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Short Communication

Isolation and characterization of a muskmelon cDNA encoding Lycopene Beta-cyclase

State Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai'an 271018, China

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Hui Hao 1, Leyuan Ma 1, Hongzi Cong, Qiang Li, Xiyan Yu

Article history: Accepted 18 April 2012 Available online xxxx Keywords: Cucumis melo CmLcyb1 Gene expression Betacarotene

Lycopene Beta-cyclase (LCY-B) is thought to play a critical role in Betacarotene synthesis in fruit. A fulllength cDNA clone encoding Lycopene Beta-cyclase was isolated from muskmelon (Cucumis melo L.) by RTPCR and RACE. The clone, designated CmLcyb1, contains 1871 nucleotides, with an open reading frame of 1512 nucleotides. The deduced 504-amino-acid sequence showed high identities with other plant Lycopene Beta-cyclases. Real time quantitative RT-PCR analysis indicated that CmLcyb1 was expressed in all tissues and organs of muskmelon inbred M01-3 with white mesocarp and, Homoka, an orange mesocarp cultivar. The expression levels of CmLcyb1 in roots, stems, leaves and owers in the two genotypes differed little. The expression level was highest in mature fruit of Homoka and was much higher than that in mature fruit of M013. Moreover, the mRNA level of CmLcyb1 was very low in fruits before fruit-size xation and increased dramatically in the size-xed fruits of these two genotypes. The mRNA levels of CmLcyb1 during fruit development of Homoka were all higher than those of M01-3. Interestingly, Betacarotene content showed almost the same change trend as mRNA levels during fruit development in these two genotypes, suggesting that Betacarotene accumulation may be linked to the CmLcyb1 transcript level in muskmelon fruit. 2012 Elsevier B.V. All rights reserved.

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1. Introduction

Muskmelon (Cucumis melo L.) is one of the economically important and widely cultivated crops in the world. Muskmelon, as one of the most consumed fruits, provides abundant Betacarotene (Lester and Eischen, 1996), which is essential in human nutrition. Beta-carotene's action can be indirect, as a precursor to vitamin-A which is important in reducing the progression of diseases such as age-related macular degeneration, certain types of cancers, and cardiovascular diseases (Aluru et al., 2008; Butrum et al., 1988; DellaPenna and Pogson, 2006; Paine et al., 2005). Thus, The US Cancer Institute has recommended that people increase their intake of high Beta -carotene content foods (Craft and Wise, 1993). The elucidation of the carotenoid biosynthetic pathway and the enormous progress in gene cloning have provided the tools necessary to embark upon genetic engineering of the pathway (Fraser and Bramley, 2004; Phadwal, 2005; Tanaka and Ohmiya, 2008; Tao et al., 2006; Tucker, 2003). The genes and enzymes involved in the


biosynthesis of carotenoid pigments are highly conserved in plants. The genes and cDNAs encoding nearly all the required enzymes in plants have been identied and sequenced, and their products have been characterized (Bartley et al., 1999; Cunningham and Gantt, 1998; Hirschberg, 2001). Lycopene Beta-cyclase catalyzes a two-step reaction that creates one Betaionone ring at each end of the lycopene molecule to produce Betacarotene. LCY-B plays a key role in the synthesis of Beta-carotene in plants (Bang et al., 2007). Recently, the LCY-B gene has been isolated and characterized from bacteria (Matsumura et al., 1997; Tao et al., 2007) and some higher plant species (Alquezar et al., 2009; Dwamena et al., 2009; Kang et al., 2010; Skelton et al., 2006). However, studies on the Lycopene Beta-cyclase gene from muskmelon have not been reported until now. In this report, we describe the molecular cloning and characterization of CmLcyb1 from muskmelon. In addition, the differential expression of CmLcyb1 was analyzed in various tissues and fruit development stages of two genotypes of C. melo. 2. Materials and methods 2.1. Plant material and tissue sampling Muskmelon inbred line M01-3 and cv. Homoka were grown in a greenhouse on an experimental farm of Shandong Agricultural University in Tai'an, China from Mar. through June 2010, with spacing of 50 cm between plants and 120 cm between rows. Average day/ night temperatures were about 30 C/20 C. Average daylight was about 12 h. Fertilizer was applied at two stages, a pre-plant broadcast application of 900 kg ha 1 of 14 N6.1 P29.9 K, followed by a

