Patent Application
20240067976
The present invention relates to: a wild-type-derived introgression line of a tomato in which the expression of tomato lycopene epoxidase BC2.1 is inhibited and the amount of beta-carotene or lutein is increased; and a transgenic tomato.
Carotenoids are organic pigments synthesized from fat and basic organic metabolites by plants, algae, bacteria and fungi, and are also called tetraterpenoids. Carotenoids are also essential and useful nutrients to animals as metabolite precursors (e.g., vitamin A and antioxidants, respectively), and have specific health benefits such as prevention of blindness and maintenance of an immune system. Animals lack the de novo synthesis ability of carotenoids and need to consume carotenoids through fruits and vegetables. For this reason, an interest on improved crops with increased carotenoid content and genetic control of carotenoid biosynthesis in plants has been increased. A provitamin A content may be greatly increased by increasing both breeding and sequestration increases through genetic engineering using genetic resources and gene silencing of heterologous or endogenous genes. Studies on beta-carotene and carotenoid biosynthesis leading to its regulation have been actively conducted. A carotenoid biosynthetic pathway from phytoene to lycopene and beta-carotene, and following desaturation, isomerization, and additional cyclization are key to carotenoid accumulation during tomato ripening. Chromoplast specific lycopene β-cyclase (CYC-B, Chromoplast specific β-cyclase) is a genetic determinant of beta-carotene accumulation during tomato ripening, and beta-carotene is not completely converted to lycopene by endogenous CYC-B overexpression.
Lutein is one of 600 naturally occurring carotenoids known to date. The lutein is synthesized only in plants and other xanthophylls, and is found in a large amount in leafy vegetables such as spinach, kale, and yellow carrots, but general lutein products contain 99% or 99% of higher of flower extracts of marigold. The lutein has been recently judged to be helpful for eye health and approved as an individual authorized ingredient, but the lutein is known to help eye health by maintaining macular pigment density, which may be reduced due to aging. However, since the lutein is not naturally produced in the body, there is a limitation that the lutein needs to be taken from the outside through foods, supplements, or the like.
The present inventors found the increased content of beta-carotene or lutein in a wild-type-derived introgression line of a tomato in which expression of BC2.1 was inhibited and a transgenic tomato and completed the present invention.
Therefore, an object of the present invention is to provide an introgression line of a tomato in which the expression of BC2.1 is inhibited and a transgenic organism.
One aspect of the present invention provides a tomato in which the expression of BC2.1 is inhibited and the amount of beta-carotene or lutein is increased.
In an exemplary embodiment of the present invention, the expression of ORF2 of the BC2.1 may be inhibited.
In an exemplary embodiment of the present invention, the expression of the BC2.1 may be inhibited to express LCY-B, thereby increasing the content of lutein.
In an exemplary embodiment of the present invention, the expression of the BC2.1 may be inhibited by administering an RNAi construct. The RNAi construct may be prepared using RNAi-F having a sequence represented by SEQ ID NO: 59 and RNAi-R having a sequence represented by SEQ ID NO: 60.
In an exemplary embodiment of the present invention, the expression of the BC2.1 may be inhibited by administering a sequence encoding BC2.1 containing a mutation to the tomato. The sequence encoding BC2.1 containing the mutation may be selected from nucleic acid sequences represented by SEQ ID NOs: 2 to 4.
In an exemplary embodiment of the present invention, the tomato may be obtained by crossing subIL2-2-1, a genetic introgression line of Solanum pennellii LA716, with IL6-3, or produced by crossing subIL2-2-1 with IL12-2.
According to the present invention, it is possible to increase the content of beta-carotene or lutein by inhibiting the expression of BC2.1, and to genetically improve the nutrients of many crops. In addition, it is possible to understand the genetic and biochemical basis of accumulation of plant carotenoids by identifying lycopene epoxidase BC2.1 and to provide a molecular tool for screening for allelic variants or genetic variants for increased provitamin A and lutein.
Hereinafter, the present invention will be described in more detail through Examples. However, these Examples are more specifically illustrative the present invention, and the scope of the present invention is not limited to these Examples.
