KR102634549B1 - Tomato lycopene epoxidase improving lutein level and uses thereof - Google Patents

Abstract

The present invention relates to an introgression line derived from a wild tomato species and a transgenic tomato with suppressed expression of BC2.1 and increased lutein content.

Description

Lutein enhancement method using tomato lycopene epoxidase {TOMATO LYCOPEENE EPOXIDASE IMPROVING LUTEIN LEVEL AND USES THEREOF}

The present invention relates to an introgression line derived from a wild tomato species and a transgenic tomato in which the lutein content is increased by suppressing the expression of BC2.1, a tomato lycopene epoxidase.

Carotenoids are organic pigments that plants, algae, bacteria and fungi synthesize from fat and basic organic metabolites, 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 certain health benefits, such as prevention of blindness and maintenance of the immune system. Animals lack the ability to synthesize carotenoids de novo, so they must consume carotenoids through fruits and vegetables. For this reason, there is increasing interest in genetic control of carotenoid biosynthesis in plants and improved crops with increased carotenoid content. Both increased breeding and sequestration through genetic engineering using genetic resources and genetic silencing of xenogeneic or endogenous genes can significantly increase provitamin A content.

Lutein is one of 600 naturally occurring carotenoids known to date. Lutein is synthesized only from plants and other xanthophylls and is found in large quantities in leafy vegetables such as spinach, kale, and yellow carrots. However, lutein products are generally released as products containing more than 99% of marigold flower extract. .

Lutein was recently judged to be helpful for eye health by the Food and Drug Administration and was approved as an individually recognized ingredient. Individually recognized ingredients are not researched and announced by the Food and Drug Administration, but are approved by the Food and Drug Administration after individuals or companies present research results. According to the Food and Drug Administration, lutein is recorded to help eye health by maintaining the density of macular pigment, which can decrease due to aging.

However, since lutein cannot be produced naturally in the body, it must be consumed from outside through food or supplements.

Korean Patent No. 10-1553642

The inventors of the present invention completed the present invention by confirming that the expression of BC2.1 was suppressed and the lutein content was increased in introgression lines derived from wild tomato species and transgenic tomatoes expressing LCY-E .

Accordingly, the purpose of the present invention is to provide a tomato transfection line and transformant in which expression of BC2.1 is suppressed.

In order to achieve the above object, the present invention provides tomatoes with suppressed expression of BC2.1 and increased lutein content.

In one embodiment of the present invention, the expression of ORF2 of BC2.1 may be suppressed.

In one embodiment of the present invention, the expression of BC2.1 is suppressed and LCY-E is expressed, thereby increasing the lutein content.

In one embodiment of the present invention, the expression of BC2.1 may be suppressed by administering an RNAi construct. The RNAi construct may be prepared using RNAi-F having the sequence shown in SEQ ID NO: 59 and RNAi-R having the sequence shown in SEQ ID NO: 60.

In one embodiment of the present invention, BC2.1 expression may be suppressed by administering a sequence encoding BC2.1 containing a mutation to tomatoes. The sequence encoding BC2.1 containing the above mutation may be selected from the nucleic acid sequences represented by SEQ ID NOs: 2 to 4.

In one embodiment of the present invention, the tomato is Solanum. It may be obtained by crossing subIL2-2-1, an introgression line of pennellii LA716, with IL6-3, or it may be produced by crossing subIL2-2-1 with IL12-2.

The present invention can increase the content of lutein by suppressing the expression of BC2.1, thereby genetically improving the nutrients of many crops. In addition, the present invention identifies BC2.1, a lycopene epoxidase, to understand the genetic and biochemical basis of plant carotenoid accumulation, and to screen for allelic mutations or genetic mutations for increased provitamin A and lutein. It can provide molecular tools for

