ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 4, с. 585-593
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
CLONING AND EXPRESSION PROFILE OF 1-DEOXY-D-XYLULOSE 5-PHOSPHATE REDUCTOISOMERASE GENE FROM AN OIL-BEARING ROSE1 © 2014 H. Wang, L. Yao
Aromatic Plant R&D Centre, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
Received April 4, 2013
The components of rose essential oil are mainly monoterpene alcohols, predominantly synthesized through the methylerythritol 4-phosphate (MEP) pathway in plants. 1-Deoxy-D-xylulose 5-phosphate reductoi-somerase (DXR) is specified to be a first committed enzyme of the MEP pathway. In order to understand better the role of DXR in the rose essential oil biosynthesis at the molecular level, the full-length cDNA of DXR sequence (designated as RhDXR) was isolated from an oil-bearing rose hybrid Rosa cv. Zizhi and characterized, and the expression profile of it was investigated. Essential oils of rose Zizhi and the other five oil-bearing roses were distilled to evaluate the relationship between the expression of DXR gene and oil yield rate. The full-length cDNA of RhDXR was 1915 bp in length, comprised an open reading frame of 1419 bp, encoding an enzyme of 472 amino acids. A comparative analysis with DXRs of selected species from bacteria to higher plants revealed three conserved domains: a conserved cleavage site for plastids, an extended Pro-rich region, and a highly conserved NADPH oxidase-binding motif existing in the N-terminal region, like in other higher plant species. The relative expression levels of the DXR gene were determined in various tissues: the receptacle, leaf, sepal, pistil, stamen, and petal (in the order of decreasing expression level), and at different flowering stages (flower bud, flower in half bloom and full bloom). Six cultivars could be classified into two groups according to flower color, and within each group there was a positive correlation between the expression level of DXR gene and oil yield rate.
Keywords: Rosa - DXR - expression profile - essential oil
DOI: 10.7868/S0015330314040204
INTRODUCTION
Rose essential oil is extracted from a type of oil-bearing rose of genus Rosa. It is widely used in perfume, culinary, and medicinal industries for its elegant fragrance and multiple pharmacological activities. But the oil content in rose flowers is low in all oil-bearing rose cultivars, which makes it one of the most expensive essential oils on the world market. The components of rose essential oil are mainly monoterpene alcohols, including citronellol, geraniol, nerol, linalool, etc. So, the research of terpene metabolic pathway and the key catalyzing enzymes is of high importance to
1 This text was submitted by the authors in English.
Abbreviations: DMAPP - dimethylallyl diphosphate; DXR - 1-deoxy-D-xylulose 5-phosphate reductoisomerase; GAPDH -glyceraldehyde-phosphate dehydrogenase; IPP - isopentenyl diphosphate; MEP - methylerythritol 4-phosphate; MVA - me-valonate pathway.
Corresponding author: Yao Lei. Aromatic Plant R&D Centre, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China. Fax: 86-021-6478-5710; e-mail: YaoLei@sjtu.edu.cn
finding out the biosynthesis mechanism and further regulation of essential oil production.
All terpenoids are derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are formed by two pathways, the mevalonate (MVA) pathway in the cytoplasm and the methylerythritol 4-phosphate (MEP) pathway in the plastids of higher plants. Monoterpenes are mainly formed through the MEP pathway [1]. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the key catalyzing enzyme of the second step of the MEP pathway, in which 1-deoxy-D-xylulose 5-phosphate (DXP) is converted into 2-C-methyl-D-erythritol 4-phosphate (MEP). Because DXP is an intermediate not only for IPP and DMAPP biosyntheses but also for thiamine and pyridoxol biosyntheses [2-4], the reaction catalyzed by DXR is actually the first committed step of the MEP pathway [5]. So, DXR is specified to be the most important upper stream catalyzing enzyme of the MEP pathway.
Until now the DXR gene has been successfully cloned fromArabidopsis thaliana [6], Mentha pipertia [7],
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Catharanthus roseus [8], Ginkgo biloba [9], Camptothe-ca acuminate [10], etc. However, there was little research about this gene in rosaceous plants.
As the most important upper stream rate-limiting enzyme of the MEP pathway, DXR is also the most important terpenoid metabolic engineering target. Overexpression of DXR in transgenic peppermint plants has led to an increase in essential oil monoter-penes [11]. This was the first report of the successful use of terpenoid metabolic engineering, which showed attractive prospects of the DXR gene use.