To evaluate introgression line (IL) fruit carotenoids and their environmental changes, tomatoes grown outdoors in Florida (spring, 2004), grown in a greenhouse in New York (fall, 2010) and outdoors in Daegu (spring, 2004) were used. Tomato mapping populations and transgenic plants were grown under standard conditions (27/19° C.; 16/8 h light/dark) in a greenhouse of the Boyce Thompson Institute for Plant Research (NY, USA). Seeds were obtained from the Tomato Genetics Resource Center at the University of California, Davis (CA, USA) (http://tgrc.ucdavis.edu/) and Hebrew University of Israel (Jerusalem, Israel).
Sequences were obtained from GenBank: bc2.1 (JX683513), Spbc2.1 (JX683514), and Sgbc2.1 (JX683515).
Genetic analysis of bc2.1 was performed by using 100 plants of an M82×IL2-2 F2 population in addition to lines derived from IL2-1 representing a subset of a 2-1 genetic introgression line (also derived from M82×IL2-1 F2) acquired from the Hebrew University of Israel (
Additional high-resolution mapping of bc2.1 was performed using 1100 F2 plants derived from M82×IL2-2 crossing. Bc2.1 flanking markers 2A and 98 (the latter was a PCR marker upstream of IL2-2 defined in an initial small mapping population) were used for recombinant screening. Eight F2 recombinants were obtained in the 1.1-Mb region and beta-carotene was assayed from mature fruits at the same developmental stage labeled by HPLC in their F3 progeny which was the same type as a bc2.1 LA716 or M82 allele (paired one-tailed t-test, P<0.05). Between markers 09 and U582871 (
g60640-F
g60640-R
P-F
P-R
indicates data missing or illegible when filed
About 100 mg of frozen and crushed pericarp from individual fruits (including biological replicates) were used for carotenoid extraction. All results from each experiment were compared with control fruit genotypes grown under the same conditions. Carotenoids and chlorophyll were extracted and quantified by HPLC.
A bc2.1-specific 282-bp DNA fragment (EST clone: TUS-64-H10) located in a 3′-UTR region of cDNA (i.e., bases 1866 to 2147 of GenBank accession number JX683513) was amplified by PCR using Phusion High-Fidelity DNA polymerase (New England Biolabs, MA, USA), gene-specific primers, RNAi-F and RNAi-R (Table 2), and EST clone as template. The purified cDNA fragment was cloned into pHellsgate 2 as an inverted repeat sequence under the control of a 35S promoter using a Gateway™ cloning system (Gateway™ BP Clonase™ Enzyme Mix, Invitrogen, CA, USA). The integrity of the structure was confirmed by sequencing, and introduced into M82 and AC tomato species using Agrobacterium tumefaciens LBA4404 by a previously published method (Fillatti, J. J. et al., Efficient Transfer of a Glyphosate Tolerance Gene into Tomato Using a Binary Agrobacterium tumefaciens Vector. Bio-Technology 5, 726-730 (1987)). Transgenic plants were confirmed by PCR using a CaMV-35S-specific primers, 35S-F and 35S-R (Table 2), and subjected to Southern blot analysis using a CaMV-35S-specific probe.
P
P
screening
indicates data missing or illegible when filed
Measurement of mRNA Accumulation by qRT-PCR
Total RNA (2 μg) was reverse transcribed with random hexamers and Superscript III (Invitrogen). Purified cDNA (2 ng) was used as a template for qRT-PCR. qRT-PCR analysis was performed with gene-specific primers (Tables 2 and 3) using an ABI PRISM 7900HT (Applied Biosystems, CA, USA) real-time thermocycler and a SYBR Green PCR master mix (Applied Biosystems).