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Abbreviations: Bp, base pair(s); cDNA, DNA complementary to RNA; cv., cultivar; DAP, day after pollination; DNA, deoxyribonucleic acid; HPLC, high-performance liquid chromatography; mRNA, messenger RNA; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; RACE, rapid amplication of cDNA ends; RNA, ribonucleic acid; UV, ultraviolet. Corresponding author at: State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, Shandong 271018, China. Tel.: + 86 0538 8242201; fax: + 86 0538 8246017. E-mail address: (X. Yu). 1 These authors contributed equally to the paper. 0378-1119/$ see front matter 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.04.059

Please cite this article as: Hao, H., et al., Isolation and characterization of a muskmelon cDNA encoding Lycopene Beta-cyclase, Gene (2012), doi:10.1016/j.gene.2012.04.059

H. Hao et al. / Gene xxx (2012) xxxxxx

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2.2. Extraction and purication of RNA Total RNA was isolated using the guanidine isothiocyanatephenol chloroform method, as described by Sambrook et al. (1989), with modication. Frozen tissue samples (510 g) were ground to powder in liquid N2 using a mortar and pestle. Samples of the homogenized, powdered tissue were transferred to 50-ml screw-cap centrifuge tubes pre-chilled on ice, followed by immediate addition of 15 ml solution D (4 M guanidine isothiocyanate, 0.025 M trisodium citrate dehydrate, 0.5% lauryl sarcosine) and of 1.5 ml of 2 M sodium acetate, pH 4.0, 15 ml of phenol equilibrated with distilled water and 3 ml of 49 chloroform:1 isopropyl alcohol (v/v). After vortexing and incubation for 15 min on ice, the samples were processed according to Sambrook et al. (1989). Crude RNA preparations were then treated with DNase (TaKaRa Bio Inc., Kyoto, Japan) to degrade genomic DNA, extracted with 1 phenol:1 chloroform (v/v), and total RNA was precipitated by addition of 1 volume isopropyl alcohol plus 0.1 volume of 3 M sodium acetate, pH 5.5. The precipitated RNA was pelleted by centrifugation, washed with cold 70% ethanol, and resuspended in diethyl pyrocarbonate-treated water. Quality of the extracted RNA was checked by agarose gel electrophoresis and RNA was quantied spectrophotometrically (UV 2450, Shimadzu Corp., Kyoto, Japan).

sidedress application of 150 kg ha 1 N at the owering stage. Irrigation by furrows was applied as needed. Freshly opened female owers were tagged on the day of hand-pollination to identify fruit of known age and one fruit per plant was allowed to develop. Fruits at different stages (Fruits of inbred M01-3 were at 5, 10, 15, 20 and 25 DAP; fruits of Homoka were at 9, 18, 27, 36 and 45 DAP) and mature fruits were harvested. Commercial maturity of M01-3 and Homokas occurs at 30 DAP and 54 DAP, respectively. Five fruits from each stage were pooled to isolate total RNA. A portion of each fruit was diced into small pieces and used for Beta-carotene determination, carried out on duplicate samples. Each experiment was repeated three times. For each replication, ve fruits were used. Vegetative tissues, including leaves, stems, and roots were sampled, along with owers at bloom, and harvested simultaneously from ve 7- to 8-week-old plants. All tissues were quickly frozen in liquid nitrogen and stored at 80 C until later use.

