacterium tumefaciens, Plant Tissue Culture Manual B7: 1-13, Kluwer Academic Publishers).

[0079] The transgenicity of the plants was verified by PCR. The use of designed primers led to the amplification of a particular DNA fragment from the nptII gene. The PCR was performed using 10 ng genomic DNA, a primer concentration of 0.2 micron at an annealing temperature of 55° C. in a Multicycler PTC-200 (MJ Research, Watertown, USA).

[0080] Verification of the flowering and bolting behavior of the transformants:

[0081] For each of the RNAi constructs five transgenic sugar beet lines were regenerated carrying the corresponding binary vector pZFN d35S RNAi FUL-AP1. The sugar beet plants were grown for several weeks in sterile culture media, propagated and then rooted together with non-transgenic isogenic controls. 7-9 plants per line and control were transferred to the greenhouse. The transgenic lines were grown in pots and then tested in different vernalization regimes to determine the bolting and flowering behavior. After an adjustment period, the plants were subjected to vernalization for three, four and six months at 8° C. in a cooling chamber (winter simulation). Subsequently, the transformants, as well as identically treated non-transgenic control plants, were transferred back into the greenhouse (25° C.). Shortly after transfer, already after 10 days, the control plants began to grow shoots. After 4 weeks the control plants began to bloom. In contrast, several transformants lines showed surprisingly no response to the shoot and flower induction by vernalization. Hereunder were lines of each used RNAi construct.

[0082] Thus, the resulting transformants behaved like not-vernalized sugar beets. They neither developed shoots nor blooms. None of the plants showed deviations from the normal phenotype. The plants were cultivated further; they continued to develop to normal beets with normal beet bodies.

[0083] These lines were again tested in a greenhouse supplied with soil for optimal root growth without temperature control in two winters (2013/2014; 2014/2015). None of the plants did bolt or flower.

[0084] Surprisingly, using the inventive approach, the vernalization or its effect, namely the bolting and flowering, were completely blocked in sugar beet.

[0085] 2. Inhibition of bolting and flowering by RNAi constructs targeted to AP1, FUL and VIL1

[0086] For this approach an RNAi construct comprising partial sequences of AP1 and FUL cDNA was extended by a third partial sequence of 399 bp based on cDNA of the BvVil1 gene from Beta vulgaris (WO 2011/032537). VIL1 was chosen due to its involvement in flower formation.

[0087] PCR product BvVil1 RNAi was amplified using a forward primer according to SEQ ID NO: 27 and a reverse primer according to SEQ ID NO: 28. BvVil1 cDNA was used as template. The amplification led to a VIL1 cDNA fragment according to SEQ ID NO: 29. Additionally, PCR product BvFUL-AP1 was synthesized and amplified using primers according to SEQ ID NO: 25 and 26. Vector pZFN as described above was used as template. The amplification led to a FUL-AP1 cDNA fragment according to SEQ ID NO: 30.

[0088] Both amplified DNAs were cloned into vector pAB70S-1 35S Ataap6 RNAi (FIG. 1). PCR product BvVil1 RNAi was cloned using Ecl136II and XhoI. PCR product BvFUL-AP1 was added using XhoI and SmaI. The resulting intermediate pAB70s-1 d35S Ataap6 RNAi vil1-AP1-FUL LF (FIG. 4) was used for another PCR step. The vector pAB70s-1 d35S Ataap6 RNAi vil1-AP1-FUL LF was used as template for forward primer according to SEQ ID NO: 31 and reverse primer according to SEQ ID NO: 32. The PCR product was an RNAi construct which then was cloned into the vector using SalI and SmaI. The resulting vector was cut using SfiI and cloned into vector pZFN resulting in vector pZFN d35S RNAi vil1-FUL-AP1 (FIG. 5).

[0089] Vector pZFN d35S RNAi vil1-FUL-AP1 was used to transform Agrobacterium tumefaciens Gv3101 pmp90 which subsequently was used to generate transgenic sugar beet lines. Transgenicity was confirmed by PCR. After regeneration nine plants of one line were rooted as described above. After vernalization in the greenhouse none of the plants did bolt or flower.

[0090] 3. Inhibition of bolting and flowering by knock-out mutants of BvAP1 and BvFUL

[0091] Mutagenization of sugar beet cells and identification of BvAP1 and BvFUL mutants:

[0092] A sugar beet mutant population has been created by treatment with different EMS concentrations for different durations of incubation. From treated cells M1 plants could be regenerated. Through selfing of the M1 plants several thousands of M2 plants were grown.

[0093] These M2 plants were screened for knock out mutations in the BvAP1 gene and the BvFUL gene. For that, DNA was been extracted from collected leaf samples and analysed by use of designed primers. Thereby, point mutations in the genes which introduce additional stop codons into the coding sequence of the genes could be identified. One plant showed a point mutation in the AP1 gene which is a nucleotide exchange from cytosine (c) to thymine (t) at position 262 of the cDNA (SEQ ID NO: 2) or at position 6999 of the genomic DNA (SEQ ID NO: 1). A second identified plant contained a point mutation in the FUL gene which is a nucleotide exchange from cytosine (c) to thymine (t) at position 316 of the cDNA (SEQ ID NO: 4) or at position 19552 of the genomic DNA (SEQ ID NO: 3).

[0094] Verification of the flowering and bolting behavior of single mutants:

[0095] Cells of the identified sugar beet mutants were cultured for several weeks in sterile culture media, propagated and then rooted together with non-mutated controls. 5 plants per mutant and control were transferred to the greenhouse. The mutant lines were grown in pots and then tested in different vernalization regimes to determine the bolting and flowering behavior. After an adjustment period, the plants were subjected to vernalization for three months at 8° C. in a cooling chamber. Subsequently, they were transferred back into the greenhouse (25° C.). Shortly after transfer, already after 11 days, the control plants as well as the mutant lines began to grow shoots. After 4 weeks all plants began to develop flowers.

[0096] Verification of the flowering and bolting behavior of double mutants:

[0097] F1 progenies of a cross of AP1 mutants with FUL mutants have been analyzed for identification of plants carrying the mutated AP1 gene and the mutated FUL gene. Two F1 plants could be detected which then were selfed to generate a F2 population. Selected plants of the F2 generation have been tested in greenhouse as described above. Shortly after transfer, already after 10 days, the control plants and most of the selected plants began to grow shoots. However, a few plants showed surprisingly no response to the shoot and flower induction by vernalization. These non-bolting plant were all homozygous for both of the identified point mutations in AP1 gene and FUL gene, expect two of the plants which showed a heterozygous genotype for at least one of the mutation.