Figure 1 shows the carotenoid profiles of IL2-1, IL2-2, subIL2-2-1, and M82: a. Ripening fruits of IL2-1, IL2-2, and M82; B. IL2-1, IL2-2, and IL2-2 grown under three different environmental conditions: trial 1 (outdoor, Florida, 2004), trial 2 (greenhouse, New York, 2010), and trial 3 (greenhouse, Daegu, 2016). Relative levels of beta-carotene in M82; (n=5) c. Carotenoid and chlorophyll profiles of M82 and subIL2-2-1 (SubIL containing overlapping introgression of IL2-1 and IL2-2) during fruit ripening (n=10). MG: mature green, BR: breaker (early ripening), B3: 3 days post breaker, B7: 7 days post breaker, B10: 10 days post breaker; d. Relative levels of carotenoids and chlorophyll in leaves and flowers of M82 and subIL2-2-1 (n=5). nd: not detected.
Figure 2 shows the positional cloning of bc2.1 : top to bottom, IL gene map of chromosome 2, beta-carotene level and statistical significance (n>3, paired one tail t -test, P <0.05), 15 ORFs (arrows) in _a 160 kb region defined by markers between U582871 and 09, and two repeat element insertions in the promoter (triangles). ) and a gene model with seven mutations in the coding region of the LA716 allele (compared to M82) (distinguished by orange arrows in the 15 ORFs). The Copia -like retrotransposon (1261 bp) and tomato-specific repeat element (553 bp) shown in green triangles were inserted into the promoter of the LA716 bc2.1 allele. The unknown fragment (53 bp) shown in the orange triangle is inserted into the bc2.1 promoter of LA317 and LA1412. Red squares in the first exon represent the putative plastid target sequence and black squares represent the eight exons. a= significantly higher than IL2-2, b= significantly higher than M82. b, Results of PCR analysis of the insertion of two repeat elements in the promoter region of bc2.1 using cis1-F and cis1-R primers (Table 2). Gene expression analysis results of BC2.1/ORF2 in M82 and subIL2-2-1 in fruit, leaf and flower tissues (n=5) at different developmental stages. Relative expression in fruit normalized to M82-BR stage.
Figure 3 shows the biochemical activities of genes encoded by the bc2.1 and BC2.1 alleles: a. Results of HPLC analysis comparing relative carotenoid profiles in ripening fruits of R bc2.1 (subIL2-2-1), Beta (IL6-3), and bc2.1 Beta (DIL26) with wild type (M82); b. Ripening fruits of M82 (WT), IL2-2-1 ( bc2.1 ), IL6-3 ( Beta ), DIL26 ( bc2.1 Beta ), IL12-2 ( Delta ), and DIL212 ( bc2.1 Delta ); c. Relative gene expression of BC2.1/ORF2 and relative levels of lycopene and beta-carotene in S. galapagense “LA317” and S. cheesmaniae “LA1412” compared to M82 (n=5). nd : not detected; d. BC2.1 and CYC-B. Immunoblot and co-immunoprecipitation analysis results showing relative association with Myc-BC2.1 , Myc-GFP , and CYC-B-Flag ; e. Results of BC2.1/ORF2 protein function analysis in E. coli , which accumulates lycopene and produces lycopene epoxide. Peak identification: I. trans -lycopene; II. lycopene epoxide; f. A new branch of carotenoid biosynthesis by bc2.1 . LCY-E: lycopene e-cyclase, CYC-B: coloroid-specific b-cyclase, BC2.1: lycopene epoxidase, MoB : Beta-modifier
Figure 4 shows the results of genetic segmentation of the bc2.1 region using 100 F2-F3 from M82×IL2-2 and subIL2-1s. Blue dotted lines represent candidate QTL regions based on genotype and phenotype.
Figure 5 shows the results of quantitative RT-PCR analysis of carotenoid biosynthesis in ripened fruits of M82 and subIL2-2-1 (n=5).
Figure 6 shows the results of Pearson correlation analysis between beta-carotene and bc2.1 expression in ripened fruits of LA716 ILs.
Figure 7 shows multiple alignment of bc2.1 homologues in plants. The red box represents the FAD binding domain, and the blue box represents a highly conserved region in the squalene epoxidase family. The green line represents the putative plastid target sequence based on TargetP 1.1 server (http://www.cbs.dtu.dk/services/TargetP-1.1/index.php), the orange line represents the putative signal peptide, and the yellow line represents the putative signal peptide. Mitochondrial targeting sequence is indicated. The protein sequences used in the analysis are as follows: SlBC2.1 (JX683513), SpBC2.1 (JX683514), SgBC2.1 (JX683515), StSQE2 (Sotub02g011310), SlSQE1 (Solyc04g077440), StSQE1 (XP_015168607), NtSQ E1a (XP_016434789 .1), NtSQE1b (XP_016502919.1), AtSQE1 (AT1G58440), AtSQE2 (AT2G22830), AtSQE3 (AT4G37760), AtSQE4 (AT5G24140), AtSQE5 (AT5G24150), AtSQE6 (AT5G24160) , AtSQE7 (AT5G24155), ZmSQE1a (ZM09G25330) , ZmSQE1b (ZM01G08660), OsSQE1a (Os03g12910), OsSQE1b (Os03g12900).
Figure 8 shows the results of phylogenetic analysis of BC2.1 analogs in plants, showing a neighbor-joining phylogenetic tree based on the estimated amino acid sequences of BC2.1 analogs in tomato, potato, tobacco, Arabidopsis, rice, and corn. will be.
Figure 9 shows the results of analysis of the intracellular location of BC2.1 in N. benthamiana . Scale bars = 5 μm.
Figures 10A-10C show alignment of bc2.1 promoter sequences in M82, LA317, LA1412 and LA716. Major insertions in wild species are indicated by bold, colored nucleotides. The start codon is underlined. Red: copia-like retrotransposon in LA716, green: tomato-specific repeat element in LA716, blue: unknown 52 bp nucleotide in LA317 and LA1412.
Figure 11 shows the results of qRT-PCR analysis showing the transcription level of bc2.1 in wild-type and T ₀ RNAi-transgenic tomatoes of ripening fruit (B+10). Transcript levels are shown relative to wild type (M82 or AC).
Figure 12 shows LC-MS/MS characterization of BC2.1 activity assay using LC-APCI-MS/MS: a. Shows an overlay of extracted ion chromatograms (EIC) from trans- lycopene (extract from E. coli transformed with empty vector, green) and lycopene epoxide (extract from BC2.1 expressing E. coli cells, red). (Figure 3e). The upper panel shows the EICs of m/z 537.44 Da [M+H] ⁺ (ppm error: 0.12) and the lower panel shows the EICs of m/z 553.44 Da [M+H] ⁺ (ppm error: 0.23) from the control. will be. b. MS-MS refined spectrum at m/z 537.44, trans -lycopene; c. MS-MS refined spectrum of m/z 553.44, lycopene epoxide.
Figure 13 shows LC-MS/MS characterization of BC2.1 activity assay using UPLC-ESI-QTOF-MS/MS: a. This shows the UV-Vis spectrum of trans- lycopene and lycopene epoxide extracted at 471 nm. b. This shows the MS spectrum of lycopene epoxide. c. This shows the MS/MS spectrum of lycopene epoxide.
Figure 14 shows the lycopene epoxide content of wild type (M82 and AC), subIL2-2-1 and RNAi transgenic tomatoes in ripened fruits (10 days post-breaker, n=5).
Figure 15 shows the subIL2-2-1 transfection location and subIL2-2-1 breeding system diagram.

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail through examples. However, these examples are for illustrating the present invention in detail, and the scope of the present invention is not limited to these examples.

Example 1

plant matter

To evaluate IL (introgression line) fruit carotenoids and their environmental changes, field cultivation was conducted in Florida (spring 2004), greenhouse cultivation in New York (fall 2010), and outdoor cultivation in Daegu (spring 2004). Tomato mapping populations and transgenic plants were grown under standard conditions (27/19 °C; 16/8 h light/dark) in a greenhouse at 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).

Access code

Sequences were obtained from GenBank: bc2.1 (JX683513), Spbc2.1 (JX683514), and Sgbc2.1 (JX683515).

bc2.1 Positional cloning

100 plants of the M82×IL2-2 F ₂ population in addition to the line derived from IL2-1, which represents a subset of the 2-1 introgression line (also derived from M82×IL2-1 F ₂ ) from the Hebrew University of Israel. Genetic analysis of bc2.1 was performed using (Figure 4). Using these plant materials, bc2.1 , corresponding to a 1.1-Mb segment of tomato chromosome 2 (encompassing the 29.9- to 31.0-Mb region as defined in tomato genome assembly SL2.40), DNA markers 2A and It was confirmed that it was located near the top of chromosome 2 between C2_At1g60640 (Figure 4).

Additional high-resolution mapping of bc2.1 was performed using 1100 F ₂ plants derived from the M82×IL2-2 cross. The bc2.1 flanking markers 2A and 98 (the latter a PCR marker upstream of IL2-2 defined in the initial small mapping population) were used for recombinant screening. Eight F2 recombinants were obtained from a 1.1-Mb region and their F ₃ progeny homozygous for the bc2.1 LA716 or M82 allele were analyzed for beta-carotene from ripe fruits of the same developmental stage labeled by HPLC (paired one-tailed t -test, P <0.05). Between markers 09 and U582871, a 160-kb introgression fragment from LA716 (Figure 2A) co-segregated with increased beta-carotene content, and this fragment contained 15 predicted ORFs. Markers used for genotyping are shown in Table 1. Candidate bc2.1 alleles from M82 and subIL2-2-1 were sequenced and gene expression was assessed by qRT-PCR. For carotenoid analysis, fruits were harvested from all genotypes at different stages of development (7 days after breaker). The pericarp tissue was immediately frozen with liquid nitrogen and stored at -80°C. The tissue was pulverized using liquid nitrogen to extract carotenoids and RNA.

Carotenoid profiling by HPLC

Approximately 100 mg of frozen and ground pericarp from individual fruits (including biological repeats) was 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.

RNAi construct development and tomato transformation

A bc2.1- specific 282-bp DNA fragment (EST clone: TUS-64-H10) located in the 3'-UTR region of cDNA (i.e., bases 1866 to 2147 of GenBank accession number JX683513) was purified using Phusion High-Fidelity DNA Polymerase. (New England Biolabs, MA, USA), gene-specific primers, RNAi-F and RNAi-R (Table 2), and an EST clone as a template were amplified by PCR. The purified cDNA fragment was cloned into pHellsgate 2 as an inverted repeat sequence under the control of the 35S promoter using the Gateway ^TM cloning system (Gateway ^TM BP Clonase ^TM Enzyme Mix, Invitrogen, CA, USA). The intact structure was confirmed by sequencing and previously published method (Fillatti, JJ et al., Efficient Transfer of a Glyphosate Tolerance Gene into Tomato Using a Binary Agrobacterium-Tumefaciens Vector. Bio-Technology 5, 726-730 (1987) ) was introduced into M82 and AC tomato varieties using Agrobacterium tumefaciens LBA4404. Plants inheriting the transgene were confirmed by PCR using CaMV-35S-specific primers, 35S-F and 35S-R (Table 2), and subjected to Southern blot analysis using a CaMV-35S-specific probe.