and reverse primer (5-CTGATCTAGAGGTACCGGATCC-3) provided with the kit. According to the manufacturer's instructions, PCR was performed under the following conditions: cDNA was denatured at 94C for 2min, followed by 35 cycles of amplication (94C for 30s, 55C for 30s and 72C for 1min) and 7min at 72C. Based on the sequence of the initial C. melo LCY-B gene, the specific primers 2, 3, 4 (primer 2 5-GCCTGGATTGTAGGGCTTATCAT-3, primer 3 5TGGTCACTCCATCATTGCAAA-3, primer 4 5GAGCTGCTTTCTATTAACCCTCCC-3) were designed to amplify the 5 end of CmLcy1 gene using 5 RACE system for rapid amplication of cDNA ends, version 2.0 (Invitrogen Corp., Carlsbad, CA). The 5 RACE was performed according to the manufacturer's instructions. All PCR products were puried, then cloned into pMD18-T vector (TaKaRa), respectively and sequenced by TaKaRa Bio Inc. The DNAman 4.0 (Lynnon Biosoft, Quebec, Canada) and NCBI web sites were used to analyze DNA and protein sequences.

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2.4. Real time quantitative RT-PCR

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Specic gene primers were designed from the cDNA sequence of CmLcyb1 to be analyzed using the Primer 5.0 software (PE Applied Biosystems) following the manufacturer's guidelines. For quantitative real-time PCR analysis, the actin gene from muskmelon (GenBank FJ763186) was used as an internal constitutively expressed control (house-keeping gene). The primers (Table 1) were synthesized by Invitrogen and used at 0.2 M nal concentration. Quantitative realtime PCR was performed using the RealMasterMix (SYBR Green) kit (TIANGEN, China) and quantied the PCR amplication according to the manufacturer's instructions. Amplication was performed on an iCycler iQ multicolor real-time PCR detection system (Bio-Rad, Hercules, USA). Three replicates were run for each sample. The PCR cycles were as follows: 1 cycle of 2 min at 95 C, followed by 40 cycles each of 15 s at 95 C, 15 s at 55 C and 31 s at 68 C. Data was analyzed using the 2 CT method (Livak and Schmittgen, 2001). 2.5. HPLC analysis of Beta-carotene The extraction and analysis of Beta-carotene were performed exactly as described by Lester and Eischen (1996) with slight modications. Samples were extracted at least twice and each analytical determination was replicated. All operations were carried out on ice under dim light to prevent photodegradation and structural change of Beta-carotene. Fruit tissue (12 g) was homogenized with ice cold acetone/tetrahydrofuran (1:v/v); 10 mg butylated hydroxytoluene; and 1.2 mg Mg2CO3. Carotenoids were partitioned into petroleum ether, dried over Na2SO4 and reduced in volume using a rotary evaporator at 32 C. Carotenoids were resuspended to 2 ml in mobile phase, ltered through a 0.45 m PTFE lter (Sigma Chemicals, St Louis, MO, USA) and stored at 80 C until HPLC analyses. Beta-carotene was separated by a 510 Waters HPLC equipped with Waters PDA detector 2487, C18 Nova-Pak (Waters, Milford, MA, USA) column (250 4.6 mm) with a mobile phase of 60% acetonitrile:10% methylene chloride:20% methanol (v/v/v) and detected at 450 nm. Quantication was performed by integrating the peak areas of the HPLC results using Millennium chromatography software