pCAMBIA2300-eGFP for carrying increased GFP under the control of a CaMV 35S promoter, was used for subcellular localization prediction analysis. Whole bc2.1 excluding a stop codon was amplified using gene-specific primers BC2.1-GFP-F and BC2.1-GFP-R. The amplified bc2.1 and pCAMBIA2300-eGFP were double-cleaved with XbaI and SalI at 37° C. for 4 hours, and then bc2.1 was ligated to a compatible XbaI/SalI site of pCAMBIA2300-eGFP using T4 DNA ligase (Promega Corporation, WI, USA). The resulting plasmid pCAMBIA2300-BC2.1-eGFP was transferred to E. coli DH5α and sequenced. Appropriate plasmids were transformed into Agrobacterium tumefaciens GV3101 by a freeze-thaw method. Agrobacterium was incubated at 28° C. until OD 600 was 0.7, resuspended in an induction buffer (10 mM MES, 10 mM MgCl2, 200 μM acetosyringone), and then incubated at room temperature for 2 hours before agroinfiltration. Young leaves of 6-week-old N. benthamiana plants were agroinfiltrated with GV3101 containing either pCAMBIA2300-eGFP or BC2.1-eGFP. The intracellular localization of a GFP fusion protein was observed by confocal laser-scanning microscopy (LSM700, Carl Zeiss, Oberkochen, Germany). Chloroplasts were identified by red auto-fluorescence at 555 nm and green fluorescence (GFP) at 488 nm.
To identify a bc2.1 locus, an F2 population obtained by hybridizing M82 and IL2-2 was constructed, and located between marker 98 and U572717 (Table 1) on chromosome 2 to breed subIL2-2-1 with an increased β-carotene content. The subIL2-2-1 introgression location and subIL2-2-1 breeding tree were illustrated in
IL6-3 and IL12-2 were obtained from the Tomato Genetics Resource Center at the University of California, Davis (CA, USA) (http://tgrc.ucdavis.edu/).
For separation of a double genetic introgression line DIL26, subIL2-2-1 was crossed with IL6-3. Among the resulting 75 F2 plants, homozygous lines containing double mutations were obtained using gene-specific PCR markers of bc2.1 (Table 1) and CYC-B. One line, DIL26, was used for further analysis. For separation of a genetic introgression line DIL212, subIL2-2-1 was crossed with IL12-2. Among the resulting 82 F2 plants, homozygous lines containing double mutations were obtained using gene-specific PCR markers of bc2.1 (Table 1) and LYC-E. One line, DIL212, was used for further analysis. Carotenoids were analyzed by HPLC in mature fruits of M82, subIL2-2-1, IL6-3, IL12-2, DIL26 and DIL212 (Table 4).
)
0.08
indicates data missing or illegible when filed
DNA Construct for Agrobacterium-Mediated Transient Expression and Co-Immunoprecipitation in N. benthamiana
Agrobacterium-mediated transient expression in N. benthamiana, co-immunoprecipitation and immunoblot were performed. Plant overexpression constructs for Myc-GFP were previously published and used (Bhattacharjee, S. et al. Virus resistance induced by NB-LRR proteins involves Argonaute4-dependent translational control. Plant J. 58, 940-951 (2009)). CYC-B and BC2.1 with an AscI site at each end were amplified with oligonucleotides (CYC-B-AscI-F+CYC-B-AscI-R and BC2.1-AscI-F+BC2.1-AscI-R, respectively, Table 2) from fruit cDNA. The amplified CYC-B and BC2.1 fragments were ligated to pER8-Flag and pER8-Myc, respectively, to generate pER-CYC-B-Flag and pER-BC2.1-Myc.
Expression of BC2.1 in Trans-Lycopene-Accumulating E. coli
PCR fragments were amplified using the BC2.1/ORF2 full cDNA of M82 and subIL2-2-1 and cloned into a pTrcHis2-TOPO vector (Invitrogen, CA, USA) using an mTrcHis2 primer (Table 2), and confirmed by sequencing. pAC-LYC was co-transformed into E. coli strain BL21-AI with the pTrcHis2-TOPO-BC2.1 construct. A control consisting of a trans-lycopene-producing strain and pTrcHis2-TOPO without BC2.1 was subjected to the same induction treatment. 3 mL of the culture incubated overnight was inoculated into 50 mL of an LB medium containing appropriate antibiotics and 0.2% glucose. The culture was grown at 28° C. until the absorbance at 600 nm was 0.5. The expression of the protein was induced by adding 1 mM IPTG and incubated overnight at 28° C. in the dark. Then, an equal volume of acetone was added and then a double volume of ethyl acetate was added. Phases were separated by adding water and an ethyl acetate phase was stored for HPLC and LC-MS/MS analysis. After centrifugation, the medium from the culture was split twice with an equal volume of ethyl acetate. HPLC fractions were dried and resuspended in ethyl acetate, and then used in HPLC as described above.