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 using an ABI PRISM 7900HT (Applied Biosystems, CA, USA) real-time thermocycler and SYBR Green PCR master mix (Applied Biosystems) with gene-specific primers (Tables 2 and 3).

Subcellular localization of BC2.1

pCAMBIA2300-eGFP, which carries increased GFP under the control of the CaMV 35S promoter, was used for intracellular organelle location prediction analysis. The entire bc2.1 , excluding the 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- digested with It was linked to the compatible Xba I/ Sal I site. The resulting plasmid (pCAMBIA2300-BC2.1-eGFP) was transferred to E. coli DH5α and sequenced. The appropriate plasmid was transformed into Agrobacterium tumefaciens GV3101 by freeze-thaw method. Agrobacterium was cultured at 28°C until OD 600 reached 0.7, resuspended in induction buffer (10mM MES, 10mM MgCl ₂ , 200μM acetosyringone), and incubated at room temperature for 2 hours before agroinfiltration. Young leaves of 6-week-old N. benthamiana plants were agroinfiltrated with GV3101 containing pCAMBIA2300-eGFP or BC2.1-eGFP. The intracellular location of the GFP fusion protein was observed using confocal laser-scanning microscopy (LSM700, Carl Zeiss, Oberkochen, Germany). Chloroplasts were identified through their red auto-fluorescence at 555 nm and GFP (green fluorescence) at 488 nm.

Development of double introgression lines

To confirm the bc2.1 locus, an F2 population obtained by crossing M82 and IL2-2 was created, and subIL2-2-1, located between markers 98 and U572717 on chromosome 2 (Table 1), has increased β-carotene content. was fostered. The subIL2-2-1 transfection location and subIL2-2-1 breeding diagram are shown in Figure 15.

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 isolation of the double introgenic line DIL26, subIL2-2-1 was crossed with IL6-3. Among the resulting 75 F ₂ plants, isogenic lines containing the double mutation were obtained using gene-specific PCR markers of bc2.1 (Table 1) and CYC-B . One line, DIL26, was used for further analysis. For isolation of the introgenic line DIL212, subIL2-2-1 was crossed with IL12-2. Among the resulting 82 F ₂ plants, isogenic lines containing the double mutation were obtained using gene-specific PCR markers of bc2.1 (Table 1) and LCY-E . One line, DIL212, was used for further analysis. Carotenoids were analyzed by HPLC in ripened fruits of M82, subIL2-2-1, IL6-3, IL12-2, DIL26 and DIL212 (Table 4).

N. benthamiana in Agrobacterium -DNA constructs for mediated transient expression and co-immunoprecipitation

Agrobacterium -mediated transient expression, co-immunoprecipitation, and immunoblotting in N. benthamiana were performed. The plant overexpression construct for Myc-GFP was used as previously published (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 AscI sites at each end were synthesized with oligonucleotides (CYC-B-AscI-F + CYC-B-AscI-R and BC2.1-AscI-F + BC2.1-AscI-, respectively). R, Table 2) was amplified from fruit cDNA. The amplified CYC-B and BC2.1 fragments were linked to pER8-Flag and pER8-Myc, respectively, to generate pER-CYC-B-Flag and pER-BC2.1-Myc.

trans -Lycopene-accumulation E. coli Expression of BC2.1 in

The PCR fragment was amplified using the BC2.1/ORF2 total cDNA from M82 and subIL2-2-1, cloned into the pTrcHis2-TOPO vector (Invitrogen, CA, USA) using the mTrcHis2 primer (Table 2), and sequenced. Confirmed. pAC-LYC was co-transformed with the pTrcHis2-TOPO-BC2.1 construct into E. coli strain BL21-AI. The trans -lycopene-producing strain and a control consisting of pTrcHis2-TOPO without BC2.1 were subjected to the same induction treatment. 3 mL of overnight culture was inoculated into 50 mL of LB medium containing appropriate antibiotics and 0.2% glucose. Cultures were grown at 28°C until the absorbance at 600 nm was 0.5. Protein expression was induced by adding 1mM IPTG and cultured in the dark at 28°C overnight. Then, an equal volume of acetone was added and a double volume of ethyl acetate was added. Water was added to separate the phases and the ethyl acetate phase was stored for HPLC and LC-MS/MS analysis. After centrifugation, the medium from the culture was split twice with equal volumes of ethyl acetate. HPLC fractions were dried, resuspended in ethyl acetate and used for HPLC as described above.

LC-MS/MS analysis of carotenoids for enzyme assays

LC-MS/MS analysis was performed by Mass Spectrometry Convergence Research Center (Kyungpook National University, Daegu, South Korea). 5 μl extracts from the enzyme assay (Figure 3e) were analyzed using an Ultimate 3000 U-HPLC system (Thermo Fisher Scientific, MA, USA) and a Q EXACTIVE ^TM Focus massspectrometry system (ThermoFisherScientific) using atmospheric pressure chemical ionization (APCI) (positive mode). ) was analyzed. Chromatographic separation was performed using a Kinetex C18 column (100 × 2.1 mm; Phenomenex, Torrance, CA). The mobile phase was methanol (A) and methyl tert -butyl ether (B) containing 0.1% ammonium acetate at a flow rate of 0.5 mL/min, flowed under conditions of 90% A and 10% B for 5 minutes at 25°C. APCI parameters are: sheath gas flow rate, 50 arbitrary units; auxiliary gas unit flow rate, 12 arbitrary units; vaporizer temperature, 300℃; capillary temperature, 320℃; discharge current, 5 mA; S-lens radiofrequency level, 95; resolving power, 70,000. For the target compound, a scan range of 520-620 m/z was selected. The automatic gain control was set to 1 × 106 and the injection time was set to 100 ms.

In addition, UPLC-ESI-QTOF-MS/MS was performed using a high-resolution 6540 QTOF-MS/MS spectrometer (Agilent, Santa Clara, CA) accompanied by electrospray ionization (ESI, positive mode) in the detection range of 500~600 m/z. , USA) was used. LC separation was performed with 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, ~200 mg of pericarp was used 7 days after fruit breaker. Extraction of squalene and 2,3-oxidosqualene was performed using 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)), quantitative analysis was performed using a 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).

Example 2

result

To identify natural variations affecting carotenoids in tomatoes, the carotenoid content of ripe fruits was evaluated in introgression lines (ILs) of S. pennellii “LA716”. Among the ILs, IL2-1 and IL2-2 (Fig. 1a) have overlapping portions of the introgressed chromosomes and the fruit color and shape are visually indistinguishable from the control S. lycopersicum “M82”, but their heritable beta-carotene content is 2.5 times higher. was higher than that (Figure 1b). In the present invention, this beta-carotene QTL (Quantitative trait locus) was designed and named bc2.1 . To study additional effects of the bc2.1 locus, HPLC carotenoid profiling was performed on subIL2-2-1. subIL2-2-1 was grown in the F3 population of M82 Compared with M82, subIL2-2-1 did not show significant changes in carotenoids or chlorophyll in leaf and flower tissues (Figure 1d). Interestingly, beta-carotene increased only during fruit ripening, and beta-carotene precursors phytoene, phytofluene, and trans -lycopene slightly decreased compared to M82 (Figure 1c). bc2.1 did not significantly change the transcript accumulation of carotenoid biosynthetic genes, suggesting that quantitative changes in beta-carotene accumulation in subIL2-2-1 tomato fruits are a novel alternative rather than changes in mRNA accumulation of already known biosynthetic genes. This clearly suggests that it is through (Figure 5). Moreover, among the genes related to carotenoid accumulation in plants, the gene located in BIN2C is not known.