2.3. Cloning of Cmlcyb1 complete cDNA

Total RNA isolated from the mature fruit of C. melo M01-3 plants was used for the initial cloning of LCY-B cDNA fragments, including products of 3 and 5 RACE (Rapid Amplication of cDNA Ends). As the initial step in cloning a C. melo LCY-B gene, degenerate primers (P1: 5-TCTYGACACGACTTGGTYCG-3, P2: 5 TGYCTCCTCCTTTCDATGGGC-3) were designed based on the conserved domain of LCY-B genes from other plants in GenBank (GenBank accession numbers for the plant LCY-B sequences are: ABM90918, AAN86060, ABD91578, ACL27573 and ACT09103). RTPCR was performed according to the protocol of RNA PCR Kit (AMV) Ver.3.0 (TaKaRa). The rst cDNA was synthesized by reverse transcription using reverse transcriptase with 1g RNA as template and with oligo (dT)-adaptor primer as rst strand primer. The RT reaction program was 42C for 30min, 99C for 5min and 5C for 5min, 1 cycle. PCR was performed by running the following program: 94C pre-denature for 5min, 94C for 1min, 55C for 1min and 72C for 1min, 30 cycles; 72C for 10min. The PCR product was separated by electrophoresis in 1.0% agarose gel. A total of 1g RNA from muskmelon fruit were used to convert mRNAs into cDNAs using a 3 RACE kit (TaKaRa) provided with AMV reverse transcriptase and a universal oligo (dT) containing adaptor primer (5-CTGATCTAGAGGTACCGGATCCT17-3). The partial CmLcyb1 gene from the 3 end was then amplied by a pair of PCR primers: the gene specic forward primer 1 (5-AAGAGTTGTTGGAATTGGTGGAAC-3)

Table 1 t1:1 Primers sequences used in Real-time quantitative RT-PCR assays of genes CmLcyb1 and actin from muskmelon (GenBank FJ763186). t1:2 Primer Sequence t1:3 CmLcyb1-F CmLcyb1-R Actin-F Actin-R 5-TTCTGCACCCCATTAGGATTC-3 5-GAAACGACAGAGCAACAGAAAC-3 5-TGGATTTGCTGGTGACGATG-3 5-ACCTCTTTTCGACTGAGCTTC-3 t1:4 t1:5 t1:6 t1:7

Please cite this article as: Hao, H., et al., Isolation and characterization of a muskmelon cDNA encoding Lycopene Beta-cyclase, Gene (2012), doi:10.1016/j.gene.2012.04.059


H. Hao et al. / Gene xxx (2012) xxxxxx

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(Waters, Milford, MA). The data were analyzed using factorial analysis of variance. 3. Results 3.1. Cloning and characterization of CmLcyb1 To isolate LCY-B cDNA from muskmelon, we rst designed a pair of degenerate primers according to the conserved domain of LCY-B amino acid sequences from other plant species. An 820 bp cDNA fragment was amplied by RT-PCR. A 740 bp and a 590 bp cDNA fragments were produced by 5 RACE and 3 RACE, respectively. After sequence conrmation, an 1871 bp cDNA clone including a fulllength coding region was isolated from muskmelon and named CmLcyb1 (GenBank accession: GU457407). The deduced amino acid sequence of CmLcyb1 contains 504 amino acid residues. The alignment analysis used by DNAman 4.0 and the NCBI web site showed that CmLcyb1 shared 95.44% identity with watermelon Lcyb (GenBank accession: ABM90918), 82.74% identity with citrus Lcyb (GenBank accession: AAN86060), 80.75% identity with papaya Lcyb (GenBank accession: ABD91578), 79.13% identity with carrot Lcyb (GenBank accession: ABB52071) and 75.89% identity with arabidopsis Lcyb (GenBank accession: AAA81880). At the amino acid level, CmLcyb1 is most closely related to watermelon Lcyb and then citrus Lcyb (Fig. 1). 3.2. CmLcyb1 expression analysis and Betacarotene content during fruit development and ripening To determine the pattern of CmLcyb1 expression in different muskmelon tissues, real-time RT-PCR analysis was performed. The result indicated that CmLcyb1 was differentially expressed in root, stem, leaf, ower and fruit tissues in both M01-3 and Homoka. For M01-3, the mRNA level was highest in the ower, then in mature fruit, leaf and stem, respectively, and was lowest in the root. However, the

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Fig. 1. Alignment of predicted amino acid sequences of LCYB genes from different plants. The GenBank accession numbers for the plant LCYB sequences are: Citrullus lanatus, ABM90918; Citrus unshiu, AAN86060; Carica papaya, ABD91578; Daucus carota, ABB52071; and Arabidopsis thaliana, AAA81880. This alignment was produced using DNAman 4.0 program. Conserved residues are shaded in black. Dark gray shading indicates similar residues in ve out of six of the sequences and clear gray shading indicates similar residues in three out of six of the sequences.