LC-MS/MS analysis was performed by Mass Spectrometry Convergence Research Center (Kyungpook National University, Daegu, South Korea). A 5 μl extract from the enzyme assay (
In addition, UPLC-ESI-QTOF-MS/MS was performed on a high-resolution 6540 QTOF-MS/MS spectrometer (Agilent, Santa Clara, CA, USA) with electrospray ionization (ESI, positive mode) in the detection range of 500 to 600 m/z. LC separation was performed on an Agilent 1290 UPLC system (Agilent) as previously published. UPLC-ESI-QTOF-MS/MS was performed by Metabolomic Discoveries GmbH (Potsdam, Germany).
Quantification of Squalene and 2,3-oxidosqualene
For extraction and profiling of squalene and 2,3-oxidosqualene, about 200 mg of pericarp was used at 7 days after fruit breaker. The extraction of squalene and 2,3-oxidosqualene was performed by a known method (Khachik, F. et al. Chemistry, distribution, and metabolism of tomato carotenoids and their impact on human health. Exp Biol Med (Maywood) 227, 845-851 (2002)), and quantitative analysis was performed using 6540 Q-TOF/LC-MS (Agilent, CA, USA) and reverse-phase C18 columns (100×3 mm, 1.8 mm, Agilent). Standard squalene and 2,3-oxidosqualene were purchased from Sigma-Aldrich (St. Louis, MO, USA).
To identify natural variations affecting carotenoids in tomato, the carotenoid content of mature fruits was evaluated in genetic introgression lines (ILs) of S. pennellii “LA716”. Among the ILs, IL2-1 and IL2-2 (
High-resolution mapping of bc2.1 was performed using the M82×IL2-2 F2 population and subIL2-1s (
The expression of BC2.1/ORF2 was significantly decreased compared to M82 not only during leaf and flower ripening but also during fruit ripening (
SQE promoted the metabolism of squalene to produce 2,3-oxidosqualene, which was a precursor to cyclic triterpenoids of all angiosperms. Carotenoid metabolizing enzymes were mostly classified as being present in chromatophores, and the metabolism of triterpenoids, including squalene, mainly remained in the ER and cytosol. Unlike Arabidopsis SQE family members, ORF2 contained a predicted chromatophore-targeting peptide in the N-terminal region (
RNAi experiments were conducted to enhance the beta-carotene content of tomato fruits by ORF2, as a mediator of bc2.1. The RNAi structure of ORF2 was introduced into M82, which had a relatively low beta-carotene content (1.5 to 2.0 μg/g fw) in fruit. The same construct was also transformed into Ailsa Craig (AC), which had a relatively high beta-carotene content (about 14 μg/g fw) in mature fruits. Six independent transgenic lines in M82 and three independent transgenic lines in AC were generated (Table 6) and showed increased beta-carotene and reduced ORF2 expression (
-carotene
2-1
4.15
±
0.43
69.42
±
3.24
3.53
±
0.29
2.36
±
0.13
2-5
4.77
±
0.64
2-6
4 ± 0.42
2-9
6.36
±
0.69
2-16
5.82
±
0.76
.22
4.28
±
0.41
2-2
4.81
±
0.86
2-1
5.19
±
0.69
76.84
±
6.02
2-5
2-6
2-9
6.48
±
1.18
2-16
6.4
±
0.63
2-2
5.44
±
0.5
6.27
±
0.11
5.42
±
0.17
28.6
±
0.66
6.86
±
0.19
indicates data missing or illegible when filed
As described above, decreased expression of ORF2 which was a SQE homolog affected the accumulation of fruit-specific carotenoids, which was suggested that there was a possible role in carotenoid metabolism. To study the molecular basis of BC2.1/ORF2, any genetic interaction with Beta was confirmed. SubIL2-2-1 was crossed with IL6-3 (including a Beta allele of CYC-B), and one homozygous line (DIL26) in which both genes were introgressed was selected from the resulting F2 population. beta-carotene constituted less than 5% of the total carotenoids in M82 and about 40% of the total carotenoids in red-orange Beta (IL6-3) fruits. On the other hand, DIL26 fruits were orange in color and beta-carotene accounted for almost 90% of total carotenoids, which had at least 24 times increased beta-carotene (