High-resolution mapping of bc2.1 was performed using the M82×IL2-2 F ₂ population and subIL2-1s (Figure 2a). The segment containing 160-kb of LA716-derived chromosomes between marker 09 and U582871 was isolated with increased beta-carotene content and contained 15 open reading frames (ORFs) (Figure 2a). The bc2.1 allele, which is the cause of beta-carotene enhancement, was confirmed to be recessively inherited through the F ₁ phenotype, and heterozygous plants showed a similar phenotype to plants homozygously carrying the M82 allele. (Figure 2, Figure 4). These results suggest that the increase in beta-carotene content in ILs is caused by a loss-of-function allele derived from LA716. Through RNA-seq (Figure 6) and analysis of genomic DNA sequences from LA716, ORF2, which is induced during fruit ripening and is highly homologous to squalene epoxidase (SQE: Figure 7), was identified as a candidate gene. In plants, SQE family members include 7 genes from Arabidopsis and 2 genes from rice, corn, and Solanaceae plants (FIG. 8).

The expression of BC2.1/ORF2 was significantly reduced compared to M82 not only in leaves and flowers, but also during fruit ripening (Figure 2c), which further supports that this gene is responsible for the bc2.1 fruit beta-carotene phenotype. . In the putative promoter sequence 1.4 kb upstream of ORF2, an insertion of 1.8 kb sequence was identified in the LA716 allele. Compared to the M82 allele, a copia -like retrotransposon and a tomato-specific repeat element were inserted into LA716 at positions 1263 and 223 bp upstream from the start codon, respectively (Figure 2B, Figure 10), which are subIL2-2 -1 could result in a decrease in ORF2 expression (Figure 2c). In S. pennellii , Copia elements located in proximal promoters lead to lower expression of these genes than in orthologous genes in S. lycopersicum without copia elements. Additionally, 6 amino acid substitutions and 1 amino acid deletion were confirmed in the coding region of LA716 (FIG. 2A, FIG. 7). As described below, variations in the coding sequence of the allele do not affect its activity (Figure 3E), suggesting that any differences in phenotype result from reduced gene expression.

SQE promotes the metabolism of squalene to produce 2,3-oxidosqualene, the precursor of cyclic triterpenoids in all angiosperms. Most carotenoid metabolic enzymes are classified as existing in plastids, and triterpenoid metabolism, including squalene, mainly remains in the ER and cytosol. Unlike Arabidopsis SQE family members, ORF2 contains a predicted plastid-targeting peptide in its N-terminal region (Figure 6). To confirm whether the reduced expression of ORF2 in subIL2-2-1 affects squalene metabolism, squalene and 2,3-oxidosqualene were quantified by Q-TOF-LC/MS (Table 5). Squalene was not detected in ripe fruits of either M82 or subIL2-2-1, and 2,3-oxidosqualene levels were very similar in both cultivars.

An RNAi experiment was conducted to determine whether ORF2, as a mediator of bc2.1 , enhances the beta-carotene content of tomato fruit. The RNAi construct of ORF2 was introduced into M82, which has a relatively low beta-carotene content (1.5-2.0 μg/g fw) in fruit. The same construct was also transformed into Ailsa Craig (AC), which has a relatively high beta-carotene content (~14 μg/g fw) in ripe fruit. Six independent transgenic lines in M82 and three independent transgenic lines in AC were generated (Table 6) and showed increased beta-carotene and decreased ORF2 expression (Figure 11). Beta-carotene content increased 2- to 4-fold in all nine RNAi lines, and other carotenoids did not significantly change in the RNAi lines (Table 6). In the M82-RNAi line, the carotenoid phenotype observed in the T0 generation was maintained in the T1 generation. (Table 6). Taken together, the above results confirmed that reduced ORF2 expression increased beta-carotene concentration in ripe fruit and that reduced expression of the LA716 allele of this gene was the fundamental determinant of the bc2.1 QTL. These results also demonstrated that BC2.1/ORF2 had a negative effect on b-beta-carotene accumulation in ripe tomato fruit.

As described above, reduction of expression of the SQE homolog ORF2 affects carotenoid accumulation in a fruit-specific manner, suggesting 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 (containing the Beta allele of CYC-B ), and one isogenic line (DIL26) in which both genes were introgressed was selected from the resulting F ₂ population. Beta-carotene constitutes less than 5% of the total carotenoids in M82 and approximately 40% of the total carotenoids in red-orange Beta (IL6-3) fruit. On the other hand, DIL26 fruits are orange in color and beta-carotene accounts for almost 90% of the total carotenoids, with beta-carotene increased more than 24-fold (Figure 3a, Table 4). These results suggest that bc2.1 exerts a genetic synergy with Beta for beta-carotene synthesis.

The carotenoid profile of the DIL26 genotype is consistent with a previously proposed two-gene model for beta-carotene content in tomato fruit, in which beta-carotene is influenced by both a modifier gene, designated as Beta and Beta -modifier ( MoB ). receive Furthermore, DIL26 has a profile of carotenoids that is very similar to that reported in S. galapagense “LA317” (previously L. chesmannii ) containing the Beta and MoB loci. To evaluate the unique nature of the bc2.1 allele in LA317, the promoter and cDNA sequences were analyzed. In the promoter, a 52-bp insertion with no clear homology to the previously known sequence was found 1,234 bp upstream from the start codon (Figure 7), and the insertion site was the location where the copia -like retrotransposon found in the LA716 bc2.1 allele was inserted. The location was similar. BC2.1/ORF2 expression in LA317 ripe fruits was reduced to a level similar to that observed in subIL2-2-1 ripe fruits, and the most abundant carotenoid in orange-colored LA317 ripe fruits was beta-carotene. The same mutation was found in S. cheesmaniae “LA1412”, which has an orange ripe fruit color and a carotenoid profile similar to LA317 (Table 7) (Figure 10). Similar to DIL26, lycopene was not detected in ripe fruits of LA317 and LA1412 (Figure 3c), suggesting bc2.1 as a promising candidate for MoB .

Consistent with the genetic interaction between bc2.1 and Beta , subIL2-2-1 was crossed with IL12-2 containing the Delta allele of lycopene e-cyclase ( LCY-E ), resulting in the doublet, designated DIL212. Introgressive lines were cultivated. DIL212 produced orange-colored fruits when compared to IL12-2 ( Delta ), which produced a dark orange color in ripe fruits, and M82, which produced a red color (Figure 3b). While a very small amount of lutein was accumulated in ripened fruits of M82, lutein (11.74±0.80 mg/g fw) in DIL212 was more than 20 times higher than that of M82. As seen in DIL26, a very small amount of lycopene was also accumulated in DIL212, suggesting genetic interaction between bc2.1 and Delta . Lutein variation in IL was not observed, because LCY-E is not expressed in the ripe fruit of cultivated tomatoes, including M82 (Figure 1c).