Please cite this article as: Hao, H., et al., Isolation and characterization of a muskmelon cDNA encoding Lycopene Beta-cyclase, Gene (2012), doi:10.1016/j.gene.2012.04.059


expression level of CmLcyb1 in mature fruit of Homoka was the highest, and more than 4 times higher than that in mature fruit of M01-3. The mRNA levels in other tissues of Homoka were similar to those of M01-3 (Fig. 2). To further investigate the developmental regulation of CmLcyb1 in fruit mesocarp tissues, real-time RT-PCR analysis was carried out using different mesocarp tissues from young to ripening fruits. The result showed that CmLcyb1 mRNA levels were low in the fruits before fruit size xation (25 DAP and 45 DAP in M01-3 and Homoka, respectively) but increased dramatically in the size-xed fruit then decreased again in the mature fruits. Overall, the expression levels of CmLcyb1 during the fruit development of Homoka were much higher than those of M01-3 (Fig. 3A). To demonstrate the relationship of CmLcyb1 expression level and Betacarotene content during muskmelon fruit development, Beta carotene content was analyzed. The results indicated that very low concentrations of Betacarotene were present in fruits before fruit size xation of both M01-3 and Homoka. However, a dramatic increase of Betacarotene in the size-xed fruits was observed, followed

Fig. 2. Patterns of CmLcyb1 expression in different tissues of Cucumis melon determined by Real-time quantitative RT-PCR. Total RNA prepared from root, stem, leaf, ower and mature fruit of M01-3 and Homoka. Root, stem, leaf and ower tissues were harvested simultaneously from ve 7- to 8-week-old plants and mature fruits were harvested as they mature from another ve 12- to 16-week-old plants.


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Beta-carotene content (g g-1 FW)







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Fig. 3. CmLcyb1 expression (A) and carotene content (B) during fruit development of M01-3 and Homoka. Fruit samples were harvested at six stages, M01-3 is 5, 10, 15, 20, 25 DAP and mature fruits, respectively and Homoka is 9, 18, 27, 36, 45 DAP and mature fruits, respectively. Commercial maturity of M01-3 and Homoka occurs at 30 DAP and 54 DAP, respectively. Five fruits from each stage were pooled to isolate total RNA. A portion of each fruit was diced into small pieces and used for -carotene determination.

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by a decrease in the mature fruits. As with the expression of CmLcyb1, Betacarotene content throughout the fruit development of Homoka was much higher than that of M01-3 (Fig. 3B). 4. Discussion

In higher plants, carotenoids provide distinct yellow, orange, and red colors to certain organs, such as owers and fruits, to attract animals for pollination and for dispersal of seeds. In these tissues, unique carotenoids synthesized as secondary metabolites accumulate to high concentrations and are stored in the chromoplasts. The Lcyb gene encodes an enzyme that determines the colors of owers and fruit by regulating the proportions of Betacarotene and its derivatives. Bang et al. (2007) demonstrated that Lcyb is the genetic determinant for canary yellow (active Lcyb) or red (reduced Lcyb) esh color of watermelon. In wild type tomato, Lcyb is expressed highly in ower, whereas in fruits expression is low and limited to a short period around the breaker stage (Ronen et al., 1999, 2000). In our study, we found that CmLcyb1 expression is higher (2 CT value is 1.84 0.41) in owers than in mature fruits (2 CT value is 1.55 0.13) of M01-3 with white mesocarp (Fig. 2). In contrast to M01-3, CmLcyb1 expression level in mature fruit (2 CT value is 6.39 0.31) of Homoka, with orange mesocarp, was signicantly higher than in ower (2 CT value is 1.96 0.26). Moreover, CmLcyb1 expression levels were much higher during the fruit development of Homoka than those of M01-3 (Fig. 3A). Betacarotene content during the fruit development of Homoka was much higher than that of M01-3 and Betacarotene content in mature fruit of Homoka was almost 4 times higher than in mature fruit of M01-3, the result is in accordance with previous studies on muskmelon (Lester and Eischen, 1996) and watermelon (Tadmor