The main composition of rose essential oil is similar to mint oil, comprising mainly monoterpenes. The difference is the storage organ and oil-bearing rate, which an average is of 0.03% (v/w) in the rose flower, while 1% (v/w) in the mint leaves. There are mainly five varieties of oil-bearing rose in cultivation in China at present: Rosa rugosa cv. Fenghua, Rosa cv. Zizhi, R.. sertata x R. rugosa Yu et Ku, R. rugosa cv. Plena, R. damascene, Rosa cv. Hetian. They are different bo-tanically and agronomically, including the flower color, flower size, the number of flower petals, flowering habit, flower output, and essential oil yield rate. We selected a widely grown perpetual-flowering rose cv. Zizhi as our plant material. The full length of the DXR gene was cloned by RACE, and its expression profile in various tissues, at different flowering stages, and in different cultivars was investigated in this research. It may provide reference for the metabolic control and further use of the gene.
MATERIALS AND METHODS
Plant materials. The plant material used in the research was the rose cultivar (Zizhi) planted on the farm of the School of Agriculture and Biology, Shanghai Jiao Tong University. cDNAs from the rose petals were used for DXR gene cloning, its expression levels were determined in different tissues, at different flowering stages, and in different cultivars. The different tissues were petal, stamen, pistil, sepal, receptacle, and leaf. The different flowering stages were petals of flower bud (S1), flower in half bloom (S2), and flower in full bloom (S3). The different cultivars were Rosa rugosa cv. Fenghua, Rosa cv. Zizhi, R. sertata x R. rugosa Yu et Ku (cv. Kushui), R. rugosa cv. Plena, R. damascena_1 (dama_1), R. damascena_2 (dama_2), of which the petals of S2 were used. These cultivars were all planted in the garden of Pingyin Rose Research Institute. In the middle of May, when most of the cultivars were in their full flowering, the flowers in half-bloom were handpicked randomly early in the morning before sunrise (6:00-7:00 a.m., to avoid of essential oil loss), quickly taken back in polyethylene bags, and distilled directly by hydro-distillation in a Clevenger apparatus. All the conditions from picking
to distillation were kept consistent as far as possible for all cultivars. There were three repeats for each cultivar, and after that an average oil yield rate was expressed as percentages (v/w).
RNA isolation and cDNA preparation. Total RNA was extracted from above-mentioned tissues, which had been deposited in ultra low temperature refrigerator after being frozen in liquid nitrogen, using the EASYspin Plus Plant RNA Quick Extraction Kit ("Aidlab biotechnologies", China) and treated with RNase-free DNaseI ("TaKaRa Biotechnology", China) to eliminate the residual genomic DNA according to the manufacturer's instructions. Total RNA was then quantified using a spectrophotometer and loaded on a denaturing agarose gel to check the concentration and integrity. Total RNA was reverse-transcribed using the HiFi-MMLV cDNA Kit ("CWBio", China) for full-length cDNA cloning and PrimeScript® RT Master Mix Perfect Real Time ("TaKaRa Biotechnology") for gene expression profile analysis.
Cloning of RhDXR full-length cDNA by RACE.
cDNA from a mixture of the petals of Rosa cv. Zizhi at different flowering stages was used as a template for amplification of the conserved region of DXR gene. Two degenerate oligonucleotide primers pRhDXR-S1 and pRhDXR-X1 (table) were designed according to the conserved sequences of other DXR genes and used for the amplification of the core cDNA fragment of RhDXR under the following conditions: 35 cycles (30 s at 94°C, 30 s at 55°C, and 1.5 min at 72°C), using Premix Ex Taq® Version 2.0 ("TaKaRa Biotechnology"). The amplification product of the conserved region of DXR was subcloned into the pMD18-T vector ("TaKaRa Biotechnology") and transformed into Escherichia coli strain DH5a followed by sequencing. The core fragment was subsequently used to design the gene-specific primers for the cloning of the full-length cDNA of RhDXR by RACE.
The RACE method was used to clone the 3'- and 5'-ends of RhDXR cDNA. The adaptors and primers used are shown in the table. The first-strand cDNA samples for 5'- and 3'-RACE were reverse transcribed using the HiFi-MMLV cDNA Kit with 701 and 702 as primers. Two 3'-gene-specic primers were designed for 3'-RACE. For the first cycle of amplification of 3'-end of RhDXR cDNA, RhDXR3-4, and UPM702 were used. For the nested PCR, primers RhDXR3-2 and 703 were used with the products of the first cycle as templates. The 5'-end of RhDXR cDNA was amplified using RhDXR5-2 and UPM702 as primers for the first cycle and RhDXR5-4 and 703 for the nested PCR. For the amplification of RhDXR cDNA 3'- and 5'-ends, the same enzyme was used as in the amplification of the core fragment, using touch-down PCR (performed with the annealing temperature progressively
The adaptors and primers used for DXR gene cloning and expression profile
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Primer Sequences (5'—3') Purpose
pRhDXR-S 1 AC(T/C)GG(T/C)T
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