Based on the increase in beta-carotene content associated with BC2.1/ORF2, the possible activity of the encoded enzyme in carotenoid metabolism was identified. Recombinant proteins (SlBC2.1 from M82 and SpBC2.1 from LA716) were transfected into previously constructed E. coli , which highly accumulates trans -lycopene. trans -lycopene accumulation When SlBC2.1 (JX683513, 75~602 aa) or SpBC2.1 (JX683514, 75~601 aa) is expressed together with the expression vector pTrcHis2-TOPO in E. coli, most of the trans -lycopene appears as a new peak. converted (Figure 3e). Despite the unavailability of standards, LC-MS/MS analysis revealed that the new peak with 553.4428 m/z was protonated lycopene epoxide (Figures 12 and 13). Lycopene epoxide was previously identified in tomato fruit and reported to be produced through catalytic and non-catalytic processes. Considering that lycopene epoxide has been identified in human plasma, its bioavailability remains to be studied. Lycopene epoxide was quantified through HPLC, and was decreased in the RNAi line and subIL2-2-1 compared to M82 or AC (Figure 14). These results indicate that BC2.1 is a lycopene epoxidase leading to a new branch of the lycopene pathway (Figure 3f).

Allelic variation within the coding region of BC2.1/ORF2 (Figure 2A) did not cause the bc2.1 fruit phenotype. Instead, this phenotype was caused by differences in expression of the BC2.1/ORF2 allele. Although the bc2.1 phenotype was restricted to fruit, the expression variant was not (Figure 2c). Based on the lycopene epoxide activity of BC2.1/ORF2 seen in the E. coli system, the bc2.1 phenotype appears to be determined by reduced maturation of LA716 allele expression and lycopene, a specific bc2.1 substrate.

As mentioned above, BC2.1 contains the predicted transit peptide (Figure 7). The BC2.1-GFP fusion protein was transiently expressed in the leaves of N. benthamiana under the control of the CaMV 35S promoter, and the intracellular organelle localization of BC2.1 was confirmed by confocal laser microscopy. The GFP fluorescence signal at 488 nm showed that the BC2.1-GFP fusion protein was localized in plastids, possibly plastoglobuli, whereas pCAMBIA2300-eGFP was localized in nuclear cells (Figure 8).

The carotenoid pathway is thought to function as a multi-enzyme complex to enable metabolite channeling, as expected in the absence of pathway intermediates and in the presence of complexes containing carotenoid biosynthetic enzymes. Flag-tagged BC2.1 (JX683513, 1~602 aa) and Myc-tagged CYC-B (AF254793, 1~498 aa) were transiently expressed in N. benthamiana . In immunoblot analysis using αMyc antibody, BC2.1 migrated as a doublet; Therefore, it was shown that BC2.1 had post-translational modifications (Figure 3d). Co-immunoprecipitation analysis showed that BC2.1 forms a complex with CYC-B, but not with GFP, which was used as a negative control (Figure 3D), indicating that BC2.1 binds CYC-B in planta . It suggests that it is related to .

Genetic and biochemical analysis of bc2.1 suggests that the lycopene epoxide activity of BC2.1/ORF2 competes with CYC-B for its substrate, trans -lycopene, and that the balance of BC2.1/ORF2 and CYC-B activities is maintained at maturity. It was shown to determine the relative amounts of lycopene and beta-carotene in fruits. Similarly, BC2.1/ORF2 also regulates lutein levels by competing with LCY-E in Delta . By analyzing naturally occurring mutations in the bc2.1 locus, new targets that regulate provitamin A were identified.