et al., 2005). Moreover, Betacarotene contents in M01-3 and Homoka reach a maximum level just before the mature stage and then decrease from 8.3 g mg 1 to 3.5 g mg 1 fresh weight for M01-3, and from 42.1 g mg 1 to 20.2 g mg 1 for Homoka (Fig. 3B). This result is similar to those described in the non-red capsicum varieties by Ha et al. (2007). In their study, they indicated that Betacarotene in the full ripe fruit appeared to be mainly converted into capsanthin and small amounts of zeaxanthin in the pepper. It is not clear about fruit carotenoids accumulation in muskmelon and we require further investigation to understand it. Cloning genes for carotenoid biosynthesis enzymes has paved the way for genetic manipulation of the pathway in crop plants to improve their nutritional value. Some successful attempts to alter the pathway have been recently reported (Jayaraj et al., 2008; Shewmaker et al., 1999; Ye et al., 2000). Diretto et al. (2007) silenced the non-heme Betacarotene hydroxylases CHY1 and CHY2 in the potato tuber, and they found that the total carotenoids and Betacarotene levels were increased dramatically in CHY silenced tuber. Ronen et al. (2000) used similar genetic techniques to increase Betacarotene in tomato fruit. Their results showed that the orange-colored Beta (B) tomato genotype accumulates high levels of Betacarotene in addition to low levels of lycopene. This phenotype is caused by higher expression levels of chromoplast-specic Lycopene Betacyclase, a single dominant gene that increases carotene in the fruit than in wild-type tomatoes, in which lycopene cyclase transcript levels are signicantly diminished during fruit ripening. Zhu et al. (2003) indicated that during ower development of Gentiana lutea, the increased transcript levels of Lycopene Betacyclase and decreased levels of lycopene -cyclase correspond well with a diversion of metabolic ow away from -carotene and its hydroxylation product lutein and into the Betabranch leading to the higher proportions of Betacarotene and derivatives. Our present results indicate that CmLcyb1 transcripts are lower in young muskmelon fruits, but rapidly accumulate in size-xed fruit and mature fruit (Fig. 3A). This pattern of CmLcyb1 expression in mesocarp tissues is in accordance with the alteration of Betacarotene accumulation during the fruit development of muskmelon (Fig. 3B). Taken together, these data led us to conclude that the Betacarotene content in muskmelon fruit is correlated to the Lcyb transcript level. Therefore, increasing Lcyb expression might be an important regulatory event of Betacarotene accumulation during melon fruit ripening. Currently we are overexpressing CmLcyb1, which will help reveal the potential application of CmLcyb1 in controlling the quality of muskmelon fruit.

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Please cite this article as: Hao, H., et al., Isolation and characterization of a muskmelon cDNA encoding Lycopene Beta-cyclase, Gene (2012), doi:10.1016/j.gene.2012.04.059

Acknowledgments We would like to acknowledge Professor Robert Turgeon in Cornell University critically reading this manuscript. This work was supported by the National Natural Sciences Foundation of China (No. 30471191 and 30871719). References
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Please cite this article as: Hao, H., et al., Isolation and characterization of a muskmelon cDNA encoding Lycopene Beta-cyclase, Gene (2012), doi:10.1016/j.gene.2012.04.059