A carotenoid profile of the DIL26 genotype was consistent with a previously proposed two-gene model for beta-carotene content in tomato fruits, and in this model, beta-carotene was influenced by both Beta and a modifier gene referred to as a Beta-modifier (MoB). Moreover, DIL26 had a very similar profile of carotenoids to that reported in S. galapagense “LA317” (previously L. chesmannii), including Beta and MoB loci. To evaluate the intrinsic nature of the bc2.1 allele in LA317, the promoter and cDNA sequences were analyzed. In the promoter, a 52 bp insertion having no obvious homology to previously known sequences was found in 1,234 bp upstream from the initiation codon (
As derived a genetic interaction between bc2.1 and Beta, subIL2-2-1 was crossed with IL12-2, which contains the Delta allele of lycopene e-cyclase (LCY-E), to breed a double genetic introgression line referred to as DIL212. DIL212 represented orange-colored fruits when compared with IL12-2 (Delta), which exhibited a deep orange color and M82, which exhibited a red color in mature fruits (
Based on an increase in beta-carotene content associated with BC2.1/ORF2, the possible activity of encoded enzymes in carotenoid metabolism was confirmed. The recombinant proteins (S1BC2.1 from M82 and SpBC2.1 from LA716) were introgressed into E. coli, which highly accumulated trans-lycopene previously constructed. In trans-lycopene accumulated E. coli, when S1BC2.1 (JX683513, 75 to 602 a.a.) or SpBC2.1 (JX683514, 75 to 601 a.a.) was expressed together with an expression vector pTrcHis2-TOPO, most of the trans-lycopene was converted to a new peak (
Allelic mutations in a coding region of BC2.1/ORF2 (
As mentioned above, BC2.1 contained a predicted transport peptide (
It was considered that the carotenoid pathway acted as a multi-enzyme complex to enable metabolite channeling, as would be expected in the absence of a pathway intermediate and in the presence of a complex comprising a carotenoid biosynthetic enzyme. Flag-labeled BC2.1 (JX683513, 1 to 602 a.a.) and Myc-labeled CYC-B (AF254793, 1 to 498 a.a.) were transiently expressed in N. benthamiana. In immunoblot analysis using an αMyc antibody, BC2.1 migrated as a doublet; Thus, it was shown that BC2.1 had post-translational modifications (
In genetic and biochemical analysis of bc2.1, it was shown that the lycopene epoxide activity of BC2.1/ORF2 competed with CYC-B for its substrate, trans-lycopene, and the balance of BC2.1/ORF2 and CYC-B activities determined the relative amounts of lycopene and beta-carotene in mature fruits. Similarly, BC2.1/ORF2 also competed with LCY-E in Delta to regulate a lutein level. A new target regulating provitamin A was identified by analyzing naturally occurring mutations in the bc2.1 locus.
Nutritional problems are a major problem emerging internationally, and continuous research is required to solve the problems. Accordingly, there is an increasing demand for genetically improving the nutrients of many crop plants. In addition, it is possible to understand the genetic and biochemical basis of accumulation of plant carotenoids by identifying lycopene epoxidase and to provide a molecular tool for screening for allelic variants or genetic variants for increased provitamin A and lutein.
Hereinabove, the present invention has been described with reference to preferred exemplary embodiments thereof. It will be understood to those skilled in the art that the present invention may be implemented as a modified form without departing from an essential characteristic of the present invention. Therefore, the disclosed exemplary embodiments should be considered in an illustrative viewpoint rather than a restrictive viewpoint. The scope of the present invention is illustrated by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0184545 | Dec 2020 | KR | national |
| 10-2020-0184546 | Dec 2020 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/019727 | 12/23/2021 | WO |