Nutrition is a major problem emerging internationally, and continuous research is needed to solve it. Accordingly, there is an increasing need to genetically improve the nutrients of many crops. Identification of lycopene epoxidase allows understanding of the genetic and biochemical basis of plant carotenoid accumulation and may provide a molecular tool for screening of allelic or genetic variants for increased provitamin A and lutein. there is.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> INTROGRESSION AND TRANSGENIC TOMATO CONTAINING INCREASED BETA-CAROTENE CONTENT <130> PN2009-396 <160> 108 <170> KoPatentIn 3.0 <210> 1 <211> 1423 <212> DNA <213> M82 <400> 1 aaataattac aagtgttatt aacattgaaa aagtccagat aattaaaatt agtacactac 60 aacaatatgt ttgtttgttc aatatttagt gtatagtaac tacgatgatt tgtttgtttg 120 tcaaatttat agttgtatag taattacaat gatatgtttg tttgccaaat tatagggtaa 180 ttgcaagtgg actatttgat tgtacaagtg taattataaa attatatgaa ttttaaaaac 240 taaaaagtta tactagataa attaatcata aaaattaaaa atacaaagtt ttaatgaaaa 300 tcatgaaata tatgtttgac aaaaaaatta atattataaa tatgttgtca taatattata 360 gaaatatttg ataaaaataa tatattaagt ctaactaaaa aaataaatta caatgtaaaa 420 tattgacgta atattcctaa aacaaataaa tttgaaaaca taacataagt tctaaattcg 480 aaataaaaaa tcaacatata atactctgat gttaaattcg acacaacata cacaaagatg 540 attttaaaag aacag gaaaa tgtatatcca taatcttatg caacaatgaa ttctatttta 600 attttaaaaa atttaaaata ataacaatgt ttatatatat ttgaaaaacg ttagaataat 660 aataaaataa atgaaaaaaa aataaagaga agaaattaaa aagtaatccc tatatgtcaa 720 aaaatcacga actcttaaaa attgaaaagt ataatt attc gtgtcaatta tctagtgatc 780 caaaataata taattatatt aaattacacc aaatttaatc aacaaaatga tgtttattcg 840 taattttgta taaagaatgt tacgataatt tatagtctaa attcaaatta tgataaagaa 900 gaattaaata tatacgtaca taaacaaaaa aaagtttaaa tcttgcaatc taggaataat 960 aatatctaat cattcgtatt ta aattgaga aatgttaata cttctttcgt tttaaaaaaa 1020 aatcttaata gtctatttaa aaaagaatga ttactttttt tttggtaata ttttaatttt 1080 ttgaataata ttttaatttt aatttttcac atgacctatt taagaccaca aggttataag 1140 atatttttat acatttaata taattttaat ttagatccac a aaattaaat attttcttta 1200 ttttcttaaa ctatatttaa aattaaacta gataactctt tttaaaagga aagaaatatt 1260 aacgaaataa ataaaatgtg ctaaataaat attcttgcat ttggcccctc ctgccaattg 1320 ccagacttga atattgtctc tcgtgttttt ttttattgtc atcctcgcaa ttttcaccca 1380 aaatcacacg gtccaattgg atccccaatt tcggattgcg atg 1423 <210> 2 <211> 1480 <212> DNA <213> LA317 <400> 2 aaataattac aagtgttat aacattgaaa aagtccagat aattaaaatt agtacactac 60 aacaatatgt ttgtttgttc aatatttagt gtatagtaac tacgatgatt tgtttgtttg 120 tcaaatttat agttgtatag taattacaat gatatgtttg tttgccaaat tatagggtaa 180 ttgcaagcgc caaccccgac tcctccacca aagaattct g gtctggagct cattccgaag 240 tggactattt gattgtacaa gtgtaattat aaaattatat gaattttaaa aattaaaaag 300 ttatactaga taaattaatc ataaaaatta aaaatacaaa gttttaatga aaatcatgaa 360 atatatgttt gacaaaaaaa ttaatattat aaatatgttg tcataatatt atagaaatat 42 0 ttgataaaaa taatatatta agtctaacta aaaaaataaa ttacaatgta aaatatcgac 480 gtaatattcc taaaacaaat aaatttgaaa acataacata agttctaaat tcgaaataaa 540 aaatcaacat ataatactct gatgttaaat tcgacacaac atacacaaag atgattttaa 600 aagaacagga aaatgtatat ccataatctt atgcaacaat gaattctatt ttaattttaa 660 aaa atttaaa ataataacaa tgtttatata tatttgaaaa acgttagaat aataataaaa 720 taaatgaaaa aaaataaaga gaagaaatta aaaagtaatc cctatatgtc aaaaaatcac 780 gaactcctaa aaattgaaaa gtataattat tcgtgtcaat tatctagtga tccaaaataa 840 tataattata ttaaattaca ccaaatttaa tcaacaaaat gatgtttat cgtaattttg 900 tataaagaat gttacgataa tttatagtct aaattcaaat tatgataaag aagaattaaa 960 tatatacgta cataaacaaa aagaagttta aatcttgcaa tctaggaata ataatatcta 1020 atcattcgta tttaaattga gaaatgttaa tacttctttc gttttaaaaa aaagatctta 1080 atagt ctatt taaaaaagaa tgattacttt ttttggtaat attttaattt tttgaataat 1140 attttaattt taatttttca catgatctat ttaagaccac aaggttataa aacaatttta 1200 tacatttgat ataattttaa tttagatcca caaaattaaa tattttcttt attttcttaa 1260 actatattta aaattaaact agata actct ttttaaaagg aaagaaatat taacgaaata 1320 aataaaatgt gctaaataaa tattcttgca tttggcccct cctgccaatt gccagacttg 1380 aatattgtct ctcgtgtttt ttttttttt tattgtcatc ctcgcaattt tcacccaaaa 1440 tcacacggtc caattggatc cccaatttcg gatttcgatg 1480 <2 10> 3 <211> 1480 <212> DNA <213> LA1412 <400> 3 aaataattac aagtgttat aacattgaaa aagtccagat aattaaaatt agtacactac 60 aacaatatgt ttgtttgttc aatatttagt gtatagtaac taagatgatt tgtttgtttg 120 tcaaatttat agttgtatag taattacaat gatatgtttg tttgccaaat tatagggtaa 180 ttgcaagcgc caaccccgac tcctccacca aagaattctg gtctggagct cattccgaag 240 tggactattt gattgtacaa gtgtaattat aaaattatat gaatt ttaaa aattaaaaag 300 ttatactaga taaattaatc ataaaaatta aaaatacaaa gttttaatga aaatcatgaa 360 atatatgttt gacaaaaaaa ttaatattat aaatatgttg tcataatatt atagaaatat 420 ttgataaaaa taatatatta agtctaacta aaaaaataaa ttacaatgta aaatatcgac 480 g taatattcc taaaacaaat aaatttgaaa acataacata agttctaaat tcgaaataaa 540 aaatcaacat ataatactct gatgttaaat tcgacacaac atacacaaag atgattttaa 600 aagaacagga aaatgtatat ccataatctt atgcaacaat gaattctatt ttaattttaa 660 aaaatttaaa ataataacaa tgtttatata tatttgaaaa acgttagaat aataataaaa 720 taaat gaaaa aaaataaaga gaagaaatta aaaagtaatc cctatatgtc aaaaaatcac 780 gaactcctaa aaattgaaaa gtataattat tcgtgtcaat tatctagtga tccaaaataa 840 tataattata ttaaattaca ccaaatttaa tcaacaaaat gatgtttat cgtaattttg 900 tataaagaat gttacga taa tttatagtct aaattcaaat tatgataaag aagaattaaa 960 tatatacgta cataaacaaa aagaagttta aatcttgcaa tctaggaata ataatatcta 1020 atcattcgta tttaaattga gaaatgttaa tacttctttc gttttaaaaa aaagatctta 1080 atagtctatt taaaaaagaa tgattacttt ttttggtaat attttaattt tttgaataat 1140 attttaattt taatttttca catgatctat ttaagaccac aaggttataa aacaatttta 1200 tacatttgat ataattttaa tttagatcca caaaattaaa tattttcttt attttcttaa 1260 actatattta aaattaaact agataactct ttttaaaagg aaagaaatat taacgaaata 1320 aataaaatgt gctaaataaa t attcttgca tttggcccct cctgccaatt gccagacttg 1380 aatattgtct ctcgtgtttt tttttttttt tattgtcatc ctcgcaattt tcacccaaaa 1440 tcacacggtc caattggatc cccaatttcg gatttcgatg 1480 <210> 4 <211> 3217 <212> DNA <213> LA716 <400> 4 aaataatta c aagtgttat aacattgaaa agtccagata attaaaatta gtacactaca 60 acaatatgtt tgttaaatat ttagtgaata gtaattacga tgatttattt gtttgttaaa 120 tttatagtgt atagtgatta caatgatatg tgttatattc taggtgtttt gatgatcctc 180 actaatgtgg ggacctggtc cctggcagag tgctttcgtc cagatacaaa ggggtacagt 240 cgtaaaagct gtcatgtcag gtgataggtg aaaagtgcag tatcatacac caatagaaaa 300 gcggacgaga ttgtatgttt cagtatct ca caaatgcagg gcttggtacc aggcagagtt 360 tcctccatca gatacataat tggttacagc tgtaaagttg tcacgtcaga ggttggtaag 420 gcgtcagtgt actacacacc aatcagaatg gaggagggtg tttgtccaac ggataataac 480 tctttatgtt tgatactttt ag catgacaa acaccatatt atattcattc atccaaagaa 540 ggaagtcagc cagcaagcct tttcaagaac tcacaaagaa gtttgcaagg gacctggtcc 600 ccgcaagact gcgaggtgaa aaggatgaaa agacagctgt catctctgat gcgataggtg 660 cacaactttt ccaacagcag gccttcagcc aatgggatgg cttcaacgaa tttgtgggaa 720 attatattat ctctttcaac aaacacaaaat tgaagacttg gaaaatta aa cttggatttt 780 ctctgaaaat tcacaagctt gaacagaaag ccatcttcaa ggaagaacat tgctcaactc 840 aacagaagga cctgatagag taaagaagaa tctttgttgt atttagagct ttgtgtctat 900 gtacttatat tgtaaatttg ttcctaatct ataaaggatt cagatatttg ttt tgggttt 960 gtaaaagtct aaatcagtta aggttaactg atttagtggg caacattgtt ttgttgctta 1020 gtcaaattct aagtaatcta gagttaggtg gtttagtggg cgatgtggta tccgcttagc 1080 aaattctaag ttatctaggg gtgatagctt agtgggcggc gttgtatccg cttaggctct 1140 tcaagtaata ggtagattac ttgatcgtga gattaataac tttttctcac attg attgta 1200 accgggtttc acttttgctt gagaagatta gtgaagacgg ttgtaaatcc tgtgcagcag 1260 gtcatggttt tactcccttg agcaaggagg tttccacgta aattgcttgt gttgtgttct 1320 tcatattt taatcttccg cactgttctt gctgtgtcaa gggacctggt ccgttgactg 1380 gtagtggacg cacatattcc aacaaatatg tttgtttgcc aaattatagg gtaattgcaa 1440 gtgtactatt tgattgcaca agtgtaatta taaaattata tgaattttaa aaactaaaaa 1500 gttatactag ataaattaat cataaaaact aaaagtacca aatttcaatg aacatcatga 1560 aatgtatgtt tgacaaaaag attaatatta taaatatgat gtcataatat tatagaaata 1620 tttgataaaa atagtatgtt aagtctaact aaaaaaataa attacaatgt aaaatatcga 1680 cgtaatattc ctaaaacaaa tgaaattaaa aatataacat aagttctaaa tttaaaataa 1740 aaaatcaata tataatatac tctgatgtca aattcaacac aacatacaca aagataattt 1800 ttttaaaaga aaaaaaaatg tatgtgcata atctt attca acaatgaatt ctattttaat 1860 tttaaaaaat taaaataatg acaatgtgta tatatatttg aaaaacatta gaataataaa 1920 aataaataat aaaataaaat ggaaaaaaaa taaagagaag aaattagaaa gtaatccctg 1980 tatgtcaaaa agtcacaaac tcctgaagag tgtaattatt cgtgtcaatt acctaataat 2040 ctaaaataaa tatgtcagac aatataatta tactaaatta caccaaattt aattaacaaa 2100 ataatgttta tttgtaattt tgtactaaga atgttacaat aatttatagt ctaaattcaa 2160 attatgataa agaagcatta aatatatacg tacataaaca aaaagtagtt taaatcttac 2220 gatctagaaa taatatctaa tcattcgtat ttaaattgag a aatgttaat actttttttc 2280 gtttaaaaaaa ttgattttat tttttttagt ttattttaaa aagaatgatt acttttcttt 2340 tttgacaata ttttaatttc tttttttggt aatatttaaa ttttaatttt ccacgtgata 2400 tgtttaagat aaaaaaatta taagatattt tggtacatat gaagctagcg tttgactata 2460 aattttcaaa tattcttgg c aaatattatt tgagtgaaaa tttggttggg aaatatattt 2520 caccttttta gaaaaatata atttataacc ataagtttta aaaactatta aaactaacta 2580 taagtttata caataacaaa tagtttccat cacactagag caatgtggta atttggagcg 2640 agtgcaacaa gtatcattcc atacat cgtg aagtagacga agcacatgat tttattacct 2700 tgagttaatt tttcatcatt ttcatcaaca atcatatcat tatttaatat tcactaaata 2760 tttcatcact attttggtgt tcacgtaaaa aaattatgta atacaacaca aacaactact 2820 aaaagggtgt ttttgtaaaa gataaaagtt tggggataaaa ttttaatatt caaaagatcc 2880 caaataatga gatttggccc aaatactaga aaaaattag t atttgggaat ttgggatatt 2940 tgccaaaaat gtttgccaaa taatgacaaa ttgtatggac aaacattaaa tatttttttc 3000 ttaaattctg tttgaaatta aattagatca ttctttttaa aacgaaaaaa tattaactaa 3060 agaaataaaa tgtgctaaat aaatattctt gcatttgg cc cctcctgcca attgccagac 3120 ttgaatattg tctctcgtgt tgtttttttt tgtcatcctc gcaattttca cccaaaatca 3180 aacggtccaa ttggatccca aatttcgtat ttcgatg 3217 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T1117-F <400> 5 caaggctgaa accaaaagac attg 24 <210 > 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T1117-R <400> 6 acatttaaat gctttgtcgt ctgc 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 97 -F <400> 7 cctgttaact acttcttgac tatg 24 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 97-R <400> 8 tcagtgtttc acaatctaag tgc 23 <210> 9 <211 > 23 <212> DNA <213> Artificial Sequence <220> <223> 98-F <400> 9 tacagccttc catctatgca aag 23 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223 > 98-R <400> 10 cctatttgtt agacggcgag g 21 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> At1g60640-F <400> 11 tctgacaggg ggaaatgaat cttc 24 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> At1g60640-R <400> 12 acttcattga aagagctatc ttcactctc 29 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 01-F <400> 13 tgtgtgctta ttgccactta tata 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 01-R <400> 14 tgttcttgag gaaaacaata gagc 24 <210 > 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 02-F <400> 15 tccaattcac gttaggtaat taag 24 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence < 220> <223> 02-R <400> 16 ctgtacctcc tcattatatt tgg 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 04-F <400> 17 ggctcaggat caagttcaat atg 23 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 04-R <400> 18 cttcacatca ctctcaggca ac 22 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 09-F <400> 19 atagatcaat ttagtgcagt agtg 24 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 09-R <400> 20 agtaattttg agtaatggag gaag 24 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 10-F <400> 21 gtgaatgtgt aagagagttg tcc 23 <210> 22 <211> 23 <212> DNA <213 > Artificial Sequence <220> <223> 10-R <400> 22 actattgtgt tgtgccgaaa tcc 23 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 13-F <400> 23 gaattattct gcttctaagg ctc 23 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 13-R <400> 24 atgtctttcg agtactagtc atg 23 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RnaB-F <400> 25 gaatatgatt tatcaacaag tatcc 25 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RnaB-R <400 > 26 gcatgattgg tgttagcggg ac 22 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TG276-F <400> 27 ctggtgctgc tcttgctcat ac 22 <210> 28 <211> 22 <212 > DNA <213> Artificial Sequence <220> <223> TG276-R <400> 28 aatccctttc gatgacgtga ac 22 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RnaP-F <400> 29 gttcatccgc aacctattgt acc 23 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RnaP-R <400> 30 tgcactctca ttagctacat cac 23 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> U582871-F <400> 31 taacagaatt aattcgtgtg attg 24 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> U582871 -R <400> 32 agcaagaaac gtgtctgttc ag 22 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 26D15-3-F <400> 33 aaagagttgc cttctttaac atg 23 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 26D15-3-R <400> 34 ggtttgtata tccctcctca tc 22 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 2B-F <400> 35 cgtggactac ctaactagca gac 23 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 2B-R <400> 36 tgtgtgttgg acgtaggcca tg 22 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> T0888-F <400 > 37 gaatcggagc aagtaactca gtc 23 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T0888-R <400> 38 gagtttgagg ttactcgtta tgtc 24 <210> 39 <211> 23 <212 > DNA <213> Artificial Sequence <220> <223> T1238-F <400> 39 gaattcttgc aagctacatg agg 23 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T1238-R <400> 40 acaagatgtc ctttcttaat agtg 24 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> U562933-F <400> 41 gcacacatgg ttacctgatt acc 23 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> U562933-R <400> 42 ctgagtgacc aaatgtgtgt acc 23 <210> 43 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 46485 -3-F <400> 43 gggagataac tcgagggctc ag 22 <210> 44 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 46485-3-R <400> 44 cgccaacttt atacccaatg aatgtgc 27 <210 > 45 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> U572717-F <400> 45 atccatccga ttatcccttc cg 22 <210> 46 <211> 22 <212> DNA <213> Artificial Sequence < 220> <223> U572717-R <400> 46 tgagactgaa ctcgccatgc tc 22 <210> 47 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> U572498-F <400> 47 gatcaattcg tgtcaattac agg 23 <210> 48 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> U572498-R <400> 48 gtgatcaagt tggctgtgtg aag 23 <210> 49 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 2A-F <400> 49 cttcatgatt acactttgcc attat 25 <210> 50 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 2A-R <400> 50 gattacctca ggagggctta acc 23 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> U569822-F <400> 51 aacggagaaa gtgggatcag c 21 <210> 52 <211> 22 <212> DNA <213 > Artificial Sequence <220> <223> U569822-R <400> 52 ttggcgcttt gtgagcctgt ag 22 <210> 53 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CT196-F <400> 53 acaacagctg ctattactac cg 22 <210> 54 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CT196-R <400> 54 ttcttgccgg agatagaggt gg 22 <210> 55 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> T0869-F <400> 55 gttacgctag ggcatttccg tc 22 <210> 56 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> T0869-R <400 > 56 gataccaatc gaagtaacag cag 23 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SSR40-F <400> 57 tgcaggtatg tctcacacca 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SSR40-R <400> 58 tgcaacaact ggataggtcg 20 <210> 59 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> bc2.1-attB1 <400> 59 ggggacaagt ttgtacaaaa aagcaggctt gaagacggag tcagattaag 50 <210> 60 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> bc2.1-attB2 <400> 60 ggggaccact ttgtacaaga aagctgggtg agtctg acta gattaaatg 50 <210 > 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> bc2.1-mTrcHis2-F <400> 61 ttcatgatgg ataagtatat tg 22 <210> 62 <211> 21 <212> DNA <213 > Artificial Sequence <220> <223> bc2.1-mTrcHis2-R <400> 62 ttaatctgac tccgtcttca c 21 <210> 63 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> qRT-F <400> 63 ctgctcttgc tcataccctt gc 22 <210> 64 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> qRT-R <400> 64 ggattgttgg cgaattgctt cag 23 <210> 65 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> cis1-F <400> 65 aaataattac aagtgttatt aacattg 27 <210> 66 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> cis1 -R <400> 66 tgtttgtatt tataagaagc atcg 24 <210> 67 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> cDNA1-F <400> 67 ttgtcatcct cgcaattttc acc 23 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> cDNA1-R <400> 68 cttaatctga ctccgtcttc acg 23 <210> 69 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CYC -B-AscI-F <400> 69 ggcgcgccat ggaaactctt ctcaagcctt tt 32 <210> 70 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CYC-B-AscI-R <400> 70 ggcgcgccaa ggctctctat tgctagattg cc 32 <210> 71 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> bc2.1-AscI-F <400> 71 ggcgcgccat gcttcttata aatacaaaca at 32 <210> 72 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> bc2.1-AscI-R <400> 72 ggcgcgccat ctgactccgt cttcacggga gg 32 <210> 73 <211> 35 <212> DNA <213> Artificial Sequence < 220> <223> bc2.1-GFP-F <400> 73 ggcgcgcctc tagaatgctt cttataaata caaac 35 <210> 74 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> bc2.1-GFP- R <400> 74 ggcgcgccgt cgacatctga ctccgtcttc acgg 34 <210> 75 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 35S-F <400> 75 cccaagaagg ttaaagatgc agtca 25 <210> 76 <2 11 > 25 <212> DNA <213> Artificial Sequence <220> <223> 35S-R <400> 76 gctttgaaga cgtggttgga acgtc 25 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223 > PSY1-F <400> 77 tggcccaaac gcatcatata 20 <210> 78 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PSY1-R <400> 78 caccatcgag catgtcaaat g 21 <210> 79 < 211> 21 <212> DNA <213> Artificial Sequence <220> <223> PSY2-F <400> 79 gttgatggcc ctaatgcatc a 21 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> < 223> PSY2-R <400> 80 tcaagcatat caaatggccg 20 <210> 81 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PDS-F <400> 81 gtgcattttg atcatcgcat tgaac 25 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PDS-R <400> 82 gcaaagtctc tcaggattac c 21 <210> 83 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ZDS-F <400> 83 ttggagcgtt cgaggcaat 19 <210> 84 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ZDS-R <400> 84 agaaatctgc atctggcgta taga 24 <210> 85 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Z-ISO-F <400> 85 tatgaggatt accaggcatc c 21 <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Z-ISO-R <400> 86 acagcgtgtg agctaagcac 20 <210> 87 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CRTISO-F <400> 87 ttttggcgga atcaactacc 20 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CRTISO-R <400> 88 gaaagcttca ctcccacagc 20 <210> 89 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CYC-B-F <400> 89 tgttatgag gaagagaaat gtgtgat 27 <210> 90 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CYC-B-R <400> 90 tcccaccaat agccataaca tttt 24 <210> 91 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LCY-B-F <400> 91 tcgttggaat cggtggtaca g 21 <210> 92 <211> 21 <212> DNA <213 > Artificial Sequence <220> <223> LCY-B-R <400> 92 agctagtgtc cttgccacca t 21 <210> 93 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CCD1A-F <400> 93 tggattatga caaacgattg acg 23 <210> 94 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CCD1A-R <400> 94 ttggatataa ccctataagt gatg 24 <210> 95 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CCD1B-F <400> 95 ggattacgat aaaaggttgc aac 23 <210> 96 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CCD1B-R <400 > 96 cttggatatg actctatatg tagc 24 <210> 97 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CRTR-b1-F <400> 97 agatgggcac acaaagcact g 21 <210> 98 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CRTR-b1-R <400> 98 gcgaaaacgt cgttcagctc 20 <210> 99 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CRTR-b2-F <400> 99 tttcagcctc cgctagttcc 20 <210> 100 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CRTR-b2-R <400> 100 cggagagaag aacagaaccg g 21 <210 > 101 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ZEP-F <400> 101 ttgatggtat ttctggcaac tgg 23 <210> 102 <211> 22 <212> DNA <213> Artificial Sequence < 220> <223> ZEP-R <400> 102 accaacagca cgtgcaagga tc 22 <210> 103 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VDE-F <400> 103 tgcctgaaac gattatacct gag 23 <210> 104 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> VDE-R <400> 104 ccgctctcct tcttccactt tc 22 <210> 105 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NCED1-F <400> 105 gggatggttt agaatatacg tcc 23 <210> 106 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NCED1-R <400> 106 ggttctctga gaatctctca cac 23 <210> 107 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 18S-F <400> 107 cggagaggga gcctgagaa 19 <210> 108 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 18S-R<400> 108 cccgtgttag gattgggtaa ttt 23

Claims (10)

Tomatoes with increased lutein content and suppressed expression of BC2.1, a gene encoding tomato lycopene epoxidase. The tomato with increased lutein content according to claim 1, wherein the expression of ORF2 of BC2.1 is suppressed. The tomato with increased lutein content according to claim 1, wherein the expression of BC2.1 is suppressed by administering an RNAi construct, and the RNAi construct is RNAi against the BC2.1 gene. The tomato with increased lutein content according to claim 1, wherein BC2.1 expression is suppressed by introgressing a sequence encoding BC2.1 containing a mutation into the tomato. The tomato with increased lutein content according to claim 4, wherein the sequence encoding BC2.1 containing the mutation is selected from the nucleic acid sequences represented by SEQ ID NOs: 2 to 4. The tomato with increased lutein content according to claim 3, wherein the RNAi structure is produced using RNAi-F having a sequence shown in SEQ ID NO: 59 and RNAi-R having a sequence shown in SEQ ID NO: 60. . The tomato with increased lutein content according to claim 1, wherein expression of BC2.1 is suppressed and LCY-E is expressed. According to claim 1, Solanum. Tomatoes with increased lutein content produced by crossing subIL2-2-1, an introgression line of pennellii LA716, with IL12-2. A method for increasing the content of lutein in tomatoes, comprising: inhibiting the expression of the BC2.1 gene encoding lycopene epoxidase. A method for producing tomatoes with increased lutein content, comprising: inhibiting the expression of the BC2.1 gene encoding lycopene epoxidase.